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+{"text": "The proteasome is a large multiprotein complex that degrades unwanted cellular proteins. The mechanisms that control this protein-eating machine are being uncovered Inside eukaryotic cells there is a massive protein complex called the proteasome whose raison d'\u00eatre is to remove unnecessary proteins by breaking them down into short peptides. The proteasome is thus responsible for an important aspect of cellular regulation because the timely and controlled proteolysis of key cellular factors regulates numerous biological processes such as cell cycle, differentiation, stress response, neuronal morphogenesis, cell surface receptor modulation, secretion, DNA repair, transcriptional regulation, long-term memory, circadian rhythms, immune response, and biogenesis of organelles . With thWith so many proteins to target for degradation, the activity of the proteasome is subject to multiple levels of regulation. In the overwhelming majority of cases, selected proteins are first \u201clabeled\u201d by the addition of several copies of a small protein tag called ubiquitin and are thus targeted for degradation in the proteasome . The ubiInterestingly, ubiquitination is a reversible process. Even when a protein has been tagged with ubiquitin, its fate is not sealed\u2014specific hydrolytic enzymes called deubiquitinases can remove the ubiquitin label intact . By deubThe proteasome itself is made up of a multiprotein core particle (CP) where proteolysis occurs and a separate multiprotein regulatory particle (RP) that recognizes and prepares substrates for degradation by the CP. A base subcomplex of the RP is pivotal in anchoring polyubiquitin chains during this process, either directly or via auxiliary ubiquitin-binding proteins . The basAn intriguing evolutionary and structural relationship between the proteasome lid and an independent complex, the COP9 signalosome (CSN), may shed light on their respective roles in regulated protein degradation. Both are made up of eight homologous protein subunits that contain similar structural and functional motifs. While a lot is still unknown, the CSN appears to mediate responses to signals  in a manner that is intimately linked to the ubiquitin\u2013proteasome system. This is accomplished, for instance, by suppressing ubiquitin E3 ligase activity or interacting with various components of the pathway . In partSurprisingly, the proteasome itself harbors intrinsic deubiquitination activity . Moreove+/JAMM motif garnered great attention. The trademark of the MPN+/JAMM motif is a consensus sequence E\u2014HxHx(7)Sx(2)D that bears some resemblance to the active site of zinc metalloproteases. Members of this family were predicted to be hydrolytic enzymes, some of which are specific for removal of ubiquitin or ubiquitin-like domains from their targets  and the auxiliary deubiquitinating enzymes (situated in the regulatory particle) is clear-cut: the latter cleave between ubiquitin domains, while the core proteolytic subunits process the target protein itself . HoweverOnce bound to the proteasome, a polyubiquitinated substrate can be unfolded by the RP and irreversibly translocated into the CP. It has been proposed that long polyubiquitin chains commit a substrate to unfolding and degradation by the proteasome, whereas short chains are poor substrates because they are edited by deubiquitinating enzymes, resulting in premature substrate release . ExtendeIdentifying Rpn11 and Csn5 as members of a novel class of metallohydrolases immediately elevates them into promising \u201cdrugable\u201d candidates. Undoubtedly, the molecular structures deciphered by the groups of Deshaies  and Bycr"}
+{"text": "Proteins may be the workhorse of the cell, but when a cell can synthesize one protein in a matter of minutes, chances are some will become obsolete. Though many proteins put in years of productive service, others quickly outlive their usefulness and can even damage the cell. Proteins that help form bone and muscle, for example, function for years while regulators of mitosis and cell proliferation might finish their jobs in seconds. Such short-timers are soon tagged as superfluous by a chain of small proteins called ubiquitin, which marks the proteins for degradation in an enzyme called the proteasome. Once in the proteasome, these proteins are broken down and can then be recycled for more productive ventures.A massive structure by cellular standards, the proteasome consists of multiple subunits, including a cylindrical core particle called 20S, which catalyzes degradation, and regulatory complexes called 19S caps, which form lid and base structures at both ends of the core. While the structure and biomechanics of the 20S core have been well characterized, much less is known about the functional mechanics of the regulatory complexes. The lid--base complex recognizes only ubiquitin-tagged proteins, which are then unfolded so they can enter the proteasome. But first ubiquitin chains must be detached from the protein, a task performed by an enzyme in the proteasome called Rpn11 isopeptidase. How the lid\u2013base complex removes the ubiquitin tag, unfolds the protein, and shuttles it into the proteasome's core is not clear. Now Raymond Deshaies and colleagues present the structure of a homolog of the 19S lid's isopeptidase enzymatic center and provide new insights into these questions.The proteasome Rpn11 subunit contains a key region called the JAMM motif, which Deshaies' lab has shown previously is required for the proteasome to remove ubiquitin tags. For the work discussed in this paper, the researchers set out to understand how the proteasome strips off ubiquitin tags from proteins about to be destroyed by determining the three-dimensional structure of the JAMM motif.Archaeoglobus fulgidus. After determining the structure of the JAMM protein , the researchers discovered that AfJAMM looks nothing like the well-known deubiquitinating enzymes. But the arrangement of a set of amino acids that binds a zinc ion and forms the proposed active site of AfJAMM does resemble that found in a well-known protein-degrading metalloprotease called thermolysin, even though in other respects AfJAMM and thermolysin have very different features. The researchers mutated amino acid residues in another JAMM protein called Csn5  and found that the residues are indeed important for Csn5 function. These results suggest that JAMM does indeed represent a novel family of metalloproteases.The researchers tested many genes to look for a JAMM-containing protein that would crystallize properly and found one in the heat-loving prokaryote As for the wider function of JAMM proteins, the researchers speculate that these proteins are likely to be involved in a variety of important regulatory systems since they appear in life forms that lack ubiquitin and ubiquitin-like proteins. The crystal structure reported in this paper will provide a valuable tool for investigations into the underlying structural and functional mechanisms of these enzymes. And it may have important therapeutic implications. Proteasome inhibitors are promising anticancer therapies\u2014fighting cancer by blocking machinery required by rapidly dividing cells. In the hopes of developing more targeted therapies, scientists are trying to fine-tune their control of the ubiquitin system and the proteasome. Inhibiting the JAMM domain of enzymes like Csn5, which remove ubiquitin-like tags from proteins upstream of the proteasome, for example, might just do the trick."}
+{"text": "The polypeptide ubiquitin is used in many processes as different as endocytosis, multivesicular body formation, and regulation of gene transcription. Conjugation of a single ubiquitin moiety is typically used in these processes. A polymer of ubiquitin moieties is required for tagging proteins for proteasomal degradation. Besides its role in protein degradation, ubiquitin is also engaged as mono- or polymer in intracellular signalling and DNA repair. Since free ubiquitin is present in limiting amounts in cells, changes in the demands for ubiquitin in any of these processes is likely to indirectly affect other ubiquitin modifications. For example, proteotoxic stress strongly increases poly-ubiquitylated proteins at the cost of mono-ubiquitylated histones resulting in chromatin remodelling and altered transcription. Here we discuss the interconnection between ubiquitin-dependent processes and speculate on the functional significance of the ubiquitin equilibrium as a signalling route translating cellular stress into molecular responses. Ubiquitin is abundantly expressed in eukaryotes and can be found in all cell types and tissues with up to 10per cell -6; Figurper cell . A largeUbiquitin contains seven lysine residues, all of which can be used to form poly-ubiquitin chains . Poly-ubA cascade of different classes of enzymes is required for identification and ubiquitylation of proteins . TheSeveral groups have described the existence of various forms of ubiquitin in eukaryotic cells, including free ubiquitin molecules, mono- and poly-ubiquitylated proteins -34. ThesTo monitor the amount of free ubiquitin in living cells the nuclear pore was used as a molecular sieve in a FLIP (Fluorescence Loss In Photobleaching) protocol wherein the GFP fluorescence in either the complete nucleus or cytoplasm was bleached and the effect of GFP-Ub fluorescence in the other compartment was measured. The rationale behind this approach is that proteins up to approximately 50 kDa can passively diffuse through the nuclear pore, whereas larger species (like conjugated ubiquitin) are excluded . The FLIGiven the availability of a small pool of free ubiquitin, free ubiquitin has to be replenished continuously by DUBs. This ubiquitin cycle is essential to supply ubiquitin to substrates in a multitude of nuclear and cytosolic processes Figure . But whaIn principle, histone deubiquitylation could be the result of enhanced deubiquitylation activity following proteotoxic stress. This was assayed using photo-activated GFP-Ub in a protocol where the fate (i.e. the off-rate) of ubiquitin fluorescence was followed in one half of the nucleus. Proteotoxic stress did not affect the off-rate of fluorescent (photoactivated) GFP-Ub from histones indicating the DUBs were not activated by proteotoxic stress . AntibodUbiquitylation of histones is one of the major (and largest) modifications in chromatin. This modification is in mammalian cells mainly found on the core histone H2A. Approximately 5\u201315% of histone H2A is ubiquitylated, and this is associated with condensed DNA and gene silencing -44. H2B Regulated protein turnover by the ubiquitin-proteasome system (UPS) is essential for the survival of eukaryotic cells. This process is required for various cellular processes such as cell cycle control, signalling pathways, transcription and protein quality control. Alterations in the UPS are correlated with a variety of human pathologies, like cancer, immunological disorders, inflammation and neurodegenerative diseases . The exaMany neurological disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are caused by an accumulation of aberrant proteins leading to the formation of protein aggregates, inclusions and plaques. It is not completely clear why the UPS is failing to clear these aberrant proteins. For polyglutamine diseases like Huntington's disease it has been demonstrated that the UPS is unable to clear inclusions -52, and The flux of free ubiquitin between different cellular processes could be a passive mechanism in which unconjugated ubiquitin diffuses intracellular until it is utilized in ubiquitylation processes. However, a recent publication suggest that active factors could actually be involved in channelling ubiquitin from one ubiquitin-dependent process to another with temporally a higher priority . It has Ubiquitin seems more then just a signalling molecule involved in the regulation of various distinct processes in eukaryotic cells. The dynamic behaviour of ubiquitin modifications creates an equilibrium which allows crosstalk between different cellular processes that may allow cells to translate cellular stress to molecular responses by affecting the transcriptome.DUBs, deubiquitylation enzymes; uH2A, ubiquitylated H2A; UPS, ubiquitin proteasome system.The author(s) declare that they have no competing interests.FAS and TAMG conceived the manuscript. JN and NPD revised the manuscript. All authors approved the final version."}
+{"text": "Lead concentration in whole blood (BPb) is the primary biomarker used to monitor exposure to this metallic element. The U.S. Centers for Disease Control and Prevention and the World Health Organization define a BPb of 10 \u03bcg/dL (0.48 \u03bcmol/L) as the threshold of concern in young children. However, recent studies have reported the possibility of adverse health effects, including intellectual impairment in young children, at BPb levels < 10 \u03bcg/dL, suggesting that there is no safe level of exposure. It appears impossible to differentiate between low-level chronic Pb exposure and a high-level short Pb exposure based on a single BPb measurement; therefore, serial BPb measurements offer a better estimation of possible health outcomes. The difficulty in assessing the exact nature of Pb exposure is dependent not so much on problems with current analytical methodologies, but rather on the complex toxicokinetics of Pb within various body compartments . If we are to differentiate more effectively between Pb stored in the body for years and Pb from recent exposure, information on other biomarkers of exposure may be needed. None of the current biomarkers of internal Pb dose have yet been accepted by the scientific community as a reliable substitute for a BPb measurement. This review focuses on the limitations of biomarkers of Pb exposure and the need to improve the accuracy of their measurement. We present here only the traditional analytical protocols in current use, and we attempt to assess the influence of confounding variables on BPb levels. Finally, we discuss the interpretation of BPb data with respect to both external and endogenous Pb exposure, past or recent exposure, as well as the significance of Pb determinations in human specimens including hair, nails, saliva, bone, blood , urine, feces, and exfoliated teeth. Over the last two decades, atmospheric concentrations of lead have decreased significantly around the globe as more and more nations have chosen to remove tetraethylead from gasoline . HoweverAfter Pb enters the body, it can travel along several pathways depending on its source and, by extension, its bioavailability. The fraction of Pb that is absorbed depends mainly on the physical and chemical form, particularly particle size and the solubility of the specific compound. Other important factors are specific to the exposed subject, such as age, sex, nutritional status and, possibly, genetic background . One of Like many other \u201cbone-seeking\u201d elements, Pb from blood is incorporated into calcified tissues such as bone and teeth, where it can remain for years . AccordiPhysiologic differences between children and adults account for much of the increased susceptibility of small children to the deleterious effects of Pb: whereas in adults 94% of Pb body burden is stored in bones and teeth, this proportion is only 70% in children . In addiBiomonitoring for human exposure to Pb reflects an individual\u2019s current body burden, which is a function of recent and/or past exposure. Thus, the appropriate selection and measurement of biomarkers of Pb exposure is of critical importance for health care management purposes, public health decision making, and primary prevention activities.It is well known that Pb affects several enzymatic processes responsible for heme synthesis. Lead directly inhibits the activity of the cytoplasmic enzyme \u03b4-aminolevulinic acid dehydratase (ALAD), resulting in a negative exponential relationship between ALAD and BPb. Pb depresses coproporphyrinogen oxidase, resulting in increased coproporphyrin activity. Pb also interferes with the normal functioning of the intramitochondrial enzyme ferrochelatase, which is responsible for the chelation of iron by protoporphyrin. Failure to insert Fe into the protoporphyrin ring results in depressed heme formation and an accumulation of protoporphyrin; this in turn chelates zinc in place of Fe, to form zinc protoporphyrin. These effects also result in modifications of some other metabolite concentrations in urine (ALA-U), blood, (ALA-B) and plasma (ALA-P), coproporphyrin in urine (CP). The activities of pyrimidine nucleotidase (P5\u2019N) and nicotinamide adenine dinucleotide synthase (NADS) are also modified in blood after Pb exposure. Levels of these various metabolites in biological fluids have been used in the past to diagnose Pb poisoning when direct Pb levels were difficult to obtain in tissues or body fluids  or as inThroughout the last five decades, whole blood has been the primary biological fluid used for assessment of Pb exposure, both for screening and diagnostic purposes and for biomonitoring purposes in the long term. Although BPb measurements reflect recent exposure, they may also represent past exposures, as a result of Pb mobilization from bone back into blood . In thosData collected as part of the U.S. National Health and Examination Survey (NHANES) give the 95th percentile for BPb as 7.0 \u03bcg/dL for children 1\u20135 years of age and as 5.20 \u03bcg/dL for adults 20 years of age and older [U.S. Centers for Disease Control and Prevention . In lighMany studies have reported statistically significant associations between BPb levels and various health effect outcomes. Some, however, have been statistically weak, with the magnitude of the effect relatively small. According to Plasma-Pb likely represents a more relevant index of exposure to, distribution of, and health risks associated with Pb than does BPb. Indeed, from a physiologic point of view, we can assume that the toxic effects of Pb are primarily associated with plasma-Pb because this fraction is the most rapidly exchangeable one in the blood compartment. In recent years increased attention has been paid to monitoring the concentration of Pb in plasma (or serum). However, research on associations between plasma-Pb and toxicologic outcomes is still sparse, and a significant gap in knowledge remains.Plasma/serum Pb levels in nonexposed and exposed individuals reported in older publications range widely, from 0.02 to 14.5 \u03bcg/L . This isThe use of advanced analytical techniques is not the only essential requirement to ensure accurate and reliable plasma-Pb data. Contamination of the specimen may occur at the preanalytical phase, namely, during collection, manipulation, or storage. Use of Class-100 biosafety cabinets and clean rooms for specimen preparation and analysis is mandatory. Moreover, all analytical reagents used must be of the highest purity grade. These conditions are far more rigorous than are typically required for clinical BPb measurements performed in a commercial laboratory. After the blood specimen has been collected, the serum/plasma separation must be performed as soon as possible because there is high potential for Pb to move from the dominant BPb subcompartment repository, namely, the erythrocytes, into the plasma via hemolysis, leading to erroneously high results for plasma-Pb. Plasma hemolysis can be estimated by analyzing hemoglobin levels in the specimen because these levels are likely to become abnormally elevated with hemolysis . MateriaCommercial evacuated blood tubes, prepared specifically for BPb measurements, are available with < 5 \u03bcg/L Pb , but it There are many reports of plasma-Pb measurements where validation data are either weak or absent. For example, some simply cite successful participation of the analyzing laboratory in quality assurance (QA) programs for whole blood Pb operated by the CDC and the College of American Pathologists , whereasSaliva has been proposed as a diagnostic specimen for various purposes, as it is easily collected . HoweverSome data suggest an association between Pb levels in saliva and those in either plasma or blood . MoreoveUncontrolled variation in salivary flow rates, lack of standard or certified reference materials, and absence of reliable reference values for human populations are major factors that limit the utility of saliva Pb measurements. In addition the very low levels of Pb present in saliva limit the range of suitable analytical techniques, thereby further diminishing the utility and reliability of this biomarker for evaluating Pb exposure.Hair is a biological specimen that is easily and noninvasively collected, with minimal cost, and it is easily stored and transported to the laboratory for analysis. These attributes make hair an attractive biomonitoring substrate, at least superficially. Because Pb is excreted in hair, many have suggested it for assessing Pb exposure, particularly in developing countries where specialized laboratory services may be unavailable and resources are limited . However, an extensive debate is ongoing about the limitations of hair as a biomarker of metal exposure generally. Here we limit our discussion to Pb exposure, although many of the issues for Pb, such as preanalytical concerns for contamination control, sampling, and reference ranges, also apply to other metals.The ability to distinguish between Pb that is endogenous, namely, absorbed into the blood and incorporated into the hair matrix, and Pb that is exogenous, namely, derived from external contamination, is a major problem. During the washing step it is assumed that exogenous Pb is completely removed, whereas endogenous Pb is not. However, no consensus exists about how removal of exogenous Pb is best accomplished. Some publications that describe the use of hair for assessing Pb exposure reference a hair washing method proposed by the International Atomic Energy Agency (IAEA) in 1978. The approach entailed washing hair specimens with acetone/water/acetone . HoweverAnother issue is the significant variation in the Pb concentration profile among various subpopulations according to age, sex, hair color, and smoking status . MoreoveRecently, the ATSDR established an expert advisory panel to review current knowledge about the use of hair analysis for trace metals in biomonitoring . The genIn addition to the preanalytical issues and the absence of reliable reference ranges, the quality of analytical techniques used for determining Pb, as well as other trace metals, in hair has been questioned. In a recent interlaboratory study of commercial laboratories that specifically market the test for trace metals in hair, interlaboratory agreement was judged very poor, with wide discrepancies observed for Pb as well as for other elements .2EDTA. In unexposed subjects BPb levels remained stable even after 5 hr of CaNa2EDTA administration. However, plasma-Pb levels in the unexposed group decreased by as much as one half, while urine-Pb increased by a factor of 10. In the Pb-exposed group the same amount of chelation therapy resulted in plasma-Pb levels increasing by a factor of 2, while BPb levels decreased by a factor of 2, with a higher urine-Pb excretion. Thus, it seems that in nonexposed subjects a major contribution for urine-Pb is derived from the Pb fraction in soft tissues that is in equilibrium with that in plasma compartment. We could speculate that the larger the amount of erythrocyte-bound Pb, the weaker the binding forces, and that a significant fraction of Pb is released from red blood cell membranes into plasma and is then filtered by the kidney. Because the amount of Pb excreted is very high, the kidneys are unable to remove it rapidly from the blood stream; this may account for the temporal elevation of plasma-Pb levels.The determination of Pb in urine (urine-Pb) is considered to reflect Pb that has diffused from plasma and is excreted through the kidneys. Collection of urine for Pb measurements is noninvasive and is favored for long-term biomonitoring, especially for occupational exposures. However, a spot urine specimen is particularly unreliable because it is subject to large biological variations that necessitate a creatinine excretion correction. Urine-Pb originates from plasma-Pb that is filtered at the glomerular level; thus, according to some authors , urine-PThe availability of reliable urine quality-control materials and reference materials certified for Pb content and participation in external quality assessment schemes for urine-Pb are important factors in assuring the accuracy of analytical results. However, the tendency for urate salts to precipitate out of urine during transit and storage can be a complicating factor in the analysis. Moreover, because only a few studies have examined associations between urine-Pb and other biomarkers, the use of urine-Pb measurements is essentially limited to long-term occupational monitoring programs, monitoring patients during chelation-therapy, and, until very recently, to clinical evaluation of potential candidates for chelation therapy.Measurement of fecal-Pb content over several days is one possible approach to estimating the overall magnitude of childhood Pb intake. According to Like hair, nails have many superficial advantages as a biomarker for Pb exposure, especially because specimen collection is noninvasive and simple and because nail specimens are very stable after collection, not requiring special storage conditions. Nail-Pb is considered to reflect long-term exposure because this compartment remains isolated from other metabolic activities in the body . BecauseLead concentration in nails depends on the subject\u2019s age , but it Because bone accounts for > 94% of the adult body burden of Pb (70% in children) , many re206Pb/204Pb isotopic ratios in immigrant Australian subjects, Australian-born subjects, and environmental samples. The immigrant population exhibited Pb isotopic ratios from 17.7 to 18.5, distinct from the ratio in Australian-born subjects (~ 17.0). This difference allowed a distinction to be drawn between current exposure acquired from Australian sources and older bone-stored Pb that was not acquired from Australian sources.As pointed by Differing bone types have differing bone-Pb mobilization characteristics. For example, the tibia principally consists of cortical bone, whereas the patella is largely trabecular bone. Pb in trabecular bone is more biologically active than Pb in cortical bone, and trabecular bone has a shorter turnover time. The endogenous contribution of Pb from bone stores is an important health consideration. The O\u2019Flaherty kinetic model can be used to indicate the quantity of Pb delivered from bone as a function of bone turnover and Pb exchange . A recenin vivo X-ray fluorescence (XRF) methods have become increasingly accepted. The technique uses fluorescing photons to remove an inner-shell electron from a Pb atom, leaving it in an excited state. The result is emission of X-ray photons that are characteristic of Pb. Measurements are performed by using one of four kinds of XRF: two involve fluorescence of the K-shell electrons of Pb (K-XRF), and the other two involve fluorescence of the L-shell electrons (L-XRF)  dose (as compared to L-XRF) (Over the last decade bone-Pb measurements based on noninvasive  (L-XRF) . Severalo L-XRF) . The radCalibration is usually performed with Pb-doped plaster-of-Paris phantoms . Method Thus, an appropriate selection of the precise bone type to be analyzed for Pb content must be made before commencing. Moreover, further research on the relationship between various bone-Pb subcompartments and other Pb measures is warranted.Like bone, teeth accumulate Pb over the long term. However, there is some evidence that teeth are superior to bone as an indicator of cumulative Pb exposure because the losses from teeth are much slower . MoreoveChronic Pb exposure from mouthing activity in early childhood may be camouflaged by \u201cdilution\u201d effects during periods of rapid skeletal growth in young children and adolescents and may not be detected by a single BPb measurement. However, most published data on tooth-Pb have been based on whole tooth analysis, with no attempt to distinguish among tooth types (different teeth are formed at different ages) or to differentiate the Pb concentration in enamel from that in dentin . The influence of age and/or sex have also not been considered . Furtherin vivo enamel biopsy of children. In this approach superficial minerals are leached from teeth and Pb is determined by electrothermal atomic absorption spectrophotometry. One important drawback to this approach is that, because an accumulation gradient for Pb has not yet been established for enamel, only biopsies of a given depth can be compared to one another. Another issue related to tooth-Pb measurements is whether Pb that accumulates in the first few micrometers of the enamel surface was incorporated posteruptively  rather than during the period when the tooth was mineralized inside the bone.Recently, in utero and thus may provide information on prenatal exposure to Pb. This information could be valuable in improving our understanding of dose\u2013effect relationships for embryonic anomalies, particularly neurotoxic dysfunction. The dentine of the primary teeth provides evidence of exposure during the early childhood years, when hand-to-mouth activity is usually an important contributor to Pb body burden .A critical question that might be asked with respect to an individual\u2019s bone-Pb measurement is what does it mean in terms of health risk or, perhaps, clinical management? To answer this question, we may need to distinguish between bone-Pb measurements in children and pregnant women, namely, those with high bone turnover rate compared to (nonpregnant) adults. In children, bone-Pb may have little effect on BPb levels, but it may help us to estimate the extent to which BPb derives from endogenous sources and the possible contribution to the labile plasma-Pb pool."}
+{"text": "The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation.The model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics.We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process. Ageing is due to the gradual accumulation of unrepaired random molecular faults, which leads to an increased fraction of damaged cells and eventually to the functional impairment of older tissues and organs [reviewed in ]. CellulTerman mainly applied his theory to the lysosomal pathway of degradation. He suggested that lysosomes gradually accumulate lipofuscin and that the lipofuscin granules attract lysosomal enzymes which, however, fail to degrade the pigment. This leads to a shortage in lysosomal enzymes, resulting in a decrease in degradation by this pathway. However, the major pathway for the rapid degradation of proteins is the ubiquitin-proteasome pathway. Proteins need to be degraded either because they are short-lived e.g. key regulatory proteins or because they have become damaged and partially unfolded. Partially unfolded proteins have exposed hydrophobic surfaces and are liable to form aggregates. Molecular chaperones bind to the exposed surfaces and an attempt is made to refold the protein. If refolding is unsuccessful then chaperones assist in the removal of these proteins and so prevent their aggregation .Interestingly, proteasomes themselves are degraded by the lysosomal system and there is evidence for cross-talk between the two degradation pathways ,9. It haIt has been reported that aggregated protein directly impairs the function of the ubiquitin-proteasome system ,20. AnotMany neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome . It has In order to be recognised and degraded by the 26S proteasome, proteins must first be tagged with chains of four or more ubiquitin molecules. Ubiquitin is a small protein found in all eukaryotic organisms. Ubiquitination occurs as a result of the sequential activity of three classes of enzymes, E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin protein ligase). In the first step, ubiquitin is activated through ATP hydrolysis mediated by an E1 enzyme. The activated ubiquitin is then transferred to an E2 (ubiquitin conjugate) enzyme. There are approximately 50 different E2 proteins in mammalian cells  that havA large number of de-ubiquitinating enzymes (DUBs) are also found within all eukaryotic cells . In addiA model of protein turnover and the role of the molecular chaperone Hsp90 in maintaining protein homeostasis has previously been developed . This moThe aim of this paper is to show that the model previously used to simulate chaperone function can be extended to model ubiquitin-mediated proteolysis, incorporating molecular details of this degradation system. We have also sought to incorporate details of the aggregation process. One approach would be to take the computer code for the chaperone model and then extend the code. However, a better approach is to build a new and separate model that can be tested individually and then linked with the chaperone model. A network diagram of the ubiquitin-proteasome model is given in Figure The model was simulated with the initial values and parameter values given in Tables k1, k2, k67, k68, and k69. Varying the parameter for protein synthesis, k1, affects the levels of native protein but only an increase in k1 has any affect on any of the other species. Increasing protein synthesis leads to a depletion in monomeric ubiquitin pools due to the increase pool of protein requiring degradation. An increase in the parameter for misfolding, k2, leads to an increase in misfolded protein, a decrease in native protein, all the ubiquitin ending up in conjugates and an increase in degradation. Conversely, a decrease in k2, leads to higher levels of native protein, more monomeric ubiquitin, less ubiquitin conjugates and less degradation. Increasing the parameter for binding of polyubiquitinated misfolded protein to the proteasome, k67, has no effect on the model output, but a decrease in k67 leads to a depletion in monomeric ubiquitin pools, an increase in unbound polyubiquitinated misfolded protein and less degradation. Increasing the parameter for the chain shortening of polyubiquitinated misfolded protein, k68, has the same effect as decreasing k67. However, decreasing k68 has little effect on model output except that nearly all the degradation reactions are via the substrates with the longest ubiquitin chain. Increasing the parameter for proteasome activity, k69, leads to more degradation and less ubiquitin conjugates, whereas decreasing k69 has the opposite effect and also results in depletion of monomeric ubiquitin due to less recycling of ubiquitin.We carried out a sensitivity analysis to see how changing each of the model parameters affects the simulation output. The only parameters which have a significant effect on the results when increased or decreased by an order of magnitude were in silico experiment of inhibiting the proteasome by setting the parameter k69 = 0. The model predicts that there is initially a build up of ubiquitinated protein and that the pool of monomeric ubiquitin is depleted. The depletion of ubiquitin is followed by a steady rise in the level of misfolded protein , the only parameters which affect aggregation are k1, k2, k3 and k61, Either an increase in protein synthesis (k1) or in the misfolding rate (k2) leads to an increase in aggregated protein which in turn leads to both an increase in sequestered aggregated protein (SeqAggP) and binding of aggregated protein to the proteasome (AggP_Proteasome). This is due to the increase level of misfolded protein which can not be degraded. On the other hand an increase in refolding (k3) leads to a reduction in aggregation. Increased refolding could occur if there was an upregulation in molecular chaperones. Increasing the rate of binding of E3 to misfolded protein (k61) also reduces the level of aggregation suggesting that, if a particular protein was involved in aggregation, then overexpressing the relevant E3 for a misfolded protein could help to prevent the accumulation of aggregates. However, aggregates are usually composed of a variety of proteins, so this approach may not be feasible. As expected, increasing the rate of aggregation by either increasing k71 or k72 leads to more aggregation, and varying the parameters k73 or k74 changes the ratio of SeqAggP to AggP_Proteasome.We also varied each of the parameters in turn to see which parameters affect the kinetics of aggregation. We found that apart from the parameters actually involved in the aggregation steps  is most sensitive with a ten-fold increase leading to a ten-fold decrease in protein half-life, and a ten-fold decrease leading to a ten-fold increase in half-life  only has an effect when decreased, with a ten-fold decrease leading to a four-fold increase in protein half-life , leads to a slight increase (\u00d7 1.4) in protein half-life . These results suggest that the availability of chaperones and E3 enzymes are important in maintaining protein homeostasis.We also examined which parameters affect protein half-life. The most important parameter in this respect was the parameter for misfolding with a ten-fold increase/decrease leading to a ten-fold decrease/increase in protein half-life. Since the misfolding reaction can be driven by the level of ROS in the cell, our model would predict a similar outcome by changing ROS levels, with an increase in ROS leading to higher misfolding and an increase in degradation, consistent with current thought linking protein oxidation and ageing . Our modThere are also many other modifications that we could make to the model. For example, we have assumed that E3 binds to misfolded protein in one simple step; however it is likely that when a native protein misfolds, the first step would be for a molecular chaperone (e.g. Hsp90) to bind to the exposed hydrophobic surface to prevent inappropriate interactions. We could easily add this detail by including the following steps:MisP + Hsp90 \u2194 Hsp90_MisP (reversible reaction)Hsp90_MisP + E3 \u2192 E3_MisP + Hsp90Similarly, we could include the possibility that chaperones aid in delivery of ubiquitinated proteins to the proteasome.Our model includes a HECT E3, but it would be easy to adapt the model for other types of E3 enzymes. For example if E3 is a RING ligase, then it has to be in complex with E2 before it can bind to the misfolded protein. This could be represented by the following reactions:E3 + E2_Ub \u2194 E2_E3_Ub (reversible reaction)MisP + E2_E3_Ub \u2192 E2_E3_MisP_UbE2_E3_MisP_Ub \u2192 MisP_Ub + E2 + E3Our model only includes one pool of long-lived proteins and so far ignores the fact that the majority of proteasome activity is involved in the turnover of short-lived regulatory proteins and also the elimination of incorrect newly-synthesised proteins. It would make the model more realistic to include an additional pool of short-lived proteins and this work is currently in progress.We have so far restricted our models of protein turnover to the ubiquitin-proteasome system; however work is currently in progress to develop other models of protein turnover. It will be important to incorporate models of lysosomal pathways since studies suggest that protein aggregates such as mutant huntingtin are removed by autophagy [reviewed in ].We have included the possibility of aggregated protein inhibiting proteasomes. Another possible cause of proteasome inhibition is via unaggregated damaged protein which could also sequester proteasomes. It would be fairly straight forward to add this extra detail to our current model. By running simulations over long time periods, the models can then be used to see how proteasome activity is affected by damaged and/or aggregated protein. This would be more realistic, than simply setting the parameter for proteasome activity to zero.Our model predicted that levels of native protein did not change when misfolded protein levels rise, but in reality we would not expect total protein levels to continually rise. This prediction was due to our assumption of a constant protein synthesis rate. We could later include detail of feedback mechanisms such as the unfolded protein response which downregulates translation when unfolded protein accumulates in the endoplasmic reticulum.We have encoded our model using SBML which not only allows for easy portability but also enables the model to be modified and extended in a very straight-forward way. The model is available on the BASIS website and can be freely accessed from the public repository, so that the interested reader can run simulations, make their own modifications, or download the SBML code. The BASIS website has a stochastic simulator and a database for storing models and simulation results.Computer models of other molecular mechanisms involved in ageing are being developed at Newcastle University, for example the role of mitochondria, the antioxidant system, and DNA damage signalling pathways. Methods to link models in an automated way are also under development which will greatly facilitate the development of integrated models. For example, our model of the ubiquitin-proteasome system could be linked to a model of the mitochondria. This would be very valuable since neurodegeneration is not only associated with an increase in aggregated protein but also an increase in damaged mitochondrial DNA .We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. We have used SBML to encode the model to ensure that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process. The model and its extensions will be an invaluable aid to further our understanding of the cellular mechanisms involved in maintaining protein homeostasis and how it is disturbed during ageing.2 at 37\u00b0C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of a 2:1 mixture of fetal bovine and donor bovine serum. ts20 cells harbouring a temperature sensitive E1 ubiquitin activating enzyme  were maintained in 5% CO2 at 34\u00b0C in DMEM as above. Inactivation of E1 was achieved by incubating the cells at 42\u00b0C for the indicated time. When proteasome inhibitor  was used, it was added to the culture medium 3 or 4 hours prior to harvesting the cells for western analysis at a final concentration of 50 \u03bcM.U87MG human glioblastoma cells, NIH-3T3 mouse fibroblasts and IMR90 human fibroblasts were maintained in 5% COCells were harvested in protein lysis buffer , 0.5 mM EDTA, 5% glycerol) containing the following protease and phosphates inhibitors: phenylmethylsulfonyl fluoride, leupeptin , aprotinin , sodium fluoride (NaF), sodium pyrophosphate (NaPPi) and N-ethylmaleimide. The cell extracts were sonicated and centrifuged at 14,000 rpm for 20 minutes at 4\u00b0C to pellet cellular debris. The soluble fractions were recovered and the protein concentration was determined using the Bradford protein assay . 40 \u03bcg of cytoplasmic extracts were then resolved on a two-phase SDS-polyacrylamide gel (15 and 8%) and electroblotted onto a hybond C nitrocellulose membrane . The membranes were autoclaved on a liquid cycle for 45 minutes to enhance the detection of poly-ubiquitinated proteins, stained with Ponceau S  and analyzed by western blotting with the indicated antibody. The proteins were visualized by a horseradish peroxidase method using the ECL kit from Kirkegaard and Perry Laboratories Inc., Gaithersburg, Maryland, USA.The rabbit polyclonal antibody used to detect ubiquitin was purchased from Dako. The mouse monoclonal antibodies recognizing actin and GAPDH were from Sigma  and Stressgen Bioreagents  respectively.The model was built by first drawing a diagram of the system , ubiquitin conjugating enzyme (E2), ubiquitin ligase (E3), de-ubiquitinating enzymes (DUB), proteasomes, ATP, ADP, AMP, reactive oxygen species (ROS), and complexes to represent binding of elements e.g. ubiquitinated protein. A full list of the species with their names, database terms and their initial amounts is shown in Table , ubiquitIn our previous model of the chaperone system , we inclwhere MisP represents the pool of misfolded protein.We now replace this single step with a more detailed representation of the ubiquitin-proteasome system.The first step in the degradation pathway is that the substrate (in this case MisP) is bound to its specific ubiquitin ligase (E3). The binding of E3 to MisP produces a complex E3_MisP. This reaction is reversible and so we have two reactions as follows:k61<#E3><#MisP>, where # denotes the number of molecules. The disassociation reaction is a first-order reaction and is given by kr61<#E3_MisP>. The values for k61 and kr 61can be estimated from knowledge of the protein half-life and the steady state level of protein in its misfolded state.We use mass action stochastic kinetics for the rate laws . The binBefore a substrate can be ubiquitinated, ubiquitin must be activated by the ubiquitin-activating enzyme (E1). This reaction requires one molecule of ATP. The binding of E1 to ubiquitin (Ub) produces a complex which we denote by E1_Ub:The activated ubiquitin is then transferred to the ubiquitin-conjugating enzyme (E2) which releases E1 making it available for further ubiquitin-activating reactions:Although it is possible that this reaction is reversible, it seems unlikely that E2 would return the Ub to E1. So we assume that the reverse reaction is negligible and do not include it in the model. Ubiquitin bound to E2 is then transferred to the misfolded protein bound to E3, releasing E2 and E3:Further ubiquitin is then attached to the monoubiquitinated protein via the action of E2. Since each ubiquitin molecule must be activated by E1 before being transferred to E2, each step in the formation of polyubiquitin chain uses one molecule of ATP. The extension of the ubiquitin chain is modelled by the following set of reactions:Each of the above reactions is reversible, with the reversible reactions requiring the activity of a deubiquitinating enzyme (DUB). We assume that the shortening of ubiquitin chains by DUBs is a step-wise process, although we could later modify the model to allow whole chains to be removed if it becomes of interest to model this. The step-wise removal of ubiquitin is modelled by the following reactions:We assume that ubiquitinated protein first binds to the proteasome and waits for degradation and that it can remain bound so long as at least 4 ubiquitin sub-units are attached. This can be modelled by the following reactions:where MisP_Ub4_Proteasome etc. represents an ubiquitinated protein bound to the proteasome.Bound ubiquitinated protein can also be de-ubiquitinated in a step-wise process, so that proteins with longer chains stay longer at the proteasome. This is modelled by the following set of reactions:When a bound protein has a chain shorter than four, then it dissociates from the proteasome. This is shown in the following reaction:Any protein which has a chain of four or more ubiquitin molecules may be degraded by the proteasome in an ATP dependent reaction:This model can either be combined with the chaperone model  or can bThe rate of misfolding depends on the level of reactive oxygen species (ROS) within the cell. We have omitted details of the chaperone activity, where a misfolded protein would bind to Hsp90 before refolding can take place. However, when we combine the models, this simple reaction will be replaced with the more detailed reactions given in Proctor et al., . In thisIf a misfolded protein is not removed immediately by refolding or degradation, then there is a chance that it will interact with another misfolded protein to form a small aggregate, or it may interact with an existing aggregate to form a larger aggregate. (Here we will not be concerned with the size of aggregates \u2013 a more detailed model is in preparation). An aggregate may be sequestered to prevent it interfering with the cellular machinery or it may bind to the proteasome. We include enough detail to be able to examine whether an increase in misfolding (for example by an increase in levels of ROS) leads to an increase in aggregation and inhibition of the proteasome which in turn leads to an even greater level of aggregated protein.The following set of reactions show how protein aggregation can be simply modelled:where AggP represents the pool of aggregates. The aggregates may be sequestered (SeqAggP) which keeps them out of harms way, or they may bind to the proteasome (AggP_Proteasome) and so inhibit its function. Ubiquitinated misfolded protein may also form aggregates but this will not normally occur unless the proteasome is inhibited. As there are many ways in which misfolded proteins with ubiquitin chains of different lengths can interact, we only list a subset of the reactions:6 cells and 61.1 pmol/106 cells respectively. From this we can calculate that there are approximately 107 ubiquitin molecules per cell . Since we are only modelling part of the total cellular protein, we have scaled down the level of ubiquitin to 500 molecules per cell. The total level of proteasomes per cell has been estimated to be 8 \u00d7 105 per cell in L929 cells [Before the model can be simulated, it is necessary to specify the initial amounts of each species and the parameter values. There is experimental data on levels of ubiquitin and proteasomes in human cells. For example, Haas et al.  measured29 cells  which giThe level of E1 has been measured in IMR90 cells by western analysis of cell lysate compared to recombinant protein  and is estimated to be 1 million molecules per cell. This is one order less than the level of ubiquitin and so we chose an initial amount of 100 for E1. We do not have data for E2 or E3 enzymes but for the sake of building the model assume that for specific substrates these would be present at roughly the same abundance as E1. We do not have any experimental data for the abundance of DUBs within cells but assume that these are less abundant than ubiquitin and set the initial amount to 200.The level of native protein, NatP, was set at 500 and at this level the ratio of native protein to ubiquitin and proteasomes enabled efficient degradation to take place. If we increased the level of NatP to 5000, then it was also necessary to increase the pool of ubiquitin and proteasomes to prevent depletion of monomeric ubiquitin under normal conditions. The levels of ATP, ADP and AMP were set at a fairly arbitrary level of 10000, 1000 and 1000 respectively and the levels were kept constant by imposing a boundary condition to these species. We chose to do this instead of allowing the levels to fluctuate as cells possess mechanisms to maintain ATP levels at fairly constant levels and it is unlikely that the ubiquitin-proteasome system would cause major changes to levels of these molecules. ROS levels were set at an arbitrary value of 10 and the level remained constant as there are no mechanisms in this model that would cause ROS levels to change. The initial amounts of all the species are shown in Table The default values for the model parameters are shown in Tables k1, k2 and k3 were set so that the half-life of the pool of protein equals about 10 hours, and under normal conditions, there is only a very low proportion of misfolded protein [k61 for E3/MisP binding is set so that the average time for one E3 to bind to MisP is about 10 minutes. The parameter kr 61is set so that the reverse reaction (dissociation of E3 from MisP) happens once every 100 minutes. So the forward reaction is 10 times stronger than the reverse reaction. The parameter k62 for E1/Ub binding is set so that about 40 ubiquitin molecules are activated every minute and the parameter k63 for E2/Ub binding is set to give about 6 reactions per minute. The parameter k64 for monoubiquitination is set so that once MisP is bound by E3 it takes about 10 seconds to receive the first Ub molecule. The parameter k65 for polyubiquitination is set so that it took about one second for each additional Ub molecule to be added to the chain. So each polyubiquitination step is about 10 times faster than the first ubiquitination reaction. The parameter k66 for the de-ubiquitination reactions is set so that it takes about 8 minutes for each reaction. It is necessary that this reaction is very slow compared to the ubiquitination reactions so that the model output fits the experimental data when E1 activity is shut down (see section 4.4). The parameter k67 for the binding of ubiquitinated proteins (with chains of 4 or more ubiquitin molecules) to the proteasome is set so that under normal conditions when there are about 200 ubiquitin conjugates present in the cell, on average about 5 reactions per second are occurring. The parameter k68 for the de-ubiquitination of proteasome-bound ubiquitinated proteins is set to be equal to k66. We assume that this reaction is slow so that proteins with long chains are highly likely to reside long enough at the proteasome for degradation to take place, whereas proteins with short chains may be shortened to below the required threshold and would then dissociate from the proteasome. The parameter k69 for proteasome activity is set so that it takes about 15 seconds to degrade each proteasome-bound ubiquitinated protein. We initially set k71 and k72 to be equal to 10-7 as in the model of Proctor et al. [k71 and k72 set equal to 10-8, The parameter k73 for sequestering aggregates is set so that if there is a low level of aggregates in the cell (1\u201310), then on average there are 0.005 reactions per second, so each reaction takes about 3 minutes for each reaction.We now describe each of the parameters in turn and give an interpretation of their values in terms of reaction kinetics. The parameters  protein . The parr et al. . Howeverk74 is set so that there is an equal probability of an aggregated protein being sequestered or binding to the proteasome when all the proteasomes are available for binding.The parameter in silico experiment of inhibiting the proteasome by setting the parameter k69 = 0. We also varied each of the other parameters in turn to see which parameters affect the aggregation kinetics.We carried out an k1, to zero we can use our model to check the half-life of the protein pool. We also varied each of the other parameters in turn to see which parameters affect the half-life. For each parameter we increased and decreased its value ten-fold, re-ran the simulations and plotted the model output.By setting the parameter for protein synthesis, in silico model by adding an event to the SBML code so that at a certain time-point the parameter for the E1/Ub binding reaction is set to zero. This event is equivalent to putting the cells at the non-permissive temperature in the wet lab experiment. We chose 30 minutes as the time-point at which this event occurred to allow time for ubiquitin conjugates to form. The details of the event are given in Table It is very straight forward to mimic this experiment with our Statistical analysis of repeated simulations was carried using the R programming language.CJP constructed and coded the models, ran simulations, plotted results and drafted the manuscript. MT designed and performed laboratory experiments and wrote sections of the manuscript. DAG designed the experiments, advised on the model construction and contributed sections of the manuscript. All authors read and approved the final manuscript.Proctor2007_UPSProteinDegradation.xml. SBML code for the ubiquitin-proteasome modelClick here for file"}
+{"text": "The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15.bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice. Posttranslational modification by ubiquitin regulates processes such as proteasomal degradation, intracellular trafficking, and transcription. Ubiquitin is attached to substrates in covalent isopeptide linkage or as an N-terminal fusion Ubiquitin-like molecules show sequence and structural similarity to ubiquitin. Unlike ubiquitination, modification by UbLs usually does not target proteins for destruction by the proteasome. A notable exception may be FAT10, a modifier that serves as a ubiquitin-independent signal for proteasomal degradation A closely related homolog of ubiquitin in vertebrates is the UbL polypeptide ISG15, an interferon-inducible gene product that is strongly upregulated following viral or bacterial infection The C-terminal six amino acids of ubiquitin and ISG15 are identical. This tail region is required for specific recognition of ubiquitin by conjugating enzymes, and also for recognition of ubiquitin adducts by isopeptidases in vitro transcription and translation (IVT) of cloned cDNAs, which affords a rapid method to generate radiochemically pure proteins. This technique allows the generation of DUBs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. To profile for DUB specificity, we used recombinantly expressed ubiquitin, SUMO1 and ISG15, and installed an electrophilic trap at their C-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (UbVME), SUMO1VME and ISG15VS, respectively bona fide ISG15-isopeptidase USP18 displayed reactivity only towards ISG15VS , validating the choice of such C-terminal modifications analysis . Whereasthe cell , similar pattern . Consist nucleus . To demous  is contrasted by the functional differences and localization of these two proteases. These enzymes provide a unique opportunity to investigate the structural features that may contribute to ubiquitin versus ubiquitin-like specificity. A characteristic of USP5 and USP13 is the tandem occurrence of a UBA domain, which has been implicated in the binding of ubiquitin The ubiquitin gene is prone to duplications and insertions, leading to the formation of new fusion proteins USP2 and USP18 cDNAs were cloned from a human kidney cDNA library . The cDNAs encoding the other human DUBs were obtained from ATCC. All cDNAs encoding full-length DUBs were subcloned into pcDNA3.1. Point mutations were generated with the Stratagene \u201cQuik Change II\u201d kit. The Genbank Identification numbers of the cloned constructs are: USP2 (GI:21361712), USP3 (GI:10720340), USP5 (GI:4507855), USP7 (GI:4507857), USP10 (GI:24307889), USP12 (GI:32698815), USP13 (GI:4507849), USP14 (GI:4827050), USP17/DUB3 (GI:37778800), USP18 (GI:32313610), USP22 (GI:113426698), USP24 (GI:113408685), USP25 (GI:6941888), USP35 (GI:89034166), USP36 (GI:35250686), USP37 (GI:32698744), Bap1 (GI:4757836), CylD (GI:14165258), CGI-77 (GI:40254879), OTU1 (GI:109148508), OTU2 (GI:12962939), UCH-L1 (GI:93279272).http://www.psc.edu/biomed/genedoc/) by K.B. Nicholas & HB Nicholas Jr., using the putative active-site cysteine as an alignment anchor. The phylogram represents a consensus tree based on 100 bootstrap iterations, calculated by the Minimum Evolution method under default parameters The protein sequences of human DUBs were obtained from the National Library of Medicine and the core domains were aligned with the CLUSTALW algorithm (EBI server) IVT was performed using the \u201cTNT-T7 Quick Reticulocyte Lysate System\u201d kit (Promega) for 30 to 45 min (0.25\u20131 \u00b5g DNA per reaction). Then, aliquots of the reaction mix were treated with RNase B  for 10 min and incubated with the probes as described below. SDS-PAGE followed by fluorography was performed as described E. coli-expressed human recombinant USP14 for the experiments shown in 2>0.99):The synthesis of human ubiquitin and UbL probes has been described 2, 20 mM DTT, 40 mM ATP) was added ISG15  or GST-SUMO1  and biotinylated peptide 7-mer . The solution was mixed thoroughly and to this was added ISG15 activating enzyme  and UbcH8 , or SUMO activating enzyme  and UbcH9 . The solution was mixed and incubated for 15 hours at 37\u00b0C. The reaction mixture was transferred to a microcentrifuge membrane filter , diluted to 600 \u00b5L total volume with 50 mM Tris, pH 7.4 and concentrated at 4\u00b0C to 50 \u00b5L. This dilution/concentration procedure was repeated six times. The products were transferred to a clean tube and diluted with 50 mM Tris, pH 7.4 to a final volume of 100 \u00b5L. The SUMO1- and ISG15-linked biotinylated isopeptide was detected after transfer to a PVDF membrane (Perkin Elmer) with Streptavidin-HRP (Amersham).To 50 \u00b5L of conjugation buffer  and ISG15 was manually modeled onto USP2 and USP14 with the SPDB-Viewer"}
+{"text": "It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton.The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways.in vivo.We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-\u03b2 (TGF-\u03b2) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB), \u03b2-catenin, AP-1, and nuclear factor-kappa B (NF-\u03baB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-\u03b2 but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/\u03b2-catenin pathway activity, it induced NF\u03baB signaling and inhibited AP-1 signaling.Pb-exposed MSCs showed enhanced basal and TGF-\u03b2/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-\u03b2, BMP, AP-1, and NF\u03baB.The  In spite of effort to reduce human exposure, lead toxicity continues to be a major environmental health concern in the United States and in other industrial countries. Although recent research has focused on understanding its toxic effects in the developing peripheral and central nervous system, Pb also impacts other developmental processes, including those that occur in the hematologic and skeletal systems . In factin vitro experiments to elucidate the underlying mechanism of toxicity during chondrogenesis and chondrocyte differentiation. We employed micromass cultures of mesenchymal stem cells, a culturing technique that has been used previously as a model to study the early events of embryonic limb development  signaling in neurologic tissues, and interfere with long-term learning and other functions  with a DME/F12 mixture (40%/60%) supplemented with 10% fetal bovine serum  as previously described  by enzymatic digestion of limb bud tissue as previously described . Brieflyescribed . One mic2O, photomicrographs of the stained cultures were obtained. Quantification of alcian blue staining was performed using NIH Image . The threshold was standardized to detect nodule area, and the area of the staining was converted from pixels to square millimeters. The area and intensity values were recorded and used to determine staining intensity.Three days after seeding of micromass cultures and treatment with various Pb concentrations and/or factors of interest, wells were washed once with phosphate-buffered saline  and then fixed for 20 min with 1 mL 10% neutral buffered formalin . The cultures were then washed three times with sterile distilled, deionized water, with the last wash being left on the cells for 15 min. Subsequently, 0.5 mL 3% alcian blue (Sigma) in glacial acetic acid was added to each well for 24 hr at room temperature. After two washes with 70% ethanol and three washes with sterile distilled, deionized H, reactions were carried out using the Rotor Gene Real-Time DNA Amplification System  according to the manufacturers instructions. cDNA samples were treated with RNase A , and 50-ng aliquots were combined in a 20-\u03bcL final volume with 0.5 mM primers and the SYBR Green PCR Master Mix . The PCR protocol began with 94\u00b0C denaturation (5 min) and then followed by 35 cycles of the following: 94\u00b0C denaturation (20 sec); 50\u201360\u00b0C annealing ; and 68\u00b0C extension (30 sec). Dilutions of the cDNA pool were used for each primer set to generate a standard curve, which was utilized by RotorGene software  to perform quantitative analyses. Data were normalized to the \u03b2-actin expression level for each sample. All samples were run in triplicate and repeated four times (n = 4).Cultures were harvested at various time points, and mRNA was prepared using Trizol reagent  as directed by the manufacturer. cDNA was generated using the Advantage RT-for-PCR kit , and real-time quantitative RT-PCR was performed as described previously , 2004. BUse of mice in this experiment was reviewed and approved by the University of Rochester Institutional Animal Care and Use Committee (IACUC). Mice were treated humanely with regard for alleviation of suffering. At 10 weeks of age, female C57/B6 mice were divided into three groups of 35 mice. Groups were exposed to 0, 55, or 230 ppm Pb (in the form of Pb acetate) in drinking water for 6 weeks before initiation of the ectopic bone formation experiment. Our group previously used an atomic absorption method to establish that these doses of Pb exposure correspond to environmentally relevant whole-blood Pb concentrations of 15 and 40 \u03bcg/dL for 55 and 230 ppm, respectively .l-glutamine, and 1 \u03bcg/mL doxycycline to repress BMP-2 expression . Nine days before implantation, doxycycline was removed from the medium to allow BMP-2 expression, which was confirmed using an ELISA kit . As a negative control, wild type C3H10T1/2 cells were cultured in basal medium Eagle (BME) with 10% FBS, 1% penicillin/streptomycin, and 2 mM l-glutamine. Cells from both lines were washed, trypsinized, counted, and resuspended in medium at a concentration of 106 cells/100 \u03bcL. Surgifoam  was cut into 2 mm \u00d7 2 mm \u00d7 2 mm segments and soaked in medium, then excess fluid was removed. Aliquots (100 \u03bcL) of the cell suspension were micropipetted into each section of gel foam and incubated for 2 hr to allow cells to adhere.Ectopic bone formation was induced in mice by the intramuscular implantation of a gel foam substrate impregnated with cells expressing BMP-2. Specifically, C9 cells derived from murine C3H10T1/2 mesenchymal stem cells, which express BMP-2 under control of a doxycycline-repressible promoter, were cultured in DMEM with 10% FBS, 1% penicillin/streptomycin, 2 mM n = 15 for statistical purposes).Using an IACUC-approved surgical protocol, we placed C9 cell-impregnated gel foam segments into a muscle pouch in the right quadricep of each mouse; C3H10T1/2-impregnated gel foam was placed in the contralateral limb as a negative control. Ectopic nodules were allowed to develop for 3\u201328 days after implantation. At each time point, tissue was harvested, fixed in 10% formalin for 3 days, embedded in paraffin, and cut into 5-\u03bcm sections. Three sections 30 \u03bcm apart were stained with alcian blue and counterstained with alcian blue/hematoxylin. Three representative sections from each sample at day 5 and day 7 were analyzed using Osteometrics software . Cartilage cross-sectional area was measured for each slide and included in statistical analysis , AP1, and cyclic adenosine monophosphate response element binding protein (CREB). Reporter and control plasmids for each of these pathways was transfected into MSCs plated as a monolayer, and then cells were trypsinized and replated as a micromass as described above. Thus, MSCs were initially plated in monolayer in 6-cm dishes at a density of 8 \u00d7 105 cells per dish. Twelve hours after plating, cells were transfected with either P3TP-luciferase (Luc), 12 \u00d7 Smad binding element (SBE)-Luc, NF-kB-Luc, TOPFLASH, AP1-Luc, or cAMP-responsive element (CRE)-Luc. These transfections were performed using Superfect  according to the manufacturers instructions. The SV40 Renilla-Luc plasmid was co-transfected to facilitate determination of transfection efficiency on a well-by-well basis, as previously described . Aliquots of protein extract (25 \u03bcg) were separated by SDS-PAGE (10% polyacrylamide) and then transferred to a polyvinylidene fluoride membrane . The blots were probed overnight at 4\u00b0C with the following rabbit anti-mouse polyclonal antibodies: phospho-Smad3 , phospho-Smad1/5/8 , or mouse anti-\u03b2-actin monoclonal antibody . All primary antibodies were applied at a 1:1,000 dilution. Blots were further incubated for 1 hr at 20\u00b0C in the presence of horseradish peroxidase\u2013conjugated secondary antibodies against rabbit or mouse  at a dilution of 1:3,000. The immune complexes were detected using ECL-Femto  and visualized via exposure of X-OMAT AR film .After rinsing the cell layer with PBS, protein was extracted from micromass cultures using Golden lysis buffer containing protease inhibitor cocktail tablets (Roche Applied Science) as previously described . The lysMSCs were placed in micromass culture and were treated with Pb in the presence and absence of BMP-2 (50 ng/mL) and TGF-\u03b2 (5 ng/mL). After 3 days in culture, treatment with Pb resulted in a stimulation of chondrogenesis as measured by the presence of alcian blue nodules . The effSox9, col2, and aggrecan were determined via real time RT-PCR. Consistent with the alcian blue staining shown in Sox9, a 5-fold increase in col2, and a 2.5-fold increase in aggrecan compared with untreated cultures  for 6 weeks. In control C3H10T1/2 cell implanted limbs, we observed proliferation of host mesenchymal cells at the periphery of the implant, but no cartilage tissue formed  induced a 2.5-fold stimulation . HoweverMSCs were transfected with the BMP-responsive reporter 12 \u00d7 SBE-Luc and treated with Pb in the presence and absence of BMP-2 (100 ng/mL). Pb itself did not alter reporter activity, but BMP-2 stimulated a 2.4-fold induction. Interestingly, Pb induced a dose-dependent inhibition of BMP-2\u2013induced signaling on the 12 \u00d7 SBE-Luc promoter, with a maximal 65% inhibition occurring at a concentration of 10 \u03bcM Pb . ConsistBecause Pb has no effect on basal TGF-\u03b2\u2013Smad and BMP-Smad signaling, and has opposite effects on these signaling pathways in the presence of their respective growth factors, it is unlikely that the induction of chondrogenesis by Pb is related to direct regulation of Smad signaling. To determine the possible involvement of other pathways known to be important in the process of chondrogenesis, MSCs were transfected with AP1-Luc, NF\u03baB-Luc, CRE-Luc, and TOPFLASH reporters, each containing multiple repeats of the respective consensus binding sites. Although there was no effect of Pb on CRE or TOPFLASH activity , Pb causThere is growing awareness of the pleiotropic effects of Pb, with targets of toxicity including multiple tissues that include the skeleton. In fact, at concentrations considered subtoxic, Pb is known to affect skeletal growth. The two most recent National Health and Nutrition Examination Surveys (NHANES II and NHANES III) have demonstrated that Pb exposure reduces skeletal growth independent of factors such as nutritional status, social/ economic class, and co-morbid diseases . Moreovea) block calcium signaling and inhibit Ca2+/phospholipid-dependent PKC signaling (b) induce ERK1/2 and p38 (MAPK) phosphorylation and activation (c) activate AP-1 signaling  indutivation ; and c) ignaling . We havecolX, Interestingly, this study also showed that inhibition of colX by TGF-\u03b2 was completely reversed by Pb. Furthermore, although neither Pb nor TGF-\u03b2 alone affected the expression of BMP-6, in combination they induced its expression 3-fold (Previous work has established that Pb regulates BMP and TGF-\u03b2 signaling pathways. Oral administration of Pb chloride has been shown to cause a reduction in the expression of TGF-\u03b2 in intestinal tissues in diabetes-prone NOD mice . In an en 3-fold . AlthougIn the present study, we found that BMP signaling is also regulated by Pb. BMP receptor signaling occurs in a manner analagous to the TGF-\u03b2 pathway, with Smad1/5/8 binding to the type I BMP receptor, followed by phosphorylation of these factors upon ligand activation . Using ain vivo appears to stimulate osteogenesis over chondrogenesis (The complex regulation of the Smad signaling molecules makes it unlikely that the induction of chondrogenesis by Pb is cause by direct alteration of these pathways. Although BMP and TGF-\u03b2 signaling pathways have antagonistic effects on some cells, including growth plate chondrocytes where TGF-\u03b2 inhibits and BMP-2 stimulates maturation, both signals enhance chondrogenesis in mesenchymal stem cell populations . An earlogenesis , in humaogenesis . Similarogenesis . Overallin vitro Pb exposure in rats results in activation of AP-1 and NF\u03baB levels in multiple regions of the brain and in astrocytes in culture (In vivo Pb exposure results in increased NF\u03baB signaling in renal tubular cells and results in nephritis in rats (To extend these signaling results, we also examined the effect of Pb on other signaling pathways. Although no effects were observed on CRE and TOPFLASH activation using the respective luciferase-based reporters, the experiments showed that Pb inhibited basal AP-1-Luc reporter activity. AP-1 activation has been shown to inhibit chondrogenesis, so it is possible that the inhibition of AP-1 could be involved in the induction of chondrogenesis by Pb . The inh culture . In vivo in rats . Similar in rats , it is uin vivo murine model of fracture healing (The induction of chondrogenesis by Pb in the current study is consistent with findings observed in an  healing . Mice wi healing . OverallIn conclusion, the present study establishes that in addition to affecting chondrocyte maturation, Pb accelerates the differentiation of mesenchymal precursors into chondrocytes. Although Pb alters BMP and TGF-\u03b2 signaling, the mechanism through which Pb regulates mesenchymal cell fate determination is complex and likely involves modulation and integration of multiple signaling pathways. Increased understanding of the mechanisms through which Pb regulates stem cell fate and subsequently affects tissue repair is critical, given the sensitivity of these tissues and the ubiquitous presence of Pb in our society and in other industrialized and developing nations."}
+{"text": "There is growing pressure for clinicians to prescribe chelation therapy at only slightly elevated blood lead levels. However, very few studies have evaluated whether chelation improves cognitive outcomes in Pb-exposed children, or whether these agents have adverse effects that may affect brain development in the absence of Pb exposure.The present study was designed to answer these questions, using a rodent model of early childhood Pb exposure and treatment with succimer, a widely used chelating agent for the treatment of Pb poisoning.Pb exposure produced lasting impairments in learning, attention, inhibitory control, and arousal regulation, paralleling the areas of dysfunction seen in Pb-exposed children. Succimer treatment of the Pb-exposed rats significantly improved learning, attention, and arousal regulation, although the efficacy of the treatment varied as a function of the Pb exposure level and the specific functional deficit. In contrast, succimer treatment of rats not previously exposed to Pb produced lasting and pervasive cognitive and affective dysfunction comparable in magnitude to that produced by the higher Pb exposure regimen.These are the first data, to our knowledge, to show that treatment with any chelating agent can alleviate cognitive deficits due to Pb exposure. These findings suggest that it may be possible to identify a succimer treatment protocol that improves cognitive outcomes in Pb-exposed children. However, they also suggest that succimer treatment should be strongly discouraged for children who do not have elevated tissue levels of Pb or other heavy metals. This study did not detect a benefit of chelation, relative to placebo, in children with blood Pb levels between 20 and 44 \u03bcg/dL . Howevera) determine whether treatment with a succimer regimen shown to produce significant reductions in blood and brain Pb levels (b) determine whether succimer produces lasting cognitive and/or affective impairment when administered in the absence of Pb exposure. Findings from the latter group are needed to gauge the safety of prolonged regimens when treating Pb-exposed children, as well as the safety of the drug for treating autistic children, as currently advocated on numerous websites by physicians, autism parent groups, and organizations such as the American College for Advancement in Medicine.Using a rodent model of early childhood Pb exposure, the present study was designed to b levels  also lesIn this article, we describe a subset of the administered tests\u2014namely, a series of visual attention tests designed to tap several functions reported to be affected in Pb-exposed children, including sustained and selective attention, inhibitory control, learning/associative ability, and regulation of arousal or emotion. Two of these tasks, the sustained attention and selective attention tasks, are similar to ones commonly used to assess attention in human subjects, such as the Continuous Performance Test and Leonard\u2019s 5-choice Serial Reaction Task , described below. The sample size for the behavioral testing was 120 animals, 20 per group. These 120 animals were the female offspring of 60 litters, one succimer-treated and one vehicle-treated animal per litter. All procedures were approved by the Cornell Institutional Animal Care and Use Committee, and the housing facilities were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.http://www.ehponline.org/docs/2006/9263/suppl.pdf).Nulliparous Long-Evans rats  were mated in the laboratory. They were treated humanely and with regard for alleviation of suffering. Twenty-four hours after birth, each litter was culled to 10 pups and assigned to one of three Pb-exposure conditions . The dams in the no-Pb group received drinking water (deionized distilled) adulterated with 300 ppm sodium acetate from postnatal day (PND)1 to PND30. Both Pb-exposed groups received water adulterated with 300 ppm Pb acetate from PND1 to PND17. On PND17, around the age when pups begin consuming water directly from the drinking water bottle, the water Pb concentration was lowered to 20 ppm for the Mod-Pb group for the remainder of the Pb exposure period (until PND30) in order to maintain the moderate level of Pb intake experienced prior to PND17 when milk provided the sole source of Pb. The High-Pb group was maintained on the 300-ppm Pb acetate drinking water until PND30. At PND30, the pups were weaned and Pb exposure was terminated. The period of Pb exposure, PND1\u201330, corresponds roughly, in terms of neural development, to the period spanning the sixth month of pregnancy until late childhood/early adolescence in humans . AdditioOn PND30, the pups in each Pb treatment group were divided into succimer  and vehicle (apple juice) subgroups. This assignment was made at the level of each litter to provide littermate controls for the vehicle\u2013succimer comparisons. Succimer or apple juice (vehicle) was administered twice daily via oral gavage from PND31 to PND52. The daily succimer dose was 50 mg/kg body weight/day during the first week of chelation, followed by 25 mg/kg/day for the second and third weeks, similar to the regimen used clinically .http://www.ehponline.org/docs/2006/9263/suppl.pdf).On PND52 (end of chelation therapy) one animal from each litter was euthanized for analysis of blood and whole brain Pb levels by inductively coupled plasma-high resolution mass spectrometry, as previously described  was mounted above each port; the illumination of one of these LEDs served as the discriminative cue in all of the tasks described here. For the selective attention task, compressed, scented air (which served as the olfactory distractors) was projected into the response ports via polyethylene tubes (see http://www.ehponline.org/docs/2006/9263/suppl.pdf) with ad libitum access to water. Testing on the four tasks described in this report was initiated, on average, at 9, 10, 13, and 15 weeks of age, respectively.Behavioral testing began on PND62. All animals were weighed and tested 6 days/week. For all tasks, a daily testing session consisted of 200 trials or 2 hr, whichever came first. All behavioral testing was conducted by individuals blind to the treatment condition of the subjects. Throughout behavioral testing, animals were maintained on a restricted food intake regimen  a delay of varying duration  was imposed between trial onset  and presentation of the cue (the illuminated LED); and b) the duration of cue illumination was shortened to 700 msec. Animals were tested on this task for 20 sessions.This task was a variation of the visual discrimination task with two modifications: The sustained attention task was identical to the previous task, except that the duration of the cue illumination varied between 200, 400, and 700 msec. Animals were tested for 10 sessions on this task.The animals were then tested for 10 sessions on a selective attention task, similar to the preceding task but with modifications of the stimulus parameters and the addition of unpredictable olfactory distractors on one-third or the trials in each daily test session. This task was preceded by three sessions on a baseline task, which had stimulus parameters identical to those in the selective attention task, but without olfactory distractors. In both tasks, the stimulus delay varied between 2 and 3 sec, and the stimulus duration varied between 300 and 700 msec.p < 0.05; p-values between 0.05 and 0.10 were considered to be trends and are discussed if they aid in clarifying the nature of the Pb and succimer effects. All statistical analyses were conducted using SAS 9.0  for Windows 2000. Additional information on the various dependent measures and statistical analyses is provided in the http://www.ehponline.org/docs/2006/9263/suppl.pdf).We analyzed the various performance measures using a generalized linear mixed model (GLMM), which correctly handled the repeated measures on each animal . DiffereBecause of space constraints, the results of behavioral testing presented here include only those instances where effects of Pb and/or succimer were seen. = 8.54; p = 0.0004] and a significant interaction of treatment and session number . The controls (no lead exposure and no succimer treatment) performed significantly better than the High-Pb (p = 0.017) and High-Pb\u2013succimer (p < 0.0001) groups. Chelation offered no benefit; the performance of the High-Pb and High-Pb\u2013succimer groups did not differ significantly. The High-Pb and control groups did not differ significantly by the sixth session, indicating that their inferior performance earlier in testing reflected impaired learning ability.Analysis of average percent errors for the first six sessions on the visual discrimination task revealed a significant main effect of treatment group ; and significant interactions involving treatment and delay , and treatment, session block, and trial block . Rats in the High-Pb group committed a significantly higher percentage of premature responses than controls for trials with the 3 and 6 sec pre-cue delays (averaged across the 20 sessions on the task)  revealed a borderline main effect of treatment , no ben = 3.24; p = 0.009]. As seen in p = 0.001), suggesting some hesitation to enter the testing alcove during this early portion of the task when all groups were performing very poorly. Succimer treatment was beneficial in that the High-Pb\u2013succimer rats did not exhibit this hesitancy in the first block of sessions: they did not differ from controls (p = 0.38) and differed significantly from the High-Pb group (p = 0.01).Alcove latency (AL) is the time elapsed between the raising of the alcove door at trial onset and entry of the animal into the testing alcove. Because the data were highly skewed, with a high proportion of identical values (the minimum latency), the dependent measures used for each animal were the percentages of ALs that fell into each of four bins of latency values: < 0.1, 0.1\u20130.5, 0.6\u20131.0, and > 1 sec. A significant treatment \u00d7 session block interaction was found in the analysis of very short ALS , and treatment, delay, and session block . The increased response latency seen early in the task for all animals (when error rate was very high) was more pronounced for the High-Pb rats than for the control and High-Pb\u2013succimer groups. This Pb effect was normalized by succimer treatment: the High-Pb\u2013succimer group was significantly faster than the High-Pb group during these early sessions and did not differ from controls .We defined response latency as the time between cue onset and a response. The data were log-transformed before analysis. Analysis of the High-Pb, High-Pb\u2013succimer, and control groups revealed significant interactions of treatment and delay  and treatment \u00d7 delay . The High-Pb group committed a higher percentage of premature responses than the controls for trials with a 3-sec (p = 0.001) or 6-sec (p = 0.05) pre-cue delay (p = 0.001) and 6-sec (p = 0.02) delays and was not different from the High-Pb group for either.Analysis of premature response rate for the High-Pb, High-Pb\u2013succimer, and control groups revealed significant effects of treatment , treatment \u00d7 trial block \u00d7 cue duration , and treatment \u00d7 trial block \u00d7 previous trial outcome (Prev) . For all groups, we found an increase in percent omission errors across each session, an effect that was most pronounced for trials that followed an error. As shown in An omission error was tallied when a rat entered the testing alcove but failed to respond within 15 sec of trial onset, indicative of missing the cue. The analysis of percent omission errors for the High-Pb, High-Pb\u2013succimer, and control groups revealed the following significant treatment-related effects: treatment \u00d7 trial block  reflected that a) the High-Pb rats committed a borderline greater percent omission errors than controls in the first block of trials in each session (p = 0.06), and b) during this first block of trials in each session , the High-Pb group benefited less than controls by the lengthening of the cue from 200 to 700 msec (p = 0.009), such that group differences were greatest on trials with the longest cue duration .Additional details of the omission error analyses are provided in the  = 4.88; p = 0.01) and a significant interaction of treatment and session . The Mod-Pb group learned significantly more slowly than controls . Chelation offered a significant benefit in that the Mod-Pb\u2013succimer group learned significantly faster than the Mod-Pb group  and did not differ from controls for any session. The three groups performed similarly by the sixth session, indicating that the observed group differences in performance reflected variations in learning ability  = 2.89; p = 0.016]. As depicted in p = 0.05), and succimer treatment offered a significant benefit . The Mod-Pb\u2013succimer and control groups did not differ for any session. The performance of the three groups did not differ during the final 10 sessions on the task (blocks 3 and 4), indicating that the observed performance differences reflected differing learning ability.The analysis of percent premature responses revealed a significant interaction of treatment and session block .Analysis of average percent errors for the first six sessions on the task revealed that the succimer-only group (treated with succimer in the absence of lead exposure) learned significantly more slowly than the controls . The groups did not differ significantly for this type of error during session block 1 (sessions 1\u20135), an early point in the learning process when most responses were made before cue onset (premature responses).The percentage of inaccurate responses  was significantly higher for the succimer-only group than for controls during session blocks 2, 3, and 4  and of treatment, cue duration, and trial block . As shown in p = 0.004). This pattern was seen only during the beginning and end of each daily session .The analysis of omission errors for the control and succimer-only groups revealed significant interactions of treatment and cue duration .The succimer-only group benefited less than controls from the lengthening of the cue, resulting in increasing group differences in percent inaccurate responses as the cue duration increased , an interaction of treatment and cue duration , and a trend toward a three-way interaction of treatment, distraction condition, and the outcome of the previous trial . The succimer-only group benefited less by the lengthening of the cue than did controls (p = 0.017), group differences were greatest for trials that both included a distractor and that followed an error , whereas for controls, the rates were similar for these two conditions , providing evidence that motivational differences did not account for the observed differences in performance .Motivation was assessed by analyzing the average rate of response trials [number of response trials per session divided by session length (minutes)]. No significant group differences were detected , measured on PND52 after cessation of succimer treatment. None of the other groups differed from controls. The slightly smaller size of the High-Pb rats likely reflects a decrease in growth hormone during the Pb exposure period . Low-level Pb exposure in children has also been associated with small but statistically significant reductions in height, in the absence of any other signs of overt toxicity  reductions in brain Pb levels lag significantly behind reductions in blood Pb following chelation or cessation of Pb exposure (b) blood and brain Pb levels were still quite elevated in the High-Pb group at the end of chelation ; and c) in a previous study from our laboratory, a second 3-week succimer regimen offered significant benefit over one regimen in terms of both blood and brain Pb reductions  blooductions . TherefoFor the four tasks presented here, the rate of all types of errors was significantly greater on trials that followed an error than on trials following a correct response. Similarly, the latency to enter the testing alcove at trial onset was significantly longer on trials following an error than on trials that followed a correct response, a pattern also seen for the latency to make a response after cue onset. The disruption produced by committing an error was significantly greater for the High-Pb animals than for controls for several dependent measures: In the sustained attention task, the percentage of omission errors was significantly higher for the High-Pb group than for controls during mid- to late-session trials following an error\u2014but not following a correct response\u2014a pattern that indicates the combined influences of the disruptive effects of committing an error and the changing motivational and attentional state of the animals across each session. Similarly, in attention task 1, the High-Pb rats took significantly longer to enter the testing alcove and to make a response than controls, but only early in the task when error rate was very high and the rats had not yet learned the task rules. Finally, the drop in performance on trials following an error was also more pronounced for the High-Pb rats than for controls in a conditional olfactory discrimination task with periodic reward omission, an additional task administered subsequently to these rats .The interpretation of the heightened sensitivity to errors of the High-Pb rats is informed by previous studies that examined performance changes as a function of an error on the previous trial, all involving human subjects. In some of these studies, the error rate on post-error trials was exceptionally low e.g., , indicatImportantly, this heightened disruption following an error was very responsive to succimer treatment: In all cases, the High-Pb\u2013succimer group was indistinguishable from controls . These rThe sustained attention task revealed two types of attentional dysfunction in the High-Pb group. First, the early-session increase in omission errors (relative to mid-session), seen for all groups across all sessions on this task, was more pronounced for the High-Pb group than for the controls, with group differences being largest for trials with the longest cue duration (700 msec). This pattern of results may indicate that lapses in attention were more common for the High-Pb rats than for controls during this early part of the session. More frequent attentional lapses in the High-Pb group would have the consequence of flattening the slope across cue duration and making group differences largest on trials for which performance of the controls was best . One interpretation of this pattern is that early Pb exposure may impair the ability to rapidly engage in a new task when transitioning between activities, manifested here as an increased tendency to miss the cue .A second type of attentional dysfunction observed in the High-Pb rats was evident later in each testing session. The incidence of omission errors increased for all groups across each daily testing session, the classic pattern seen when sustained attention is taxed . HoweverThe effectiveness of succimer chelation varied for these different types of attentional deficits. As discussed above, the heightened attentional disruption seen in the High-Pb rats following an error was completely normalized by succimer treatment, as the chelated High-Pb rats differed significantly from the High-Pb group and did not differ from controls. In contrast, succimer treatment only partially alleviated the attentional lapses seen early in each test session; the High-Pb\u2013succimer group was intermediate to the other two, not differing from either.In the sustained attention task, the percentage of premature responses was significantly higher for the High-Pb rats than for controls, indicating deficient inhibitory control. Converging evidence for this area of dysfunction was provided by the higher percentage of premature responses committed by the High-Pb rats in the final block of trials of attention task 1, a point at which the basic rules of the task had already been learned. Succimer treatment was ineffective in alleviating this area of dysfunction: In both instances, the percentage of premature responses for the High-Pb\u2013succimer rats was significantly greater than that of the control rats, and not different from the High-Pb group.The efficacy of the succimer treatment varied as a function of both the level of Pb exposure and the specific functional domain tested. Interestingly, the pattern seen for succimer efficacy parallels the apparent sensitivity of specific functional domains to the Pb exposure. Learning was impaired by both Pb exposure regimens, whereas impaired regulation of arousal or emotion was seen only in the High-Pb group, suggesting that disruption in learning occurs at lower exposures than dysregulation of arousal or emotion. The efficacy of succimer across these domains corresponded to the degree to which chelation reduced brain Pb levels in the two Pb exposure groups. For the Mod-Pb group, succimer treatment almost completely removed Pb from the brain and effectively alleviated the learning dysfunction. In contrast, brain Pb levels were still moderately elevated in the High-Pb group following chelation; these chelated High-Pb rats exhibited the same pattern of dysfunction as the unchelated Mod-Pb group, which also had moderately elevated brain Pb levels on PND52. Both groups also exhibited impaired learning but not affective dysfunction. This constellation of findings suggests that succimer treatment of the High-Pb rats reduced tissue  Pb levels below that which produces lasting impairment in emotion regulation, but not enough to alleviate the learning dysfunction.The mechanistic basis for this pattern of effects is unknown, but likely reflects complex interactions between the timing of the Pb exposure and succimer treatment relative to the ontogenetic stage of specific neural systems, the extent to which succimer treatment reduced Pb levels in specific neural systems , and theThe present study also revealed the unexpected finding that a single 3-week course of succimer treatment during early development produced lasting dysfunction in cognition and arousal regulation in rats not previously exposed to Pb. Note that these four behavioral tests were administered across a 7-month period following cessation of succimer treatment, suggesting lasting brain changes. These results corroborate preliminary results from a similar study in nonhuman primates . The sucThe mechanism(s) responsible for these adverse succimer effects is not known. One possibility is that succimer, a metal-chelating agent, altered essential metal homeostasis and increased metal diuresis, as has been suggested by a number of clinical and nonhuman primate studies .http://www.ehponline.org/docs/2006/9263/suppl.pdf), direct comparison of blood Pb levels across species should be cautioned. The available evidence indicates that the blood Pb levels required to produce overt toxicity  or lasting neurobehavioral effects  are higher in both rats and nonhuman primates than in humans (Both Pb-exposed groups exhibited low blood Pb levels (\u224825\u201335 \u03bcg/dL) during the first 3 weeks of the 4-week exposure period. During the fourth and final week of exposure, both groups experienced an increase in tissue Pb levels because of the direct ingestion of Pb-adulterated water. Although the blood Pb levels produced during this final week of exposure were higher than commonly reported for children  succimer-induced reductions in blood Pb overestimate reductions in brain lead (b) the succimer treatment protocol used in the TLC study achieved relatively modest group differences in blood Pb levels (relative to placebo), averaging only 4.5 \u03bcg/dL over the 6 months following treatment  study, the one clinical trial in Pb-exposed children that included cognitive outcomes . There aain lead  and b) treatment . In compreatment . Second,In conclusion, this study demonstrates that it is possible for chelation therapy to alleviate certain types of Pb-induced behavioral/cognitive dysfunction in rats. This is an important demonstration because Pb-induced behavioral dysfunction is generally considered to be irreversible e.g., . The preIn the original manuscript published online, the symbols for the control and succimer-only groups were reversed in"}
+{"text": "In the Funding section, the number of the US National Institutes of Health grant to SST was listed incorrectly. The correct number is: GM34921."}
+{"text": "Occupational and environmental Pb-exposure is associated with protein aggregation diseases which typically present in elderly populations (Parkinsons and cataract). Post-translational processing of crystallins, the major structural proteins of the lens, is altered with short-term Pb-exposure in Fisher 344 rats. In addition, lenses from aged rats become opaque upon long-term exposure to Pb in organ culture. To explore the route to lens opacification in the presence of Pb, cultured lenses from young rats which exhibit higher metabolic activity in lens culture and are more susceptible to experimental cataract in vivo and in vitro were exposed to Pb and evaluated for morphological and biochemical alterations.Following culture in Pb (as lead nitrate) for four days , lenses were examined for clarity, integrity of epithelial layer, and molecular stability including crystallin post-translational modification and choline transport. Clarity of lenses cultured with/without Pb for up to 8 days was assessed to determine if Pb exposure would accelerate opacification.Lenses cultured in Pb for four days exhibited epithelial abnormalities including epithelial cell multilayering and nuclei abnormalities with extension of the nucleated epithelial cells past the bow region. Alterations in crystallin post-translational modifications and decreased membrane transport of choline were noted without corresponding lens opacification or altered \u03b1-crystallin chaperone activity. Lenses treated with Pb according to the same exposure protocol with subsequent challenge by hydrogen peroxide became opaque while the contralateral control lenses did not. Lenses which were cultured in the presence of Pb for longer periods with no subsequent oxidative insult exhibited lens failure at earlier time points than did the controls.These data indicate that Pb-exposure can accelerate the degradation of the cultured lens through induction of epithelial cell abnormalities, induce structural protein modifications before opacity, and predispose the lens to opacification with subsequent oxidant challenge. The banning of Pb, a potent environmental toxin, as a pigment, pesticide, and fuel additive has led to decreases in the Pb burden of the general population and of children specifically -3. HowevThe mechanisms related to lenticular opacity associated with Pb-exposure have been explored. Pb accumulation in normal healthy lenses during organ culture or in vivo after oral dosing may be directly related to Pb binding to the lens capsule ,14. Pb hWith the association of Pb exposure and decades later cataract formation, it seems likely that Pb exposure acts in conjunction with later systemic stressors to induce opacification. The current study examines whether Pb acts as a predisposing effector for lens opacity in conjunction with a secondary oxidative challenge in cultured lenses from young rats (approximately 4\u20136 weeks of age). Additionally, this study explores the effect of Pb exposure on epithelial transport/cellular structure and crystallin protein homeostasis as possible mechanisms through which Pb may induce opacity.Pb nitrate, sodium nitrate, urea, dithiothreitol (DTT), depleted \u03b1-lactalbumin, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), trichloroacetic acid, trifluoroacetic acid, \u03b1-cyano-4-hydroxycinnamic acid and sequencing grade trypsin were purchased from Sigma Chemical Company . Immobilized pH gradient (IPG) strips were purchased from BioRad . Bis-Tris gels, MOPS running buffer, SilverQuest and Colloidal Blue staining kits were purchased from Invitrogen .2) for 2 h; lenses that were damaged during dissection were identified and discarded during this period by measuring protein leakage into the medium. After the equilibration period, surviving \u201ccompetent\u201d lenses were cultured with 1\u00a0\u00b5M Pb(NO3)2 (~20\u00a0\u03bcg/dl) or unmodified media. The media was changed every other day for a period of 3\u20139 days. An additional group, 2\u00a0\u00b5M NaNO3 (n=3), was initially included as a counter ion control exposure grouping to assess the impact of nitrate concentration on lens competence. As TC-199 media contains ferric nitrate, the additional nitrate added through addition of the Pb salt doubled the media nitrate content. The lenses that were cultured in NaNO3 did not vary from the lenses cultured in unmodified media in hydrogen peroxide clearance, choline uptake, protein disulfide formation or in morphology. The data from the NaNO3 (n=3) and unmodified media (n=6) for these assays were combined and represented as the Control group. Subsequent analyses, including the 2D gels and \u03b1-crystallin chaperoning assay, used lenses cultured in unmodified media as controls and without the inclusion of a NaNO3 group since the added nitrate salts did not affect lens competence.Eyes were obtained from Sprague-Dawley  rats (4\u20136 weeks of age) immediately after euthanasia following the NIH Animal Research Advisory Committee guidelines. The competent lenses were cultured in modified TC-199 media as previously reported . The osm3)2 , the Pb containing media was replaced after 3 days with modified TC-199 supplemented with 250\u00a0\u00b5M hydrogen peroxide and changed daily. One hundred microliter aliquots of the medium were removed to assay for hydrogen peroxide clearance. H2O2 concentration was measured after 2 h using a Model 2700 Biochemistry Analyzer . After 2 days of hydrogen peroxide exposure the lenses were collected and photographed to document alterations in lens clarity. The experiment was repeated 3 times for a total of 12 lenses per group.For the hydrogen peroxide challenge of lenses cultured in Pb. After 4 h, the lenses were removed from culture, rinsed with PBS, blotted, and weighed. The lenses were homogenized in 1\u00a0ml of cold 10% trichloroacetic acid and the soluble fraction was collected. One hundred microliters of the lens supernatant and 100 ul of the media were counted on a scintillation counter. The lens water (L) to medium (M) concentration ratios (L/M) were calculated as previously published [Tracer levels (0.2\u00a0\u00b5Ci per well) of [ublished . In brieFollowing 4 days of culture in the presence or absence of various concentrations of Pb nitrate, lenses were collected and homogenized in 500\u00a0\u00b5l cold 10% TCA. The protein pellets were washed three times with 10% TCA and then resuspended in 10\u00a0mM Tris pH 7.4. One hundred microliters of resuspended pellet was mixed with 10\u00a0\u00b5l of 2.5% sodium borohydride in 0.01 N NaOH. The mixture was incubated for 1 h at 37\u00a0\u00b0C. Ten percent TCA was added to a final concentration of 1% to stop the liberation of thiols. The freed thiol levels (nmol/mg) were measured by the Ellman\u2019s assay as previously described . A totalThree lenses per group were cultured for 3 days in the presence or absence of Pb nitrate as above, fixed in 2.5% glutaraldehyde in cacodylate buffer for 4 h, and transferred to 10% buffered formalin. The fixed samples were embedded in methylmethacrylate, sectioned and stained with hematoxylin and eosin (H&E). The experiment was repeated twice for a total of 9 lenses per group. Three sections per lens were examined for histological alterations.3. Following centrifugation at 10,000\u00d7 g for 30 min at 4\u00a0\u00b0C, the sample was loaded on a Superose-12 column and eluted with the same buffer with 1\u00a0ml fractions collected. The \u03b1-crystallin peaks were identified by SDS\u2013PAGE and pooled for reinjection on the same HPLC system [3).Three to four lenses per group (control and 1\u00a0\u00b5M Pb nitrate) were homogenized in 0.5 M Tris buffer (pH 7.4) with 0.1 M KCl, 1\u00a0mM EDTA, 10\u00a0mM 2-mercaptoethanol, and 0.2% NaNC system . The finThe first dimension isoelectric focusing was performed using a BioRad Protean IEF Cell with 7 cm 3\u201310NL immobilized pH gradient strips. Aliquots of the purified \u03b1-crystallin or whole lens samples  were homogenized on ice with 8 M urea containing 4% CHAPS and 100\u00a0mM DTT (rehydration buffer). No insoluble material was visible. Four micrograms of lens protein was loaded onto the IPG strips with rehydration buffer containing 2% ampholytes and bromophenol blue for a total volume of 125\u00a0\u03bcl. IEF strips were rehydrated for 12 h and then a three step program was used including Step 1: initial removal of salts (500 V for 500 V-h); Step2: an intermediate step (1000 V for 2000 V-h); and Step 3: the final protein separation according to pI . Following protein IEF focusing, the IPG strips were equilibrated with 100\u00a0mg/10\u00a0ml DTT and 400\u00a0mg/ml iodoacetamide and the second dimension SDS\u2013PAGE was performed using 12% pre-cast Bis-Tris gels and MOPS as the running buffer. The gels were fixed and stained with either silver or colloidal Coomassie blue G-250. The gels were scanned with a Molecular Dynamics Personal Densitometer and analyzed using Phoretix image analysis software . Six samples from the 1\u00a0\u00b5M Pb and 6 samples from the control group were analyzed in duplicate.Protein Prospector algorithm.Protein spots of interest were cut from the gel, destained in 50% methanol in 20\u00a0mM ammonium bicarbonate buffer, and digested overnight with 10 ng/ul trypsin . Peptide\u03b1-Crystallin and apo-\u03b1-lactalbumin (1:3 weight ratio) were dissolved in 50\u00a0mM phosphate buffer (pH 6.82) containing 0.1 M NaCl and 2\u00a0mM EDTA. DTT at a final concentration of 50\u00a0mM was added to initiate denaturation of the lactalbumin. Aggregation was monitored as a function of lactalbumin aggregation as previously described .t-tests were calculated  to determine statistical significance of the mean \u00b1standard deviation of the Pb groups versus the control groups for all assays.Student\u2019s To examine whether longer term culture of young lenses in the presence of Pb would lead to lens opacity formation (as does long-term culture of older lenses) without subsequent oxidative challenge, lenses were cultured up to 9 days in the presence or absence of Pb nitrate. The lens clarity was monitored and graded daily according to the chart shown in For control lenses, at culture day 5, roughly 80% of the lenses were at Stage 1, 10% at Stage 2, and 5% at Stages 3 and 4. By culture day 8, 50% of the lenses had progressed to Stage 2, with approximately 7% at Stages 3 and 4. This indicates that 8 days in culture is approaching the outer limits of lens viability under the conditions employed. In contrast, by day 5 in culture with Pb only 30% of the lenses remained at Stage 1. At culture day 8, the Pb exposed lenses have all advanced to Stage 3 and 4 indicating that lenses cultured in the presence of Pb  fail at an accelerated rate as compared to the control lenses.To examine whether lenses cultured in the presence of Pb are predisposed to failure, lenses were cultured for 3 days in the presence or absence of Pb nitrate and then challenged for 2 days with 250\u00a0\u00b5M hydrogen peroxide. Lenses cultured with Pb exhibit a higher clearance of hydrogen peroxide from the media than the control lenses  at 2 h aThe degree of protein mixed disulfides present, a measure of protein oxidation and oxidative stress, was increased in Pb-exposed lenses 4 days; . In FiguAlterations in the lens epithelium following culture in 1\u00a0\u03bcM Pb for 3 days as compared to the control cultured lenses  include ins-crystallin form, which is present in rodents but not humans or other primates [\u03b1-Crystallin, a molecular chaperone and major lens structural protein, is believed to maintain lens transparency through prevention of protein aggregation. Post-translational modification of \u03b1-crystallin may negatively affect maintenance of lens clarity through decreased chaperone function. To examine the degree of post-translational modification of \u03b1-crystallin in Pb exposed lenses, \u03b1-crystallin was purified from lenses cultured for 4 days in the presence or absence of Pb and separated by two dimensional gel electrophoresis with the individual protein spot intensities quantified by densitometry. A comparison of the 2D gel spot patterns obtained from 10\u00b5g of protein from control and Pb exposed lenses is shown in primates .Six forms of \u03b1B-crystallin are visible on the gels shown in ins-crystallin isoform, spots \u03b1A8- and \u03b1A9-crystallin are increased in abundance in the Pb cultured lenses when normalized against the dominant \u03b1A1-crystallin spot. The spot \u03b1A7-crystallin does not change significantly in the Pb cultured lenses as compared to the control cultured lenses. This indicates alterations in the normal processing of \u03b1A-crystallin do occur in optically transparent lenses which have been cultured in the presence of Pb nitrate. It is also interesting that the total amount of \u03b1Ains-crystallin appears to increase in lenses cultured with Pb nitrate since there is approximately 5%\u201310% more of two of the three spots identified as \u03b1Ains-crystallin for a total change in the amount of \u03b1Ains-crystallin by 10%\u201320%. This result was not statistically significant but the trend was evident in nine out of twelve individual lenses examined in the Pb exposed group.Quantitation of the relative spot densities of the different forms of \u03b1-crystallin is shown in Three forms of \u03b1B-crystallin which are present in both the control and Pb cultured lenses, representing more acidic  and cleaved forms , have significant increases in relative abundance in the Pb cultured lenses when normalized against the dominant spot form of \u03b1B-crystallin , is mosThe current study also examined whether lenses from young rats (4\u20136 weeks) cultured in the presence of Pb for longer periods of time would develop opacities. The significantly increased rate of failure of Pb cultured lenses was associated with development of lenticular opacities, severe abnormalities of the epithelial layer, increased weights, and pronounced acidification and degradation of crystallins (data not shown). Lenses cultured in unmodified media exhibited the same types of abnormalities upon failure, though failure was at a later time point. Thus Pb may accelerate the decline in lens membrane function ultimately leading to cataract formation in both young lenses and in the older, less metabolically active lenses (from 4.5 month old rats) from our prior study . We propThis study has demonstrated the ability of Pb to compromise lens integrity following short-term in vitro challenge and to predispose the lenses from 4 to 6 week old rats to opacities induced by subsequent oxidative challenge. Pb-exposure also negatively impacted lens clarity at longer exposure times which may point to a direct cataractogenic effect of Pb. Coupled with our previous in vivo data demonstrating alterations in lens homeostasis following short-term oral Pb-exposure in young rats, these data support the hypothesis that Pb-exposure is causally related to lens opacification. While there is a lack of knowledge of the type of cataract associated with Pb-exposure in humans, the current study raises the question of whether cortical opacities and lens epithelial cell biochemical abnormalities may be linked to in vivo Pb-exposure in humans."}
+{"text": "Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an  Osteoarthritis (OA) is characterized by the progressive degradation and loss of articular cartilage . OA is tin vitro chondrocyte and explant cultures  with specific activity of 90 curies per millimole was obtained from American Radiolabeled Chemicals, Inc. .NZW rabbits (2.2 to 2.9 kg) of either gender were killed by intravenous injection of Beuthanasia-D special . The chondrocytes were isolated as described previously . The via5 cells per well in 1 ml of complete media. Chondrocytes were allowed to attach for 3 to 5 days, and media were changed every 3 days. Confluent cells in multiwell plates were labeled with 1 to 2 \u03bcC/well with [3H]-proline during the last 24 to 48 hours of cell culture. The cell monolayer was washed at least four to five times with warm HBSS by flipping the plates to remove unincorporated proline from the matrix. Albumin- or serum-free EBSS was added to wells. Experiments were carried out in triplicate wells. The test reagents were added, and the total volume was adjusted to 0.5 ml with EBSS. The cultures were incubated at 37\u00b0C in a humidified 5% CO2 incubator for 4 to 24 hours. [3H]-proline release was measured in cell supernatant and cell lysates. A 100-\u03bcl aliquot was removed and processed for scintillation counting. The plastic-bound [3H]-proline-labeled matrix  was solubilized with 0.5 M NaOH and counted. Percentage release of total [3H]-proline-labeled collagen was calculated.Primary rabbit articular chondrocytes were distributed into 24-well plates at a concentration of 1 to 2 \u00d7 102+ (copper ion) or 5 \u03bcM Cu2+ and 50, 5, or 0.5 mM glucosamine. Data are expressed as malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) equivalents in nanometers.The very-low-density lipoprotein and low-density lipoprotein (LDL) fractions were isolated from serum by ultracentrifugation at a density of 1.063 g/ml and were kindly provided by Vincent A. Rifici and Avedis K. Khachadurian from the Department of Medicine of our medical school . Lipoprog for 10 minutes at 25\u00b0C, and the absorbencies of the supernatants were measured in a spectrophotometer at 532 nm. TBARS are expressed as nanomoles of malondialdehyde equivalents of lipoprotein protein compared with tetramethoxypropane standard -proline-labeled collagen as compared with the background amount of collagen released by unstimulated control chondrocytes. In the presence of 25 mM concentrations of glucosamine hydrochloride or glucosamine sulfate, there was statistically significant inhibition of the release of labeled collagen at 4 hours. In comparison, N-acetyl glucosamine and N-acetyl mannosamine did not result in inhibition of collagen degradation. The data indicate that glucosamine hydrochloride and glucosamine sulfate have specificity and significantly inhibit collagen degradation by activated chondrocytes.We tested the effect of glucosamine hydrochloride and glucosamine sulfate on chondrocyte-dependent collagen degradation in the previously described ro model . For comAs shown in Figure We monitored conjugated diene formation as an indicator of lipid peroxidation in activated chondrocytes and purified lipoprotein oxidation with or without glucosamine hydrochloride . As showWe investigated the effect of glucosamine hydrochloride on TBARS formation in Cu-induced oxidation of lipoproteins. As shown in Figure We tested the effect of glucosamine hydrochloride on aldehyde-protein adduct formation in control and stimulated chondrocytes. Protein gel electrophoresis and immunoblot analysis using MDA2, specific for MDA-modified lysine of chondrocyte extracts, is shown in Figure We tested the effect of glucosamine hydrochloride on lipoprotein protein oxidation using the identification of protein carbonyls as one of the modifications as described in oxidized proteins ,34. The in vitro model of chondrocyte activation-dependent collagen degradation, we show that glucosamine specifically and significantly inhibited collagen degradation. Inhibition of collagen degradation by glucosamine was not mediated by inhibiting the chondrocyte lipid peroxidation process but by inhibiting advanced lipoxidation reactions. Specifically, glucosamine inhibited purified lipoprotein protein oxidation and aldehydic oxidation of chondrocyte matrix.Using this in vitro model, we had previously shown [in vitro model to human OA pathogenesis was demonstrated by detection of in vivo molecular imprints of lipid peroxidation in which OA and normal cartilage tissue sections were studied [in vivo role of lipid peroxidation in the OA pathogenesis [Using this ly shown ,35,36 thly shown ,35. Thisogenesis . CollectWe investigated the effect of glucosamine in our assay system. As shown, only glucosamine hydrochloride or glucosamine sulfate specifically and significantly inhibited collagen degradation by activated chondrocytes and the effect was dose-dependent. Similar effects by both agents (glucosamine hydrochloride and glucosamine sulfate) excluded the possibility that the inhibition observed was mediated by the sulfate moiety in the latter compound. Glucosamine hydrochloride had little or variable effect on hydrogen peroxide-induced collagen degradation, suggesting that it did not inhibit oxygen radical/hydrogen peroxide-mediated collagen degradation (data not shown).in vitro model for studies on lipoxidative modification of proteins [Since the mechanism of collagen degradation in this model appears to involve the activation of lipid peroxidation in chondrocytes, it raises the possibility that glucosamine was acting like a chain-breaking antioxidant similar to vitamin E. However, glucosamine had no discernable effect on conjugated diene formation by activated chondrocytes, suggesting that its mechanism of action was not due to chain-breaking antioxidant activity. As expected, vitamin E inhibited conjugated diene formation by chondrocytes. To further confirm these findings, we tested the effect of glucosamine in a purified lipoprotein oxidation model system, a commonly used proteins . Again, The inhibition of collagen degradation by glucosamine was manifested even when the addition of glucosamine was delayed in activated chondrocyte cultures, indicating that its mechanism of action involved downstream events of chondrocyte activation rather than interfering with or blocking the early events of chondrocyte activation by calcium ionophore. We tested the effect of glucosamine on TBARS formation by Cu-induced oxidation of purified lipoproteins. Glucosamine in a dose-dependent manner inhibited malondialdehyde formation by oxidized lipoprotein. The data suggest that glucosamine either inhibited or scavenged aldehydic products of lipid peroxidation. However, glucosamine did not interfere in the detection of control malondialdehyde in TBARS assay, suggesting that most likely glucosamine inhibited advanced lipoxidation reactions rather than scavenging aldehydic products.in vitro model system.The identification of aldehydic adducts provides a molecular clue of chondrocyte matrix damage mediated by lipid-free radicals . On immuInhibitors of advanced lipoxidation reactions such as aminoguanidine and pyridoxamine have been evaluated in animal models of diseases such as diabetes ,39. Thesin vivo tissue levels of glycosaminoglycans in cartilage are hundreds perhaps thousands of folds higher than in serum or joint fluids, glucosamine, which is a structural component of aggrecan, may locally provide an antioxidant environment that may protect cartilage collagen from oxidative damage.The pharmacokinetics of oral administration of glucosamine sulfate show that plasma levels increase more than 30-fold from baseline and peak at approximately 10 \u03bcM with the standard 1,500-mg once-daily dosage . We postin vitro model system may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.Our data suggest that the decrease in collagen degradation by glucosamine observed in this in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction, glucosamine decreases collagen degradation by inhibiting advanced lipoxidation reaction and thus prevents the oxidation and loss of collagen matrix from labeled chondrocyte matrix.In an AGE = advanced glycation reaction; BSA = bovine serum albumin; Cu = copper; DMEM = Dulbecco's modified Eagle's medium; DNP = dinitrophenyl; EBSS = Earl's balanced salt solution; ECL = enhanced chemiluminescence; FBS = fetal bovine serum; HBSS = Hanks' balanced salt solution; HRP = horseradish peroxidase; IL-1 = interleukin-1; LDL = low-density lipoprotein; OA = osteoarthritis; PBS = phosphate-buffered saline; TBARS = thiobarbituric acid-reactive substances; TBS = Tris-buffered saline.The authors declare that they have no competing interests.MLT developed the study experimental protocol. All authors participated in conducting and analyzing the experiments. All authors were involved in the drafting, review, and final approval of the manuscript."}
+{"text": "Candida) in the elderly. The subjects were 64 elderly subjects with fixed prostheses and 49 who wore removable partial dentures aged over 65 years. We used one-way ANOVA to test for overall differences of the number of MT among 5 oral environmental factors. The significant differences were observed in the lactobacilli counts for different number of MT. The number of MT increased with an increase in the lactobacilli counts with removable denture. In conclusion, for the patients wearing removable dentures, increasing number of MT was associated with an increase in the lactobacilli counts in saliva. For the patients with crowns and fixed partial dentures, the number of MT was not significantly affected by salivary mutans streptococci, lactobacilli, and Candida counts.The purpose of this study was to investigate the relationship between the number of missing teeth (MT) and the statuses of oral environmental factors (the stimulated salivary flow rate, buffering capacity, and the counts of mutans streptococci, lactobacilli, and  Caries activity test using saliva is believed to provide useful information for selecting the type of prosthesis. The oral environmental factors considered in the caries activity test include both host and microbial factors associated with caries. We investigated the differences between the statuses of the oral environment factors in elderly individuals with fixed and those with removable prostheses. The results of our study revealed that the amount of cariogenic bacteria in persons with fixed prostheses is different from that of elderly persons with removable dentures . HoweverThe population of the elderly has been increasing worldwide. There is an increase in the number of risk factors of oral diseases and MT in the elderly. The type of prosthesis was decided by the number of MT in most cases. However, the distal extension missing must place removable denture even if the number of MT is little. Therefore, the oral environment should examine not only the statue of the prosthesis, but also the number of missing teeth. To prevent an increase in the number of MT, maintenance of oral environmental factors is valuable and important. The purpose of this study was to investigate the relationship between the statuses of oral environmental factors and the number of MT in elderly persons with different types of prosthesis.This study protocol was screened and approved for its ethical acceptability by the Committee on Experimental Research on Humans of the Osaka Dental University. The volunteers selected included 113 elderly aged over 65 years divided into 2 groups: The volunteers were divided into the crowns and fixed partial denture group (Dentate group), in whom MT were restored only by using fixed partial dentures, and into the removable denture group (Denture group), in whom MT were restored using removable partial and complete dentures.The one group comprised 64 dentate elderly with fixed prosthesis alone, with a mean age and the number of remaining teeth of 69.8 \u00b1 4.4 years and 26.7 \u00b1 2.8 (Dentate group). The other group comprised 49 elderly with removable prostheses, with a mean age and the number of remaining teeth of 69.4 \u00b1 6.1 years and 15.3 \u00b1 6.6 (Denture group). Candida (CA).We examined their oral conditions and assessed the oral environmental factors. We selected the following 5 factors for the evaluation of the oral environment: the stimulated salivary flow rate, buffering capacity, and the counts of mutans streptococci (SM), lactobacilli (LB), and The number of MT and the kind of prosthesis  were determined by oral examination.The saliva test was performed using the Dentocult series , and saliva was collected more than 1 hour after breakfast, between 9:30\u2009am and 11:30\u2009am. The stimulated salivary flow rate was measured by spitting out saliva after chewing a 1\u2009g paraffin pellet for 5 minutes. The stimulated salivary flow rate was the volume of whole stimulated saliva collected per minute. The stimulated saliva buffer capacity was measured by Dentocult buff strip. The amount of bacteria in the saliva was counted using simple culture kits, Dentocult SM, LB, and CA. After culture, the SM, LB, and CA counts were determined by a comparison with model charts attached to the kits \u20134. The fWe examined the relationship between the number of MT and oral environmental factors in individuals with different types of prosthesis (Dentate and Denture group) using one-way ANOVA. The significance level was set to 0.05.Dentists should select restorations so as to avoid secondary caries and periodontitis, which will in turn prolong the survival of the remaining teeth. Increase in the number of missing teeth necessitates extensive and large number of restorations. Therefore, it is important to compare the number of MT with the bacterial counts with different types of prostheses.In general, the volume of stimulated salivary flow is decreased in the elderly. However, the number of MT did not influence the amount of the stimulated salivary flow rate in each group. The results regarding buffer capacity may have been influenced by the saliva volume. The bicarbonate level in saliva is strongly dependent on the amount of saliva secreted. Bicarbonate is one of the main factors determining the buffer capacity of saliva. A significant effect on the number of missing teeth was not detected for SM and CA counts in each group. The Dentate group had a lot of low risk level for SM, LB, and CA counts. Therefore, we think that there was no difference. In the Denture group, SM and CA counts have been distributed at each level. However, the number of MT was significantly affected only by LB. LB is associated with dental caries (open carious lesions), poorly executed restorations, and poor oral hygiene , 6. ThusSM increase caries activity in the acid stress , whose cCA coexist with SM in denture plaques and readily adhere to resin-base dentures . CA counWe think that in routine clinical practice, significant microbial counts should be considered in designing prosthesis. Fixed partial and removal dentures are the 2 main methods for restoration of MT. Removal of dentures is indispensable in cases of a high number of MT, but it is associated with a risk for developing caries. We think that it is preferable to use a fixed partial denture than a removable denture when fixed restoration is possible.In conclusion, for the patients wearing removable dentures, increasing number of missing teeth was associated with an increase in the lactobacilli counts in saliva. For the patients with crowns and fixed partial dentures, the number of missing teeth was not significantly affected by salivary mutans streptococci, lactobacilli, and Candida counts."}
+{"text": "Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders.Apicomplexa. For each of the 13 species investigated, a set of proteins predicted to be involved in the ubiquitylation pathway has been identified and demonstrates increasing component members of the ubiquitylation pathway correlating with organism and genome complexity. Sequence homology and domain architecture analyses facilitated prediction of apicomplexan-specific protein function, particularly those involved in regulating cell division during these parasite's complex life cycles.Here we describe the use of an HMM search of functional Pfam domains found in the key components of the ubiquitin-mediated pathway necessary to activate and reversibly modify target proteins in eight apicomplexan parasitic protozoa for which complete or late-stage genome projects exist. In parallel, the same search was conducted on five model organisms, single-celled and metazoans, to generate data to validate both the search parameters employed and aid paralog classification in This study provides a comprehensive analysis of proteins predicted to be involved in the apicomplexan ubiquitin-mediated pathway. Given the importance of such pathway in a wide variety of cellular processes, our data is a key step in elucidating the biological networks that, in part, direct the pathogenicity of these parasites resulting in a massive impact on global health. Moreover, apicomplexan-specific adaptations of the ubiquitylation pathway may represent new therapeutic targets for much needed drugs against apicomplexan parasites. Plasmodium, Toxoplasma and Cryptosporium. Toxoplasma gondii and Cryptosporium parvum are the etiological agents of toxoplasmosis and cryptosporidiosis, respectively, which are predominantly opportunistic infectious agents responsible for severe mortality amongst immuno-suppressed patients such as those infected with HIV. The human malarial parasite Plasmodium falciparum, which is responsible for over a million deaths annually P. falciparum has evolved resistance to many front-line antimalarial drugs P. falciparum but other apicomplexan parasites as well.Apicomplexans are obligate protozoa intracellular parasites responsible for several major human diseases prevalent in the developing world. These include organisms belonging to the genera P. falciparum and the rodent malaria parasites P. yoelii, P. berghei and P. chabaudi have been published P. vivax well underway. In addition, the complete annotated genomes of T. gondii, C. parvum and Cryptosporium hominis have been recently released P. falciparum malaria Genome sequencing projects are available for several apicomplexan parasites, with many of them completed. The full genome sequence of the human malarial parasite via covalent conjugation to ubiquitin (or more often polyubiquitin chains) is a well-established signal for proteosomal destruction P. falciparum has been established Here we describe a comparative analysis of one of the essential post-translational regulatory networks commonly found in eukaryotic cells\u2013the ubiquitin/proteasome system (UPS). Modification of proteins via an activation and transfer cascade via conjugation to the E3) to form an isopeptide bond. Since ubiquitin contains several lysine residues, it can itself be ubiquitinylated, leading to the formation of polyubiquitin chains. Differences in affinity for ubiquitin/UBLp by the component parts of the cascade, as well as a hierarchical increase in the numbers of these proteins , drive the transfer of ubiquitin/UBLp through the cascade with the final target specificity mediated through the E3 complex.Specificity in the conjugation of ubiquitin and ubls to their final target is elegantly achieved Ubiquitin, a highly conserved 76 amino acid peptide, was first described in 1974 via a transesterification reaction relies on additional motifs present in E1. The E2 protein contains a single motif that mediates interaction with both E1 and E3, signifying the \u201cshuttle\u201d status of E2 in the transfer of ubiquitin/UBLps between activation and subsequent ligation to their final target. E2s are present as multiple isoforms, each with distinct roles. E2s exist for each UBL modifier, with multiple E2s capable of accepting ubiquitin. However, even within the ubiquitin E2 isoforms, there is functional divergence in the specific E3s they interact with, and thus the cellular processes they are involved in. For example, Rad6p and Cdc34p E2 isoforms deliver ubiquitin to E3s that ultimately target proteins involved in the regulation of DNA repair and cell cycle progression, respectively Analysis of the E1-activating enzymes indicates that they share sequence homology to MoeB/ThiF domains of prokaryotic biosynthetic proteins involved in sulphur donor systems e.g. MDM2 known to target p53) and U-box. Two sub-classes of RINGs have further been defined: RING in between RING-RING (RIR) and Cullin-RING ligases (CRL), which are multi-protein complex E3s , DNA-repair (ubiquitylation of p53 by the RING finger MDM2) and transcriptional regulation (activation of NF-kB by the RING finger TRAF6).E3 ubiquitin ligases are very diverse. They have been classified into three main classes according to the presence of specific domain motifs: Homologous to E6-associated protein C-terminus (HECT), Really Interesting New Proteins  Conversely, de-ubiquitinylation enzymes (deubiquitinases or DUBs) specifically remove ubiquitin/UBLps. DUBs are a large group of cysteine proteases or zinc-dependent metalloproteases that specifically cleave after the terminal carbonyl of the last residue of ubiquitin adducts. Compared to the proteins involved in the activation, conjugation and ligation of ubiquitin/UBLps relatively little is known about the functional role of DUBs. However, evidence suggests that DUBs are key regulators of the ubiquitin system; DUBs are functionally similar to protein phosphatases in the phosphorylation system. Based on their sequences similarities, structural studies and potential mechanism of action, DUBs fall into at least six distinct subfamilies: the ubiquitin C-terminal hydrolases (UCH-Peptidase_C12), the ubiquitin specific proteases (USP-UCH), otubains (OTU), the ataxin-3/Josephin ubiquitin protease (MJD), the JAMM isopeptidase (Mov34) and the recent Pseudomonas syringae has been shown to induce sensitivity in tomato plants by targeting a host kinase, Fen, to the proteasome, which leads to the inhibition of the Fen-activated immunity-associated programmed cell death Salmonella enterica), which causes gastroenteritis in humans, has similarly been implicated in its virulence The UPS is known to play important roles in modulation of immune and inflammatory responses. Deregulation of the UPS can lead to the development of inflammatory and autoimmune diseases, such as inflammatory arthritis, psoriasis, allergy and asthma . With regards to the five eukaryotic model organisms that were used, the threshold E-value \u22640.5 gave the most consistent results when compared to previously published results. The number of UPS-related proteins in A. thaliana and the other model organisms has previously been analyzed, particularly the number of E2 and E3 enzymes that are found in A. thaliana, H. sapiens, C. elegans and S. cerevisiae . In H. sapiens, 162 DUBs/DUBLs were found although a previous publication only identified 95 putative DUBs/DUBLs from which 79 exhibited conserved catalytic residues H. sapiens and D. melanogaster genomes contain multiple isoforms for some families of DUBs and DUBLs. Furthermore, the Hidden Markov Model that we used to search for UPS components compiled more complete datasets than many other search approaches would do. For example, while our HMM search identified domain OTU-carrying proteins in apicomplexan parasites (OTU is a major sub-class of DUB) none was reported in a recent publication on parasitic protozoa deconjugating enzymes where the authors used a more selective BLASTP homology search Amongst the 13 proteomes investigated in this study, a total of 4453 proteins were identified as carrying one or more of the 24 selected Pfam domains . For exae.g. 43% in A. thaliana) while only few of them were identified in apicomplexan organisms. F-box-containing proteins are adaptor proteins in Cullin-RING-Ligase complexes (CRLs), and are involved in direct and specific substrate recognition. Previous authors have hypothesized that the very high number of F-box proteins in A. thaliana suggests that plants can assemble numerous CRLs, which could control a wide array of substrates With regards to the relative abundance of each domain family, a striking observation is that a high proportion of F-box-carrying proteins are present in multi-cellular organisms  in triangular distance matrices. Results of this analysis are shown in A. thaliana UCHs and deSUMOylases. In the other subclasses, relative divergences exist within and between species. An increased divergence can be observed in the metalloprotease (JAMM/Mov 34). The function of the WLM family, usually found only in plant and fungus, in Plasmodium and Toxoplasma thus deserves to be fully investigated.With regards to the eight subclasses of DUBs analyzed, the Josephins (MJD), UCHs (peptidase_C12), autophagins (peptidase_C54) and deSUMOylases (peptidase_C48) families are well conserved within and between species with the exception of a clear differential expansion in the http://lerochlab.ucr.edu/UPS_prediction_data). When possible, the complete dataset was used to predict apicomplexan functional homologs of known UPS components from data available from the five model organisms investigated here. For each domain, dendrogram trees were built with all the 13 species that we used in this study. For purpose of clarity, only apicomplexan data are presented here. However, our complete results are available for download on the laboratory website (P. chabaudi and P. yoelii) contain partial ubiquitin sequences; however it was impossible to definitively assign the final ubiquitin gene based on the sequence available. In addition to ubiquitin/UBLps, several genes were identified by the HMM search that contain the highly related ubiquitin-binding domain (UBD). These typically N-terminal located domains are found in proteins that have evolved to adopt the ubiquitin domain in a non-conjugated role in processes such as signal transduction and proteasomal delivery Ubiquitin is a 76 amino acid protein extensively conserved between all eukaryotic sequences, with similarities in excess of 98% between humans, yeast and apicomplexan parasites de novo synthesis of ubiquitin and recycling of ubiquitin following cleavage from their target proteins. During periods of stress, elevated demands for ubiquitin are met, in part, by increased levels of polyubiquitin expression P. falciparum indicates all three ubiquitin genes are expressed throughout the parasite's life cycle As in all eukaryotes, ubiquitin is encoded by one of three types of fusion-protein precursors in the apicomplexans investigated here . AlthougPlasmodium spp., single copies of genes encoding SUMO have been identified across all the apicomplexan organisms investigated here , is most similar to ubiquitin at the primary sequence level. NEDD8 typically accumulates in the nucleus where its only known target, cullin, is found S. cerevisiae, conjugation to proteins involved in mRNA and pre-mRNA splicing have been described, and may more likely reflect the role of HUB 1 in apicomplexans Escherichia coli sulphur carring proteins ThiS and MoaD involved in thiamin and molybdopterin synthesis, respectively S. cerevisiae, URM1 has only been found to conjugate to alkyl hydroperoxide reductase 1 (AHP1), suggesting some role in adaptation to oxidative stress may similarly operate in apicomplexans Single copies of genes encoding the less characterized UBLps URM1 and HUB1 are found throughout the apicomplexan lineages investigated here . Both UBS. cerevisiae identified two UBLps involved in this system, termed ATG8 and ATG12 Leishmania spp. and play a key role in parasite virulence P. falciparum as being an ATG12 paralog .The autophagy system facilitates degradation of the cytoplasm following engulfment in a vesicle followed by fusion to lysosomes, a process necessary for both cell differentiation and response to starvation. Analysis of mutations in autophagy in P. falciparum and T. gondii), suggests that these UBLps are expressed at all the life stages investigated. These data suggest that ubiquitin/UBLps are essential components in controlling cellular processes throughout apicomplexans complex parasitic life cycles.A number of UBLps typical of higher eukaryotes  have not been found in this analysis, nor that previously described by Ponder and Bogyo (2007). Although some UBLps may not be expected based on their predicted roles in immune system regulation in higher eukaryotes, their absence, coupled with that of SUMO variants and ATG12 in apicomplexans suggest a more restricted role for UBLps in apicomplexan cell biology. However, analysis of gene expression data (microarray and proteomics) for SUMO, NEDD8, HUB1, URM1 and ATG8, where available  can deliver activated ubiquitin to several E2 isoforms, the E1s responsible for activating the UBLps SUMO and NEDD8, termed UBA2 and UBA3, respectively, only transfer to a single cognate E2 (see below). UBA1 has two UBA domains on a single polypeptide. UBA2 and UBA3, each only have one UBA domain containing the active site cysteine required for the covalent attachment of activated SUMO and NEDD8, and actually represent one part of a E1 heterodimer complex with AOS1 or APPBp1, respectively P. falciparum of UBA2 and UBA3 with those of the unassigned UBA containing proteins, as well as searching for existing characterized yeast two hybrid interactions, did not provide any additional clues in defining AOS1 or APPBp1 paralogs E1 proteins are characterized by the presence of the ubiquitin activating (UBA) Pfam domain. Additional motifs in E1 are responsible for the correct selection of ubiquitin/UBLp for activation and subsequent E2 to which transfer the activated ubiquitin/UBLp In addition to those described above, three additional E1 proteins are indicated in P. falciparum provide extensive evidence for the ubiquitous expression of E1s throughout the parasite's life cycle T. gondii similarly suggest constitutive expression of E1s throughout apicomplexan life cycles.Proteomic and transcriptomic profiling data available for via a transesterification reaction, is buried in a shallow groove Eukaryotes express a number of E2 isoforms, typically of between 17\u201322kDa  are only found in Plasmodium spp. and T.gondii in this analysis and are atypical E2s of up to 54kDa with a long N-terminal extension. N- and C-terminal extensions in E2 are thought to play key roles in recognition and association with E3s and their subsequent protein target and thus these atypical E2s may reflect a specific adaptation in the Plasmodium and Toxoplasma lineages.The apicompexan parasites investigated here have eight to fourteen E2 proteins, similar to the 14 described for the only other single cell eukaryote revisiae . The numP. falciparum indicates a clear association of the UBC13 and UEV paralogs in this organism Different isoforms of E2 have distinct roles in regulating downstream functions through specific interaction with distinct E3s . While sP. falciparum E2s suggest a diverse pattern of steady state mRNA accumulation at different stages of intraerythrocytic development. The fact that different E2 isoforms are expressed at distinct stages in the parasite's life cycle suggests that a temporal profile of delivering ubiquitin/UBLps to different E3s exists, which highlights a potential additional level of temporal control in the UPS system during apicomplexan parasite's life cycles.Extensive gene expression data for nine of the fourteen P. falciparum, and their homologs in T. gondii, C. parvum, and yeast. There are three superfamilies of E3 ubiquitin/UBL ligases. HECT ubiquitin ligases have a direct role in catalysis during ubiquitylation, whereas RING  finger and U-box E3s are involved in multi-protein complexes. RING finger E3s are the most abundant ubiquitin/UBL ligases.E3 ubiquitin/UBL ligases are a very diverse group of proteins involved in specifically transferring ubiquitin/UBLps to a given substrate. In all organisms, 48% of the predicted UPS components identified belong to the E3 ubiquitin/UBL ligase family. This high percentage of E3 reflects the specificity that is required for specific substrate recognition. P. falciparum, and other apicomplexans. Three of them have a homolog in S. cerevisiae: TOM1, UFD4, and HUL5 (HUL5 has unknown functions). The fourth HECT-domain protein that we identified in apicomplexans does not match any protein from yeast but is similar to UPL5 in A. thaliana  has been recently described as being involved in cell cycle arrest after DNA damage, mediating CDC6 ubiquitylation, a protein essential to initiation of DNA replication P. falciparum, and 80.m02344 in T. gondii) is involved in the ubiquitin fusion degradation pathway (UFD pathway) in vivo half-life of a protein to the identity of its N-terminal residue; for example, in eukaryotes, a protein with an isoleucine at its N-terminal end will be targeted to the proteasome more rapidly than a protein with a glycine, which is a stabilizing residue; see Our search identified four HECT domain-containing proteins in iana see . UPL5 haS. cerevisiae UFD2  and PRP19  homologs  contains WD40 repeats, which are known to be involved in a wide array of cellular processes ranging from signal transduction and transcription regulation to cell cycle control and apoptosis PRP19 is an oligomeric U-box-containing E3 ligase Plasmodium species  and shares extensive homology with the human protein CHIP . Like UFD2, CHIP has been described as being an E4 ubiquitin ligase in human. A particular feature of CHIP is that its catalytic activity requires its homodimerization through the U-box domain . Interestingly we also identified CHIP homologs in P. berghei, P. chabaudi, P. vivax and P. yoelii  whereas no homolog was found in yeast Cryptosporidium spp. and T. gondii. Thus, it can be hypothesized that CHIP homologs in Plasmodium are involved in the hepatic infection stage, although a precise role remains to be determined.A third U-box containing ubiquitin ligase, that is absent in yeast, has been identified in P. falciparum, and 80.m02207 in T. gondii). The SCF contains at least four subunits: CDC53 (Cullin1) which is stabilized by the ubiquitin-like modifier NEDD8, the RING E3 RBX1, the adaptor protein Skp1 and a F-box protein for substrate recognition , the cullin classically involved in the APC/C (APC2) was not found in apicomplexan parasites. The role of CUL8 is not well known. Previous data suggest that it may be involved in anaphase progression P. falciparum is not entirely unexpected. Cell division during schizogony is apparently asynchronous in P. falciparum, that is, several rounds of DNA replication/DNA division occur before final cytokinesis, instead of the paradigm of successive cycles of alternating DNA replication/DNA division/cytokinesis . The most famous CRLs are the Skp1-Cullin1-Fbox (SCF) complex, containing the cullin protein CDC53 in yeast, and the Anaphase Promoting Complex/Cyclosome (APC/C), containing the cullin protein APC2 in yeast. Both the SCF and the APC/C are involved in cell-cycle progression . For example, we have found potential RAD16, RAD5, and TFB3 homologs , which are known to be involved in nucleotide excision repair (NER) P. falciparum) or pre-mRNA maturation  P. falciparume.g. PfBre1 PFF0165c in P. falciparum, 35.m01589 in T. gondii), or chromatin remodeling and silencing . Recent data have increasing shown that epigenetic mechanisms play a key role in the control of gene expression in Apicomplexa. In Plasmodium, chromatin modeling is one of the proposed mechanisms that control allelic exclusion of the var virulence genes T. gondii, acetylation of histone H4 or acetylation on lysine 9 of histone H3 as well as trimethylation of lysine 4 of histone H3 were shown to be located close to the 5\u2032 UTR of the active genes RING finger and RING-like E3 ubiquitin ligases are the largest group of E3s. They do not have a direct catalytic role in linking ubiquitin and ubiquitin-like modifiers. They rather act as adaptor partners: RING E3s interact with both an E2 ubiquitin-conjugating enzyme (that is carrying ubiquitin) and its given specific substrate, bringing the substrate in close proximity with ubiquitin. Since RING and RING-like E3s are involved in substrate recognition, and given the diversity of proteins that are targeted by the UPS, a large diversity of RING and RING-like E3s is expected , RING/helicase, RING/forkhead-associated domain (FHA), and RING/in between RING (IBR). It is interesting to observe that predicted Plasmodium RING E3s with a single RING domain appear more abundant than those of yeast. Several of these proteins carry several coiled-coil regions, e.g. up to six in PFF0165c. Coiled-coil domains are known to be involved in regulation of gene expression and many other biological processes . Membrane bound E3 ubiquitin ligases have been shown implicated in the regulation of immune recognition during virus infections  were recently identified in in vivo has been challenging in other model organisms. It is possible that in vivo protein localization and the presence of adaptors such as E3 ligases can increase specific interactions Determination of target specificity P. falciparum, respectively) and could be potentially involved in the regulation of parasite virulence genes, principally the var gene family in P. falciparum.Increasing evidence suggests that several DUBs are implicated in remodeling chromatin structure, transcriptional regulation and gene silencing. In yeast, UBP8 and UBP10 have been implicated in the dynamic histone monoubiquitination of H2B. The presence of UBP8 correlates with changes in transcriptional regulation Plasmodium and a locus containing a DUB related to USP7 DUBs are also implicated in the endocytic pathway and intracellular traffic P. falciparum life-cycle, providing additional evidence for a functional role in the parasite's life cycle.Expression profiles analysis P. falciparum and other Apicomplexa. All apicomplexans possess the complete machinery that is required to ubiquitylate proteins . Ubiquitin and the common UBL modifiers SUMO, NEDD8, HUB1, URM1, and ATG8 were identified in apicomplexans. However, several UBLps are missing, such as SUMO variants and ATG12, although it has been suggested that autophagy is one of the parasite's death pathway P. falciparum and T. gondii have up to triple the molecular weight of E2s found in other organisms, which could reflect adaptation of Plasmodium and Toxoplasma lineages. Finally, the superfamily of E3 ubiquitin ligases is very diverse, and several of the E3s predicted in apicomplexans do not find homolog in other eukaryotic organisms. Such proteins could have a cellular role directly linked to parasitic processes, such as invasion. The over-representation of RING E3 ligases that contain coiled-coil domains, known to play important role in host-pathogen interactions, supports this hypothesis.The present study allowed for the identification of up to 114 proteins that are predicted to be involved in the UPS of Plasmodium genome. The presence of a modified APC complex in the atypical erythrocytic Plasmodium cycle is not surprising. The cell cycle in Plasmodium can be closely related to the early embryogenesis division observed in D. melanogaster with a depletion of key cell cycle regulators. During the first 13 cell divisions of a D. melanogaster zygote, the divisions are rapid, synchronous and occur in the absence of cytokinesis and detectable gap phase. These divisions result in the formation of a syncytial cell containing a large number of nuclei in a single cytoplasm. In Plasmodium, divisions during schizogony are rapid and asynchronous with up to four or five rounds  of DNA synthesis and mitosis during the trophozoite and early schizont stages. Cytokinesis takes place late in the cycle during the mature schizont stage. This is a major divergence from the classical cell-cycle events that consists of a linear succession of G1/S/G2/M phases. A proposed model for the Plasmodium cell-cycle control is presented in An example of apicomplexan adaptation in the UPS is an apparently modified APC/C . In yeast, SCF and APC are known to play a fundamental role in cell-cycle control. All proteins known to be involved in the SCF machinery are present in the Plasmodium and other apicomplexa is a good estimate when compared to other eukaryotic organisms. Our results demonstrate that E3 ubiquitin/ubl ligases remain one of the most specific components of the UPS. The presence of numerous and diverse E3s, and in particular RING domain-containing proteins, suggests that target-specific ubiquitylation via E3 ligases is a complex and important part of cellular regulation in eukaryotic cells, including apicomplexan parasites. In terms of sequence homology, when counterparts exist, most of the ubiquitin ligases identified in Apicomplexa display divergences from their human-host counterpart. The domain architecture analysis for P. falciparum's RING finger E3s revealed an abundance of coiled-coil domain. Since such domains have been shown to play a major role in signal transduction during the molecular cross-talk that occurs during a viral or a bacterial infection, our hypothesis is that E3 ubiquitin/UBL ligases in P. falciparum and other Apicomplexa are involved in pathogen virulence and/or pathogenicity.The number of genes that our work predicted to encode components of the UPS in via drugs that target the proteasome component of the UPS has been established in P. falciparum. However, this potential to exploit these drugs is limited. The latest generations of drugs that target the UPS have focused more on the specificity of the action of E3 ligases and DUB/DUBLs. Given the apparent apicomplexan diversity in these proteins, opportunities to develop small molecule inhibitors specific against the the apicomplexan-specific E3 ligase and DUB/DUBLs offer some possibility for new, much needed, therapeutics for these devastating global diseases.The potential to treat apicomplexan parasites P. falciparum, P. vivax, P. yoelii, P. berghei, P. chabaudi, C. parvum C. hominis, Toxoplasma gondii, S. cerevisiae, C. elegans, D. melanogaster, A. thaliana and H. sapiens. Additional information regarding data sources and release information are given in http://pfam.janelia.org .Full proteome datasets from the following thirteen organisms were downloaded: http://hmmer.janelia.org. The program hmmsearch was used to analyze domain distribution among all proteome datasets and to extract sequences that carry the Pfam domains described above. HMM searches were run using a series of incrementally increasing threshold E-values, from E-value \u22641 to E-value \u22640.1, and results were checked for false positives. Threshold E-value \u22640.5 gave the best quality results, and thus was used in the present study.Components of the HMMER 2.3.2 package (released in October 2003) were used throughout the study Proteins carrying same domain motifs were pair aligned by BLASTP. Output bits scores were used as protein distance values: the higher the values are, the more proteins are similar and therefore could be evolutionary closed. All scores below 20 were set to 20. Values were scaled in the range  by normalizing using a global transformation (d\u200a=\u200aminimum score/score). A color-mapping matrix, from red to blue, was built, using MATLAB\u00ae 7, from the normalized scale. The red color means \u201chighly divergent\u201d, and blue means \u201chighly conserved\u201d.Protein sequences were aligned using ClustalW P. falciparum and yeast RING and RING-like E3 ubiquitin ligases were searched for functional domains using SMART Our datasets of Table S1Exhaustive list of the proteins identified by the HMM search.(0.37 MB XLS)Click here for additional data file.Table S2http://orthomcl.cbil.upenn.eduComponents of the Cdc48-Npl4-Ufd1 escort pathway in P. falciparum, predicted by BlastP, and their orthologs retrieved from (0.04 MB XLS)Click here for additional data file.Table S3Homologs of Skp1 in apicomplexan parasites.(0.03 MB XLS)Click here for additional data file.Table S4Protein dataset sources and release information.(0.02 MB XLS)Click here for additional data file.Figure S1By-domain dendrogram trees of the predicted apicomplexan proteins(1.64 MB ZIP)Click here for additional data file."}
+{"text": "Long-lasting insecticidal nets are an effective tool for malaria prevention, and \"universal coverage\" with such nets is increasingly the goal of national malaria control programmes. However, national level campaigns in several countries have run out of nets in the course of distribution, indicating a problem in the method used to estimate the quantity needed.A major reason for the shortfall in estimation is the mismatch between the quantification factor used to plan procurement and the allocation algorithm used at community level, in particular the effect of needing to add an additional net to households with an odd number of inhabitants. To solve this problem a revised quantification factor is suggested.Based on data from a broad range of household surveys across Africa, the effect of odd-numbered households on numbers of nets distributed is estimated via two frequently used allocation methods. The impact of these algorithms on the proportion of households reaching a person to net ratio of 2:1, a frequently used marker of universal coverage is then calculated.In order to avoid stock-outs of nets during national coverage campaigns, it is recommended to use a quantification factor of 1.78 people per net, with an additional allocation factor suggested to account for other common problems at the community level resulting in a final recommended ratio of 1.60 people per net. It is also recommend that community level allocation procedures be aligned with procurement estimates to reduce shortages of nets during campaign distributions. These analyses should enable programme managers to make evidence-based decisions and support a more efficient and effective use of LLIN distribution campaign resources. Insecticide treated nets (ITN) are an effective tool for preventing the transmission of malaria . This isCentralized mass distribution campaigns have served as the cornerstone of efforts to achieve universal coverage . Recent ). A similar campaign in Uganda noted that the actual number of nets required for universal coverage was 30% greater than the estimate obtained using the WHO calculation .Inadequate procurements may be one reason that these campaigns fail to provide households with a sufficient number of nets. The World Health Organization/Global Malaria Programme (WHO/GMP) recommends dividing the estimated total population by a factor of two when calculating the total number of nets needed to achieve the desired ratio of 1 net per every two people . In pracInefficient household allocation strategies may be a second factor limiting the ability of these campaigns to achieve universal coverage. Countries use a variety of approaches for allocating nets to households during their distribution campaigns. In some countries, campaigns distribute a fixed number of nets, typically two or three nets, to each household. In other countries, the number of nets allocated to a household varies by the size of the household or by the number of sleeping places in the household. This variation in approaches illustrates the lack of clarity in the current understanding regarding how best to allocate nets in support of universal coverage objectives.Drawing on existing household survey data, this paper provides empirical support for an improved algorithm for estimating the number of LLIN needed to achieve universal coverage within a given population and evaluates the various approaches for allocating LLIN to specific households. These analyses should provide guidance to programme managers to make evidence-based decisions and support a more efficient and effective use of LLIN distribution campaign resources.The current algorithm for calculating the number of LLIN necessary to provide one net for every two people in a population simply divides the total population size by a factor of two . Howeverde jure members  received a quantity of nets equal to half of the household size. The number of nets allocated to households with an uneven number of de jure members differed between the two scenarios. In Scenario A, households received a quantity of nets equal to half of the household size minus 1; while in Scenario B, households received a quantity of nets equal to half of the household size plus 1. Table A more accurate algorithm would use a population-level divisor that accounts for the one additional LLIN required by households with an uneven number of members. To identify this divisor and assess the additional coverage that would result from its use, two scenarios were used to simulate the allocation of LLIN to households sampled in 12 Demographic and Health Surveys (DHS)  and six Once households are allocated a quantity of nets according to the rules described in Table Strategies to allocate nets to households are guided by two primary questions. First, should the campaign allocate a fixed or varying number of nets to each household? And, if households should receive a varying number of nets, should the number of nets allocated to the household be based on the number of people or the number of sleeping places within the household? At present, there is little empirical data upon which program managers can base their decisions to these questions.The decision to allocate a fixed number of nets to each household seems based on the assumption that a single quantity can be identified that provides a substantial number of households with the correct number of nets, while minimizing the proportion of households that receive too many nets or too few nets. To test this assumption, the results of two fixed allocation campaigns, one providing two nets to each household and the other providing three nets, were assessed using the distribution of household sizes in the 18 datasets. Households were classified as receiving the correct number of nets if they received one net for every two people in the household . They were classified as receiving too many nets if they had fewer household members and were classified as receiving too few nets if they had more household members.When the decision is made to vary the number of nets allocated to a household, the number of sleeping places within the household appears to be the logical metric to use to ensure that a mosquito net covers every person sleeping in the household. In practice, this approach presents some concerns. First, where individuals sleep on a variety of surfaces, the definition of a sleeping place may include areas in which several people sleep and that may be too large to be enclosed by a single net. Second, households from lower wealth quintiles may be more likely to have more individuals sharing fewer sleeping places. Use of sleeping places to allocate nets then may result in an inequitable distribution of nets favouring households from higher wealth quintiles. To investigate these concerns, data from five surveys that measured both the number of household members and the number of sleeping places were analysed.Recent experience suggests that dividing the total population by two underestimates the total number of LLIN required to achieve universal coverage, defined as at least one net for every two people in a household. To test the hypothesis that this underestimate is attributed to the additional net required by households with an uneven number of members, simulated allocations of LLIN to households either ignored this additional need (Scenario A) or met this additional need (Scenario B). It was expected that Scenario B would be more likely than Scenario A to reach the household threshold of universal coverage and that the mean number of persons per net in Scenario B would serve as a more effective population divisor for calculating the total number of nets needed to reach universal coverage.de jure members received a quantity of nets equal to half of the household size minus 1. Following this allocation rule, only 50 to 60% of households would receive at least one net for every two people and meet the threshold required for universal coverage. The mean number of persons per net ranged from 2.02 to 2.27, with a median score of 2.19. This \"LLIN allocation factor\" is slightly higher than the number (2.0) currently recommended by WHO, suggesting that the current approach should result in approximately 60-70% of households reaching the threshold of one net for every two people that is required for universal coverage.Table de jure members are allocated an additional net to accommodate the extra person, all households meet the threshold of one net for every two people that is required for universal coverage. In this scenario, the mean number of persons per net ranged from 1.64 to 1.85, with a median score of 1.78. This indicates that dividing the total population by 1.78 would provide enough nets for a campaign to provide each household with at least one net for every two people in the household. The mean number of persons per net in Scenario B remains remarkably consistent across countries, regardless of the level of urbanization and the mean household size in each country (Figure The results from the Scenario B simulation are presented in Table These results are nearly identical to those obtained from intensive micro-census investigations in Mozambique and Cambodia. In Mozambique, information on the number, gender, and age of household members was used to determine the likely sleeping arrangements of the household members and identified that 1.75 persons per net was necessary to achieve universal coverage . A similThe second hypothesis examined the relative effectiveness of using a fixed allocation of nets to achieve universal coverage. It was expected that the variation in household sizes would limit the effectiveness or efficiency of this approach, suggesting the use of tailored allocations as the recommended approach for determining the number of nets to be given to households.As expected, allocating a fixed number of LLIN to each household does not appear to be an effective approach for achieving universal net coverage or an efficient way to allocate nets to households Table . When twSince this suggests allocating a varied, rather than fixed, number of nets to households, the remaining question concerns the use of household members or sleeping places to determine the amount of nets required by each household. It was hypothesized that the subjective definition of a \"sleeping place\" may result in an identification of spaces in which multiple individuals sleep and which may be too large to be covered by a single LLIN. It was further hypothesized that the number of sleeping places in a household would be positively related to household wealth and that allocating LLIN by sleeping places would disproportionately favour the distribution of nets to wealthier individuals.Figure de-jure residents did not, resulting in a decreasing mean number of persons per sleeping place with increasing wealth (Table Data from Northern Bahr el Ghazal in Southern Sudan (unpublished data: Malaria Consortium, see annex for details on survey and methodology [Additional file th Table . Using tth Table . In contThese results recommend revising the current approach for calculating the number of LLIN necessary for achieving universal coverage through a centralized mass distribution campaign. Rather than dividing the total population by a factor of 2, a new divisor of 1.78 is necessary to account for the additional LLIN needed in households with an uneven number of members. This revised divisor is very similar to that suggested by \"bottom up\" approaches of micro-planning at the household level.It should be noted, however, that these simulations are unable to account for some additional logistical hurdles experienced by distribution campaigns. First, campaigns often need to pre-position stocks of LLIN to adjust for inconsistencies between the census estimates used for planning and the actual numbers obtained during registration exercises. Second, since LLIN are packaged in bales of 50 or 100 and are difficult to transport once opened, campaigns typically round-up the number of LLIN sent to the distribution points to the nearest bale. Both practices result in a certain number of LLIN being \"stuck\" in the distribution chain, resulting in an additional demand of 5% to 10% of initial estimates.The obvious solution is to include a buffer in the quantification that will compensate for these logistical \"losses.\" While there is little empirical information regarding the extent of this margin, reasonable expectations suggest it is between 5% and 15% of the total need. Applying these rates to the recommended population divisor of 1.78 results in the corrected factors presented in Table These results also recommend allocating LLIN to households based on their household size, without an upper limit for larger households, rather than using a fixed number of nets per household. Using the number of household members, rather than sleeping places, appears more likely to provide households with a sufficient number of nets to achieve universal coverage and to result in a more equitable distribution of LLIN across all wealth quintiles. However, if sleeping places are to be used to determine the number of nets required by a household, efforts should be made to confirm that the mean number of people per sleeping place is close to 2.0 and the definition of a sleeping place needs to be clearly articulated and standardized. One possible approach would be to define sleeping places as \"a place where people sleep and that can be covered by a single net.\"Whether household size or sleeping places are used to allocate nets to households, a similar metric should be used to quantify the total number of nets needed for the campaign. If the method of quantification differs from that used for actual net allocation during the campaign, the allocation of nets may require a greater quantity than were estimated by the quantification method. In Kano, Nigeria, LLIN needs were quantified at two nets per household using the standard definition of \"people eating from the same pot,\" but were allocated using the household definition of \"wife with her dependents\" in polygamous families. Instead of delivering two nets per household, the outcome of the post-campaign survey showed that only 1.7 nets were delivered among those who attended the distribution, with 28% of households receiving only one net .The authors declare that they have no competing interests.AK drafted the manuscript, provided data form the sub-national studies and did part of the statistical analysis. MB provided the statistical analysis of the DHS data and contributed to the manuscript development. HK provided input on structure and campaign logistics, and ML helped draft and edit the manuscript. All authors read and approved the final manuscript.AK is director monitoring & evaluation for the Malaria Consortium; MB is Assistant Professor at the Department of Health, Behavior & Society, Johns Hopkins Bloomberg School of Public Health; HK is senior program officer at the Center for Communication Programs, Johns Hopkins Bloomberg School of Public Health; ML is Director of the Global Program on Malaria at the Center for Communication Programs, Johns Hopkins Bloomberg School of Public HealthNet tracking survey Northern Bahr el Ghazal, Southern Sudan. Details of the study design, methodology and analysisClick here for file"}
+{"text": "The accumulation of mutant protein is a common feature of neurodegenerative disease. In Huntington's disease, a polyglutamine expansion in the huntingtin protein triggers neuronal toxicity. Accompanying neuronal death, mutant huntingtin aggregates in large macromolecular structures called inclusion bodies. The function of the machinery for intracellular protein degradation is linked to huntingtin toxicity and components of this machinery colocalize with inclusion bodies. An increasing body of evidence implicates the ubiquitin-proteasome pathway in the failure of cells to degrade mutant huntingtin. A number of potential mechanisms that link compromised ubiquitin-proteasome pathway function and neurodegeneration have been proposed and may offer opportunities for therapeutic intervention."}
+{"text": "The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable diversity with respect to UDP function. Here, we give a short summary of the biochemical and physiological roles of the UDPs, which have been linked to human diseases including neurodegeneration and cancer.Publication history: Republished from Current BioData's Targeted Proteins database .   Ubiquitin is a small and phylogenetically conserved eukaryotic protein known to covalently modify proteins and thereby mark them for destruction by the 26S proteasome . This prUbiquitin ligation is accomplished in multiple steps , the firThe formation of polyubiquitin chains is a reversible process and several deubiquitylating enzymes (DUBs) play important roles in trimming the chains on target proteins .Different ubiquitin lysine residues are used in the formation of polyubiquitin chains, with various outcomes. For example, whereas Lys48-linked polyubiquitin chains target proteins for degradation by the 2.5 MDa 26S proteasome in an ATP-dependent manner .Ubiquitin is a stable and compact protein consisting of two \u03b1-helices and five \u03b2-strands arranged in the order \u03b2\u03b2\u03b1\u03b2\u03b2\u03b1\u03b2 to form the ubiquitin superfold . RecentlThe UDPs are responsible for recruitment of ubiquitylated substrates to the proteasome  and bindUDPs that contain one or more ubiquitin binding UBA (ubiquitin-associated) domain figure  in additin vitro[in vitro by biochemical experiments, which showed that UBL/UBA proteins are essential for degradation of a proteasome substrate [Precipitation experiments have shown that the UBL domain of both Rad23 and its human homologue (HHR23) interacts directly with the 26S proteasome in vitro, while tin vitro. UBL/UBAin vitro. This shubstrate . Since mubstrate , are proDespite the fact that none of the UBL/UBA proteins are essential in yeast, combined loss of Rad23, Dsk2 and the proteasome's ubiquitin receptor Rpn10/S5a results in mitotic arrest . This inIn precipitation experiments the UDPs Rad23, Dsk2 and their human orthologues HHR23 and ubiquilin-1 interact with the 26S proteasome in a UBL domain-dependent manner ,18,19. Hin vitro[Both Ufd2 (an E4) and the 26S proteasome associate with the Rad23 UBL domain in a mutually exclusive manner in vitro. One migin vitro, a protein involved in the development of the neurodegenerative Machado-Joseph disease [In addition to the 26S proteasome and ubiquitylated substrate, HHR23 also interacts with ataxin-3  disease . Similar disease ,25. Sinc disease  and perh disease ,23. Therubiquilin-1 gene are proposed to substantially increase the risk of developing Alzheimer's disease [presenilin-1 and presenilin-2 lead to an increased ratio of \u03b2-APP42 to \u03b2-APP40  from amyloid \u03b2-precursor protein [in vitro translated presenilins, SDS-PAGE of the precipitates revealed a slowly migrating smear. It is therefore likely that only ubiquitylated presenilins can interact with ubiquilin-1. It is also possible that ubiquilin-1 only interacts with ubiquitylated \u03b3-aminobutyric acid A (GABAA) receptors [Ubiquilins 1\u20134 are human homologues of yeast Dsk2. Ubiquilin-1 and ubiquilin-2 both bind the proteasome  and, int disease . The gen protein . Accumul protein . The pre protein . Howevereceptors .A receptor at inhibitory synapses is tightly regulated [A receptors and facilitates their membrane insertion by increasing the stability of the intracellular pool [A receptors. As proteasomal degradation of the receptors was not observed, ubiquilin-1 may therefore also function in endocytosis [In order to efficiently transmit synaptic signals, the amount of GABAegulated ,33. Thisegulated . Ubiquillar pool . Based oocytosis . Accordiocytosis . Eps15 cocytosis . Althougocytosis . This isocytosis . It is pocytosis . These socytosis . Once thocytosis .Ubiquilin-4 is linked to spinocerebellar ataxia type 1 (SCA1) , an inhePARK2 gene encoding PRKN2  [PARK2 impair its catalytic activity [Autosomal recessive juvenile Parkinsonism (AR-JP) is an early onset and slowly progressing disease, the most common cause for which is mutations in the  parkin) -43. PRKN parkin) . Accordiactivity -47. Seveactivity ,48-51. Tactivity . Moreoveactivity .in vitro precipitation assays failed to confirm the interaction [in vivo[Although most of the pathogenic missense mutations cluster to the RING domain-containing C-terminus of PRKN2, a few mutations have also been identified in the UBL region. NMR interaction studies have identified an interaction between the PRKN2 UBL domain and the proteasome subunit Rpn10/S5a, an observation not made with the pathogenic PRKN2 mutant R42P . Howevereraction , its sig [in vivo. Though  [in vivo,55, this [in vivo, indicat [in vivo. Upon tr [in vivo.PRKN2 can also be destabilized by pathogenic mutations within its UBL domain . HoweverVHL (Cullin\u2013Elongin-BC\u2013VHL) [Von Hippel-Lindau (VHL) disease is an autosomal dominant cancer, which manifests as angiomas of the retina, hemangioblastomas of the CNS and renal clear cell carcinomas . Patient-BC\u2013VHL) . In addi-BC\u2013VHL) .VHL catalyses the ubiquitylation of hypoxia-inducible transcription factor \u03b1 , the murine orthologue of RAD23B (HHR23B) [Yeast has proved to be an excellent model system for the study of the UBL/UBA proteins. However, developing transgenic mouse models will further reveal important functional aspects of their molecular functions. A knockout is already available in (HHR23B) , which d(HHR23B) .Park2 knockout mouse models have been reported [Park2 knockout, which additionally lacks the PRKN2 co-regulated gene Pacrg[quaking mutants are characterised by dysmyelination in the CNS, resulting in phenotypes including movement disorders, tremors and seizures [To date, two reported ,67, bothene Pacrg. Homozygseizures .Drosophila, the loss of park (homologue of PARK2) results in reduced cell size and number, infertility, reduced lifespan, movement and flying disorders, and increased sensitivity to oxidative stress [park mutant flies do not display age-dependent neuron loss in the brain.In e stress . HoweverDrosophila PRKN2 models will serve as an invaluable tool for understanding the biological role of PRKN2 and may provide important clues to the molecular mechanisms of Parkinson's disease.Collectively, the mouse and Inhibitors of the 26S proteasome, such as bortezomib, are successfully being used in the treatment of certain cancers . HoweverXenopus extracts performed by Verma et al. at Caltech have identified a group of relatively small molecules, called ubistatins, which inhibit the turnover of cyclin-B and the cdk-inhibitor Sic1 in vitro by inhibiting the binding of ubiquitylated proteins to substrate shuttles [Devising efficient inhibitors for non-enzymatic proteins such as the UBL/UBA proteins is more difficult than for proteins with enzyme activity, which can be assayed in high-throughput screens. Nonetheless, recent chemical genetic screens in shuttles . AccordiPARK2 gene with a wild-type copy could perhaps provide a cure in the future.As for many loss-of-function mutations in disease-connected E3 enzymes, it is difficult to envision small molecules that could reactivate the mutant PRKN2 and thereby promote neuronal survival in patients suffering from AR-JP. However, a gene therapeutic approach to replace the dysfunctional In the case of von Hippel-Lindau disease, small molecule activators of mutant VHL may be able to sustain HIF\u03b1 degradation and prevent tumour angiogenesis. However, it was found that the molecular chaperone Hsp90 protects certain VHL substrates from proteasomal degradation  and cyclIt is evident that the UBL/UBA proteins and PRKN2 appear to be directly involved in the etiology of cancer and neurodegenerative diseases. Although this is by no means a general feature of the UDPs, it highlights the importance of the ubiquitin system in maintaining an appropriate intracellular protein milieu.With respect to the UBL/UBA proteins, one major issue that remains unresolved relates to their specificity. Though some details of their substrate selectivity have emerged , a more In relation to PRKN2, identifying novel substrates of its E3 activity and resolving its proteasome-dependent and/or -independent functions  may shed some light on the molecular mechanism of Parkinson's disease and hopefully lead to novel therapeutic approaches.A, \u03b3-aminobutyric acid A; HIF\u03b1, hypoxia-inducible transcription factor \u03b1; SCA1, spinocerebellar ataxia type 1; UBA, ubiquitin-associated domain; UBL, ubiquitin-like; UDP, UBL domain protein; UIM, ubiquitin-interacting motif; VHL, von Hippel-Lindau.A\u03b2, amyloid \u03b2-protein; AR-JP, autosomal recessive juvenile parkinsonism; CBC, Cullin\u2013Elongin-BC; DUB, deubiquitylating enzyme; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GABAThe authors declare that they have no competing interests.).    Republished from Current BioData's Targeted Proteins database (TPdb;"}
+{"text": "In late 2006, the seaside community in Esperance, Western Australia, was alerted to thousands of native bird species dying. The source of the lead was thought to derive from the handling of Pb carbonate concentrate from the Magellan mine through the port of Esperance, begun in July 2005. Concern was expressed for the impact of this process on the community.This study was designed to evaluate the source of Pb in blood of a random sample of the community using Pb isotope ratios.The cohort comprised 49 children (48 < 5 years of age) along with 18 adults (> 20 years of age) with a bias toward higher blood lead (PbB) values to facilitate source identification.n = 49; geometric mean, 6.6 \u03bcg/dL), with four children whose PbB was > 12 \u03bcg/dL. The isotopic data for blood samples lay around two distinct arrays. The blood of all children analyzed for Pb isotopes contained a contribution of Pb from the Magellan mine, which for young children ranged from 27% up to 93% . Subtraction of the ore component gave a mean background PbB of 2.3 \u03bcg/dL. Several children whose PbB was > 9 \u03bcg/dL and most of the older subjects have complex sources of Pb.Mean PbB level of the children was 7.5 \u03bcg/dL (range, 1.5\u201325.7 \u03bcg/dL; The death of the birds acted as a sentinel event; otherwise, the exposure of the community, arising from such a toxic form of Pb, could have been tragic. Isotopic data and mineralogic and particle size analyses indicate that, apart from the recognized pathway of Pb exposure by hand-to-mouth activity in children, the inhalation pathway could have been a significant contributor to PbB for some of the very young children and in some parents. In late 2006, the community of about 14,000 people in the town of Esperance in Western Australia  was alerThe source of the Pb ore concentrate was thought to derive from the handling of Pb concentrate at the port that began in July 2005. The Pb concentrate originated at the Magellan mine some 600 km north of Esperance and was transported by road and rail to the port . Other pAfter the birds began dying, state and local government authorities began several investigations, although shipping of the ore concentrate continued for several months. Rainwater from household tanks is one of the main sources of drinking water in Esperance. The local Shire Council and Western Australia Department of Health (DoH) undertook measurements of Pb and Ni in the rainwater tanks and initially found that 10% of residences had Pb levels above the World Health Organization guidelines of 10 \u03bcg/L and 30% had Ni levels above the guidelines (20 \u03bcg/L). The DoH then undertook a blood Pb (PbB) survey of any resident who wished to be tested, and the Department of Environment and Conservation (DEC) undertook environmental investigations. After results from these investigations were obtained, shipping of the concentrate through the port was suspended.The Pb concentrate being shipped through the port was Pb carbonate, which is an unusual mining product; the most common Pb ore is galena [lead sulfide (PbS)], from which Pb carbonate is derived by oxidation over geologic time. Unfortunately, Pb carbonate is more toxic than PbS: A study by When the first author heard about the problem in Esperance, he alerted the DoH and DEC to the highly unusual Pb isotope signature in the ore concentrate, measured before mining by colleagues in the Commonwealth Scientific and Industrial Organisation (CSIRO), and suggested that the Pb isotope method would be a powerful tool in source apportionment investigations. Some questions that could be, and were, addressed using the Pb isotope method were as follows: Is the Pb in the bird livers derived from the Magellan concentrate? If so, what is the pathway from the concentrate to the birds, especially because they were dominantly nectar feeders? Was the Pb adsorbed on the vegetation? Are the elevated Pb levels in rainwater derived from the Magellan concentrate? What are the sources of Pb in soil in parks and residences? What is the contribution of Magellan Pb to PbB of children and adults in Esperance? In this article, we report the results of the isotopic investigation of blood in children and adults. Because the environmental investigations are the subject of litigation, they will be reported at a later date, although the The DoH undertook opportunistic sampling, taking blood from any resident who wished to have his or her PbB measured. All subjects lived within 3 km of the port. None of the subjects were occupationally exposed to Pb, although some of the adults may have undertaken activities in which they were exposed to Pb, such as renovating older houses with Pb paint or making fishing sinkers (Esperance is a seaside town). DoH took most of the samples in a clinic especially set up for the purpose, following standard protocols; a few samples were taken by general medical practitioners in the town. They collected the samples mostly in March through May 2007, after loading of the Pb concentrate had been suspended. Most were venous samples taken with Vacutainer devices, but sometimes by finger prick or heel prick if the parent expressed a preference or they could not obtain a venous sample. Samples we analyzed for Pb isotope ratios were generally for subjects with PbB values > 5 \u03bcg/dL, because other studies have pointed out difficulties in assigning sources when PbB values were < 5 \u03bcg/dL . For exa206Pb is derived by radioactive decay from 238U, with a half-life of about 4,500 million years; the decay of 207Pb from 235U is more rapid, with a half-life of about 700 million years, whereas the decay of 208Pb from 232Th is much slower, at about 14,000 million years. The other low-abundance isotope, 204Pb (~ 1%), has no known radioactive parent and is thought to represent Pb present at the time of formation of the earth, some 4,550 million years ago ; it is used as a reference isotope. Hence, Pb mineral accumulations  of different geologic ages have different isotopic abundances. The simplest explanation for the different abundances is that when the deposit formed, the Pb was separated from its parent Th and U isotopes, so no further radioactive decay or changes in the isotope abundances occurred. In this way, ore formed, say, 1,800 million years ago had more primordial Pb and relatively fewer decay products compared with ores formed only 400 million years ago; this also includes Pb formed in the intervening 1,400 million years. Isotopic investigations present data as ratios of abundance of one isotope to the other, such as 208Pb/204Pb, 207Pb/204Pb, and 206Pb/204Pb, or any combination of these. In the earlier days of use of Pb isotopes, because of the difficulty in measurement of the low-abundance 204Pb isotope, studies reported data as 206Pb/207Pb ratios. As an example of the differences in isotopes from different ore deposits, the geologically ancient so-called massive sulfide Pb\u2013zinc\u2013silver Broken Hill and Mt. Isa deposits in Australia formed about 1,700\u20131,800 million years ago and have a 206Pb/204Pb ratio of 16.0 or 16.1, respectively, whereas geologically younger deposits of similar composition in eastern Australia that formed 400\u2013500 million years ago have a 206Pb/204Pb ratio of about 18.1. The Pb isotope method makes use of the variations, arising from radioactive decay throughout geologic time, in abundances of three of four Pb isotopes, the relative concentrations of Pb, thorium, and uranium, and the time when the ore was formed. Pb has four naturally occurring isotopes, three of which are the stable end products of radioactive decay of U and Th. For example, However, other types of Pb deposits formed by a different mechanism, called Mississippi Valley deposits (found in the Tri-State District of the United States) or sandstone-type deposits . Their isotope ratios are very different from those for the massive sulfide deposits, and their data plot away from the growth curves. The Magellan mine is an example of the sandstone type and is thought to have formed about 1,650 million years ago . To accoThermal ionization mass spectrometry requires good separation of Pb from other elements that may inhibit the ionization of Pb in the mass spectrometer and to avoid interference from ions with the same mass/charge ratio as some of the Pb isotopes. Although Pb levels in the environment have decreased since Pb was removed from gasoline, the processing blank is still a major concern in the design of chemical separation procedures. All reagents we used were ultra-pure, and containers were either Teflon or polypropylene. Under best conditions, we did all processing in class 350 clean rooms with further handling in class 100 laminar flow workbench stations.202Pb-enriched tracer to determine the Pb concentration in the sample (the isotope dilution method). The procedural blank is about 50 pg (50 \u00d7 10\u221212 g), which has an insignificant effect on the measured ratios.Approximately 0.3\u20130.5 g of blood was digested with concentrated nitric acid/hydrogen peroxide and the Pb separated from other elements on a column of Pb-selective resin . We adde206Pb/204Pb ratios can be obtained, and much better precision can be obtained for the more abundant ratios 208Pb/206Pb and 207Pb/206Pb. To enable comparisons of data across laboratories, we normalized the isotope ratios to the accepted values of the NIST standard SRM 981.We dissolved the sample in water and evaporated a small volume onto a cleaned rhenium filament along with silica gel/phosphoric acid to enhance ionization. We placed the filaments in a VG Sector 354 multicollector thermal ionization mass spectrometer  and measured 250 Pb isotope ratios. From replicates of the U.S. National Institute of Standards and Technology  standard reference material SRM 981 and natural samples, a precision of better than \u00b1 0.05% (2\u03c3) for the We present our results in 207Pb/204Pb versus 206Pb/204Pb graph , and Mt. Isa and Cannington in Queensland, have 206Pb/204Pb ratios almost identical with Magellan because they formed at much the same geologic time. However, these other deposits have 207Pb/204Pb and 208Pb/204Pb ratios different from Magellan\u2019s, because the U, Th, and Pb were partially mobilized at Magellan before the ore body was formed. Using the ratios 207Pb/204Pb versus 206Pb/204Pb on a graph allows us to discriminate Magellan deposits from these other deposits, and this combination provides better visual resolution than does a graph of 208Pb/204Pb versus 206Pb/204Pb or even of the more abundant ratios 208Pb/206Pb versus 207Pb/206Pb.The isotopic signature of the Magellan ore is shown by the black cross on the left side of It is possible to calculate the contributions of PbB from different sources using well-established methods from isotope geochemistry assuming two-component mixing . More coa) most of the PbB data that intersects the Magellan ore value and includes the subjects with the lowest PbB of < 1.5 \u03bcg/dL  another array  end point is uncertain because no attempt so far has been made to identify and isotopically characterize other Pb sources such as paint, hobbies, and auto repairs in Esperance. The low-Magellan end point has been arbitrarily assigned a value of 17.8 for the n = 49) and geometric mean of 6.6 \u03bcg/dL. This compares with the mean value of 3.0 \u03bcg/dL obtained for 404 children < 5 years of age in the wider DoH survey , or myriad others. Unfortunately, no environmental samples from individual houses were available for isotopic analysis.The isotopic data for three children lie between the Magellan and older arrays. PbB levels in these three children ranged from 12 to 25 \u03bcg/dL, which suggests that there is another source (or sources) of Pb that has an isotopic composition probably lying on the older array with a R2 = 0.12, p = 0.018) of increasing PbB and proportion of Magellan Pb . The data for two subjects, however, could possibly lie on the Magellan array. The isotopic compositions suggest that another source(s) of Pb is present, such as Pb from maternal bone, paint, roof flashing, tank water, hobbies, do-it-yourself activities, unusual diet, medicines, cosmetics, or occupational activities. No environmental samples from individual houses were available for isotopic analysis.The data for 10 subjects, whose PbB ranged from 1.8 to 14.2 \u03bcg/dL, lie on the Magellan array and indicate that in some cases they have been exposed to Magellan Pb; the contribution of Magellan Pb to PbB ranged from 29% to 77%. The data, however, for two of these subjects, with low Magellan contributions of 29% and 40%, lie equally well on the older array, along with the data for subjects W725 and W747.The data for the cord blood samples are consistent with those found in other studies; that is, the isotope ratios in the cord and mothers\u2019 blood are similar . DespiteIt is possible to estimate a background PbB value expected in Esperance if there had been no significant Pb exposure from a Magellan source. Excluding the four children . Twenty-two wipe samples were taken from a variety of locations on or outside dwellings, including windows, doors, beams, cubbyhouses, and other outdoor structures. Pb content in these wipes ranged from 0.16 to 34 \u03bcg/cm2 . There are no guidelines for dust wipes apart from those promulgated by U.S. Department of Housing and Urban Development (HUD)  and strong ubiquitous winds have resulted in widespread contamination, although only limited environmental sampling of houses has been undertaken. Limited soil sampling around residential parks and school playgrounds undertaken by the Western Australia DEC showed a highest reading of 88 mg/kg, and most were < 10 mg/kg . The DoHnt (HUD)  for clea2/month were reported from August 2005 to August 2006 (2/30 days (geometric mean).The high percentage of Magellan Pb in one 6-month-old child (93%) and in both parents (~ 76%) would suggest that the inhalation pathway of Pb may have been a significant contributor to the overall PbB level. Furthermore, per unit body weight, ventilation rates are much higher in infants than among children or adults. The family lived within 200 m and downwind of the Pb loading shed. The child was not crawling and exhibited limited if any hand-to-mouth activity, according to the parents. The inhalation pathway is unusual these days, apart from occupational exposure, in contrast to the 1970s and 1980s when leaded gasoline was in use. The hypothesis of an inhalation pathway to complement hand-to-mouth activity in many of the children is reinforced by the nature of the Magellan ore concentrate and the high levels of Pb in air measured by the Port Authority using deposition gauges, which measure the particle deposition from the air over a 30-day period. These gauges showed very high levels of Pb, especially during periods of loading the concentrate onto ships. Values ranging from 14 to 42 mg/must 2006 . No guidThe Pb concentrate from the Magellan mine was originally supposed to be transported by rail as pellets or agglomerates, but the company argued against this because of problems with handling on the wharf . We subjElevated PbB levels are commonly found in mining and smelting communities, although those producing Pb carbonate and/or subject to transportation are less common and isotopic studies even more so. One example where Pb carbonate was produced is in the Pb\u2013zinc\u2013silver mines of Broken Hill, NSW, Australia. Mining began in the 1880s with open pit methods, initially of carbonate ore, which later changed to underground mining of sulfide ore. An inquiry into the prevalence of Pb poisoning and deaths among the Broken Hill community was reported in 1893 . A PbB pp = 0.02). Some of the \u201ccontrol\u201d children had higher PbB than the children of mine employees, probably from exposure to leaded paint, because six of eight houses for the control children were > 50 years of age. Five of eight children of mine employees had PbB contributions from mine Pb > 20%. However, the other three children of mine employees had contributions to their PbB from sources other than mine Pb . This study showed that houses of employees from a Pb mine can be variably contaminated by mine Pb even if they are not near the mine.In an example of \u201ctake-home\u201d Pb dust, Pb isotope and concentration data were obtained from venous blood and environmental samples  for eight children of six adult male employees and the adults who worked in a Pb\u2013zinc\u2013copper mine located approximately 40 km from the town . These dn = 20) whose PbB levels were > 19 \u03bcg/dL lived in Callao, a port that stored and shipped mineral concentrate; the isotopic results were compared with another group of 10 children from outside Callao whose PbB levels were < 10 \u03bcg/dL. The isotopic results for the two groups of children were very different, and both were different from five samples of gasoline; the results for the children from Callao were almost identical to those in the mineral concentrate.This incidence of avian Pb poisoning in a pristine environment came as a shock to the community of Esperance. Although the deaths of so many native birds is a tragedy, their role as \u201ccanaries in the coal mine\u201d is fortunate for the community, which may otherwise have been left with a legacy so common in many mining and smelting environments. This would have been exacerbated by the Pb concentrate being in the form of Pb carbonate and of such fine particulate size, combined with the ubiquitous strong winds coming off the Great Southern Ocean, resulting in wide dispersion of the material.The other fortunate aspect of this event is the unusual Pb isotopic signature of the Pb concentrate, which has allowed unequivocal identification of the source and contribution of the Pb concentrate to the blood of children and adults in the community, to the birds, and to the environmental samples. If the signature were less distinct, it would not be possible to apportion sources of the Pb with any certainty where PbB levels are < 5 \u03bcg/dL, a potential problem noted previously by The major limitation of this investigation is the lack of isotopic data for environmental samples within individual houses, which would have allowed identification of the other sources of Pb. This is especially the case for the children with the highest PbB levels of > 15 \u03bcg/dL and whose isotopic results showed that the overwhelming contribution of Pb in their blood was from sources other than the Pb concentrate. Another limitation is the lack of knowledge of potential sources of Pb besides from the concentrate. None of the subjects were occupationally exposed to Pb, although some of the adults may have undertaken activities that exposed them to Pb, such as renovating older houses with Pb paint or making fishing sinkers.This study has shown that the isotopic analysis of environmental and blood samples can contribute significantly to the investigation of elevated Pb levels. Investigation of such episodes should include the early development of protocols and procedures to enable systematic and valid sampling of environmental and blood specimens for isotopic analysis."}
+{"text": "Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10-5 to 10-3 M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10-3 to 10-4 M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum.Electron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. After 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER) with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells.A. sativum exposed to lower Pb have a rapid and effective defense system, but with the increased level of Pb in the cytosol, cells were seriously injured.Cell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress. Vacuoles are ultimately one of main storage sites of Pb. Root meristematic cells of  Lead (Pb) exists in many forms in natural sources throughout the world. According to the USA Environmental Protection Agency, Pb is one of the most common heavy metal contaminants in aquatic and terrestrial ecosystems and can have adverse effects on growth and metabolism of plants due to direct release into the atmosphere . There hIt is well known that the roots are the main route through which Pb enters plants , and aboAllium sativum L. is a potential plant for absorption and accumulation of heavy metals [-5, 10-4 and 10-3 M) of Pb on growth for 20 d were investigated in hydroponically grown A. sativum. Pb had significant inhibitory effects on shoot growth at high concentrations (10-3 M), on roots at 10-3 and 10-4 M during the entire experiment [A. sativum.Although there is extensive literature relating to cellular levels and physiological studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not been fully explained yet ,15,17. Ay metals ,23. In aA. sativum grown in control solution and in solutions containing 10-4 M Pb for different durations of time revealed extensive differences. Control cells had typical ultrastructure. Plasma membrane was unfolded with a uniform shape in all parts. Large amounts of rough ER, dictyosomes, mitochondria and ribosomes were immersed in dense cytoplasm. The nuclei with well-stained nucleoplasm and distinct nucleolus were located in the center of cells, whereas vesicles were distributed in root tip cells , indicating that Pb accumulated primarily in roots; the concentration in bulbs and shoots was much lower [Zea mays [Allium cepa [In previous work, the uptake and accumulation of Pb in ch lower . When Pbch lower . The ultZea mays  and Alliium cepa  revealedium cepa .A. sativum. Pb retention in the roots is based on binding of Pb to ion-exchange sites on the cell wall and extracellular precipitation, mainly in the form of Pb carbonate deposited in the cell wall [Our results here indicated that Pb ions were localized and accumulated in cell walls and vacuoles in ell wall . Once exell wall . Plasma ell wall .It is well known that the ER is the principal site of membrane synthesis within the cell. It appears to give rise to vacuolar and microbody membranes, as well as to the cisternae of dictyosomes in at least some plant cells . Our resThe vacuole is the final destination for practically all toxic substances that plants can be exposed to, and the vacuoles of root cells are the major sites of metal sequestration . CytoplaSalvinia minima was a direct response to Pb accumulation, and PCs participate as one of the mechanisms to cope with Pb in this Pb-hyperaccumulator aquatic fern [Tolerance to metal stress relies on the plant's capacity to detoxify metals that have entered the cell. Inside cells, plant protection against metal toxicity involves synthesis of PCs and related peptides, organic acids and their derivatives . Chelatitic fern . PC bindtic fern .Stichococcus bacilaris, PCs were detected after only 30 min of Cd exposure. In the presence of excess metals, PCs are formed and effectively capture metals [Pisum sativum, Vicia faba and Phaseolus vulgaris [P. sativum, despite the fact that this plant had a medium-tolerance index value, while the concentration of PCs in the roots of V. faba was much lower but their induction took place after only 2 h. The results showed that the rapid initiation of this cytoplasmic detoxification system, which consists of PCs, could transport Pb-PC complexes through the cytosol into vacuoles at lower concentrations of heavy metals [The histochemical test by Gomori-Swift reaction is highly sensitive and allows the detection of cysteine-rich proteins where toxic elements were usually detected . Evidence metals . Piechalvulgaris . They foy metals . Thus thy metals .A. sativum exposed to low Pb concentrations have a rapid and effective defense system, but at increased levels of Pb in the cytosol, cells are seriously injured.The results of the present and previous studies strongly suggest that: (1) cell walls, a first barrier against Pb stress, can immobilize some Pb ions. The cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction; (2) the morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress; and (3) vacuoles are ultimately one of the main storage sites of Pb. Thus, root meristematic cells of Allium sativum L. were chosen and allowed to form roots in containers of modified Hoagland's nutrient solution [3)2, 5 mM KNO3, 1 mM KH2PO4, 50 \u03bcM H3BO3, 1 mM MgSO4, 4.5 \u03bcM MnCl2, 3.8 \u03bcM ZnSO4, 0.3 \u03bcM CuSO4, 0.1 mM (NH4)6Mo7O24 and 10 \u03bcM FeEDTA at pH 5.5. Pb was provided as lead nitrate (Pb(NO3)2). The controls were grown on Hoagland solution alone. Seedlings were exposed to 10-4 M Pb for 1, 2, 4, 8, 12, 24, 36, 48 and 72 h.Healthy and equal-sized cloves of solution . Plants The terminal portion (about 2 mm) of each root of the control and the treated groups were cut and fixed in a mixture of 2% formaldehyde and 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 2 h and then thoroughly washed with the same buffer three times. This was followed by post-fixation with 2% osmium tetroxide in the same buffer for 2 h. They were dehydrated in an acetone series, and embedded in Spurr's ERL resin. For ultrastructural observations, ultrathin sections of 75-nm thickness were cut on an ultramicrotome  with a diamond knife, and were mounted in copper grids with 300 square mesh. The sections were stained with 2% uranyl acetate for 50 min and lead citrate for 15 min. Observation and photography were accomplished by transmission electron microscopy .The Gomori-Swift test was used in the present investigation to detect whether cysteine-rich protein was induced under Pb stress.Sections of 100-nm thickness from fixed material were cut and mounted on gold grids. The Gomori-Swift reaction was performed in the solution obtained by mixing two components just before staining. Solution A containing 5 mL of 5% silver nitrate and 100 mL of 3% hexamethylenetetramine, and solution B consisting of 10 mL of 1 \u00d7 44% boric acid and 100 mL of 1 \u00d7 9% borax were prepared. The final stain was obtained by mixing 25 mL of A, 5 mL of B and 25 mL of distilled water ,36.The grids were floated in the silver methenamine solution for 90 min at 45\u00b0C in the dark, and then washed four times for 2 min. The grids were then floated on 10% sodium thiosulfate solution for 1 h at room temperature to dissolve metallic silver and rinsed in deionized water four times for 2 min. The sections were continuously stained with uranyl acetate and lead citrate.Controls were carried out to block SH and SS groups by the reduction of disulfide bonds in benzylmercaptan, followed by alkylation of SH groups in iodacetate boric acid. The procedures were described by Swift  and Liu WJ carried out the present investigation, participated in sample preparation and observation and drafted the manuscript. DL conceived the study, and participated in its design and coordination and revised the manuscript. All authors read and approved the final manuscript."}
+{"text": "The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain.In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome.We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting. Targeted protein degradation by the ubiquitin-proteasome pathway is a key means of regulating a wide variety of cellular processes, ranging from cell cycle progression  to antigRecent work indicates that ubiquitin receptors, which bind ubiquitin but are not intrinsic subunits of the proteasome, facilitate the docking of ubiquitinated substrates to the proteasome -8. The bEach Rad23 family member has a ubiquitin-like (UBL) domain that binds proteasomes -13 as weTo determine how UBL and UBA domain interactions contribute to Rad23/HHR23A function in ubiquitin-mediated proteolysis, we identified mutations that disrupt UBL-UBA binding, then tested the ability of the mutant proteins to bind components of the ubiquitin-proteasome pathway and to mediate delivery of a ubiquitinated substrate to the proteasome. Our results show that the interactions of the UBL and UBA domains with each other and with other proteins are interdependent, and that modulating proteasome-binding is important for the role of Rad23/HHR23 in proteasomal targeting.To identify UBL mutations that affect UBA-binding, we established an affinity column chromatography assay using resin-bound UBA domains and mobile ligands. To demonstrate that our assay can distinguish proteins based on their relative affinities for the UBA domains, control experiments were performed with ubiquitin and SUMO. Both proteins are similar in size and structure to the UBL domain but ubiquitin binds the UBA domains whereas SUMO does not ,15,21. ETo abrogate UBL-UBA binding, we mutated residues located on the UBA-binding surface of HHR23A  that areTo determine the affinity of these UBL mutants for UBA domains relative to wild-type UBL, UBL domain constructs were mutated accordingly and tested in our affinity column chromatography assay. As shown in Figure The ubiquitin-binding surface on the UBA domains overlaps significantly with that involved in binding the UBL domain ,20. DisrThe HHR23A UBL mutants were then tested as competitors in the GST-pulldown competition assay. All the bands in the polyubiquitin Western blot are conjugates of free ubiquitin, but rather than quantify a smear, we quantified the specific band corresponding to tetra-ubiquitin, which is the minimum length required for proteasomal targeting . The amoIn contrast, the UBA domain double mutant L198A/L355A did not compete with GST-HHR23A for polyubiquitin binding. The UBL domain mutants L10E/K47E and K47E/T77E, which showed reduced UBA-binding Figure , competeIn addition to regulating ubiquitin-binding, UBL-UBA domain interactions could also affect proteasome-binding such that it is enhanced when the HHR23 proteins are bound to ubiquitinated proteins. For this hypothesis to be true, the proteasome must bind a surface on the UBL domain overlapping with that which binds the UBA domains. Indeed, the UBL domain is necessary and sufficient for interaction with the proteasome  and the Saccharomyces cerevisiae as a model system as it is better-characterized and easier to manipulate genetically.Just as ubiquitin itself binds multiple subunits of the proteasome, including Rpn10/S5a and Rpt6/S6' ,27, so c32P-labelled Rad23 proteins and purified yeast proteasomes in a electrophoretic mobility shift assay. In the presence of proteasomes, the mobility of wild-type Rad23, but not Rad23~\u0394UBL, decreased, as indicated by the arrowheads in Figure in vitro. This finding provides strong evidence that Rad23 binds the proteasome via the same surface that is responsible for S5a- and UBA-binding in HHR23A.L10, K47 and T77 in HHR23A correspond to F9, K43 and S73 in Rad23 respectively, as determined by sequence alignment. We mutated these Rad23 residues to glutamic acid as we had done for HHR23A. To test proteasome-binding, we used recombinant, purified In addition to the proteasome, the Rad23/HHR23A UBL domain binds other proteins involved in ubiquitin-mediated degradation, including the ubiquitin ligase Ufd2 ,28. We tin vitro in the presence of a 20S proteolytic inhibitor such as epoxomicin, and can be used as a functional readout for recruitment of model ubiquitinated substrates to purified 26S proteasomes ATP (NEN) and heart muscle kinase according to the manufacturer's instructions (Pharmacia Biotech). The radiolabelled proteins were cleaved from the resin with thrombin or PreScission protease, then quantified by scintillation counting and by Bradford assay. The specific activities of the proteins were normalized and mixed with proteasomes in a 1:50, 1:20, and 1:10 molar excess of proteasome over proteins. After incubation at 30\u00b0C for 15 minutes, the mixtures were resolved by 3.5% native PAGE essentially as previously described [Proteasomes were purified from escribed . All Radescribed ,36, thouGlutathione sepharose was saturated with purified GST-Ufd2. Purified Rad23 proteins were then added together with 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 2% Triton X-100, 0.2 mg/ml BSA, and protease inhibitor cocktail (Roche). After mixing at 4\u00b0C for 60 minutes, the resin was washed with PBS and 0.1% Tween-20. The resin-bound Rad23 proteins were detected by Western blotting using an antibody against Rad23 (a gift from Kiran Madura).in vitro deubiquitination assay was performed as previously described [S. cerevisiae and preincubated with 100 \u03bcM epoxomicin for 45 minutes at 30\u00b0C to inhibit the protease activity of the proteasome. Ubiquitinated MbpSic1 was then added to the proteasome and its deubiquitination was analyzed by Western blotting for Sic1. Rad23 was added to the deubiquitination assay where indicated.The escribed . EssentiS. cerevisiae, Saccharomyces cerevisiae.UBL, ubiquitin-like; UBA, ubiquitin-associated; His, polyhistidine; Ub, ubiquitin; HHR23, Human Homolog of Rad23; in vitro deubiquitination studies and helped to revise the manuscript. RJD and DF participated in the design of the study and helped to revise the manuscript. PMH participated in the design and coordination of the study and helped to revise the manuscript. All authors read and approved the final manuscript.AMG participated in the design of the study, carried out experiments and drafted the manuscript. KJW created the models of the UBL domains, participated in the design of the study and helped to revise the manuscript. SE participated in the design of the study, contributed reagents, and helped to revise the manuscript. RV performed the Retention of proteins on the GST-HHR23A~\u0394UBL column corresponds to their ability to interact with UBA domains. (A) Purified GST-HHR23A~\u0394UBL protein was used to saturate glutathione-sepharose resin, which was in turn used to pack an HR10/10 column. Equal amounts of HA-tagged SUMO (HA-SUMO), polyhistidine-tagged ubiquitin (Ub-His) and FLAG-tagged UBL (UBL-FLAG) were loaded simultaneously onto the column and the column was resolved in PBS. Fractions were collected and analyzed by Western blotting using epitope-specific antibodies. (B) Purified GST was bound to glutathione-sepharose resin in excess, which was then used to pack an HR10/10 column. Equal amounts of HA-SUMO, Ub-His and UBL-FLAG were mixed, loaded onto the column and analyzed as described above.Click here for file"}
+{"text": "Creatinine kinase BB (CK-BB) is elevated in many tumours including those of the breast. We have recently described a new, highly sensitive and specific method for measuring CK-BB, based on monoclonal antibodies and time-resolved fluorometry. Using this method, we quantitated CK-BB in 172 breast tumour cytosols and examined the associations between CK-BB and other clinicopathological variables and patient survival. High CK-BB levels were seen more frequently in tumours from patients who were younger (age < 50 years), patients who qualified for chemotherapy and patients with oestrogen receptor-positive tumours. No association was seen between CK-BB and tumour stage, grade, size, histological type or the progesterone receptor. In univariate analysis, the risk of relapse or death was higher in the group with tumours containing high CK-BB levels but the difference did not reach statistical significance. In multivariate analysis, the risk of death was statistically significantly higher in the high-CK-BB group. Analysis of subsets of patients revealed that patients with oestrogen receptor-negative cancer have higher risk of death if their tumours contain high levels of CK-BB. Our data suggest that, in general, CK-BB is associated with more aggressive tumours but its value as a prognostic indicator is limited. CK-BB content of breast tumours may be more useful as an aid in selecting therapy directed at inhibiting this enzyme activity and thus depriving tumour cells of their energy source."}
+{"text": "A few recent studies have demonstrated heightened hypothalamic\u2013pituitary\u2013adrenal (HPA) axis reactivity to acute stress in animals exposed to heavy metal contaminants, particularly lead. However, Pb-induced dysregulation of the HPA axis has not yet been studied in humans.In this study, we examined children\u2019s cortisol response to acute stress (the glucocorticoid product of HPA activation) in relation to low-level prenatal and postnatal Pb exposure.Children\u2019s prenatal blood Pb levels were determined from cord blood specimens, and postnatal lead levels were abstracted from pediatrician and state records. Children\u2019s adrenocortical responses to an acute stressor were measured using assays of salivary cortisol before and after administration of a standard cold pressor task.Pb exposure was not associated with initial salivary cortisol levels. After an acute stressor, however, increasing prenatal and postnatal blood Pb levels were independently associated with significantly heightened salivary cortisol responses.Our results suggest that relatively low prenatal and postnatal blood lead levels\u2014notably those below the 10 \u03bcg/dL blood lead level identified by the Centers for Disease Control and Prevention for public health purposes\u2014can alter children\u2019s adrenocortical responses to acute stress. The behavioral and health consequences of this Pb-induced HPA dysregulation in children have yet to be determined. Recent research has evaluated the effects of lead exposure on responses of the hypothalamic\u2013pituitary\u2013adrenal (HPA) axis to acute stress. These responses have been considered in animals, through assessment of glucocorticoid blood levels before and after the onset of an acute stressor. Several studies , 2004 haSeveral studies have considered factors that affect children\u2019s adrenocortical responses to acute stress. Such research has generally focused either on intrinsic differences such as temperament  and attaPb-induced increases in HPA reactivity in children are likely to have far-reaching consequences. The association of trace metal concentrations in blood with cardiovascular disease has been assessed in various epidemiologic studies. Such studies suggest positive associations between Pb and blood pressure , left veIn addition to cardiovascular effects of Pb exposure, low-level Pb exposure is associated with cognitive deficits in children . In somen = 25) or had missing salivary cortisol samples (n = 8). Reasons for not being tested included inability to schedule within the testing window (n = 16), technical problems (n = 4), and refusal (n = 5). Prenatal Pb levels were available for 154 children and postnatal Pb levels were available for 120 children. The child\u2019s response to an acute laboratory stressor was assessed within 2 weeks of attaining 9.5 years of age, and the family was paid $60 for participation in the current visit.Participants were recruited in the context of an ongoing longitudinal study of the effects of environmental toxicants on development . Of the p > 0.50). After the cold pressor task, we administered two cognitive stressors, mirror tracing and reaction time tasks . As previously reported  an initial rest period (10 min); b) a cold pressor task (1 min of submerging the dominant arm in ice water followed by a 2-min recovery); c) an intertask rest (8 min); d) a choice reaction time task (3 min); e) an intertask rest (8 min); f ) a mirror-tracing task ; and g) a final recovery/rest period (10 min).On the day of testing, the participant arrived at the laboratory at about 1630 hours  and before beginning read and signed an assent form, while his or her parents read and signed a separate consent form approved by the Institutional Review Board of SUNY Oswego. The laboratory session began with measurements of the child\u2019s height and weight. Each experimental session comprised the following: 2EDTA) evacuated glass tubes. Over the course of the study, both Vacutainer  and Monoject  tubes were used. However, each lot of blood tubes and needles used, regardless of source, was prescreened for Pb contamination and was certified for blood Pb measurements in the study by the analyzing laboratory. Specimens were shipped to the New York State Department of Health\u2019s Wadsworth Center and were analyzed for Pb and erythrocyte protoporphyrin, a biochemical marker of Pb exposure. Each specimen was analyzed for Pb in duplicate, using a method based on electrothermal atomic absorption spectrometry (ETAAS) with longitudinal Zeeman-effect background correction. The method has been fully validated and is described in detail elsewhere  age of 2.62 \u00b1 1.20 years, through the children\u2019s pediatricians and county public health agencies. New York State law requires blood Pb testing for all children before entering kindergarten, and only those laboratories certified by the New York State Department of Health\u2019s Wadsworth Center can accept and analyze blood Pb specimens drawn in New York State. Although more than 75 laboratories are certified to perform blood Pb testing in New York State, blood specimens are typically sent to a variety of clinical laboratories in both the commercial and public health sectors, depending on local screening practices and health insurance coverage. Analytical techniques used for blood Pb are ETAAS and anodic stripping voltammetry (ASV). Specific laboratories used to analyze specimens from children enrolled in the Oswego Children\u2019s Study included Quest Diagnostics Clinical Labs in Norristown, Pennsylvania, formerly SmithKline Beecham ; Wadsworth Center, Albany, New York ; Oswego Hospital Laboratory, Oswego, New York ; A. Lee Memorial Hospital Laboratory, Fulton, New York ; Quest Diagnostics Inc., Teterboro, New Jersey ; and SUNY Upstate Medical University Clinical Pathology Laboratory, Syracuse, New York . These laboratories participate regularly in the New York State proficiency testing program that is organized by the Wadsworth Center and are certified under both New York State and federal regulations . Blood Pn = 76) or two (n = 35) blood Pb tests; however, a few children had three (n = 9) or more (n = 2) tests before 9 years of age. The median value was used for the children with more than one draw. Blood Pb levels in the Oswego cohort ranged from 1.5 to 13.10 \u03bcg/dL, with only six children having blood Pb concentrations > 10 \u03bcg/dL, the level defined by the Centers for Disease Control and Prevention (CDC) as elevated for public health purposes , and during recovery (60 min after the cold pressor task). Following in vitro diagnostic measure of adrenal function . The test used 25 \u03bcL saliva, had a lower limit of sensitivity of 0.007 \u03bcg/dL, a range of sensitivity from 0.007 to 1.8 \u03bcg/dL, and average intra-and interassay coefficients of variation of < 5% and 10%, respectively. All samples were assayed in duplicate, and with the average of the duplicates used for all test results. Cortisol units are expressed in micrograms per deciliter. To create a more normal distribution, we eliminated a single outlier  and used a logarithmic transformation  to avoid dairy products for 30 min before collections (restriction based on evidence that some bovine hormones cross-react in immunoassay), b) to rinse their mouths with water 10 min before sample collections (no snacks were provided between sample collections), and c) to not brush their teeth within 1 hr of testing, so as to avoid blood contamination in saliva  and were given instructions to have no snacks during the 1 hr preceding the testing. If the child reported having had a recent snack, the session was either rescheduled or briefly delayed, depending on the timing of the snack. In addition, participants were instructed ng 48 hr .2). A complete list of these variables is provided in To strengthen our inferences regarding children\u2019s blood Pb levels, we considered various potential confounding variables, including characteristics measured during pregnancy, at birth, at 7 years of age, and at 9.5 years of age. Measures included paternal height and weight, maternal prepregnancy weight and height, maternal weight gain during pregnancy, maternal reported illness during pregnancy, obstetric complications , head circumference at birth, birth weight, gestational age, maternal substance use during pregnancy , the Home Observation for Measurement of the Environment (HOME), the Clinical Epidemiological Studies\u2013Depression CES-D;  inventorbis(p-chlorophenyl)ethylene , and hexachlorobenzene. We assessed mercury levels from maternal hair cuttings, with position along hair strands used to differentiate mercury levels during the first and second halves of pregnancy. Further details on these other toxicant measures can be found in prior publications , 1,1-dichoro-2,2-ications .We computed change scores for cortisol by subtracting initial levels from the task means. Therefore, our primary outcome variable was change in cortisol at 21, 40, and 60 min after the initiation of the cold pressor task.p < 0.20) to cortisol responses served as covariates in all analyses. Several studies  between exposure and outcome by \u2264 10%, is an extremely effective and rigorous means of controlling residual bias in multivariate correlational data sets  of that analysis. Only relationships that were statistically significant after this two-tiered approach were considered meaningful.Every analysis, therefore, included all covariates related to the outcome at n = 37), 1.1\u20131.4 \u03bcg/dL , 1.5\u20131.9 \u03bcg/dL , and 2.0\u20136.3 \u03bcg/dL . For postnatal Pb exposure, quartiles corresponded to the following blood Pb levels: 1.5\u20132.8 \u03bcg/dL , 2.9\u20134.1 \u03bcg/dL , 4.2\u20135.4 \u03bcg/dL , and 5.5\u201313.1 \u03bcg/dL . In the analyses of quartiles, we used SAS PROC GLM (SAS Institute Inc.) with a linear contrast to test the effects of increasing blood Pb levels controlling for all potential confounds .Although the distribution of pre-natal and postnatal blood Pb levels was nearly normal, regression models can be sensitive to the presence of outliers. In addition, use in the analyses of a value of one-half the MDL for postnatal blood Pb levels reported as below the MDL, resulted in differing Pb values, as a function of the testing laboratory\u2019s MDL (either 3 or 5). Therefore, as was done previously , we focuF  = 4.79, p < 0.005, as well as postnatal blood Pb levels, F  = 7.35, p < 0.0001. The mean values from these analyses are shown in F  = 3.19, p < 0.05] and postnatal  Pb exposure. To further analyze this interaction, we first considered initial adrenocortical levels as a function of Pb exposure.As a preliminary analysis, we conducted a 4 (blood Pb quartile) \u00d7 4  mixed factorial analysis of variance, with the saliva sample being a repeated measure. This analysis was initially performed for prenatal and postnatal Pb using a test for the linear contrast across quartiles, but without covariate control. These analyses revealed a significant interaction between the time of the cortisol sample and prenatal blood Pb levels, F  = 0.01, p > 0.25. Similar results were obtained when prenatal Pb levels were analyzed as a continuous variable, F  = 0.24, p > 0.25. After adjustment for covariates, post-natal Pb quartiles were not significantly associated with initial cortisol levels , nor were postnatal Pb levels when analyzed as a continuous variable . Having established that initial levels of salivary cortisol were unaffected by prenatal and postnatal Pb exposure, we next analyzed changes from initial  as a function of Pb exposure.After adjustment for covariates, pre-natal Pb quartiles were not significantly associated with initial cortisol levels, F  = 11.78, p < 0.001 (for linear contrast). This association was equivalent for samples collected at 21, 40, and 60 min . The adjusted means for this analysis are shown in F  = 18.20, p < 0.0001.Prenatal Pb exposure had a significant effect on cortisol responses to acute stress at 9.5 years of age, with increasing Pb associated with an increasing cortisol response, F  = 11.21, p < 0.005. This association was again equivalent for samples collected at 21, 40, and 60 min . The adjusted means for this analysis are shown in F  = 4.06, p < 0.05.Postnatal Pb exposure had a significant effect on cortisol response to acute stress at 9.5 years of age, with increasing Pb associated with an increasing cortisol response, t  = 2.97, p < 0.005], 40 min , and 60 min . Similarly, postnatal Pb exposure was significantly or marginally significantly associated with cortisol reactivity at 21 min , 40 min , and 60 min , as shown in the lower row of p > 0.10).We next performed regression analyses, with covariates entered first, followed by Pb levels as a continuous variable. As shown in the upper row of p > 0.25). In addition, the interaction between Pb exposure timing  was not significant ; however, we do illustrate the additive nature of prenatal and postnatal Pb exposure on mean cortisol reactivity (adjusted for covariates) in Finally, we tested SES and sex as potential effect modifiers, as found in previous research on Pb-induced changes in glucocorticosteroids . NeitherIn the present study we examined the relationship between children\u2019s prenatal and postnatal Pb exposures and subsequent adrenocortical activity at rest as well as in reaction to acute stress, measured at 9.5 years of age. We found that initial cortisol levels were not significantly associated with either prenatal or postnatal Pb exposure. This null association with basal glucocorticosteroid levels is consistent with findings in Pb-exposed birds , althougThe mechanism(s) for Pb effects on the HPA axis are not well understood. Pb exposure has been shown to affect the brain\u2019s mesocorticolimbic dopaminergic system  and coulA few concerns related to the current study should be noted. First, postnatal Pb was measured in various laboratories that used an MDL of either 3 or 5 \u03bcg/dL, whereas prenatal Pb was measured in just one laboratory using a single, well-characterized method  with an MDL of 1 \u03bcg/dL. Therefore, the effect of prenatal Pb exposure could have been stronger relative to postnatal Pb effects, because a more sensitive and precise measurement method (and corresponding reductions in measurement uncertainty) was used in a single laboratory for the prenatal exposure assessment.The second concern is the consistent effect of Pb exposure across the 21-, 40-, and 60-min cortisol specimens. If Pb exposure affects only adrenocortical reactivity, and if 60 min is a sufficient length of time for recovery, then Pb exposure effects should have become attenuated by 60 min after the acute stress task. However, the effects of prenatal and postnatal Pb exposure on cortisol reactivity were not significantly different for the three cortisol samples. There are two possible explanations for this pattern of findings. Either 60 min was insufficient for the children\u2019s recovery, or else our initial level did not truly represent resting adrenocortical activity. Although it is not uncommon for salivary cortisol changes to persist even after 60 min e.g., , recoverThat our assessment of postnatal Pb occurred approximately 7 years before our assessment of adrenocortical activity is a third concern. However, multiple measures of Pb over time have been shown in prospective studies of children to be highly correlated e.g., . FurtherThe present study demonstrated that prenatal Pb exposure was associated with a significant increase in the child\u2019s cortisol reactivity to acute stress at 9.5 years of age. Comparable but independent effects on cortisol reactivity were observed for postnatal Pb exposure. Although the long-term implications for this association are unclear, they are undoubtedly complex. Increased HPA axis activity is associated with a number of emotional, behavioral, and physical problems in children . However"}
+{"text": "SDF1 gene variation, plasma SDF-1alpha level, and the number and function of circulating EPCs. We also tested whether EPC number and function was related to CXCR4 gene variation.Stromal cell-derived factor-1 (SDF1) and its receptor CXC chemokine receptor 4 (CXCR4) play a critical role in progenitor cell homing, mobilization and differentiation. It would be interesting to assess the predictive value of SDF-1alpha level for EPC number, and to ascertain whether there is a relationship between SDF1 and CXCR4 genes, and measured blood SDF1\u03b1 level as well as EPC number and function. SDF1\u03b1 levels were correlated with age, gender, alcohol consumption, circulating reticulocyte numbers, and concentrations of matrix metalloproteinase-9, C-reactive protein, cystatin C, fibrinogen and homocytein. In blood samples taken in 2005, EPC number was inversely associated with SDF1\u03b1 level (p<0.001). EPC number in 2005 was also inversely associated with SDF1\u03b1 level in 2000 (p\u200a=\u200a0.009), suggesting a predictive value of plasma SDF1\u03b1 level for EPC number. There was an association between the SDF1 gene rs2297630 SNP A/A genotype, increased SDF1\u03b1 level (p\u200a=\u200a0.002) and lower EPC number (p\u200a=\u200a0.006).We genotyped a cohort of individuals who participated in the Bruneck Study for single nucleotide polymorphisms (SNPs) in the Our data indicate that a SDF1 gene variation (rs2297630) has an influence on SDF1\u03b1 level and circulating EPC number, and that plasma SDF1\u03b1 level is a predictor of EPC number. Asahara and colleagues in 1997 demonstrated for the first time that purified CD34 positive haematopoietic progenitor cells from peripheral blood could differentiate, ex-vivo, into an endothelial phenotype and were named Endothelial Progenitor Cells (EPCs)SDF1 or CXCR4 gene in mice led to early embryonic lethality due to abnormality in the cerebellar and gastrointestinal vasculature and in hematopoiesis developmentSDF1 gene transfer using the adenovirus infection techniqueAnimal studies have indicated that SDF1  and its receptor CXC chemokine receptor 4 (CXCR4) plays a critical role in progenitor cell homing, mobilization and differentiation. Inactivation of the SDF1 gene can have an influence on SDF1\u03b1 levels+ cell mobilizationSDF1 gene variation, SDF1\u03b1 level and circulating EPC number. In addition, we tested whether there was also a relationship between variation in the gene encoding the SDF1\u03b1 receptor CXCR4 and EPC number.There is emerging evidence indicating that variation in the human SDF1 and CXCR4 genes. Blood samples taken in 2000 (n\u200a=\u200a684) and 2005 (n\u200a=\u200a574) respectively were used for measurements of SDF1\u03b1 levels. EPC number and function were assessed in blood samples collected in 2005 (n\u200a=\u200a571 for EPC number and n\u200a=\u200a542 for EPC colonies number respectively). Subjects with and those without EPC number and EPC function assessments did not differ in age, sex and cardiovascular risk factors. The appropriate ethics committees (Autonome Provinz Bozen-Sanitatsbetrieb Bozen Ethikkomittee) approved the study protocol and all study subjects gave their written informed consent before entering the study.The subjects of this study were residents of the Bruneck area in the Bolzano Province of Italy, who participated in the Bruneck StudySystolic and diastolic blood pressure was taken with a standard mercury sphygmomanometer after at least 10 min of rest (mean of three independent measurements). Hypertension was defined as blood pressure \u2265140/90 mm Hg or the use of anti-hypertensive drugs. The average number of cigarettes smoked per day was noted for each smoker and ex-smoker. Diabetes was diagnosed according to ADA criteria. Assessment of regular alcohol consumption was performed with a standardized questionnaireand quantified in terms of grams per day (g/day).EPC numbers in the blood samples were determined as described previouslyThe EPC-CFU assay performed as described previouslyCommercially available SDF1\u03b1 and matrix metalloproteinase-9 (MMP9) ELISA kits  were used to determine plasma SDF1\u03b1 and MMP9 levels. All ELISA tests were carried out at room temperature on freshly thawed plasma samples. The concentration was determined by comparison with a standard curve, following manufacturer's instruction. Other laboratory parameters and vascular risk factors were all examined by standard methodsSDF1 and CXCR4 genes from the HapMap database (data release in June 2005). Three SNPs in SDF1  and two in CXCR4 (rs16832740 and rs12691874) to capture SNPs with a minor allele frequency >0.2 at these loci and R2>0.8 were typed in this study. In addition, we studied the rs266085 SNP which had been reported to influence SDF1 expressionWe selected tagging SNPs in the eB-transformed. Non-parametric tests yielded very similar findings (data not presented). Associations of EPC number, EPC-CFU and SDF1\u03b1 level with SDF1 and CXCR4 genotypes were assessed using generalized linear models adjusted for age and sex. Multivariate models additionally included standard cardiovascular risk factors and the factors associated with SDF1\u03b1 levels . A Bonferroni correction was performed to account for the multiple comparisons (5 SNPs in SDF1 and 2 in CXCR4) performed.The data were analyzed using the SPSS 15.0 and BMDP software packages. Continuous variables were presented as means\u00b1SD or medians (interquartile range), and dichotomous variables as percentages. Correlations between EPC number and EPC-CFU, SDF1\u03b1 level and other parameters were estimated by calculation of crude and partial (age/sex-corrected) Pearson correlation coefficients. Variables with a skewed distribution were logB. Kolmogorov-Smirnov p>0.05). Mean SDF1\u03b1 levels in 2000 and 2005 were 2652 pg/ml and 2565 pg/ml, with a range of 880 pg/ml to 5595 pg/ml and 1322 pg/ml to 4174 pg/ml, respectively. SDF1\u03b1 levels in 2000 and 2005 were highly correlated , indicating that the SDF1\u03b1 level is relatively consistent over time. As expected, both plasma SDF1\u03b1 level measured in 2000  and 2005  increased with age , indicating that SDF1\u03b1 has an influence on EPC number in the general population. Importantly, EPC number in 2005 was also associated with SDF1\u03b1 level in 2000 (p\u200a=\u200a0.009), indicating a long-term predictive value of SDF1\u03b1 level for circulating EPC number (EPC number and EPC-CFU number (per 1 ml blood) displayed a non-normal distribution, with the majority of subjects (more than 75%) having 0 to 1000 EPC and 0 to 340 EPC-CFU, respectively. SDF1\u03b1 levels were significantly and inversely associated with EPC number (p<0.001) in samples collected in 2005 1C, indSDF1 and CXCR4 SNPs examined in this study are shown in . The genotype distributions were consistent with Hardy-Weinberg equilibrium.Allele and genotype frequencies of the SDF1 rs2297630 SNP was associated with plasma SDF1\u03b1 level (p\u200a=\u200a0.002) and circulating EPC number (p\u200a=\u200a0.006), with the A/A genotype associating with higher SDF1\u03b1 level and lower EPC number  and the relationships of the SDF1\u03b1 levels at these different time points with circulating EPC numbers. A key finding from our study is the high degree of correlation the two SDF1\u03b1 measurements in blood samples taken in 2000 and 2005 respectively, suggesting that although SDF1\u03b1 levels are correlated with cardiovascular risk factors, the levels in an individual are relatively consistent over time. In addition, the study revealed an association between SDF1\u03b1 level in 2000 and EPC number in 2005, suggesting a long-term predictive value of SDF1\u03b1 level for EPC number. Another important, novel finding of this study is that there is an association between variation in the + cells in peripheral blood was lower but the level of SDF1\u03b1 was much higher at 14 days after ischemia, as compared with control miceSDF1 is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells and is expressed also by stromal cells of various tissuesSDF1 gene. In particular, we found that SDF1\u03b1 level and circulating EPC number are associated with the SDF1 gene rs2297630 SNP in the Bruneck study cohort. A potential interpretation for this novel finding is that variation in the SDF1 gene can influence EPC number via an effect on the level of SDF1\u03b1. The rs2297630 SNP is located in intron 3 of the SDF1 gene. It is possible that the association of this SNP with SDF1\u03b1 level and circulating EPC number has arisen from a direct functional effect of this SNP on SDF1 expression or mRNA splicing. Alternatively, the rs2297630 SNP might be a functionally neutral marker that is in linkage disequilibrium with a functional polymorphism located elsewhere at the SDF1 locus.A recent study showed that there is significant correlation in the number of circulating EPCs between parents and their offsprings, leading to the hypothesis that EPC number is, at least in part, genetically regulatedSDF1 SNPs, rs266085 and rs1801157, have been reported to be associated with SDF1 levels+ cell mobilizationSDF1 locus due to linkage disequilibrium. This possibility is consistent with the fact that the SDF1 gene is transcribed into two isoforms, SDF1\u03b1 and SDF1\u03b2, and that the rs1801157 SNP is located in the 3\u2032 untranslated region of the SDF1\u03b2 transcript but not in the SDF1\u03b1 transcriptTwo other Some particular attentions should be drawn regarding our findings. Firstly, the consideration of the nature of EPC is important, since there is no a unique criteria for identification of EPC, yet. Immense attention regarding the definition of EPC has recently been drawn due to the fact that the characterization and function of EPC isolated with different methodologies were quite different from each otherIn summary, our data indicate that although the SDF1 level and EPC number may both increase in response to acute ischaemic events, the relationship between blood SDF1 level and EPC number is complex and they are inversely correlated in the normal situation as observed in the Bruneck study cohort which was recruited from the general population. Our data also indicate that circulating EPC number is influenced by variation in the SDF1 gene likely via an effect of the genetic variation on SDF1\u03b1 expression. These findings help understand the mechanisms underlying the inter-individual variability in EPC number which has an implication in the pathogenesis of atherosclerosis and other cardiovascular diseases."}
+{"text": "The Impact Pathway Approach (IPA) is an innovative methodology to establish links between emissions, related impacts and monetary estimates. Only few attempts have so far been presented regarding emissions of metals; in this study the external costs of airborne lead (Pb) emissions are assessed using the IPA. Exposure to Pb is known to provoke impacts especially on children's cognition. As cognitive abilities  are known to have implications for lifetime income, a pathway can be established leading from figures for Pb emissions to the implied loss in earnings, and on this basis damage costs per unit of Pb emission can be assessed.Different types of models are here linked. It is relatively straightforward to establish the relationship between Pb emissions and consequent increase in air-Pb concentration, by means of a Gaussian plume dispersion model (OML). The exposed population can then be modelled by linking the OML-output to population data nested in geo-referenced grid cells. Less straightforward is to establish the relationship between exposure to air-Pb concentrations and the resulting blood-Pb concentration. Here an Age-Dependent Biokinetic Model (ADBM) for Pb is applied. On basis of previous research which established links between increases in blood-Pb concentrations during childhood and resulting IQ-loss we arrive at our results.External costs of Pb airborne emissions, even at low doses, in our site are in the range of 41-83 \u20ac/kg emitted Pb, depending on the considered meteorological year. This estimate applies only to the initial effects of air-Pb, as our study does not address the effects due to the Pb environmental-accumulation and to the subsequent Pb re-exposure. These are likely to be between one and two orders of magnitude higher.Biokinetic modelling is a novel tool not previously included when applying the IPA to explore impacts of Pb emissions and related external costs; it allows for more fine-tuned, age-dependent figures for the external costs from low-dose exposure. Valuation of additional health effects and impacts e.g. due to exposure via ingestion appear to be feasible when extending the insights from the present pilot study. While environmental scientists tend to use physical units in order to measure and describe risks and impacts, the policy-making process is faced with trade-offs, that are often economic in nature. Attempts to express the environmental impacts of emissions in monetary terms have been presented in recent years -3. Measux and SO2, e.g. from energy sources such as power plants. More recently modern sources of emissions such as WtE plants, which emit certain persistent micro-pollutants , have been explored, but authoritative figures for the external costs are still lacking. The micro-pollutants represent a matter of concern due to their high toxicity and persistence in environmental media. WtE plants provide energy and heat from the combustion of municipal waste: they are often located in urban areas and the concentration of micro pollutants in their stack emissions depends on the municipal waste composition, which may be varying, and is therefore sometimes unpredictable ['Monetization' of the impacts of emissions is not always feasible, and is associated with a great deal of uncertainty. The Impact Pathway Approach (IPA) for assessment of the 'external costs' of air emissions, developed under the ExternE project series, has however gained recognition for reducing the biases associated with more pure, preference based willingness-to-pay estimates . 'Externdictable . For sucThis study explores the possible external costs associated with atmospheric emissions of lead (Pb) from a WtE plant located in a suburb of Copenhagen, Denmark. Pb is a substance for which the negative impacts on human health have been established in numerous studies reported in the scientific literature -9, and fWe take our point of departure in a site-specific study of Pb-emissions from a large WtE plant, but we argue that the findings have general validity beyond the specific site, provided that the specific circumstances of population density and the relative monetary values in Denmark are adjusted to other sites.The IPA structure consists of four consecutive steps : 1) Emisair. The emission of Pb from the plant raises the existing level of Pbair. The concentration contribution from the source to the background level is defined as a 'delta concentration': \u0394-Pbair. It is described as a \"marginal\" contribution, because the source is small compared to the sum of all the other existing sources contributing both to Pbair and to Pb exposure. The Danish Gaussian plume dispersion model (OML) [air values . The plant considered in this study emitted 969 kg Pb in year 2000 in stack gases . The Pb el (OML) ,15 has b32 coordinates. A calculated \u0394-Pbair value is assigned by the model to each grid cell in the model domain. Mean values of \u0394-Pbair are 1 \u00d7 10-5 \u03bcg/m3 and the range comprises values of between 1 \u00d7 10-4 and 1 \u00d7 10-6 \u03bcg/m3. Values are calculated for three different years: 2000, 2001 and 2002 based on the same emissions but with different meteorological data for the three years. The concentration contribution from the point source (\u0394-Pbair) is therefore, on average, two orders of magnitude lower than the background concentration of Pbair, as reported in Denmark's Air Quality Monitoring Programme [10 (atmospheric particles with aerodynamic diameter less than 10 \u03bcm) was measured for traffic and urban background conditions. Values of Pbair = 0.0079 (\u03bcg/m3) for traffic and Pbair = 0.0039 \u03bcg/m3 for urban background conditions respectively have been measured in the city of Copenhagen [The domain of the model covers 50 \u00d7 50 km around the point source giving a total number of 1 \u00d7 1 km grid cells of approx. 2,500, all geo-referenced in UTMrogramme  where thpenhagen .air data. The population data for the age distribution of the inhabitants in each grid cell have been retrieved from the Danish CPR-registry . The population under exposure can be derived by crossing the two distributions . In this way, the grid cells where no additional Pbair occurs or no population is reported will not contribute to the calculation of external costs.Population data for Denmark have been distributed on the same grid cells as applied in the calculation of the \u0394-PbThe damage window for the impact of Pb explored in this study is cognitive impairment in children resulting from Pb exposure. Neurodevelopment is critical during childhood, especially the first 2-3 years of life ,16,17. Fblood). Pb is known to provoke cognitive impairment in children; such impact is measured in terms of Intelligence Quotient (IQ) loss in children and related to increases in the Pbblood [air and the corresponding increase in Pb impacts.The Pb concentrated in the air is inhaled and then absorbed in the body, and increases the blood Pb concentration  can then be calculated as the product of two functions (1):The CRF linking Pbblood - Pbair (\u03bcg/dl Pbblood/\u03bcg/m3 Pbair), the relationship between an increase in Pbair and the corresponding increase in Pbblood.1) Pbloss - Pbblood (IQ lost point/\u03bcg/dl Pbblood), the relationship between an increase in Pbblood, and a loss of IQ points.2) IQValues and references for the two functions 1) and 2) are presented in detail in the following sections (cf. sect. 2.2.2. and 2.2.3.).blood - Pbair function by means of statistical techniques assessing the relationship between measured media concentration and bio-monitoring values [blood after defined periods of exposure and for different age categories or physiological characteristics. In this specific case-study we only include the contribution from inhalation to the resulting concentration in blood. The amount of Pb that is daily absorbed in the body (Pbabsorbed), expressed in \u03bcg/day, is the input to the ADBM model and has been estimated according to equation (2):Previous studies determined the Pbg values ,21. In tg values  for Pb fg values -24. The inhalation is the breathing rate in m3/day, and AFPb is the Absorption Factor for Pb via inhalation expressed as percent of Pb intake.Where IR3) inhaled daily by children and adults have been adopted as reported by US EPA [air (cfr. sect.2.1.). The relation between Pbair and Pb intake is then, for the range of concentrations here considered, assumed to be linear for marginal increases in Pbair.The breathing rates and Pb absorption rates are the key parameters determining the actual Pb absorption needed as input for the ADBM. Reference values for age-dependent breathing rates, representing the amount of air  of exposure, and the Pbair. The model is nonlinear for constant values of Pbair . When age/time exposure conditions are instead fixed, Pbblood is a linear function of Pbair.Pbair - Pbblood varies greatly, for children, according to the age of the subject. It is then necessary to use different functions according both to the age of the children population under exposure and to the duration of the exposure.The ADBM shows that the Pbair, is the result of an annual Pb emission and because our interest is in the annualized external costs. Three separate age classes have been considered: children aged between 0 and 1 year, between 1 and 2 years, and between 2 and 3 years respectively.Three different scenarios for exposure have then been explored with reference to children. Duration of exposure is set to one year in all the scenarios, because the increment of Pb in the air, \u0394-Pbblood - Pbair functions determining the level of Pbblood in children exposed for one year to a constant value of Pbair have been calculated with the ADBM as 1.97 \u03bcg/dl/\u03bcg/m3 for children aged between 0 and 1 year, 2.85 \u03bcg/dl/\u03bcg/m3 for children aged between 1 and 2 years, and 3.27 \u03bcg/dl/\u03bcg/m3 for children aged between 2 and 3 years.The age-dependent PbDue to different indications in the literature ,16,17, tblood in children, even at very low Pbblood levels (Pbblood < 10 \u03bcg/dl) [blood and is assumed to be linear without threshold [blood are estimated to be between 2-4 \u03bcg/dl in Europe [loss - Pbblood function for marginal increases of Pbblood are then assumed. Grosse [loss - Pbblood function. The author also notices how the function shows a greater slope for lower values of Pbblood in children (Pbblood < 10 \u03bcg/dl) than for higher values (10 \u03bcg/dl <Pbblood < 20 \u03bcg/dl). Following the approach already proposed by Grosse [loss - Pbblood parameter: lower bound, upper bound and base case (Table loss- Pbblood slope proposed by Schwartz [Pb has a negative impact on children's neurodevelopment. Numerous studies conducted since the late 1980s to date have shown how cognitive impairment is inversely associated with increases in Pb0 \u03bcg/dl) -19,28-30hreshold ,31. Actun Europe . In this. Grosse  reports y Grosse , three lSchwartz  is derivA single pollutant can provoke multiple impacts both with regard to the environment and humans, and not all of them can be expressed in monetary terms. In the specific case of Pb, relevant impacts on human health are those related to anaemia in adults and loss of IQ in children . Monetarair value for each selected grid cell i  is multiplied by the CRF function that relates Pbblood to Pbair in children aged between 0 and 1 year, in order to obtain the marginal increase \u0394-Pbblood (3).The procedure behind IPA can be described with a number of equations. First, the \u0394-Pbblood is then multiplied by the value of the IQloss - Pbblood slope and the marginal loss \u0394-IQloss is obtained (4).\u0394-Pbloss is multiplied by the cost of one point of IQloss to obtain the marginal monetary loss \u0394-\u20acloss (5).\u0394-IQair is now associated to each considered grid cell i. This value needs to be multiplied for the number ni of children aged between 0 and 1 years present in the cell. The total cost for each single cell is obtained. This needs to be summarized across all the considered cells. The total cost of the Pb emissions Total_ \u20acloss is then obtained (6).A monetary value related to the increase in Pbloss by the total amount of Pb emitted, in kg (7).In order to obtain a cost per kg it is necessary to divide the Total_ \u20acblood to Pbair in children aged between 1 and 2 years. The two values for external costs are then added in order to obtain the total external cost of the Pb emission from the source via the inhalation pathway.The external cost per kg of emitted Pb for the population aged between 0 and 1 year is thereby determined. With the same procedure, the external cost aggregated over the population aged between 1 and 2 years is calculated, using the CRF that relates Pbloss- Pbblood slope, discount rate of 3%, and damage window for Pb impacts of 3 years from birth (0<age<3). The value of 83 Euros is the cost of each extra kg of Pb emitted, when the population is exposed via the inhalation pathway.Table loss- Pbblood slope, discount rate, and Pb impacts damage window. This approach on the variability of parameters and results cannot be considered a complete sensitivity analysis, but represents an indication of the sensitivity and uncertainty involved.Values of external costs of Pb emissions, in Euro per kg Pb emitted, for exposure via inhalation and for the different meteorological conditions of the years 2000, 2001 and 2002, are reported in Table The values of 83, 59 and 41 Euro/kg Pb emitted for the meteorological years 2000, 2001, 2002 respectively  based o-) Emission and dispersion: the measurements determine only \"Total Pb\" in flue gases. Pb is adsorbed on PM and the dispersion model does not account for differences in the dispersion between various species of Pb.-) Exposure: while the breathing rate is independent from the Pb species, the absorption rate is based only on the bio-available fraction of Pb. Not all the inhaled Pb is assumed to be absorbed. In this way a pooled estimate of the relevant Pb species appears.-) Bio-Accumulation: the ADBM parameters have been calculated from bio-monitoring data and consider \"Total Pb\" in the different organs/tissues as a pooled figure for the relevant Pb species.blood function: is in the literature calculated from bio monitoring data reporting the total (pooled) Pb content in blood.-) IQ loss - Pbloss - Pbblood function, several studies [blood in children aged 6 months to 3 years). The meta-review of Schwartz [loss - Pbblood function affects the final results only to some extent; the selection of damage window contributes more strongly, evidently because definition of the number of children exposed is involved. By using the ADBM we can give a good definition of the Pbblood - Pbair function for different ages and times of chronic exposure, but then we need to select the exposed population carefully. The state of the art of current research does not give precise indications of the damage window for Pb-related neurodevelopmental impacts. The above cited studies regarding the loss of IQ related to Pb in blood have been performed measuring IQ value  at the minimum age of 6 months, and focusing mainly on the influence of covariates such as gender, sex, and level of Pbblood, without trying to identify a specific moment when impact is supposed to occur. In general, a valid assumption, considering that the process of neurodevelopment in children is higher in the first 2-3 years immediately after birth, is that the impacts of potential impairment due to Pb should be greater in these early years. The scenarios considering 0 < age <2 and 0 < age < 3 years as the damage window are included to demonstrate the relative significance of the age parameter choice. In comparison the different spatial dispersion of the contaminant, pending only on meteorology and resulting in a different amount of population under exposure, displays some significance too.The topic of IQ loss from Pb exposure in childhood remains of concern, perhaps in particular in developing countries. With regard to the IQ studies ,28,29,31 studies , with a Schwartz , howeverSchwartz . Schwartblood- Pbair function of 5 \u03bcg/dl/\u03bcg/m3 for incremental exposure to Pb in ambient air, citing a reference from a UK study. The number of children under exposure is assumed to be the 1.1% of the total population, regardless of any damage window for Pb impact. The monetization is determined using the relationship between loss in IQ and decrease in income. The values for external costs reported by Spadaro are two orders of magnitude higher than the values here reported for Denmark. They are reported at an EU level and they are not limited to inhalation but include also ingestion of Pb, because the measured Pbblood- Pbairfunction implicitly accounts for the ingestion pathway also. The difference between the inhalation and the ingestion pathway is significant. If inhalation is assumed to account only for 4% of the total intake of Pb belonging from the source, as has been suggested above, then it is reasonable to expect a value for the external costs regarding the total Pb intake  that is somewhere between one and two orders of magnitude higher than when referring to inhalation only.It is interesting to compare the results obtained in the present study with findings from earlier studies. As the main references for comparison we can take the work performed in the Espreme project  and the blood, and in particular to relate such increment to the specific considered source of emission. The ADBM has been here used in order to isolate and specify the relationship existing between Pbair and Pbblood and separate this from other intake pathways, so the model allows for a better quantification of this source and this exposure pathway contribution to the overall impact. However, the total impact is likely to be much greater. Under natural background exposure conditions the ingestion of Pb is, in terms of Pb intake (\u03bcg/day), around two orders of magnitude greater than the inhalation [soil - Pbblood by using the ADBM and applying the same approach that has been explained above for inhalation. Ideally this could allow for accounting of the incremental impact relating to the ingestion of Pb emitted from the source and consecutively deposited in the soil. The problem is that, at the moment, the uncertainties in the establishment of a multimedia link between source and impact via ingestion, considering the marginality of the source, appear to be quite high. We provide only a partial result, but the methodology based on ADBM piloted here would allow for exploring also the ingestion pathway and so arrive at more robust figures for the external costs of Pb exposure. A multimedia approach will be required as an extension of the modelling framework presented here.In our study we chose to focus on the inhalation pathway, because the link between air emission and inhalation is direct and no multimedia distribution is involved. It is also difficult to distinguish the contribution of different exposure pathways to the total Pb exposure and to the increase in Pbhalation . This doloss - Pbblood function, the damage window for Pb impacts, and the discount rate has been discussed in a critical perspective. This approach considers only the initial effects via inhalation, and consequently accounts for only a part of the total costs of Pb emissions. Even these conservative values suggest that policies to reduce Pb exposure deserve careful considerations in the policy making process. The importance of assessing the contribution of the ingestion pathway, even if under conditions of greater uncertainty, has been stressed and will be the topic of further research.External costs for marginal Pb emission originating from a waste-to-energy plant have been assessed according to the impact pathway approach and extended with a detailed atmospheric dispersion model and a biokinetic model. The assessment of exposure to Pb for the most sensitive segment of the population has been determined carefully using spatially defined data as well as reference values for Pb intake and Pb impacts. The influence of some key parameters such as the IQADBM: Age-Dependent Biokinetic Model; AF: Absorption Factor; CBA: Cost Benefit Analysis; CPR: Personal Registration Code; CRF: Concentration Response Function; ICRP: International Commission for Radiological Protection; IPA: Impact Pathway Approach; IQ: Intelligence Quotient; IR: Intake (Breathing) Rate; LCA: Life Cycle Assessment; NERI: National Environmental Research Institute; OML: Operational Meteorological air-quality Model; PM: Particulate Matter; US- EPA: United States Environmental Protection Agency; WtE: Waste-to-Energy plants.The authors declare that they have no competing interests.MP carried out the determination of population under exposure, age-dependent CRF using the ADBM model and calculation of external costs, and drafted the manuscript. MSA and MT participated in the design and coordination of the study and supervised the work, then contributed to the drafting and revising of the manuscript. LMF carried out the air dispersion modelling and provided the relative data. All authors read and approved the final manuscript."}
+{"text": "Overexpression of neuronal calcium sensor 1 in cortical neurons can help restore axonal plasticity and regeneration following axonal injury in adult rats, and can also improve behavioral function. Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system. Following trauma to the central nervous system , neurons show very little capacity to re-grow their axons, which can lead to a permanent loss of function in those regions. In this study, we show that this failure of axon re-growth is associated with low intracellular levels of a small molecule called neuronal calcium sensor-1 (NCS1). We modified a non-replicating virus in two ways so as to increase the level of NCS1 in neurons while simultaneously labelling them with a green fluorescent protein, which allowed us to track neuronal growth. Using this virus to increase the level of NCS1 in a particular group of neurons that communicate between the brain and spinal cord, we showed that new axonal growth occurred in the spinal cord with or without injury to the neurons. Electrophysiological assessments demonstrated that these new processes formed functional connections in the spinal cord, and behavioural experiments revealed that this recovery also helped the animals move their limbs more effectively. Furthermore, an increase in NCS1 protected these neurons, such that fewer of them died after the injury. These findings demonstrate that increasing the intracellular levels of NCS1 in neurons can aid in the recovery from central nervous system injury, and can help improve behavioural function. Spinal cord injury is a significant clinical problem that produces life long disability, although in a minority of patients some degree of recovery can occur spontaneously without any therapeutic intervention There is a growing literature suggesting pharmacological interventions can enhance both axonal regeneration Recently, we have found that following injury, the lentiviral overexpression of retinoic acid receptor \u03b22 (RAR\u03b22) induces regeneration in sensory and central axons NCS1 is highly conserved across species and emerges as a key intracellular calcium signalling component in a number of regulatory pathways in neurons Here we show using lentiviral vectors that NCS1 overexpressed in primary cultured adult cortical neurons increases neurite sprouting. Following corticospinal tract (CST) denervation by unilateral pyramidotomy, axons of uninjured corticospinal neurons (CSN) overexpressing NCS1 sprout across the midline to form functional connections in the CST-denervated spinal cord. In axotomized CSN, overexpression of NCS1 induces axonal sprouting and regeneration and also neuroprotection. These studies reveal NCS1 as an important intracellular molecule for promoting anatomical plasticity following CNS injuries in the adult.To transduce adult cortical neurons with NCS1 at high efficiency and to enable visualisation with an extrinsic marker, we constructed a minimal human immunodeficiency virus (HIV) based lentiviral vector expressing NCS1 and GFP under cytomegalovirus (CMV) and spleen focus-forming virus (SFFV) promoters, respectively, termed HIV-GFP-NCS1 . A contrThe type of neurite that has undergone sprouting with NCS1 overexpression was further investigated using the specific dendritic immunomarker microtubule-associated protein 2 (MAP2). MAP2 has been previously shown strongly and weakly to immunolabel dendrites and axons, respectively These data indicate that primary cultured adult cortical neurons overexpressed with NCS1 have significantly more neurites and sprouts from dendrites and axons than the control neurons.NCS1 has been shown to induce neuronal survival via the activation of the PI3K/Akt pathway These data indicate the levels of phospho-Akt are elevated in neurons with overexpressed NCS1 and blockade of Akt production reduces neurite sprouting in these neurons.In Western blots, the levels of NCS1 were significantly higher in cortical neurons transduced with HIV-GFP-NCS1 compared with the controls both in vitro and in vivo . This inWith the demonstration that NCS1 overexpression induces neurite sprouting in primary cultured adult cortical neurons, it was next determined whether this also occurred in vivo. HIV-GFP-NCS1 or control HIV-GFP lentivector was injected into the forelimb and hindlimb regions of the left sensorimotor cortex of adult rats. High efficiency was achieved of both GFP and NCS1 expression in the CSN at 3 wk with HIV-GFP-NCS1 . SimilarAdult Wistar rats received unilateral intracortical injections of either HIV-GFP-NCS1 (NCS1-transduced) or the HIV-GFP (control) lentivector 3 wk before receiving on the contralateral side a unilateral pyramidal tract lesion which, in turn, causes CST-denervation of the contralateral side of the spinal cord . The lesDuring the 6-wk period after CST-injury, behavioural testing was used to assess sensorimotor function in control (HIV-GFP) and NCS1-transduced (HIV-GFP-NCS1) rats. The sensorimotor function was assessed using the staircase reaching apparatus to test for forelimb extension and grasping movements and the grid exploration test to test for sensory motor function of fore- and hindlimbs. The CST-denervated forelimb of the control and NCS1-transduced groups showed a significant impairment in eaten or food pellet displacement compared to the sham group 2 d post-CST-injury . HoweverIn the grid exploration test, a significant difference in limb misplacement was observed in the control group compared with the sham throughout the behavioural testing period . In contTo demonstrate further that overexpressed NCS1 had induced axon collateral sprouting from the intact CST, extending into the CST-denervated side of the spinal cord, a subsequent lesion of the intact pyramidal tract (termed second lesion) was made at medullary level . This seIt is well established in the anesthetised rats that stimulation of the forelimb area of the motor cortex results in contralateral forelimb movements To investigate whether a delayed NCS1 overexpression in CSN can also promote axon collateral sprouting to the same extent as priming the CSN for 3 wk, adult Wistar rats received unilateral intracortical injections of either HIV-GFP-NCS1 or the control HIV-GFP lentivector 2 d after receiving on the contralateral side a unilateral pyramidal tract lesion . At 4 wkThese data show that delaying the overexpression of NCS1 in CSN after injury can still induce intact CST axons to sprout into the CST-denervated side of the spinal cord.To investigate whether a delayed NCS1 overexpression of axotomized CSN can promote axonal sprouting and regeneration, adult Wistar rats received unilateral intracortical injections of either HIV-GFP-NCS1 or the control HIV-GFP lentivector 2 d after receiving on the ipsilateral side a unilateral pyramidal tract lesion at the medullary level, approximately 2 mm rostral to the decussation . At 4 wkThese data show that delaying the overexpression of NCS1 in axotomized CSN can induce axonal sprouting and axonal regeneration.It has been shown that a pyramidal tract lesion in adult hamster causes CSN to become atrophied after 2 wk post-injury These data suggest NCS1 overexpression in CSN exerts a neuroprotective effect on axotomized CSN.This present study demonstrates that the intracellular levels of NCS1 in adult cortical neurons can be significantly elevated by transduction with a lentiviral vector. In culture, neurons overexpressing NCS1 develop extensive neurite sprouting which by immunocytochemistry and Western blotting was shown to be via Akt phosphorylation. Similarly, analogous experiments conducted in vivo show that CSN overexpressing NCS1 with intact CST axons can undergo distal collateral sprouting and cross the midline into the CST-denervated side of the spinal cord. This anatomical plasticity is also functional as demonstrated by the behavioural and electrophysiological outcomes in NCS1-transduced adult rats. Furthermore, studies on the axotomized CSN show that NCS1 overexpression not only induces axonal sprouting and regeneration at the lesion site but also exerts a neuroprotective affect on injured CSN.To date, several therapies for spinal cord injury models have shown both significant axonal regeneration In addition to observing neurite sprouting in vitro, we demonstrated an NCS1 mediated axon collateral sprouting in vivo following unilateral pyramidotomy. The HIV-GFP-NCS1 lentivector injected into the cortex enabled GFP labelling of neurons overexpressing NCS1. The GFP labelling allowed visualisation and quantification of sprouting of the CST axons without the need of applying an independent tracer, and such labelling of axons and their collaterals can be detected as far distal as the lumbar region.We report for cervical region the number of GFP-positive collateral fibers from uninjured CST axons that have crossed the midline into the CST-denervated side of the spinal cord, as measured per 40 \u00b5m section, is 1\u20132 for the control and 4\u20135 for the NCS1-transduced rats. It is of interest to consider the possible total number of crossing fibers over the relevant cervical region. The length of the adult rat C5\u2013C8 cervical cord containing the majority of forelimb motoneurons is approximately 11 mm We have previously induced axonal regeneration by lentivector overexpression of retinoic acid receptor \u03b22 (RAR\u03b22) The successful regenerative responses of CSN after pyramidotomy with delayed post-injury NCS1 overexpression suggests axonal sprouting and regeneration can occur without the need to prime with overexpression of NCS1. These data provide promising support for NCS1 overexpression as a possible therapeutic treatment for CNS injury in a clinical setting.This study also reveals a neuroprotective feature of NCS1 overexpression in reducing cell shrinkage due to retrograde effects of axotomy. Others have shown that neurotrophic factors can prevent atrophy or death of axotomized CSN Our study establishes that NCS1 is an important intracellular component in the regulation of axonal sprouting and regeneration, and neuroprotection in central neurons of an adult mammalian nervous system, as recently shown for the peripheral nervous system in chick embryo studies on dorsal root ganglion (DRG) neurons In summary, the limited ability of adult CST neurons to undergo functional sprouting may be due to low endogenous levels of NCS1. Thus NCS1 emerges as a potential intracellular target for therapeutic intervention following injury to the central nervous system.5\u2032-ATGGGGAAATCCAACAGCAAG-3\u2032; and the reverse primer, 5\u2032-CTATACCAGCCCGTCGTAGAG-3\u2032 then cloned into a pCR2.1-TOPO vector (Invitrogen). The ncs1 gene was inserted under the control of a CMV promoter in a human immunodeficiency virus type 1 (HIV1) vector containing a 5\u2032 central polypurine tract (cPPT) element and a 3\u2032 woodchuck post-transcriptional regulatory element (WPRE) enhancer. To allow for coexpression of NCS1 and the enhanced green fluorescent protein (GFP), the eGFP gene was inserted into the Cla1 site under the control of a SFFV promoter. Viral vector stocks, pseudotyped with the VSV-G envelope glycoprotein, were prepared by triple plasmid transient transfection of HEK293T cells as previously described 8 TU ml\u22121 and pRRL-CMV-eGFP (for simplicity termed HIV-GFP) was 4.7\u20134.8\u00d7108 TU ml\u22121 determined by transient transfection of the HEK 293T cell line and analysed by flow cytometry.The complete cDNA sequence of the rat NCS1 was generated by PCR from adult rat cortex using the following forward primer; Adult cortical neurons were cultured as previously described For phospho-Akt studies, the reversible PI3K/Akt inhibitor LY294002  or DMSO alone at a final concentration of 0.01% was added to the cultures at 1 DIV and 2 DIV. The neurons were harvested at 3 DIV.After 3 d in culture, neurons were fixed with 4% paraformaldehyde for 20 min and then permeablized with cold methanol for 3 min. The neurons were washed three times for 5 min with 0.01 M phosphate-buffered saline (PBS) before 2 h incubation with chicken anti-GFP , rabbit anti-NCS1 , rabbit anti-phospho-Akt , or rabbit anti-MAP2 . The cover slips were washed 3\u00d75 min with PBS and then incubated with donkey anti-rabbit Alexa Fluor 546 and goat anti-chicken Alexa Fluor 488 secondary antibodies  for 45 min. After 3\u00d75 min PBS washes, they were mounted with FluorSave reagent containing 0.5 \u00b5l DAPI (10 \u00b5g/ml) to visualise cell nuclei. The staining with phalloidin-TRITC  was carried out 1 h before immunostaining with other antibodies was carried out.Image analysis and quantification was made with the observer blinded to the group assignment as previously described 3VO4, 5 mM EDTA, 1% Nonidet P-40 and 1\u00d7 protease inhibitor cocktail; Roche). To obtain sufficient protein, the same 250 \u00b5l lysis buffer was used in three cultured wells and the lysates rotated for 2 h at 4\u00b0C. After centrifugation at 13,500 g for 15 min at 4\u00b0C, the supernatant was collected and total protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce).Western blots were carried out as previously described n\u200a=\u200a4\u20135 per group) were carried out as described below. To determine the role of Akt activation, the PI3K/Akt inhibitor LY294002 (100 mM) or vehicle (DMSO) was injected intracerebroventricularly via an externalised catheter on every other day of the third post-injection week. At the end of the third week, rats were sacrificed and the injected region of the cortex was freshly and quickly removed and stored at \u221280\u00b0C until further processed. The protein obtained for Western blotting was extracted as described above.Intracortical injections of either HIV-GFP-NCS1 or HIV-GFP lentivector in adult male Wistar rats (\u03b2-mercaptoethanol) before re-blotting with rabbit anti-Akt . Western blotting was carried out with 3\u20135 independent samples.Fifteen micrograms of total protein were electrophoresed on 12% acrylamide gel before transfer onto Hybond P membranes (Amersham) and incubated overnight at 4\u00b0C with rabbit anti-phospho-Akt , rabbit anti-NCS1 , or mouse anti-\u03b2 III tubulin . Visualisation was performed using secondary antibodies, donkey anti-rabbit IRDye-800CW, and goat anti-mouse IRdye-680CW (LI-COR Biosciences). Fluorescent blots were imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences). To allow for visualisation of the total Akt on the same blot as phospho-Akt (both antibodies were raised in the same species), the blot was first stripped with buffer  were anaesthetized using a combination of ketamine and medetomidine, then fixed in a stereotaxic frame. The skull was exposed and injections were made at a depth of 2 mm dorsoventrally into the sensorimotor cortex region using the injection coordinates as determined from a microstimulation mapping study The surgery was performed aseptically in accordance with UK Home Office regulations as previously described n\u200a=\u200a5\u20136 per group) received intracortical lentiviral injections 2 d after a unilateral pyramidal tract lesion as described above. To study the sprouting effect of delayed lentivector transduction on uninjured and injured CST axons, the lentiviral injections were administered into the sensorimotor cortex corresponding to the unlesioned and lesioned pyramidal tract, respectively. After 4 wk post-surgery, the rats were perfused transcardially with 4% paraformaldehyde and tissue collected for histology.In the delayed lentivector transduction studies, adult Wistar rats (n\u200a=\u200a4\u20135 per group) received intracortical lentiviral injections 1 wk before receiving on the ipsilateral side a unilateral pyramidal tract lesion as described above. Using a microinfusion pump, Fast Blue tracer  was administered at a rate of 0.2 \u00b5l/min into the lesion site via a finely pulled glass micropipette and left in situ for a further 1 min. Unlesioned rats without intracortical lentiviral injection had Fast Blue tracer  injected into the pyramidal tract at the medullary region. Care was taken to minimise axonal damage by the injection process. After 2 wk post-injection of tracer, the rats were perfused transcardially with 4% paraformaldehyde and tissue collected for histology.In the neuroprotection study, adult Wistar rats . The next day, the sections were washed in 3\u00d75 min PBS-T before incubation with goat anti-rabbit biotin  for 1 h 30 min. After 3\u00d75 min PBS-T washes, sections were incubated in avidin-biotin-peroxidase complex  for 30 min at room temperature. Following 3\u00d75 min washes in PBS-T, sections were incubated in tyramide  for 10 min. After a further 3\u00d75 min wash in PBS-T, the sections were incubated with extra-avidin FITC  for 2 h before the sections were washed, mounted, and cover slipped. For quantitative analysis, five to six sections per animal (experimenter was blind to group assignment) stained for GFP were selected and captured as a complete spinal cord image at 10\u00d7 magnification with five z-axis planes  using a Zeiss Axioplan 2 microscope and Axiovision V4.6 program. The number of GFP positive fibers in the CST was taken as the number of stained axons. The number of CST axon collaterals in the gray matter of the CST-innervated and -denervated side was assessed by counting the number of fibers crossing a virtual line at each 100 \u00b5m interval lateral from the midline.Axon collateral sprouting of the intact CST across the midline into the CST-denervated side of the cord was assessed by staining for the GFP immunopositive fibers. Forty \u00b5m transverse sections of the cervical (C5\u2013C8 level) and lumbar (L3\u2013L6 level) spinal cord were cut using a vibratome. Sections were collected in cold 0.01 M PBS with 0.1% sodium azide with the format of one section in each of 32 wells and this was repeated so each well had at least six sections containing the corresponding spinal cord levels. Sections chosen from randomly selected wells were rinsed in 3\u00d75 min PBS-T (0.01 M PBS with 0.3% Triton X-100), then incubated with 0.3% HThe immunostaining for PKC\u03b3 in the spinal cord involved washing the sections in 3\u00d75 min PBS-T before overnight incubation at room temperature with rabbit anti-PKC\u03b3 . The next day, sections were washed with 3\u00d75 min PBS-T, then incubated with goat anti-rabbit Alexa Fluor 488  for 3 h. Following a 3\u00d75 min PBS-T wash, sections were mounted and cover slipped.Axon collateral sprouting of the injured CST across the lesion site at the caudal medullary level was assessed by staining for the GFP positive fibers. Forty \u00b5m horizontal sections of the caudal medulla oblongata including the lesion site were cut using a vibratome. The immunostaining for GFP positive fibers in the brainstem was carried out with the tyramide signal amplification procedure as mentioned above. After the sections were immunostained for GFP, they were then incubated overnight with rabbit anti-GFAP antibody  to further highlight the lesion site with immunostained reactive astrocytes. The next day, sections were washed 3\u00d75 min in PBS and then incubated in donkey anti-rabbit Alexa Fluor 568  for 3 h at room temperature. Following a further 3\u00d75 min wash in PBS, sections were mounted and cover slipped.z-axis planes  using a Zeiss Axioplan 2 microscope and Axiovision V4.6 program. The number of CST axon collaterals was assessed by counting the number of fibers crossing a virtual line at each 200 \u00b5m interval rostral and caudal to the lesion site and within the ipsilateral side demarcated by the reactive astrocytes and midline, respectively.For quantitative analysis, five to six horizontal sections per animal at the level of the pyramidal tract lesion stained for GFP and GFAP were selected (experimenter was blind to group assignment) and captured as a complete horizontal section of the medulla oblongata at 10\u00d7 magnification with four n\u200a=\u200a4\u20137 per group).The expression of NCS1 and GFP in the cortex of whole rats was assessed by immunohistochemistry. Forty \u00b5m coronal sections of the cortex between bregma \u22122.5 mm and +3.0 mm were cut using a vibratome. Sections were collected and processed as described above. To visualise NCS1 using rabbit anti-NCS1  in histological sections, an antigen retrieval step was required using the antigen unmasking solution  according to manufacturer's instructions. Chicken anti-GFP  was used to detect GFP positive neurons. Standard immunohistochemical procedures were followed with donkey anti-rabbit Alexa Fluor 568 and goat anti-chicken Alexa Fluor 488 secondary antibodies. Dual or triple colour images of the layer V cortical region were captured at 20\u00d7 magnification and analysed using the AxioVision V4.6 program. At least six transduced sections were analysed and quantified per animal (n\u200a=\u200a4\u20135 per group). A total of over 2,400 neurons were analyzed.The analysis of atrophy in CSN was carried out as previously described Following unilateral pyramidotomy, functional recovery was assessed behaviourally using the staircase reaching and grid exploration tests at 2 d post-lesion and then weekly for 6 wk as described previously n\u200a=\u200a4) received intracortical injections of HIV-GFP while NCS1-transduced rats (n\u200a=\u200a4) received HIV-GFP-NCS1 followed by a unilateral pyramidotomy on the right side 3 wk later as described above. All electrophysiological measurements were performed at least 6 wk post-injury under urethane  anesthesia. Following tracheotomy, the rat was fixed into a frame by ear bars and spinal clamps such that the forelimbs were fully pendent. A pair of hooked stainless steel wires insulated to the tip was inserted into the tricep brachii of both forelimbs approximately 6 mm apart for EMG recording. The area of the sensorimotor cortex where lentiviral vectors were injected was exposed by craniotomy, covered by mineral oil, and stimulated through a flat ended silver wire electrode (0.5 mm diameter) ensheathed with plastic to its tip to minimize surface spread of the stimulating current. A 2 mm diameter anode was placed on the skull periosteum rostral to the stimulating electrode. The stimuli repeated at 1 Hz consisted of 1 to 4 pulses, 3 ms apart, and 0.1 ms duration from an isolated stimulator (Digitimer DS2A). The final stimulation site was selected after systematic mapping with varying stimulus parameters until a discrete contralateral (left) forelimb movement was observed with a clear EMG response and a threshold below 25 V for the least number of effective pulses. The EMG was amplified  and digitized using a CED 1401 interface with a sampling rate of 10 kHz. The area of the EMG response (Vs) was measured from 20 averaged responses using Spike 2 V5.0 software.Control rats  Primary cultured adult cortical neurons transduced with control HIV-GFP lentivector showing low levels of NCS1 immunostaining and limited neurite sprouts after 3 d in vitro. (G\u2013L) Neurons transduced with HIV-GFP-NCS1 have higher levels of NCS1 immunostaining and a greater number of neurite sprouts . (M\u2013N) Quantification data show NCS1-transduced neurons (red bar) have a larger number of neurites from cell body and sprouts from longest neurites compared to control HIV-GFP-transduced neurons (black bar). Data are expressed as mean \u00b1 SEM from 3 independent experiments. Scale bars:  100 \u00b5m;  20 \u00b5m.(3.10 MB TIF)Click here for additional data file.Figure S2Further examples of NCS1 overexpression promoting neurite sprouting via Akt activation in vitro. (A\u2013D) Primary cultured adult cortical neurons transduced with HIV-GFP-NCS1 have high phospho-Akt levels and more extensive neurite sprouting when treated with vehicle (0.01% DMSO). (E\u2013H) Control GFP-transduced neurons have low phospho-Akt levels and few neurite sprouts when treated with vehicle. Phospho-Akt, red; GFP, green; DAPI, blue. Inserts are higher magnification of corresponding panels. Scale bars: 50 \u00b5m.(1.90 MB TIF)Click here for additional data file.Figure S3Further examples of NCS1-induced neurite sprouting reduced by inhibiting Akt activation in vitro. (A\u2013D) Primary cultured adult cortical neurons transduced with HIV-GFP-NCS1 have reduced phospho-Akt levels and fewer neurite sprouts in the presence of LY249002. (E\u2013H) Control HIV-GFP-transduced neurons continue to express low phospho-Akt levels and fewer neurite sprouts in the presence of LY249002. Phospho-Akt, red; GFP, green; DAPI, blue. Inserts are higher magnification of corresponding panels. Scale bars: 50 \u00b5m.(1.63 MB TIF)Click here for additional data file.Figure S4Schematic diagrams illustrating the design and sequences of the in vivo experimental studies. The green circle and line indicate the lentiviral vector transduced neurons and GFP positive labelled axons and collaterals, respectively. The white circle and line indicate untransduced neurons and axons. The black circle and line indicate the CST, which when transected rostral to the pyramidal decussation leads to degeneration of the main dorsal CST on the opposite side and a corresponding CST-denervation of the spinal cord (black dashed line). (A) A 3 wk primed NCS1 overexpression in the transduced non-axotomized corticospinal neurons (CSN) is predicted to promote axon collateral sprouting on the same side of the intact CST and into the CST-denervated side of the spinal cord, but not in the control group (thin green lines). To establish if the functional recovery is dependent on collateral sprouting from the intact CST into the CST-denervated side, a second lesion of the intact pyramidal tract then causes CST-denervation of the postulated source of sprouting resulting in loss of the previously achieved recovery in behaviour and EMG activity (dashed green lines). (B) A 2 d delayed NCS1 overexpression in the transduced non-axotomized CSN after a pyramidotomy is predicted to promote axon collateral sprouting on the same side of the intact CST and into the CST-denervated side of the spinal cord, but not in the control group (thin green lines). (C) A 2 d delayed NCS1 overexpression in the transduced axotomized CSN after a pyramidotomy is predicted to promote axonal sprouting and regeneration into and around the lesion site at the medullary level, but not in the control group (thin green lines). (D) A 1 wk primed NCS1 overexpression in the transduced axotomized CSN after a pyramidotomy is predicted to prevent cell atrophy, but not in the control group.(2.00 MB TIF)Click here for additional data file.Figure S5Assessment of the pyramidotomy lesion. (A\u2013B) Macroscopic images of a unilateral left pyramidal tract lesion rostral to the decussation in the lower medulla. The lesion (arrow) generates contralaterally a CST-denervated side of the spinal cord. (C\u2013D) Photomicrographs of reactive astrocytes (GFAP immunopositive) demarcating the lesion site and the absence of PKC\u03b3 immunostaining caudal to the lesion (arrow). Arrowhead and arrow indicate the intact and lesioned pyramidal tract, respectively. (E\u2013F) Macroscopic images from a rat containing a previous unilateral pyramidotomy (arrow) with a subsequent lesion of the intact pyramid (arrowhead) resulting in complete CST-denervation of both sides of the spinal cord. (G\u2013H) Macroscopic images of a sham operated rat that received a unilateral pyramidal tract lesion in a subsequent operation (arrowhead). Higher magnification in panels B, F, and H are indicated by dashed boxes in panels A, E, and G, respectively. Scale bars:  5 mm,  1 mm.(8.28 MB TIF)Click here for additional data file.Figure S6Complete transverse section of cervical spinal cord from a pyramidotomized control GFP-transduced rat. (A\u2013C) Rat transduced with the control HIV-GFP lentivector has GFP positive fibers present in the CST-innervated side of the spinal cord (CST (A), dorsal horn (B), ventral horn (C)). (D\u2013F) In contrast, few GFP positive fibers were detected in the CST-denervated side of the spinal cord , intermediate laminae (E), ventral horn (F)). Scale bars: (A\u2013F) 200 \u00b5m.(5.70 MB TIF)Click here for additional data file.Figure S7Complete transverse section of cervical spinal cord from a pyramidotomized NCS1-transduced rat. (A\u2013C) Animal transduced with the HIV-GFP-NCS1 lentivector has GFP positive fibers present in the CST-innervated side of the spinal cord (CST (A), dorsal horn (B), and ventral horn (C)). (D\u2013F) On the CST-denervated side of the spinal cord, extensive amounts of GFP positive fibers have crossed the midline with axon collaterals detected in the dorsal horn (D), intermediate laminae (E), and ventral horn (F). Scale bars: (A\u2013F) 200 m.(5.13 MB TIF)Click here for additional data file.Figure S8Confirmation of pyramidotomy lesion using PKC\u03b3 immunostaining. (A\u2013D) Photomicrographs show the presence of bilateral PKC\u03b3 immunostaining in the intact dorsal CST in sham rats (arrows). (E\u2013L) Following a unilateral pyramidotomy, PKC\u03b3 immunostaining is only present in the intact dorsal CST (arrow) and absent in the lesioned dorsal CST (arrowhead) in rats injected with either the control (GFP) or NCS1 overexpressing lentivector. The numbers at bottom left indicate the rat identity from which the tissues were taken. Scale bars: 200 \u00b5m.(3.32 MB TIF)Click here for additional data file.Figure S9Unilateral pyramidotomy does not affect the CST-innervated limbs in behavioural tasks. (A\u2013B) In the staircase reaching test, the CST-innervated forelimb for both transduced groups showed no significant reduction in numbers of food pellets displaced or eaten compared to sham group following unilateral pyramidotomy. (C\u2013D) In the grid exploration test, the CST-innervated fore- and hindlimbs showed no significant difference in the number of misplacements compared to the sham operated group following unilateral pyramidotomy. (A\u2013D) One week after a subsequent operation to lesion the intact pyramidal tract, the previously CST-innervated forelimb showed a reduced capability to displace or to eat food pellets in the staircase reaching test, and the number of misplacements in the grid exploration test was increased. Black line, CST-innervated limb of GFP rats; red line, CST-innervated limb of NCS1 rats; blue line, limb of sham rats.(0.37 MB TIF)Click here for additional data file.Figure S10The effects of increasing the number of stimulating pulses to the NCS1-transduced sensorimotor cortex on the EMG activities of the forelimbs. (A\u2013B) With 1 pulse, neither movement nor little or no EMG activity was observed in either forelimbs (ripple is attributed to low threshold neck muscle activity). (C\u2013D) With 2 pulses, forelimb movements occurred and EMG responses were observed in both forelimbs, with delayed latency on the CST-denervated side. (E\u2013H) With 3 or 4 pulses, the EMG responses further increased in size in both forelimbs. (I\u2013J) After transection of the intact dorsal CST at C4 spinal level, the EMG response from the forelimb on the now acutely CST-denervated side was all but abolished and it was lost altogether from the chronically CST-denervated forelimb.(0.59 MB TIF)Click here for additional data file."}
+{"text": "In March 2006, Australia introduced graphic pictorial warnings on cigarette packets. For the first time, packs include the Quitline number.To measure the combined effect of graphic cigarette pack warnings and printing the Quitline number on packs on calls to the Australian Quitline service.Calls to the Australian Quitline were monitored over 4 years, 2 years before and after the new packets were introduced.There were twice as many calls to the Quitline in 2006 (the year of introduction), as there were in each of the preceding 2 years. The observed increase in calls exceeds that explained by the accompanying television advertising alone. While call volume tapered back in 2007, it remained at a level higher than before the introduction of new packets. No change was observed in the proportion of first time callers.Introducing graphic cigarette packet warnings and the Quitline number on cigarette packets boosts demand for Quitline services, with likely flow on effects to cessation. In March 2006, graphic health warnings were included on cigarette and other tobacco packs in Australia. In addition, and for the first time, the Australian Quitline number was printed on packets. Prior to 2006, Australia had text-based warnings. There was an infoline number printed in small text on the side of the pack. This number diverted to the Quitline.www.quitnow.info.au\u201d is also included on the back of all packs. The Quitline number is also \u201cstamped\u201d on top of the graphic image on the backs of packs. Regulations prescribe the details of the size of the elements.Like the text-based warnings that preceded them, the graphic health warnings are mandated under Australia\u2019s Trade Practices Act,A series of mass media campaign activities accompanied the introduction of the new cigarette packet warnings. The Australian Government screened an awareness raising campaign in February 2006.Australia is not the first country to introduce a Quitline or smokers\u2019 helpline number on cigarette packets. In 2002, a smoking cessation message and quit line number were included on Dutch cigarette packets, along with prominent text warnings. This led to a 3.5-fold increase in calls to the Dutch Quitline.\u2013It is well established that television advertising to promote quitting can increase calls to Quitlines8The Australian Quitline can be accessed from anywhere in Australia by dialling 131848 or 13 QUIT (137848) for the price of a local call. The Telstra Analyser , software of the telecommunications provider, provides data on volume of calls, call source (broken down by state and region), time and duration of calls.Individual states and territories have their own databases of caller details. These data were examined in one jurisdiction , where callers who spoke to a counsellor  were asked routinely whether they had called the Quitline before.Television anti-smoking advertising is quantified using target audience rating points (TARPs), provided by a media agency ACNielsen . TARPs are a standard measure of television advertising weight. TARPs are used to indicate the number of people within a certain demographic group that were exposed to an advertisement within a given period of time. For example, 100 TARPs for 1 week is equal to an average of 1 exposure per person in the target population within that week of the campaign. In the present study, the TARPs relate to the target audience of Australians aged \u2a7e18 years.Data analyses were conducted with SPSS V. 15 . Linear regression analyses were used to estimate the effect on calls to the Quitline of television advertising and the introduction of graphic pack warnings using data from January 2004 to December 2007 inclusive. In regression modelling, calls to the Quitline were the dependent variable, TARPs were a continuous independent variable and separate dummy variables were created for 2006 and 2007. Although data were not distributed normally, data were not transformed as this did nothing to strengthen the resulting model.Calls increased markedly when new cigarette packet warnings were first introduced. Call volume levelled off in the weeks following the initial launch but built up again in subsequent months when the accompanying quit campaign TVCs were launched.2\u200a=\u200a0.657). The model predicted a base number of calls ; a significant linear relationship between every 100 TARPs and calls to the Quitline ; and separate independent increases in calls were observed for years 2006 ; and 2007 , above what was explained by TARPs alone. Call volume was still elevated in 2007, compared to 2004 and 2005, although there was erosion in call volume from 2006.The linear regression model showed significant relationships between the independent variables and the dependent variable overall and had good overall explanatory value , the year in which new pack warnings including the Quitline number were introduced, compared to 2005 (78%).Australia is a \u201cmature\u201d tobacco control market where most forms of tobacco promotion are banned, increasing the significance of the packet as a medium for marketing.Many countries are moving to introduce graphic cigarette packet warnings; some with a Quitline or helpline number.However, the impact on calls to the Quitline of graphic (in contrast to text-only) warnings with accompanying Quitline number has not previously been quantified.This study shows that even in a \u201cmature\u201d tobacco control environment such as Australia, such an intervention has considerable positive impact on demand for a Quitline, with positive implications for quitting.Since the 1980s, most Australian state and territories have established strong anti-tobacco (quit) mass media campaigns, supported by the Australian Quitline. Because of the clear relationships between high-quality mass media campaigns, calls to the Quitline and quitting behaviour,However, the rise in calls to the Australian Quitline service observed in this study was substantial and sustained. The size and timing of the rise in calls, compared to the previous 2 years, indicates that this is highly likely to be due to the introduction of the new graphic cigarette packet warnings that included the Quitline number. The regression analysis also demonstrates that it is very unlikely that mass media alone explained the observed increase in calls because the introduction of the warnings had an independent effect. Further evidence that mass media quit campaigns were not the primary cause of increased calls is the fact that some of the increase in calls was observed prior to the launch of the quit campaigns. The Quitline number is a prominent but integrated component of the new-style warnings on Australian cigarette packets. There was no prominent display of the Quitline number on Australian cigarette packets prior to this, only the low-profile infoline number. Therefore it is not possible to separate the contributions of the components of the new warnings: namely the visual image, the large warning text, the detailed warning on the back of the packet or the Quitline number. Their impact has been measured as a whole.There was no change in the proportion of first time callers, compared to the previous year, indicating that the intervention had a positive effect upon new quitters and repeat callers.The observed increase in call volume did persist in the year following the introduction of the warnings (2007). Although there are 14 different warnings, with a scheduled rotation mechanism, it is likely that the reduction in call volume was due to a degree of \u201cwear out\u201d. This provides another example of a health promotion intervention having a positive effect more akin to a spring than a screw.In conclusion, the Australian Quitline experienced a doubling of calls upon introduction of graphic cigarette packet warnings that included a prominent Quitline number. Other countries with mature tobacco markets could expect a similar impact upon introduction of graphic warnings, especially if accompanied by reinforcing mass media activities. The flow-on effects in terms of quitting are likely to be substantial. Previous research has demonstrated that at 12 months, around 30% of callers to the Australian Quitline have succeeded in quitting smoking,"}
+{"text": "Alzheimer's disease (AD) is characterized by the deposition of beta-amyloid (A\u03b2) peptides in the brain extracellular matrix, resulting in pathological changes and neurobehavioral deficits. Previous work from this laboratory demonstrated that the choroid plexus (CP) possesses the capacity to remove A\u03b2 from the cerebrospinal fluid (CSF), and exposure to lead (Pb) compromises this function. Since metalloendopeptidase insulin-degrading enzyme (IDE), has been implicated in the metabolism of A\u03b2, we sought to investigate whether accumulation of A\u03b2 following Pb exposure was due to the effect of Pb on IDE.in vitro studies, choroidal epithelial Z310 cells were treated with Pb for 24 h in the presence or absence of a known IDE inhibitor, N-ethylmaleimide (NEM) to assess IDE enzymatic activity and subsequent metabolic clearance of A\u03b2. Additionally, the expression of IDE mRNA and protein were determined using real time PCR and western blots respectively.Rats were injected with a single dose of Pb acetate or an equivalent concentration of Na-acetate; CP tissues were processed to detect the location of IDE by immunohistochemistry. For Immunohistochemistry and confocal imaging revealed the presence of IDE towards the apical surface of the CP tissue with no visible alteration in either its intensity or location following Pb exposure. There was no significant difference in the expressions of either IDE mRNA or protein following Pb exposure compared to controls either in CP tissues or in Z310 cells. However, our findings revealed a significant decrease in the IDE activity following Pb exposure; this inhibition was similar to that seen in the cells treated with NEM alone. Interestingly, treatment with Pb or NEM alone significantly increased the levels of intracellular A\u03b2, and a greater accumulation of A\u03b2 was seen when the cells were exposed to a combination of both.These data suggest that Pb exposure inhibits IDE activity but does not affect its expression in the CP. This, in turn, leads to a disrupted metabolism of A\u03b2 resulting in its accumulation at the blood-CSF barrier. Studies in literature have shown that more than 90% of Alzheimer's disease (AD) cases are sporadic in nature , suggestOne of the target regions in the brain where Pb has shown to accumulate substantially is the choroid plexus (CP), a tissue lining the brain ventricles and separating the blood from the CSF. This finding is supported by evidence from an autopsy study which showed an age-related accumulation of Pb in the CP of the brains of 51 human subjects who had lived in New York City . This obRecent studies from this laboratory have demonstrated that Pb accumulation in the CP results in a marked increase of A\u03b2 within the tissue . Since tIDE, a 110-KD zinc metallopeptidase, is known to participate extensively in the clearance of insulin, glucagon and A\u03b2 -21. The in vivo affected the subcellular distribution of IDE, (3) investigate whether Pb exposure altered the IDE activity and/or the expression of mRNA or protein levels in the CP, and (4) determine whether altered IDE function led to the abnormal accumulation of A\u03b2 in the BCB. The outcomes of this study will establish the role of IDE in the metabolic clearance of A\u03b2 at the BCB and illustrate a mechanism by which Pb alters A\u03b2 homeostasis in the CSF, potentially contributing to the etiology of AD.Considering the substantial accumulation of Pb and the abundant presence of IDE in the CP, there was sound reason to hypothesize that accumulation of Pb may alter the enzymatic activity or expression levels of IDE in the CP, leading to alterations in A\u03b2 metabolism at the blood-CSF barrier (BCB). Thus, the purpose of this study was to (1) identify the subcellular location of IDE in the CP, (2) determine whether Pb exposure Chemicals and assay kits were purchased as follows: ELISA kit and ultra purified A\u03b2 (1-40) ; mouse anti -IDE antibody ; IDE activity kit ; Alexa-labeled secondary anti- mouse antibody ; enhanced chemiluminescene reagent (ECL) and ECL films ; Dulbecco's modified essential medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and gentamycin ; PCR buffer, dNTP, Oligo dT and MuLV reverse transcriptase ; the ABsolute QPCR SYBR green Mix kit ; the primers for real time RT-PCR ; \u03b2-actin, 2-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), polyacrylamide, tetramethyl-ethylenediamine (TEMED), n-ethylmaeimide (NEM) and all other chemicals . All reagents were of analytical grade, HPLC grade or the best available pharmaceutical grade.Male Sprague-Dawley rats were purchased from Harlan Laboratories  and were 8-9 weeks old (250-300 g) at the time they were used. The animals were housed in a temperature-controlled, 12 h:12 h light/dark room, and were allowed free access to tap water and food. Rats received an i.p. injection of 50 mg/kg Pb acetate  or an equivalent molar concentration of Na-acetate  as control. Twenty-four h post injection, the rats were anesthetized with an injection of ketamine/xylazine  and euthanized by exsanguination to remove excess blood. The lateral ventricle CP was then isolated for further experimentation. Animal protocols pertinent to this study were approved by the Purdue University Animal Care and Use Committee.2 at 37\u00b0C and were passaged twice a week. The cells were seeded at a density of 1 \u00d7 106 in a 100-mm culture dish. Twenty-four h after initial seeding, the cells were treated with 0 or 10 \u03bcM Pb for 24 or 48 h, and the following studies were performed.The methods and characteristics of immortalized rat choroidal epithelial Z310 cells have been described previously . Brieflyin vitro studies, Z310 cells were seeded on a 35-mm dish, and treated with 10 \u03bcM Pb for 24 or 48 h. At the end of Pb treatment, the culture medium was removed and replaced with 0.5-1 mL PBS. The tissues or cells were washed with PBS three times, fixed with 3% paraformaldehyde/0.25% glutaraldehyde in PBS for 10 min, and permeabilized with 0.5% Triton X-100 for 30 min at room temperature, followed by 3-5 washes of PBS. After blocking with 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature, the tissues or cells were double-immunostained with mouse anti-IDE (1:500) in 1% BSA overnight at 4\u00b0C, washed the next day with PBS in 1% BSA, and then incubated with goat anti- mouse Alexa-488 conjugated secondary antibody (1:7500) in 1% BSA at 37\u00b0C for 60 min. After further washing in PBS with 1% BSA, the tissues or cells were observed using an inverted confocal fluorescent microscope. Negative controls were treated similarly except that they were not exposed to any of the primary antibodies.Immunohistochemistry was performed on both rat CP tissues and the Z310 cells following Pb exposure. For fresh tissue studies, the CPs were isolated from the rat following the exposure condition described above and transferred to a 35-mm glass bottomed dish containing PBS. For To acquire images, the dish containing the CP specimen was mounted on the stage of an Olympus, FV1000 inverted confocal laser-scanning microscope and viewed through a 40\u00d7 water-immersion objective (numeric aperture = 1.2), with a 488-nm laser line for excitation (Ar-ion laser). Low laser intensity was used to avoid photo bleaching. The CP was examined under a reduced transmitted-light illumination. An area containing undamaged epithelium with underlying vasculature was selected. For each image acquired , four areas of the specimen were selected for image collection. Data reported, unless otherwise stated, are representative of 3-4 replicate experiments.6 in 100-mm plates for 24 h; they were then incubated with 0, 5, 10, 25 or 50 \u03bcM of NEM overnight for 16 h and the cell viability was determined using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Based on our results and previously published data [To screen for a suitable NEM concentration at which cell death was minimal, Z310 cells were seeded at a density of 1 \u00d7 10hed data , a conce6and treated with Pb for 24 or 48 h in the presence or absence of NEM. The four treatment conditions were as follows: Group 1 served as an untreated control (Pb-/NEM-), group 2 was treated with 10 \u03bcM Pb for 24 h in the absence of NEM (Pb+/NEM-), group 3 was treated with only 10 \u03bcM NEM for 16 h in the absence of Pb (Pb-/NEM+), and group 4 with 10 \u03bcM NEM for 16 h followed by 10 \u03bcM Pb for 24 h (Pb+/NEM+).To assess the IDE activity, the cells were seeded at a density of 1 \u00d7 10The principle of the assay is based on measuring the IDE activity using a fluorescence resonance energy transfer (FRET) substrate which is cleaved by IDE to release the fluorophore from the quenching molecule, resulting in an increase in fluorescence. The activity was determined as per manufacturer's protocol . Briefly, Z310 cells were lysed with RIPA buffer and centrifuged at 10,000 rpm for 10 min and the supernatant was used for analysis. Total protein (80 \u03bcg/well) was loaded into in a 96-well plate, which contained an affinity-purified polyclonal antibody that recognizes human, mouse, and rat insulysin; the antibody was immobilized on the plate in order to capture the IDE enzyme. Following 1 h incubation, the plate was washed with sample buffer, and an aliquot (100 \u03bcL) of substrate was added and incubated for 2-4 h at 37\u00b0C in the dark. The fluorescence was measured using an excitation wavelength of 320 nm and an emission wavelength of 405 nm. The results are reported as IDE fluorescence intensity in arbitrary units (a.u.)/mg of total proteins in the cells.4 cells/well and treated as the four groups described above. The cells were washed with PBS and incubated with 200 \u03bcL of 2 \u03bcM ultrapure A\u03b21-40 solution in serum free medium for 1 h. The medium was then removed, cells washed 3 times with PBS, and fresh serum-free medium added for an additional hour to allow for metabolic clearance of A\u03b2 by IDE. The cells were then washed with PBS, scraped and lysed using 60 \u03bcL of RIPA buffer. The intracellular A\u03b2 was determined using a colorimetric ELISA kit (Invitrogen KHB3481). All data reported from the ELISA were normalized to the total amount of protein in the cells as determined by the Bradford assay.Cells were grown on 24 well plates at a density of ~4 \u00d7 10NM_013159) and rat glyceraldehydes-3-phosphate dehydrogenase (GAPDH) used as an internal control, had a forward primer 5'-CCT GGA GAA ACC TGC CAA GTA T-3' and a reverse primer 5'-AGC CCA GGA TGC CCT TTA GT-3' (Genbank Accession No. NM_017008).The transcription of the gene encoding IDE was quantified using RT-PCR as described in . BrieflyThe plexus tissues or Z310 cells were homogenized  on ice in a buffer containing 20 mM Tris (pH 7.5), 5 mM EGTA, 1% TritonX-100, 0.1% SDS, 50 \u03bcM phenylmethylsulphonylfluoride (PMSF), 15 mM 2-mercaptoethanol and a protease inhibitor cocktail containing 500 \u03bcM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 150 nM aprotinin, 1 \u03bcM E-64, 0.5 mM EDTA, 1 \u03bcM leupeptin . Samples were sonicated using a Model 500 Sonic Dismembrator (Fisher Scientific) at duty cycle 20% and output 4-6 for 30 pulses. Following centrifugation at 10,000 g at 4\u00b0C for 10 min, aliquots of supernatants were assayed for protein concentrations by the Bradford method. A volume of protein extract (40 \u03bcg of protein) was mixed with an equal volume of 2\u00d7 sample buffer , loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and then transferred to a PVDF membrane. The membrane was blocked with 5% dry milk in TBST  at room temperature for 1 h and immunoblotted with an antibody directly against IDE (1:250). The membrane was stained with a horse-radish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5000) at room temperature for 1 h and developed using ECL reagent. The exposure time varied from 30 sec to several min depending on the signal strength. \u03b2-actin (42 kD) (1:2000) was used as a loading control; the corresponding secondary antibody (1:2000) for \u03b2-actin was HRP-conjugated goat anti-mouse IgG. All band intensities were quantified using Scion Image software .To determine the Pb concentration at which it altered A\u03b2 metabolism in the CP in the absence of nonspecific cytotoxicity, three general cytotoxicity assays were conducted including the MTT cell viability assay, cell membrane permeability assessment (lactate dehydrogenase or LDH assay), and cellular oxidative stress estimation (superoxide dismutase or SOD assay). The MTT assay was performed by culturing Z310 cells at a density of 15,000 cells/well in a 96 well plate for 2-3 days until they reached 70% confluence. The medium was then replaced with fresh medium containing different concentrations of Pb as Pb-acetate . The cells were incubated for an additional 24 h, followed by adding an aliquot of MTT stock solution (2 mg/mL in PBS) to each well. The absorbance of the converted dye was measured at a wavelength of 570 nm. To determine the LDH activity, Z310 cells were treated as described above and LDH released into the culture medium was determined using the assay kit according to manufacturer's protocol. Finally, to determine oxidative stress, the cells were exposed to Pb (10 \u03bcM) for 24 h and SOD activity was determined according to the instructions in the assay kit.post hoc comparisons using the Dunnett's test or paired t-tests . All data are expressed as mean \u00b1 SD. Differences between two means were considered significant when the p value was equal or less than 0.05.Statistical analyses were carried out by a one-way ANOVA with in vivo exposure to Pb did not alter the subcellular localization of IDE, nor did it seem to affect the intensity of IDE staining  compared to untreated controls   and was comparable to the group treated with Pb in the absence of the inhibitor . When the cells were exposed to a combination of Pb and NEM , the IDE activity was reduced by a significant 29.3% (p < 0.001) relative to untreated controls , by 11.7% (p < 0.05) relative to the cells exposed to Pb alone , and by 11.8% (p < 0.05) relative to the NEM alone group . NEM did not cause any significant cell death at the concentrations used, as determined by the MTT assay. These results suggested that exposure to Pb (10 \u03bcM) results in a decrease in IDE activity; the effect of Pb on the enzymatic activity of IDE was exacerbated in the presence of IDE inhibitor, NEM.For the IDE activity study, NEM, an effective IDE inhibitor, was used as a positive control in the presence or absence of Pb exposure. Cells exposed to Pb for 24 h in the absence of NEM  showed a significant decrease of 17.6% in their IDE activity (M-) Fig. . Treatmep < 0.01, Fig. p < 0.05). Treatment with NEM alone  resulted in a 7 fold increase in intracellular A\u03b2 concentrations compared to untreated controls (p < 0.01).To determine if the inhibition of IDE activity led to cellular accumulation of A\u03b2, Z310 cells were incubated with A\u03b2 (2 \u03bcM) following treatment with Pb and/or NEM. Analysis using a one way ANOVA revealed an overall significant difference between the groups (p < 0.01). This finding supports a synergistic effect between Pb and NEM; both may possibly alter the IDE activity and subsequently decrease A\u03b2 metabolism in the CP.When both Pb and NEM  were added to the culture system, the intracellular A\u03b2 levels were increased by nearly 12 fold  IDE is located towards the apical membrane in CP tissues and the distribution does not appear to be altered by Pb exposure; (ii) Pb exposure results in a significant decrease in IDE activity; (iii) the decrease in IDE activity by Pb may lead to an accumulation of A\u03b2 in the CP possibly due to a reduced metabolic clearance at the BCB; and (iv) Pb exposure does not alter the mRNA or protein expression of IDE.The observation that the subcellular location of IDE was concentrated towards the apical membrane has significant implications. Earlier studies in our laboratory have demonstrated that the direction of A\u03b2 transport by the CP is predominantly from the CSF to the blood . The preRecent studies in our laboratory have shown that exposure to Pb results in the accumulation of intracellular A\u03b2 ; the stuin vivo Pb exposure. As a future direction, it would be of interest to know how our in vitro studies correlate with in vivo IDE activity in the CP.Results from this study indicated that while Pb exposure caused a 1.5 fold increase in A\u03b2, the group treated with NEM alone caused a 7 fold increase in A\u03b2. This may be, in part, due to a non-IDE-related effect of NEM, since NEM action involves a general covalent binding to cysteine residues. One strategy for future studies would be to knock-down IDE using short interference RNA and to more specifically determine the contribution of IDE. Another approach would be to up-regulate IDE in these cells and analyze A\u03b2 accumulation in the presence or absence of Pb exposure. We would also like to point out that due to a small tissue mass, we were unable to assess the IDE activity in the CP tissue following The current study raises several interesting questions. By what potential mechanism does Pb exert its effects on IDE? Extensive literature suggests that Pb is capable of binding to Zn finger proteins and subsequently altering their function, specifically at the sp1 binding domain -40. IDE Upstream of binding to the Zn finger, Pb might also influence the protein kinase C (PKC) pathway. Studies in literature suggest that IDE activity is decreased by an energy-dependent ATP mechanism . PKC is Another question is how much of a role does IDE play in Pb-mediated accumulation of A\u03b1? We should point out that this study does not imply that IDE is the single most important factor in A\u03b2 regulation at the BCB. Other mechanisms, particularly receptor-mediated A\u03b2 uptake and clearance in the CP including the involvement of A\u03b1 transporters such as low density lipoprotein receptor protein 1 (LRP1), receptor for advanced glycosylation end products (RAGE) and p-glycoprotein must also be considered.Finally, what is the consequence of A\u03b2 accumulation in the CP following Pb exposure? A build up of A\u03b2 in the CP could either be a result of overproduction of A\u03b2 in the CNS under the interference of Pb, or maybe due to a compromised function of the CP in clearing A\u03b2 from the CSF. Data from the current study indicate that Pb may alter the A\u03b2 breakdown mechanism at a critical site in the brain that is often overlooked. A disruption in A\u03b2 clearance, in turn, might lead to elevated A\u03b2 accumulation in the brain's internal milieu. Although this study provides some evidence for an IDE-mediated metabolic clearance, further studies are warranted to explore this mechanism in-depth.In summary, our results provide evidence that Pb exposure increases A\u03b2 accumulation at the BCB at least in part, by altering the activity of IDE and thereby potentially decreasing the metabolic clearance of A\u03b2. In addition, we provide evidence that IDE in the CP is localized towards the apical membrane, which is consistent with the large capacity of the CP to take up A\u03b2 from the CSF. Finally, our study suggests that Pb is a potential environmental trigger in the dysregulation of A\u03b2 homeostasis, and may subsequently contribute to the etiology of AD.A\u03b2: beta-amyloid; AD: Alzheimer's disease; BCB: blood-CSF barrier; CP: choroid plexus; CSF: cerebrospinal fluid; ELISA: enzyme-linked immunosorbent assay; IDE: insulin degrading enzyme; LDH: lactate dehydrogenase; MRI: magnetic resonance imaging; MTT: methylthiazolyldiphenyl-tetrazolium bromide; NEM: n-ethylmaleimide; Pb: lead; PKC: protein kinase C; RT-PCR: reverse transcription polymerase chain reaction; SOD: superoxide dismutase.The authors declare that they have no competing interests.MB participated in designing the study, performing the experiments, analyzing and interpreting the data and drafting the manuscript. YZ participated in designing the study, assisted in carrying out the experiments and analyzing the data. WZ (senior author) participated in designing the study, interpreting the data and revising the manuscript. All authors have read and approved the final manuscript."}
+{"text": "Malaria remains a major public health problem in Sub Saharan Africa, where widespread poverty also contribute to the burden of the disease. This study was designed to investigate the relationship between the prevalence of childhood fever and socioeconomic factors including poverty in Nigeria, and to examine these effects at the regional levels.Determinants of fever in the last two weeks among children under five years were examined from the 25004 children records extracted from the Nigeria Demographic and Health Survey 2008 data set. A two-level random effects logistic model was fitted. About 16% of children reported having fever in the two weeks preceding the survey. The prevalence of fever was highest among children from the poorest households (17%), compared to 15.8% among the middle households and lowest among the wealthiest (13%) (p<0.0001). Of the 3,110 respondents who had bed nets in their households, 506(16.3%) children had fever, while 2,604(83.7%) did not. (p=0.082). In a multilevel model adjusting for demographic variables, fever was associated with rural place of residence , sex of child: female  and all age categories (>6months), whereas the effect of wealth no longer reached statistical significance.While, overall bednet possession was low, less fever was reported in households that possessed bednets. Malaria control strategies and interventions should be designed that will target the poor and make an impact on poverty. The mechanism through which wealth may affect malaria occurrence needs further investigation.  Malaria is one of the most important challenges to public health with about 300 to 500 million cases reported annually. More than 1 million people die from the disease, most of them children under age 5 years. Over 90.0% of the cases and 75% of the deaths occur in sub-Saharan Africa (SSA). These childhood deaths, resulting mainly from cerebral malaria and anaemia, constitute somewhere between 20% and 25% of child mortality in Africa [African countries south of the Sahara bear the heaviest burden of malaria. These countries are among the poorest in the world and widespread poverty on the continent continues to play a role in the burden of the disease. Malaria cases and deaths have risen steadily in sub-Saharan Africa since the late 1970s, especially in Nigeria. The emergence of resistance to insecticides and chloroquine, the cheap anti-malarial treatment widely used for clinical management of uncomplicated malaria, has been held as a major factor in this trend, aided by a general weakening of health systems. This effect was exacerbated by economic stagnation and decline, which has implications for growth and welfare. For instance, malaria is responsible for about a 1.3 per cent reduction in the average annual rate of economic growth for those countries with the highest burden. In Nigeria, malaria is the major cause of morbidity and mortality, especially among children below age five .Given the above scenarios, the drive of the government to achieve the targets of the National Economic Empowerment and Development Strategy (NEEDS), Seven-Point Agenda and the MDGs is currently threatened by the seemingly intractable trend of the ancient scourge which has compounded national and household poverty through intensive loss of productive time to attack and death in extreme cases. This is compounded by the development of resistance to the traditional first-line drug (chloroquine), while the newly introduced therapy (ACT) would be largely beyond the reach of many due to poverty, if it was not heavily sponsored by international organizations. Given that malaria is endemic throughout Nigeria, and that over 50.0% of the country's population are living below poverty line, malaria incidence may increase significantly in Nigeria because many may not be able to afford the newly introduced drugs due to poverty.There are quite a good number of attempts to analyse the economic effects of malaria in the literature ,3,5-7. MThere are many pathways through which the relationship between malaria and poverty operates. At the household level, poor housing can expose people to contact with infective mosquitoes. Simple preventive measures such as insecticide-treated bed nets are unaffordable to the poorest if they must pay for them. Lack of resources prevents people from seeking timely health care. Often, peak malaria transmission coincides with harvesting time, which is a factor that affects risks at the village level. A few days of work lost to illness can mean food insecurity for the entire family.A meaningful relationship between malaria and poverty should consider the effect of both individual and cluster where individual belongs. This approach requires a multilevel analysis incorporating variables at different levels of aggregation. In this report, the relationship between poverty and malaria in Nigeria was analyzed using a multi-level logistic model. The independent variables considered were bed net ownership, type of place of residence, sex and age of child, and literacy level of mother.This report utilizes secondary data from the Nigerian Demographic and Health Survey . DemograThe study population in this analysis was the 25,004 under five children extracted from the records of 104,808 respondents who responded to the household questionnaire. The outcome measure in this analysis was occurrence of fever in children less than five years of age in the last two weeks preceding the survey. The explanatory variables were sex of child, age of child, whether or not household possess a bed net, whether child slept under bed net night before survey, type of bed net (treated or untreated), type of place of residence  and wealth index; a measure of poverty. This index serves as a proxy for measuring the long-term standard of living. It was based on data from the household's ownership of consumer goods; dwelling characteristics; type of drinking water source; toilet facilities and other characteristics that are related to a household socio-economic status such as car, radio, television, gas cooker, iron, boat/ship, telephone, source of water, electricity supplies, and type of wall, floor and roof, etc. To construct the index, each of these assets was assigned a weight (factor score) generated through principal component analysis, and the resulting asset scores were standardized in relation to a standard normal distribution with a mean of zero and standard deviation of one . Each hoFrequency tables were generated for relevant variables. Descriptive statistics such as means, medians and standard deviations were used to summarize continuous variables. Relationships between the outcome measure (occurrence of fever) and each explanatory variable was first explored using the chi squared test in bivariable analysis before a multilevel logistic regression was carried out. Explanatory variables found to be associated with the outcome were included in the logistic model based on the magnitude of the chi squared values. Odd ratios and 95% CIs were computed. A random effect logistic model was fitted that included fixed effects and group-level intercepts as random effects . This alMulti-level models allow the additional information provided by knowing which cluster a child comes from to be taken into account in modeling the relationship between independent and dependent variables. This model was run using the GLLAMM routine in Stata. In using this approach, the correlation between children from the same cluster or region arises from their sharing specific but unobserved properties of the respective regions. A random effect logistic regression model for the data is given by1, b2, b3 and b4 represents fixed effects and Ui represents a random effect. Ui is a random variable with a mean of zero and constant variance. Ui, is the estimation of the variance across all of the regions involved in the study. If the variance is large then the outcome of interest is dependent on the region, if the variance is small then the variations in outcome of interest may be explained by the measured characteristics alone.Where bi, the responses from the same regions are mutually independent that is the correlation between children from the same regions is completely explained by them having been observed in the same regions. The variance \u03c42 measures the degree of heterogeneity in the probability of experiencing fever that cannot be explained by the classification into the 2 categories (fever: yes Vs no)Given UAn important measure that describes these dependences in the data is called the intra-class correlation coefficient (ICC); this statistic measures the extent to which individuals within the same group are more similar to each other than they are to individuals in different groups. The intraclass correlation (ICC), \u03c1 was calculated using:\u03c4 = estimated variance and \u03c0 = 3.142Where This is a stata procedure for fitting generalized linear models, a class of regression models for univariate responses with density from an exponential family . The logxi: gllamm fever i.place of residence i.sex of child i.childsage i.wealthnw, i(region ) family link(logit) adapt nip(5), eformThe study sample consisted of 25,004 children. In this sample, 3,965(15.9%) children had fever in the two weeks prior to the survey. The mean age of the children was 27 months (SD = 17.2 months).Table A total of 2,068(16.3%) male children had fever compared to 1,897(15.4%) females. (p = 0.034). More children had fever in the rural areas compared with the urban areas (16.8% and 13.3% respectively) (p < 0.0001). Children aged 6-11 months and 12-23 months reported more fevers (19.9% and 21% respectively) than other children (p < 0.0001). In addition, fever prevalence decreased significantly as wealth quintile increased. A total of 1,106(17.04%) children from the poorest  household had fever, 978(16.6%) from poorer households  had fever, while 460(13.1%) from the richest households had fever (p < 0.0001).After controlling for all the predictors simultaneously in a multi-level model, fewer predictors remained significant Table . Only agThe overall prevalence of fever found in this survey is quite high when compared with the prevalence of fever in other SSA countries. Studies have shown that the prevalence of fever is less than 4.2% in the developed countries . The regThe analyses also showed that fever is higher in rural than urban areas. This may be associated with the high level of poverty in the rural areas. Poor people live in dwellings prone to mosquito proliferation. It has been shown that the characteristics of wall construction are associated with malaria prevalence . In anotThe results of this analysis suggest that the possession of bed nets is not associated with fever. In addition, no association exists between \"whether child slept under bed net and type of bed net child slept under\". However, households that had bed nets reported less fever than those who did not though the difference did not achieve significance. This result is in line with other findings in other parts of Africa on the use of insecticide treated bed nets, and their effectiveness when studied on a population basis. A study conducted by Lengeler and Snow  showed tIn the bivariate analysis, fever was found to be highest among the poor, followed by middle group, followed by the wealthiest group. On the basis of country-level data it was estimated that 57.9% of all deaths from malaria in the world in 1990 occurred among the poorest 20% of the world's population . In TanzWhile, overall bed net possession was low, less fever was reported in households that possessed bed nets. Malaria control strategies and interventions should be designed that will target the poor and make an impact on poverty. The results suggest that regional factors in Nigeria may be more influential in the risk of developing childhood fever than risks related to household wealth status. The mechanism through which wealth may affect malaria occurrence needs further investigation.The authors declare that they have no competing interests.All the authors conceived the study. OO was the principal investigator for Nigeria. DP and DB were collaborators from John Hopkins School of Public Health and contributed to the revising of the manuscript. OY conducted the analysis, BA and OO, contributed to data analysis and interpretation. OY and BA prepared the initial draft. All authors contributed to the draft. All authors read and approved the final manuscript."}
+{"text": "Uncaria tomentosa in minimizing the side effects of chemotherapy and improving the antioxidant status of colorectal cancer (CRC) patients, a randomized clinical trial was conducted. Patients (43) undergoing adjuvant/palliative chemotherapy with 5-Fluorouracil/leucovorin + oxaliplatin (FOLFOX4) were split into two groups: the UT group received chemotherapy plus 300\u2009mg of Uncaria tomentosa daily and the C group received only FOLFOX4 and served as a control. Blood samples were collected before each of the 6 cycles of chemotherapy, and hemograms, oxidative stress, enzymes antioxidants, immunologic parameters, and adverse events were analyzed. The use of 300\u2009mg of Uncaria tomentosa daily during 6 cycles of FOLFOX4 did not change the analyzed parameters, and no toxic effects were observed.To evaluate the effectiveness of  Howeverecursors  and the  . Howeve+ and CD4+ T cells (Tregs) levels, shows a correlation with survival. The CD4+/CD8+ ratio of the tumor infiltrating lymphocytes is significantly associated with colorectal cancer prognosis [\u03b3 production through the action of tumor-specific CD8+ T cells that infiltrate the tumor, and it promotes T cell-dependent antitumor responses in vivo [The immune status of patients, particularly CD8rognosis . Treatme in vivo . Again,  in vivo .Uncaria tomentosa  has antioxidant properties [Ut extract promotes proliferation of myeloid precursors through the increase in serum colony stimulating growth factors (CSFs). Other preclinical experiments have demonstrated the positive effect of aqueous Ut extract on leukocyte counts over a period of eight weeks in healthy animals [Ut could minimize the undesirable effects of chemotherapy and might improve the balance between stress and antioxidants in cancer patients. This clinical study aimed to evaluate the effect of coadjuvant treatment with Ut compared with conventional chemotherapy for colorectal cancer. The investigation evaluated the effect of Ut on oxidative stress and its consequences in relation to neutropenia, other hematological parameters, immune system, safety, and side effects.For these reasons, it is important to search for complementary treatments, including phytotherapic plants, that minimize the neutropenia associated with colon cancer chemotherapy. operties  and can operties  and myeloperties . Eberlinoperties  showed t animals  and afte animals . Given tWe performed a randomized interventional study of colorectal cancer patients who were submitted to chemotherapy treatment. Hospital Universit\u00e1rio de Santa Maria, Brazil. The study was carried out with 43 patients  who had undergone complete resection of their colorectal cancer, which was of histologically scored as stage IIB, III, or IV, and who were going to begin adjuvant/palliative chemotherapy with FOLFOX4 at the Ut, and the control group (C) received only FOLFOX4. Patients remained on study during 6 chemotherapies cycles, of 15 days each. The doses of medication in UT group were as follows: Oxaliplatin, 85\u2009mg/m\u00b2 on day1; 5FU, 1\u2009g/m\u00b2 on days 1 and 2; Leucovorin, 200\u2009mg/m\u00b2 on days 1 and 2; and Ut (Unha de Gato Herbarium), 3 tablets daily from day 3 to day 15. The dose of Ut is similar to that used in previous study, using 250\u2013350\u2009mg C-MED-100, an aqueous extracts of Ut [Patients were randomly grouped into two groups, according to the date of treatment start as follows: the first patient who agreed participating in the study was included into UT group, the second into the C group, and successively until the end. The UT group was treated with FOLFOX4 plus ts of Ut . No chan\u03b1) of 5%, and statistical power of 90% (\u03b2 10%), using as a reference the study of Sheng et al. [The calculation for estimating the sample size required for a randomized clinical trial was according to Greenberg et al. , with cog et al. .Universidade Federal de Santa Maria approved this study, and informed consent was obtained from all participants (protocol n. 0169.0.243.000-07).The Human Ethics Committee of the Ut extract. Biological materials used in the tablets were derived from plants in their natural habitat. The Ut extract was prepared by ultra-turrax extraction  from ground bark (Centroflora) using 70% ethanol . The HPLC analysis of Ut dry extract presents the content of 2.57% pentacyclic oxindole alkaloids (POAs), which were calculated with reference to external calibration curves of mitraphylline. The analysis of extract showed absence of tetracyclic oxindole alkaloids in the sample, allowing its use for therapeutic and research purposes in accordance with U.S. Pharmacopeia.Each tablet of Unha de Gato Herbarium contained 100\u2009mg dry Blood was collected in citrated, EDTA, heparin, and without anticoagulant Vacutainer tubes, before chemotherapy and after each of the 6 cycles. CAT and SOD activities were determined using whole blood diluted in a 1\u2009:\u200920 saline solution.A COBAS INTEGRA system was used for the quantitative determination of the chemical constituents of the blood, and data were acquired using a COBAS INTEGRA 400 plus apparatus (USA).The carbonylation of serum proteins was determined through a modification of the Levine method .Lipid peroxidation was estimated by measuring TBARS in plasma samples according to a modification of the method of Jentzsch et al. .The determination of CAT activity level was carried out in accordance with a modification of the method of Nelson and Kiesow . SOD actBlood samples were analyzed using a Pentra apparatus (France). The lowest values were confirmed by observation of slides using May Gr\u00fcnwald-Giemsa stain and optical microscopy.ELISA assays of IL-6 were carried out according to a previously published method  at room Alkaline comet assays were performed as described by Singh et al. . One hun+ cells.Samples were collected in EDTA, and analyses were performed using a three-color fluorescence-activated cell sorter  and Multiset software (Becton Dickinson). FITC-conjugated anti-CD4, PE conjugated anti-CD8 and PerCp conjugated anti-CD3 were used. Immune subpopulations were measured as a percentage of the total number of CD3The Common Terminology Criteria for Adverse Events (AE) v3.0 (CTCAE) from the National Institutes of Health/National Cancer Institute-EUA  has beent-test and were expressed as a mean \u00b1 SD. When the variances were not homogenous and ANOVA was not appropriate , Wilcoxon two-sample test was used to evaluate the data. P < 0.05 was considered statistically significant.Data were analyzed using the EpiInfo computer program, version 3.5.1 from the CDC, USA. The data were evaluated using analysis of variance (ANOVA) and The general characteristics of colorectal cancer patients included in this study are shown in One aim of the study was to evaluate the neutropenia, thrombocytopenia, and anemia. The hemograms were analyzed each 15 days, and there were no significant differences in hematological parameters Tables  and 3 beUt supplementation did not change oxidative stress values or activity of the antioxidant enzymes. Similarly, the comet assay, a sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell, demonstrated no differences between groups  used for the evaluation of the immune status of CRC patients were not statistically different following Ut supplementation , and bilirubin levels, and kidney function is evaluated by dosage of urea, metabolic parameters , and constitutive parameters (weight loss) (data not shown). There was a small difference in creatinine levels between groups before treatment , which remained at the sixth cycle of treatment .The assessment of adverse events (AEs) related to treatment was conducted by interviewing the patients during each cycle of chemotherapy and through analysis of laboratory tests and observation of abnormal symptoms presented by patients. AEs were classified according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE v3.0) . Since ts Tables  and 7. AComplementary and alternative medicine (CAM) has been used by a large number of cancer patients worldwide. Cultural, socioeconomic, and spiritual differences affect the rate of use. For instance, there are high rates of use in Mexico (97.2%) and China (97%) , 24, an Ut in minimizing the main side effects of chemotherapy, a decrease in neutrophil and platelet counts, hemograms were analyzed before each FOLFOX4 cycle . Treatment with Ut was suspended on the days that patients received chemotherapy, because some antineoplastic drugs and CAMs are metabolized through the cytochrome P450 family, and an interaction could alter the metabolism of patients [Ut did not improve the WBC, RBC, or platelet counts, because there were no differences between groups (UT versus C). In vivo assays in rats undergoing chemotherapy demonstrated that neutrophils recover significantly sooner with Ut supplementation [Ut on WBC is due both to the stimulation of myeloid precursors [Ut. In a human volunteer study, Ut extract was given at 250 or 350\u2009mg/day for 8 consecutive weeks to healthy adult. There were no statistically significant differences among the groups in WBC [Ut daily. In this study, we found significant differences between the group that received Ut and the control group; the Ut group showed higher neutrophils counts compared with the control group (unpublished data). The differences in the drugs used in the treatment of breast cancer  versus CRC  have to be considered, as do the differences in time between cycles of chemotherapy (21 versus 15 days). We must also consider the fact that all CRC patients in the present study underwent colectomy, which could interfere with the absorption of Ut.To evaluate the effectiveness of patients . Treatmeentation . This efecursors  and the ecursors  and inhiecursors . We did s in WBC . Our groUt, and its potent radical scavenger activity was confirmed by several assays including the following: the capacity to reduce the free radical diphenylpycrilhydrazyl (DPPH assay) [Ut extracts was further assayed through determination of TBARS production (using rat liver homogenates and sarcoplasmic reticulum membranes) and by the inhibition of free radical-mediated DNA-sugar damage [in vitro tests with one in vivo test [Ut, as assessed by lipid peroxidation (TBARS) and protein carbonyls. In addition, no differences were observed in the antioxidant enzymes SOD or catalase. Many previous studies have clearly shown the potential of the antioxidant H assay) , 31, theH assay) , and hydH assay)  as well H assay) , 32, andH assay) . The antr damage , 33. TheUt caused a significant decrease in DNA damage and a concomitant increase in DNA repair in volunteers. However, in our study, the comet assay did not demonstrate a significant difference in the group that received Ut. Ut extract was prepared through an extraction of ground bark with 70% ethanol. This process altered the composition of the extract  compared with an aqueous extract . Howeveg et al. . FurtherUt did not show an effect on the analyzed immunologic parameters, the CD4+ T cell-CD8+ T cell count and the IL-6 levels despite the in vitro evidence [Similar to oxidative stress, evidence .Ut extract at a repeated dose of 300\u2009mg/day for 12 consecutive weeks (Unha de Gato Herbarium), when judged in terms of clinical symptoms, serum clinical chemistry, whole blood analysis, and leukocyte differential counts. Similar results have been shown in previous studies with volunteers [There were no drug-related toxic effects observed for lunteers , 36. Natlunteers  has repoAdverse events related to antineoplastic drugs  are well known  and are Ut at dose 300\u2009mg dry extract daily is not effective in reducing the most prevalent adverse events due to treatment with 5FU/Leucovorin and oxaliplatin in patients with advanced CRC. No toxic effects related to Ut were observed in the group that received 300\u2009mg dry extract daily for 12 weeks. Additional studies are needed to evaluate under which conditions, drugs, or types of cancer Ut might have a positive effect on treatment, in decreasing neutropenia and thrombocytopenia, or in improving the immune response.All the authors deny any conflict of interests. This work had a financial support from the government agencies CNPq and CAPES."}
+{"text": "Across the first year of life, infants achieve remarkable success in their ability to interact in the social world. The hierarchical nature of circuit and skill development predicts that the emergence of social behaviors may depend upon an infant's early abilities to detect contingencies, particularly socially-relevant associations. Here, we examined whether individual differences in the rate of associative learning at one month of age is an enduring predictor of social, imitative, and discriminative behaviors measured across the human infant's first year. One-month learning rate was predictive of social behaviors at 5, 9, and 12 months of age as well as face-evoked discriminative neural activity at 9 months of age. Learning was not related to general cognitive abilities. These results underscore the importance of early contingency learning and suggest the presence of a basic mechanism underlying the ontogeny of social behaviors. During the first year of life, human infants develop remarkable social skills such as an emerging understanding of others' thoughts and intentions Associative learning via classical eyeblink conditioning is an ideal strategy to examine the relations between contingent learning and later social behavior development in human infants. It has been extensively used to examine learning in early infancy F4,276\u200a=\u200a24.75, P<.001, At one month of age, infant associative learning was measured using a delay eyeblink conditioning paradigm in which infants were presented with several pairings of a tone followed by a puff of air presented to the eye r51\u200a=\u200a.39, P<.01, r45\u200a=\u200a.36, P<.05). Infants who learned more rapidly at one month of age displayed heightened contingent social positivity at 5 months of age.At five months of age, the puppets and peek-a-boo tasks were administered r50\u200a=\u200a.37, P<.01, At 9 months of age, the still-face and modified peek-a-boo tasks were used to examine social contingency detection. Detection of social contingencies in these tasks was measured by examining observed changes in infant behavior following violations of social expectation. A single Social Contingency Detection score was computed, averaged across both tasks. Remarkably, longitudinal stability of the predicted relations between 1-month early associative learning and 9-month social contingency detection was found, with one-month Learning Slope significantly positively correlated with Social Contingency Detection  were recorded while infants viewed images of their mother's face and a stranger's face Ps>.20; Infant cognitive performance on the Mullen Scales of Early Learning (MSEL) The current study is the first to directly assess predictive relations between early associative learning and the emergence of social behaviors over the first year of life. Data reveal that individual differences in associative learning measured at one month of age relate to later social behavior across a variety of tasks and ages. Moreover, we discovered a significant neural correlate by demonstrating a relation between 1-month associative learning rate and neural responses to familiar versus unfamiliar face stimuli. These relations were specific to social behaviors and were not the result of individual differences in general cognition, suggesting that early associative learning may serve as a major building block for the development of social behavior.These behavioral findings are consistent with the basic neurodevelopmental concept that both circuits and skills are built from simple to more complex, the latter being highly dependent upon the former The University of Maryland Institutional Review Board approved all procedures. Prior to data collection, written informed consent was obtained from a parent or caregiver during each visit.Seventy full-term healthy infants  participated in the current study. Families were contacted by mail using commercially available lists of names and addresses compiled from local hospitals and infant registries. Prior to the laboratory visit at one month, parents completed a brief phone survey. Infants were excluded from participating in the study if they were born prior to 38 weeks of gestation, had reported birth complications or injury, serious illness, or diagnosed syndromic disorder.ts<1; Ps>.2).The population of infants used in the current study was representative of the greater Washington, DC area with 51.4% Caucasian, 21.4% African American, 5.7% Hispanic, 4.3% Asian, and 17.1% mixed ethnicity. The infants' mothers were well educated with 41.1% completing a graduate degree, 38.6% completing a college degree, 4.3% completing a professional or trade certificate, and 14.3% completing a high school degree. Of the 70 families that visited the laboratory at one month, 10 did not return for follow-up visits for a permanent attrition rate of 15%. Of the remaining 60 families who continued to participate in the study, 55 returned for the 5-month visit, 51 returned for the 9-month visit, and 48 returned for the 12-month visit for attrition rates of 8%, 15%, and 21% respectively. Learning abilities of infants who participated in the follow-up assessments did not differ significantly from those who did not participate in the follow-up assessments  and auditory conditioned stimulus (CS). The airpuff was presented through tubing that was attached to a flexible plastic arm connected to the left speaker. The arm was positioned approximately 1 inch from the infant's left eyelid.th trial was an airpuff-alone trial to test the somatosensory response and the 10th trial was a tone-alone trial to test for a conditioned response. Stimuli were presented for a total of 15 blocks .Trials consisted of the presentation of a 1000-ms, 1000-Hz tone that overlapped and co-terminated with a 100-ms airpuff, yielding a 900-ms delay interval. In each block of 10 trials, the 6The raw electromyographic (EMG) signal was amplified using a custom bioelectric amplifier  with a gain of 1000 Hz and filtered using high and low pass filters of 1 and 1250 Hz respectively. The amplified signal was digitized at a sampling rate of 512 Hz using a 12-bit A/D converter (\u00b12.5 V input range) and Snap-Master data acquisition software . Prior to recording EMG from each participant, a 50 \u00b5V 10 Hz signal was input into the channel and the amplified signal was recorded for calibration purposes. The raw signal was processed and analyzed offline using the EMG Analysis System from James Long Company . The signal was filtered digitally offline with a high-pass filter of 28 Hz, a low-pass filter of 250 Hz, and a digital band-stop filter (50\u201370 Hz) was used to remove 60-Hz noise. The signal was rectified and smoothed by using moving averages with a 20-ms window. Baseline EMG value was defined as the average activity recorded during the 20 ms prior to CS onset.Tone-alone trials were examined for the occurrence of conditioned responses (CRs). Each trial was visually examined for the occurrence of an eyeblink response that was defined as a rapid deflection in the EMG signal that was at least 1 SD above the mean of the baseline and occurred between 800 and 1500 ms after tone onset. It should be noted that previous studies examining eyeblink conditioning in awake adults have defined CRs as blink responses that are at least 5 SD above a mean baseline The percentage of CRs (%CR) across conditioning trials was used as the primary measure of learning and was computed across the 15 blocks in aggregates of 3 trials for a total of five 3-trial bins. To examine the relation between individual differences in learning and later social behaviors, the individual slope of the learning curve was computed for each infant by regressing %CR on Bin and used as the predictor variable in all subsequent analyses.During the 5-month assessment, two tasks were administered and maternal report on infant temperament was obtained. The tasks included were the peek-a-boo and puppets games The peek-a-boo and puppets tasks were specifically designed to elicit individual differences in displays of social positivity During the puppets game z scores and averaged across both the peek-a-boo and puppets games. Of the 55 infants who participated in these tasks, 4 were unable to be coded due to technical difficulties with the video recording and were not used in the current analysis.During the peek-a-boo and puppets games, behavioral positivity was coded separately including smiling intensity (0\u20133), intensity of vocalizations (0\u20133), and intensity of positive motor acts (0\u20132). The peek-a-boo game was divided into nine 10-s epochs for which the scores of each behavior were recorded. The puppets game was divided into a total of 5 epochs with four epochs equaling the time between each tickle and the last epoch occurring when the puppets were placed in front of the infant. In addition, presence of looking at the mother (0\u20131) was also coded during the puppets game as an index of social referencing. Two independent coders who were blind to the infants' 1-month learning abilities achieved sound inter-rater reliability on 20% of the data. Kappas ranged between .78 and .97 for behavioral positivity measured during the peek-a-boo game and .67 and .94 for the puppets game. The kappa for frequency of looking at the mother was .86. Individual measures for behavioral positivity were averaged across epochs for each game separately. The frequency of looking at the mother during the 5 epochs of the puppets game was computed. To obtain an overall Social Responsivity Score, behavioral positivity scores and frequency of social referencing were converted to Maternal report of infant temperament was obtained using the IBQ During the 9-month assessment, four tasks were administered in order to obtain individual differences in performance across several aspects of social behavior including social contingency detection, imitation, and social discrimination. These tasks include the still-face task During the still-face task In order to obtain a negativity proportion score for each interaction phase, the frequency of protest and withdrawn behavior was summed separately for each phase and divided by the total duration of each phase. The classic still-face response is a decrease in positivity and increase in negativity from the face-to-face interaction phase to the still-face phase During the modified peek-a-boo game z-scores and averaged separately across the trials in which the mother appeared behind the door and the trials in which the mother did not appear behind the door in order to obtain separate Social Positivity scores during mother-present and mother-absent trials. To examine individual differences in social contingency during the modified peek-a-boo task, a peek-a-boo Social Contingency Detection score was computed and defined as the difference between mother-present Social Positivity scores and mother-absent Social Positivity scores.Behavioral scores were converted to To examine imitation at 9 months, an experimenter presented the infant with four novel age-appropriate toys which were first described in studies conducted by Meltzoff During the mother-stranger face discrimination task Testing occurred while the infant sat on his or her mother's lap in a dimly lit room. Stimuli were presented using E-Prime software . Faces were presented on a black background and in the center of the screen. The computer monitor was 34 cm wide and 27 cm high. Infants viewed images at a distance of 60 cm. A camera mounted above the monitor allowed for simultaneous video recording of the infant's face during the experiment. The experimenter presented images during the experiment only when the infant was attending to the monitor. Trials were marked for deletion if the infant looked away during presentation of an image. Infants were presented 60 images of the mother's face and 60 images of the stranger's face. Faces were presented pseudo-randomly such that within every four presentations, the infant was randomly presented two images of the mother's face and two images of the stranger's face. Stimuli were presented for 500 ms followed by an inter-stimulus interval of at least 1000 ms during which time the screen was black with a white cross in the center.ERPs were recorded using a 64-channel HydroCel Geodesic Sensor Net . Signals were amplified using an EGI NetAmps 200 amplifier and sampled at 250 Hz with a band-pass filter of 0.1\u2013100 Hz. Once the impedance values were reduced below 100 k\u03a9, data acquisition was started. EEG was recorded continuously and referenced to Cz, and after acquisition, data was re-referenced using an average reference.Data were filtered offline using a 30-Hz lowpass and a 1-Hz highpass filter. Trials consisted of a 400-ms baseline period and 600-ms period following stimulus onset. Data were baseline corrected to the average voltage during the 400 ms prior to stimulus onset. Data were segmented and visually inspected for EOG and motion artifact. Data from individual sensors were rejected if there was artifact resulting from poor contact or movement. The entire trial was excluded if more than 15 sensors had been rejected, or if an eyeblink or other significant movement artifact had occurred. Of the trials that were not rejected, individual channels containing artifact were replaced using spherical spline interpolation. Individual subject averages were constructed separately for the mother and stranger faces.r-values between Learning Slope and the Nc mean amplitude difference at each electrode site were subsequently submitted to EEGLAB software N\u200a=\u200a4) or becoming too upset and unable to finish the task (N\u200a=\u200a3). These infants were excluded from the current ERP analysis.Of particular interest to the current study was examination of the Nc component, a fronto-central negative deflection that reflects some aspect of visual attention During the 12-month assessment, one task was administered to examine infant joint attention and one task was administered to examine infant cognitive abilities. The tasks included were the Early Social Communication Scale (ESCS) rs were .767 and .921 for IJA and RJA, respectively. A total Joint Attention score was computed by averaging standardized scores (z-scores) of IJA and RJA.The ESCS The MSEL Repeated measures ANOVA with Bin as the within measure was used to determine if infants' learning increased across the course of the experiment. Pearson's correlations were used to determine if there were significant relations between individual differences in 1-month learning and 5-, 9-, and 12-month social outcome measures and 12-month cognition."}
+{"text": "Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet.Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase ( Eleusine coracana) constitutes \u223c12% of the global millet area and is cultivated in more than 25 countries in Africa and Asia. It is an important food crop in India, Nepal and several African countries Finger millet , from Escherichia coli is known to catalyze mannitol production in various transgenic plants engineered with this gene and also these plants showed enhanced abiotic stress tolerance mtlD gene targeted to chloroplast showed increased hydroxyl radical scavenging capacity and retention of chlorophyll in leaf tissue under methyl viologen-induced oxidative stress Mannitol, a sugar alcohol, is an important osmolyte and scavenger of reactive oxygen species (ROS). This compound has been reported to impart tolerance to abiotic stresses in different plant species Agrobacterium tumefaciens (here after referred as Agrobacterium)-mediated transformation in finger millet. Also, by using this protocol mtlD gene expressing transgenic plants were developed and their tolerance under drought, salinity and oxidative stresses was studied.Finger millet is known to accumulate proline under stress The finger millet (var. GPU 28) seeds were obtained from all India coordinated research project , University of Agricultural Sciences, GKVK Bangalore India. For the seedling level experiments, seeds were germinated on moist filter paper at 30\u00b0C and 80% relative humidity. Seedlings (1.5 to 2 cm length) were used for stress experiments. For plant level experiments, plants were grown in plastic pots with the potting mixture consisting red soil: sand: vermiculite in the ratio of 3\u22361\u22361 by volume (22% water holding capacity). GPU28 was used for all experiments described in this manuscript except for optimization of hormonal concentrations for callus induction and plant regeneration in MS medium.Tobacco (var. KST 19) plants were grown in the greenhouse and \u223c30\u201335 day old plants were used for stress experiments unless otherwise specified.\u22122 s\u22121.For both plant species, recommended fertilizers including micro and macronutrients were provided. Prophylactic measures were taken to maintain the plants disease and pest free. Plants were grown in the greenhouse at 22\u201327\u00b0C with 60% relative humidity and a 12 h photoperiod with mild-day light intensity of \u223c500 \u00b5mol mUidA) mtlDHptII). These constructs were mobilized into Agrobacterium strain EHA105 and used for plant transformation experiments.The binary vectors pCAMBIA 1301 having beta-glucuronidase (Seeds were surface sterilized with 0.1% mercuric chloride for 5 min and then rinsed for four times in sterile de-ionized water. Sterilized seeds were placed on Murashige and Skoog (MS) medium Agrobacterium strains EHA105 harboring the binary constructs were grown on AB minimal medium with 50 mg/L kanamycin for 16 h at 28\u00b0C. Cultures (OD600\u200a=\u200a0.1) were resuspended in liquid co-cultivation medium supplemented with 100 \u00b5M acetosyringone . Calli (45 day old) were incubated with bacterial (carrying mtlD or UidA gene construct) suspension medium for 10 min. They were then blotted dry on sterile filter paper to remove excess Agrobacterium and co-cultivated for 48 h at 25\u00b0C in dark. Subsequently, they were washed using sterile water containing 300 mg/L cefotaxime for four to five times, blotted on sterile filter paper and inoculated on antibiotic selection medium . This step was repeated if Agrobacterium growth occurred.After three to four weeks of incubation the surviving calli were transferred to regeneration medium (MS with 0.5 mg/L BA) with hygromycin for plant-let growth and maintained in light for 20 days. Later, the shoot-lets were sub-cultured to rooting medium (MS with 0.5 mg/L BA and 0.1 mg/L NAA) for two weeks. Rooted plants were then transplanted in to soil mixture for further growth. Overall steps involved in mtlD transgenic plant development are described in Finger millet seeds from T1 generation were germinated on moist filter paper at 30\u00b0C for 36 h and uniform seedlings were acclimated with respective stress  for 8 h. Then, the seedlings were transferred to Petri plates with filter paper containing polyethylene glycol  or NaCl (300 and 400 mM) or the naptho-quinone compound, menadione , solution. PEG, NaCl and menadione induces osmotic, salinity and oxidative stresses respectively and solutions were prepared as described in previous literatures Fifteen day old plants were planted in pots filled with potting mixture of known weight. Pots were irrigated until all the soil macro and micro pores were filled and excess water was drained. Based on water holding capacity for this soil mixture, total weight of pot with soil mix for 100% field capacity (FC) was arrived. Plants were maintained at 100% FC (control) until imposition of drought stress. Drought stress was imposed by following gravimetric method as described previously \u22122 s\u22121 light for 2 h for stress acclimation. They were then exposed to high light (700\u2013800 \u00b5mol m\u22122 s\u22121) with 2 \u00b5M methyl viologen for 6 h. Methyl viologen accepts electron from the last step of photosynthetic electron transport chain and produces free radicals Leaf segments (1 cm diameter) were taken from 45 day old transgenic and wild-type tobacco or finger millet plants grown under non-stress condition. The leaf segments were initially incubated in lower concentration (0.5 \u00b5M) of methyl viologen  with 100 \u00b5mol mUidA gene 5\u2032-GGG CAG GCC AGC GTA TCG TG-3\u2032 and 5\u2032-GTC CCG CTA GTG CCT TGT CCA GTT 3\u2032 and primer pairs 5\u2032-ATC CGC TGC CCC TCT CT-3\u2032 and 5\u2032-ACA TGG CCT TTC AGC TGC GTG GTA-3\u2032 that amplify 500 bp fragment of the mtlD gene. Primer pairs 5\u2032- GAG GCT ATT CGG CTA TGA CTG-3\u2032 and 5\u2032-ATC GCG AGG GGC GAT ACC GTA-3\u2032 were used to amplify 750 bp fragment of hptII gene. Further, primer pairs 5\u2032-TCC ATA ATG AAG TGT GAT GT-3\u2032 and 5\u2032-GGA CCT GAC TCG TCA TAC TC-3\u2032 were used to amplify 300 bp fragment of Actin gene.The genomic DNA was isolated from the leaves of putative transformed and wild-type plants. The transformed plants were confirmed by PCR using primers specific to UidA or mtlD genes were analyzed for integration and copy number of the transgene. Genomic DNA from the transgenic and wild-type plants were restrict digested (XbaI), resolved on 0.8% agarose gel and transferred to positively charged nylon membrane. The membrane was exposed to UV (1200 \u00b5J for 60 s) for cross linking. The blot was probed with the respective radio labeled (32P dCTP 3000 Ci mmol) inserts. Pre-hybridization was done at 42\u00b0C for 2 h and hybridization was done at 55\u00b0C overnight with blocking solution . The hybridized blot was washed in the following sequence with 6\u00d7 saline sodium citrate (SSC) for 10 min at 37\u00b0C, 4\u00d7 SSC for 10 min at 37\u00b0C; 2\u00d7 SSC for 10 min at 37\u00b0C; 2\u00d7 SSC for 15 min at 55\u00b0C; 0.2\u00d7 SSC for 10 min at 55\u00b0C. The hybridization signals were detected following exposure of Kodak X-ray film to the membrane at \u221270\u00b0C for 24 h and developed by autoradiography.Putative finger millet transgenic plants having Hpt) was prepared by labeling it with 32P dCTP as described under Southern blot. The blot was hybridized using the protocol described under Southern blot and developed by autoradiography.Genomic DNA (50 ng) from finger millet plants was spotted on the nitrocellulose membrane and fixed by exposing to UV (1200 \u00b5J for 60 s) in an UV cross linker . Probe  and first strand cDNA was synthesized by oligo (dT) primers using Molony Murine Leukaemia Virus reverse transcriptase  according to manufacturer's instructions. The cDNA pool was used as a template to perform RT- PCR analysis using the following primers 0 generation) from the PCR positive transgenic plants were incubated at 37\u00b0C in GUS staining solution  for 8 h. After incubation they were transferred to 80% (v/v) ethanol/water solution to remove the chlorophyll, and later photographed. Similarly, seedlings (T1 generation) were stained with GUS solution and photographed.GUS activity in putative transformed callus was assayed as described in the literature th leaf was excised from stressed and well-watered plants and sampled at midday, quickly sealed in plastic bags, and kept on ice. These leaves were used to determine the relative water content (RWC) and solute potential.From the drought exposed  finger millet plants 5After determining the fresh weight, the leaf segments were floated on deionized water for 5 h to determine their turgid weight. The dry weight was determined after oven-drying to a constant weight. The RWC was calculated using the formula:Leaf samples were frozen in liquid nitrogen, thawed, and centrifuged for 5 min at 20 000 g. The \u03a8s of the extracted sap was measured by VAPRO vapor pressure osmometer . S100 - control leaf \u03a8S100From the values of RWC and solute potential of control and stress-grown plants, the osmotic adjustment was calculated using the formula: Osmotic adjustment (OA) \u200a=\u200a drought leaf \u03a8Chlorophyll was extracted from 100 mg of leaf tissue in 1 ml of acetone and dimethyl sulphoxide  mix. The absorbance was recorded at 663 and 645 nm using UV\u2013visible spectrophotometer Model DU800 . Total chlorophyll was estimated as described previously The standard graph was developed using Mannitol  using the previously described method Cell viability was measured by 2,-3,-5-triphenyltetrazolium chloride  as described in the previous literature Leaf segments (1 cm diameter each having dry weight (0.27 mg) were incubated in 1 ml of K-phosphate buffer  containing 500 \u00b5M of XTT  along with methyl viologen and exposed to high light stress. XTT is a sodium 3\u2032[1--bis [4 methoxy-6-nitro] benzene sulfonic acid hydrate]. Later, increase in absorbance at 470 nm of incubation medium was measured using a micro titer plate reader  as described in the previous literature P\u22640.05) or Duncan's multiple range test or students t-test. Statistical significance of values in graphs was indicated either as asterisks or as letters.Analysis of variance (anova) was performed using MS Excel software, and data points were analyzed for Fisher's least significant difference  seeds were used as explant . Among 1Agrobacterium strain EHA105 harboring pCAMBIA 1301/UidA constructs were co-cultivated for 48 h. The Agrobacterium culture was incubated with 100 \u00b5M of acetosyringone for virulence gene induction \u22122 s\u22121) for 10 days. The surviving calli were then transferred to regeneration medium (MS+0.5 mg/L BA+30 mg/L hygromycin and 300 mg/L cefotaxime). Later, as per previously standardized protocol  showed single copy integration of UidA gene in the finger millet genome  selected on the hygromycin  putative transgenic plants expressing mtlD were developed. Hygromycin resistant putative transgenic finger millet seedlings  from inde plants . Furtheric lines . Furtherern blot . The expy RT-PCR . The segy RT-PCR . Unlike Drought stress and salinity stress induces higher levels of free radical production in finger millet 1, mtlD-3 T1 and mtlD-5 T1) showed moderate level of stress tolerance under PEG-induced osmotic stress  were grown for 15 days in pots and used for testing their drought tolerance. Initially the plants were subjected to gradual acclimation stress and later maintained at severe stress levels for seven days. Phenotypic observations were made from the stressed plants (mtlD-1 T1) and wild-type. At 50% FC stress for seven days, wild-type and the transgenic plants did not show visible wilting phenotype. At much higher stress levels (20% FC) the transgenic plants showed improved tolerance to drought stress than wild-type plants (1 and mtlD-5 T1) exposed to 50% FC stress maintained higher osmotic adjustment and \u223c10% less chlorophyll degradation in all the three transgenic lines than wild-type plants  germinated on hygromycin medium (2 seedlings showed no significant improvement in stress tolerance under PEG stress (2 seedlings showed only slight improvement in growth under PEG-induced osmotic stress (2 generation) were used for mannitol quantification. Results showed that mannitol content was high in all the tested seed lots (The Tn medium  were traG stress . Similarc stress . The trac stress . Stress c stress . In ordeeed lots . Similareed lots . This ca1). Wild-type tobacco plants are more susceptible to stress compared to wild-type finger millet In order to understand the reason for only marginal improvement in oxidative stress tolerance levels in transgenic finger millet, transgenic tobacco plants expressing mtlD was used and compared their stress tolerance increase with finger millet plants  of chlorophyll maintenance over wild-type under oxidative stress (mtlD gene showed only slight improvement (<3%) in oxidative stress tolerance  which were confirmed by PCR and Southern blot were grown in greenhouse and seeds were collected. For further experiments, out of 20 putative transgenic events, four T0 transgenic events were used. These four events were designated as mtlD-1, mtlD-3, mtlD-5, mtlD-16 and taken forward. Seeds obtained from three (T0) events  were used for further experiments. Seeds from each event were bulked and used for seedling experiment  or plant level experiment. Plants obtained from individual seeds representing each (T1) event were assessed for presence of mtlD gene by PCR and then seeds were collected from these plants. These seeds were designated as mtlD-1-2 T2, mtlD-3-1 T2 and mtlD-5-1 T2 and used for mannitol quantification. A sub-set of seedlings obtained from these seeds were used for stress experiments.(PDF)Click here for additional data file.Figure S2Germination of seeds obtained from UidA gene expressing finger millet transgenic plants on hygromycin. Wild-type (var. GPU28) finger millet seeds were germinated in petri dishes on filter paper with different concentration of hygromycin for 5 days at 30\u00b0C with 70% relative humidity in dark and germination percentage was recorded at the end of treatment period (A). Similarly, seeds obtained from the GUS positive transgenic finger millet plants  and wild-type (var. GPU28) were germinated on hygromycin (60 mg/L) and number of seedlings survived was recorded (B). Asterisks indicate values are statistically significant  versus corresponding wild-type. Each bar represents the mean of standard error values (n\u200a=\u200a10).(PDF)Click here for additional data file.Figure S31 generation) and wild-type seedlings on hygromycin medium.Growth of mtlD expressing finger millet transgenic  and wild-type  were inoculated on the MS medium containing hygromycin (20 mg/L or 40 mg/L) for five days. Seedling growth on antibiotic medium was photographed at the end of treatment period.(PDF)Click here for additional data file.Figure S4Performance of mtlD expressing finger millet transgenic seedlings under osmotic, salinity and oxidative stress. Finger millet transgenic (T1 generation) and wild-type (var. GPU28) seedlings (1.5 cm length) were initially acclimated with lower concentration of corresponding stresses  for 8 h and then subjected to indicated concentrations of respective severe stress levels for 48 h. Seedlings were allowed to recover for two days and recovery growth of osmotic stress (A), salinity stress (B) and menadione induced oxidative stress (C) were measured and percent reduction in growth over corresponding control was calculated. The percent relative water content was measured from the plants exposed to 100%, 50% and 30% field capacity (D). Each bar represents the mean of standard error values (n\u200a=\u200a20). Experiments were repeated twice. Alphabets above bar indicates the statistical significance (ANNOVA). Same alphabets indicate no significant difference (p<0.05).(PDF)Click here for additional data file.Figure S52 generation) and wild-type seedlings on hygromycin medium.Growth of mtlD expressing finger millet transgenic (T Seeds from mtlD expressing transgenic finger millet (mtlD-1-2 T2) and wild-type  were incubated on the MS medium containing hygromycin (20 mg/L or 40 mg/L) for five days. Seedling growth on antibiotic medium was photographed at the end of treatment period.(PDF)Click here for additional data file.Figure S6Mannitol accumulation in mtlD gene expressing finger millet germinating seedlings. Mannitol content in the seedlings obtained from T2 generation plants were estimated as described in material and methods. Alphabets above bar indicates the statistical significance (Duncan's multiple range test). Same alphabets indicate no significant difference (p<0.05).(PDF)Click here for additional data file.Figure S7Chlorophyll retention in tobacco and finger millet plants expressing mtlD gene under methyl viologen coupled with high light-induced oxidative stress. Leaf segments were taken from transgenic tobacco, finger millet (mtlD-5) and corresponding wild-type plants grown under non-stress condition. These leaf segments were exposed to high light (800 \u00b5mol m\u22122 s\u22121) stress with methyl viologen as described in materials and methods section. At the end of stress period, total chlorophyll was measured and percent reduction in total chlorophyll over their corresponding non-stress control was calculated. Values are mean of three replications and the error bar represents standard error. Alphabets above bar indicates the statistical significance (ANNOVA). Same alphabets indicate no significant difference (p<0.05).(PDF)Click here for additional data file.Figure S8Comparison of different features of genetic transformation protocols in finger millet reported in previous literature. In the literature, three research groups have demonstrated genetic transformation in finger millet. First, particle gun-mediated protocol was developed by Latha and co-workers. Second, Agrobacterium-mediated protocol was developed by Antony Ceasar and Ignacimuthu. Both protocol uses shoot apex as explants. Third, Sharma et al., and Jagga-Chugh et al., have demonstrated UidA gene expression. In this pictorial representation, different features of these protocols are compared with the protocols developed from this current study.(PDF)Click here for additional data file.Table S1Standardization of hormonal concentrations for efficient callus induction in finger millet (var. Indaf 9).(PDF)Click here for additional data file.Table S2Standardization of hormonal concentrations for efficient shoot induction from the callus in finger millet (var. Indaf 9).(PDF)Click here for additional data file.Table S3Efficiency of Agrobacterium-mediated finger millet transformation.(PDF)Click here for additional data file.Table S4Comparison of mtlD gene expressing plant species reported in the literature with their corresponding wild-type plants for their abiotic stress tolerance.(PDF)Click here for additional data file."}
+{"text": "Clinical handover is an important process for the transition of patient-care responsibility to the next healthcare provider, but it may divert the attention of the team away from active patients. This is challenging in the Emergency Department (ED) because of highly dynamic patient conditions and is likely relevant in conditions that requires time-sensitive therapies, such as sepsis. We aimed to examine the management and outcomes of patients presenting with sepsis and septic shock to the ED during nursing handover.This retrospective cohort study was conducted at a 115-bed ED and more than 200,000 annual ED visits, within a 900-bed academic tertiary care center. Data on Surviving Sepsis Campaign (SSC) bundle elements and hospital mortality were collected for all \u226514-year-old patients who presented to the ED with a diagnosis of sepsis and septic shock between January 1, 2011 and October 30, 2013. Our primary outcome was time to antibiotics, were other SSC bundle elements and mortality counted as secondary outcomes. Patients were divided into two groups: 1) handover time group, comprising patients who presented an hour before or after the start of handover time (6\u20138\u00a0AM/PM), and 2) non-handover time group, comprising patients who presented over the remaining 20\u00a0h.P\u2009=\u20090.07), median time to serum lactate result  and median time to obtain blood culture , and hospital mortality rate .During the study period, 1330 patients presented with sepsis or septic shock . No significant differences were found between the handover time and non-handover time groups, respectively, in median time to antibiotic administration (100 [interquartile range (IQR) 57\u2013172] vs. 95 [IQR 50\u2013190] minutes; No significant differences were found in median time of SSC bundle elements or hospital mortality between patients who presented during the handover and non-handover times. Handover is an essential process in the Emergency Department (ED), as patient care is provided in a continuum around the clock manner; it follows, staff must work in shifts . HandoveNursing handover is a complex process that requires effective transfer of all required patient information in the most time-efficient manner. This process needs good communication skills and time management. Trivial miscommunication may lead to delivery of inaccurate or incomplete data, resulting in delayed care or other adverse effects , 4. NursReviewing the literature, we found ED handover studies that focused on evaluating the quality of care transfer, the need to initiate a standardized tool to aid the process, and on reporting handover-related errors and adverse events. Interestingly, we did not find any studies that investigated the effect of handover time on the patients with time-sensitive disorders who visited the ED during the nursing handover time. Sepsis is an important time-sensitive condition in which delays in providing care, such as delays in antibiotic administration, are associated with adverse outcomes. In particular, delays in antibiotic administration have been shown to be associated with a 7.6% decrease in survival for each hour of delay in antimicrobial administration during the 6\u00a0h after the first hour of documented hypotension . In thisThe study was performed in a large, urban, tertiary-care ED with an Emergency Medicine Residency Program. The ED is staffed with board-certified emergency medicine physicians, and has 115 beds. The number of annual ED visits range from approximately 200,000 to 214,000 per year at this 900-bed academic tertiary care center. In this study, we used data from the Sepsis Database, collected as part of a quality improvement project conducted by the Intensive Care and Emergency Medicine Departments. The Sepsis Database used the 2008 and 2012 Surviving Sepsis Campaign (SSC) tools.We included all patients aged \u226514\u00a0years who were admitted to the ED with a diagnosis of sepsis and septic shock between January 1, 2011 and October 30, 2013. In our institution, nursing shifts are 12-h based in all departments. Nursing handover process is done at the bedside using both verbal and written forms. The form used in our institution is SBAR  , which mWe extracted the following data from the Sepsis Database: ED arrival time, source of infection, physical examination findings, laboratory findings, time-to-antibiotic administration , time-to-lactate results and time-to-obtaining blood culture, mechanical ventilation requirement, and hospital mortality . The prit-test, and categorical values were compared using the chi-square test. All data management and analysis was performed with SAS .Because of the skewed data distribution, we have presented data as median and inter-quartile range for continuous variables, and frequency and proportion for categorical variables. Continuous variables were compared between two groups using the Over the study period, 1330 patients fulfilled the diagnostic criteria for sepsis and septic shock, and were included in the final analysis: 228 patients in the handover time group and 1102 patients in the non-handover time group.The presenting characteristics of patients who arrived during the handover time and those who arrived during the non-handover time are presented in Table\u00a0P\u2009=\u20090.07). The distribution of median time-to-antibiotic administration by hour of day is presented in Fig.\u00a0P\u2009=\u20090.33). The median time-to-obtaining blood cultures in the handover time group (54 [IQR 36\u2013119] minutes) was not significantly different from that in the non-handover time group . The hospital mortality rate in the handover time group (29.4%) was not significantly different from that in the non-handover time group .All patients received antibiotics, and median time-to-antibiotic administration showed a tendency of being longer in the handover time group (100 [IQR 57\u2013172] minutes) as compared to the non-handover time group (95 [IQR 50\u2013190] minutes; Handovers are pivotal junctures and integral process in the continuity of care in every patient\u2019s clinical course. To the best of our knowledge, the idea of handover time as a possible distractor that might delay urgent patient care is not addressed in current literature. We aim to shed light on the duration of handover process as a possible time where patient care is affected. In this study, we evaluated the direct effect of nursing handover process on patient care. We used a sepsis database to compare ED processes and outcomes between patients who arrived at the ED during nursing handover time and those who arrived during non-handover time.Our results showed a trend of longer time-to-antibiotic administration in handover group, however this was not clinically nor statistically significant. Nonetheless, the clinical value is unconvincing; as 5\u00a0min\u2019 difference, might be minor when it comes to antibiotic delivery. It follows, additional studies with another time sensitive assessment tools are needed to address the clinical outcome of this delay, and how to prevent it. We found no significant association between ED nursing handover and time-to- lactate results or time-to-obtaining blood culture, or hospital mortality in patients admitted with a diagnosis of sepsis and septic shock. This could be explained by our ED nurses are vigilant with alerts and our institution was conducting SSC with constant reminders of early management and septic alerts . ConsequA prospective observational study addressing ED handover problems revealed deficiencies in the handover processes . These dThe main strengths of our study include the numbers of patients included, detailed data collection, the tertiary academic setting with numerous complex and critically ill patients, and standardized data collection using the SSC tools. As a retrospective cohort study, the present study has some important limitations. Foremost, the study aimed to evaluate the direct impact of handover time on time to time-to-antibiotic administration, time-to-lactate results, and time-to-obtaining blood culture, as surrogate indicators of the quality of sepsis and septic shock management. Nonetheless, other important measures in sepsis management were not investigated such as time to effective fluid resuscitation. Additionally, there is inherent variation and subjectivity in the handover process among ED nursing staff might have underpowered our results. Lastly, because of the retrospective nature of this study and the fact that it was conducted in a busy ED, others factors, such as ED overcrowding and boarding patients in ED, could have affected the study results.This is one of the first reports of the impact of ED nursing handover on time-sensitive interventions that involve multiple tasks performed by ED nurses. Due to the retrospective nature, patient population with single pathology, and our structured handover process that might have reflected on the results of this study. Future studies are still needed to explore ED functionality during the handover time."}
+{"text": "The effect of swap disorder on the physical properties of the quaternary Heusler alloy PdMnTiAl is described from first principles. Y-type1, in good agreement with the Slater\u2013Pauling rule. From the band structure and the density of states, it is predicted that this Y-type1 configuration is a new gapless semi-metal material. Furthermore, it was discovered that the Pd\u2013Mn swap-disordered structure is more stable than the Y-type1 structure. The present work provides a guide for experiments to synthesize and characterize this Heusler alloy.Heusler alloys crystallize in a close-packed cubic structure, having a four-atom basis forming a face-centred cubic lattice. By selecting different composite elements, Heusler alloys provide a large family of members for frontier research of spintronics and magnetic materials and devices. In this paper, the structural, electronic and magnetic properties of a novel quaternary Heusler alloy, PdMnTiAl, have been investigated using a first-principles computational materials calculation. It was found that the stable ordered structure is a non-magnetic  The Y-type1 structure with space group b), the Y-type2 and Y-type3 configurations are found from our calculations to have relatively higher total energies. Both VASP and AkaiKKR were used to optimize the structures of these three types of alloy to obtain the equilibrium lattice parameters. Similar trends were observed, such that the Y-type1 configuration had the smallest lattice parameter and Y-type3 the largest. A comparison of the two different calculation methods is shown in Table 1VASP and AkaiKKR predictions for the magnetic moments. The Y-type1 configuration of PdMnTiAl was found to have zero magnetization by both methods. To confirm the DFT calculation results, we checked the magnetic moments with the Slater\u2013Pauling (SP) rule , Ti (s2d2), Mn (s2d5) and Al (s2p1). Thus the structure of our Y-type1 configuration complies perfectly with the Slater\u2013Pauling rule.First, the ordered quaternary Heusler alloy PdMnTiAl is considered. Fig. 1VASP and AkaiKKR. Figs. 2AkaiKKR as with VASP. Fig. 2Y-type structures have bands which cross the Fermi level, thus indicating metallic behaviour. The nonmagnetic Y-type1 structure has a pseudo-gap-like DOS at the Fermi level, indicating a semi-metal, whereas the magnetic structures Y-type2 and Y-type3 have Fermi levels located near DOS peaks. This may help to explain the energetic instability of the Y-type2 and Y-type3 configurations. In Fig. 3From the optimized structures, we calculated their density of states (DOS) and band structures using Y-type structures have different total energies and magnetic moments, and PdMnTiAl is more likely to have the Y-type1 structure. The Y-type2 and Y-type3 structures have higher energy, larger lattice parameters and greater magnetic moments than the Y-type1 structure. To investigate the atomistic disordered configurations of these quaternary Heusler alloys further, we carried out advanced calculations using AkaiKKR. Based on the above ordered structures, we considered a number of possible swap disorder types by intermixing between any two of the Pd, Ti, Mn and Al atoms. In the following, we use the numerals I, II, III, IV, V and VI to represent interchanges, or swaps, between Ti\u2013Mn, Pd\u2013Mn, Ti\u2013Al, Pd\u2013Al, Pd\u2013Ti and Mn\u2013Al, respectively, and VII, VIII, IX, X, XI and XII to represent Pd\u2013Mn, Ti\u2013Al, Pd\u2013Ti, Mn\u2013Al, Ti\u2013Mn and Pd\u2013Al swaps, respectively. For example, the M point in Fig. 40.7Mn0.3)Ti(Mn0.7Pd0.3)Al with a Pd\u2013Mn swap. Fig. 4Y-type1. Moreover, the Pd\u2013Mn swap not only lowers the total energy, but also gives rise to significant magnetization. The new ground-state disordered structure (Pd0.7Mn0.3)Ti(Mn0.7Pd0.3)Al has an energy decrease of 0.092\u2005eV per formula unit compared with the ordered Y-type1 PdMnTiAl, with its total magnetic moment enhanced to 0.964\u2005\u00b5B. The half-and-half Pd\u2013Mn randomly disordered configuration (Pd0.5Mn0.5)Ti(Mn0.5Pd0.5)Al is 0.047\u2005eV lower in energy per formula unit and has the maximum magnetic moment of 1.132\u2005\u00b5B. In the other cases of disorder, the total energies are increased by the disordering effect and the swap disorders tend to introduce finite total magnetization. We also calculated the DOS for all the different dis\u00adordered structures, as presented in Fig. S1 in the supporting information.From the above calculations the ordered VASP to verify the validity of the corresponding AkaiKKR results. In a double-sized supercell with 32 atoms (eight formula units), 25% swap disorders were simulated by exchanging two of the eight Pd atom positions and two of the eight Mn atom positions. Similarly, 50% swap disorders were simulated by exchanging four of the eight Pd atoms and four of the eight Mn atom positions. Four possible disordered supercell structures for the 50% configurational swap disorder are shown in Fig. S2 in the supporting information. We found the swap-disordered structures to be more stable than the ordered PdMnTiAl Y-type1 structure and have also verified qualitatively the correctness of the magnetic moments. The results, shown in Table S1 in the supporting information, suggest that much larger supercells might be necessary to achieve a better quantitative comparison between AkaiKKR disorder calculations and VASP supercell calculations. This comparison is beyond our current computational resources. However, experimental work is expected to synthesize and characterize the proposed PdMnTiAl quaternary Heusler alloy for eventual confirmation of our prediction.We attempted to construct larger supercells and to use 4.Y-type1 is more stable than those of Y-type2 and Y-type3. The semi-metallic Y-type1 configuration shows zero magnetic moment, in good agreement with the Slater\u2013Pauling rule. Interestingly, we discovered that the Pd\u2013Mn swap-disordered structure is more stable than the ordered Y-type1 configuration, and that the total magnetizations of these disordered (Pdx1\u2212Mnx)Ti(Mnx1\u2212Pdx)Al compounds are dependent on the degree of Pd\u2013Mn swap, 0 < x < 1. We hope these findings will stimulate further investigation into spintronics materials and devices.The structure and electronic and magnetic properties of the quaternary Heusler alloy PdMnTiAl have been investigated by first-principles calculations. In these compounds, the ordered configuration of 10.1107/S205225251700745X/ct5004sup1.pdfAdditional table and figures. DOI:"}
+{"text": "Prognosis for metastatic melanoma has improved significantly with the use of immune checkpoint inhibitors. Given improvements in survival, aggressive surgical treatment may be considered in patients with life-threatening complications from their disease that would not otherwise be considered in advanced disease. Patients with preexisting autoimmune diseases or prior immune-related adverse events from therapy are largely excluded from clinical trials. Concerns exist that immunotherapy in these patients could worsen autoimmune disease or increase the risk of developing additional immune-related adverse events on therapy. We present a case of a patient with rheumatoid arthritis that presented with obstructive heart failure secondary to melanoma that had metastasized to the right atrium. After aggressive surgical resection to stabilize him from his life-threatening heart failure, he was treated with ipilimumab, which was stopped due to an immune-related adverse event. He was then started on pembrolizumab and had a durable response to therapy. Aggressive surgical treatment should be considered in patients with a cancer that may respond to immunotherapy. Furthermore, some patients with preexisting autoimmune disease may be safely treated with checkpoint inhibition therapy, and patients with a severe immune toxicity from one class may successfully be treated with an alternate class. Melanoma is an aggressive cutaneous malignancy that accounts for 1 to 2 percent of all cancer-related deaths annually [Immune checkpoint inhibition therapy for metastatic melanoma has been shown to improve survival. Monoclonal antibodies targeting the cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed death-1 (PD-1) pathways inhibit downregulation of the immune system, thereby allowing an enhanced T-cell immune response. These pathways are essential regulators in immune tolerance tissue, and their inhibition could lead to a myriad of autoimmune conditions known as immune-related adverse events (irAEs). Patients with preexisting autoimmune diseases were excluded from clinical trials of these therapies, and only one trial included patients with a prior irAE . Here, wA 54-year-old white male with a past medical history of rheumatoid arthritis on anti-TNFalpha therapy with etanercept was admitted to the hospital with a 3-month history of dyspnea on exertion, fatigue, and lower extremity edema after a transthoracic echocardiogram (TTE) revealed a reduced ejection fraction of 40% with a large right atrial mass. Cardiac magnetic resonance imaging (MRI) identified a 5.4\u2009\u00d7\u20095.3 centimeter lobulated right atrial mass  with extThe patient was stabilized and discharged with outpatient medical oncology follow-up to discuss treatment. However, days prior to his appointment he returned to the Emergency Department with worsening dyspnea due to the right atrial mass. Although he had not received treatment for his metastatic melanoma, heart failure due to obstructive cardiac metastasis is generally a poor prognostic indicator. Consequently, the benefits and risks of the procedure were extensively discussed between the medical oncologists and cardiothoracic surgeons. It was determined to proceed with aggressive measures, given the potential for long-term durable responses from immune checkpoint inhibitor therapy. He underwent a radical resection of the right atrial mass  and recoAfter recovery from surgical resection of the metastatic heart lesion, the patient was started on immunotherapy. The patient's rheumatoid arthritis was previously well controlled with etanercept monotherapy, which was stopped prior to treatment. He was started on 3\u2009mg/kg dose of ipilimumab (anti-CTLA4) every 3 weeks. After 3 doses, he developed grade III acute kidney injury, nephrotic-range proteinuria, and anasarca requiring hospitalization. Renal biopsy demonstrated minimal change disease with acute interstitial nephritis. His creatinine increased to 5.22\u2009mg/dl from a baseline of 1\u2009mg/dl. He was treated with pulse methylprednisolone at 1\u2009gm/day and intravenous diuretics with gradual improvement in his kidney function. He did not require hemodialysis, and his steroids were successfully tapered over the next 3 months. Following his steroid taper, imaging demonstrated tumor progression in his peritoneal cavity despite improvement in his liver lesions and no recurrence in his heart.Given clear evidence of progression outside of the pseudoprogression window, he was started on 2\u2009mg/kg pembrolizumab (anti-PD-1) every 3 weeks. He had an objective response to treatment with reduction in tumor burden in his liver and peritoneal cavity. Before treatment with pembrolizumab, the patient's rheumatoid arthritis was controlled with prednisone 5\u2009mg daily. After pembrolizumab treatment, he began experiencing diffuse arthralgias. Although arthralgias are a known side effect of anti-PD-1 inhibitors, he experienced significant morning stiffness and wrist swelling, indicating that he was having a true flare of his autoimmune disease. He was started on hydroxychloroquine for symptom control. With low-dose prednisone and hydroxychloroquine, his synovitis remained stable and low grade. Despite concurrent ongoing treatment with hydroxychloroquine and low-dose prednisone, his disease continued to respond to immunotherapy. The patient was treated with pembrolizumab for approximately 19 months, and he is currently living nearly 3 years after diagnosis in clinical remission and off therapy. He had no recurrence of kidney injury on PD-1 monotherapy.While metastatic melanoma frequently involves the heart, it is rarely diagnosed because it is typically asymptomatic . PatientMultiple studies have demonstrated the importance of the CTLA4 and PD-1 pathways in immune tolerance. Both play a critical role in immune regulation, and decreased gene expression is associated with an increased risk of autoimmune disease \u20139. PatieOur patient's durable response to anti-PD-1 therapy with minimal side effects is impressive given his history of rheumatoid arthritis and prior high-grade irAE. Successful treatment of a patient with anti-PD-1 therapy following high-grade irAE from ipilimumab therapy, without recurrence of that irAE, suggests that it may be possible for patients to be safely treated with immune checkpoint inhibitors of an alternate class following resolution of immune toxicity from the first agent.A recent retrospective study evaluated the safety of anti-PD-1 use in 119 patients with either a preexisting autoimmune disease or an irAE related to prior ipilimumab treatment . 38% of Our patient's clinical course is consistent with the emerging evidence that anti-PD-1 therapy is less toxic and yields better outcomes in comparison to anti-CTLA4 therapy . Despite"}
+{"text": "Many genetic causes of focal segmental glomerulosclerosis (FSGS) have been described. A paradox is that the science in the molecular biology, which generally appears of high quality, is not mirrored by a similarly critical analysis of the renal pathology. FSGS has been applied to such a wide range of conditions that it can reasonably be said to have no useful meaning. Attempts to refine the term have been largely ignored. Study of 252 papers on genetic causes of FSGS found various clinical features. Many papers took the reported diagnosis without question. Few papers reported a pathological review, almost half reported FSGS and up to six other conditions caused by any particular gene, some reported FSGS with recognisable glomerular disorders, over 80% did not apply the Columbia classification, and in nearly all with photomicrographs, the images were not useful for refinement of FSGS. Some workers commented on a lack of genotype-phenotype correlation. One reason is a disregard of the principle that scientific investigation requires an unambiguous definition of the condition studied, to allow others to replicate or refute the findings. Genetic studies of FSGS should use a similarly rigorous approach to renal pathology to that used in molecular biology.The online version of this article (10.1007/s00467-018-4161-6) contains supplementary material, which is available to authorized users. Many genetic causes of focal segmental glomerulosclerosis (FSGS) have been described. As examples, Gast et al.  gave a lThe molecular biology in papers on genetic causes is presumably rigorously considered by referees and editors before acceptance by journals. Mutation of a single base pair in an exon of a gene is commonly reported as a significant finding, although other genetic features such as polymorphisms are also said to be associated with FSGS. The mere finding of an alteration in a gene is no longer enough by itself to justify the conclusion that the name of the gene can be added to the list of those in which a mutation can cause FSGS. Various conditions should be satisfied before this can be done, listed and discussed in detail by Lovric et al.  and otheA scientific paradox considered in this review is the discrepancy between the rigour, precision, close control, reproducibility and generally high quality of the molecular biology and if applicable the experimental work in studies of genetic causes of FSGS and the widespread lack of any of these properties in the selection of cases that are included in reports.FSGS continues to be used as a diagnosis even though virtually every paper or review accepts that the term has no useful, definable meaning applicable to a single condition , 11\u201313. FSGS is often defined tautologously as the finding on microscopy of sclerosed areas, meaning areas that appear solid and stain black with periodic acid-methenamine silver, that are segmental, meaning in only part of a glomerular tuft, and focal, meaning not in every glomerulus. There is a view that more detailed pathological study does not contribute to understanding of FSGS. As examples, comments can be found that \u2018At a practical therapeutic level all FSGS should be considered equal\u2019  and \u2018TheEven the common attempt at refinement by the division into primary and secondary types of FSGS is crude, imprecise and unsatisfactory. What do these terms mean? Is FSGS caused by a genetic disorder primary or secondary or neither? FSGS of presumed genetic cause is often said to be not primary, and so is presumably or explicitly secondary , 13, 18,The Columbia classification of FSGS was an attempt by pathologists to reduce the ambiguity of the term FSGS by the introduction of five types or variants, called cellular, collapsing, hilar, tip and not otherwise specified (NOS) . Even thThere was a range of ways in which people were identified and reported to have a genetic cause of FSGS. Most were found by study of groups with features in common, but some were explicitly reports of a single case or more than one case. The distinction between a case report and a series was not always easy to make, especially in papers about families with an apparently inherited condition. Of the 252 papers, 199 were regarded as series in the broadest sense, including most with familial cases, and 53 as case reports. Familial cases were reported in 71 papers, sporadic cases in 25 and both familial and sporadic cases in 56, while 100 papers did not say whether cases were familial or sporadic. There was a range of ages studied. Only children were included in 119 papers, only adults in 50 and both children and adults in 74, while nine papers did not specify the age of subjects.Clinical features of cases varied. There were three reasons why there was difficulty in determining precise numbers of subjects with different clinical features. At least 17 papers included patients reported in previous studies, sometimes without identifying which ones were included. Patients with different features were grouped in some papers. Some papers did not even give clinical details. The nephrotic syndrome was a common clinical finding and a common reason for inclusion in series, but sometimes, this was specifically only steroid-resistant nephrotic syndrome, sometimes both steroid-resistant and steroid-sensitive nephrotic syndrome, sometimes unspecified nephrotic syndrome and sometimes not differentiated from non-nephrotic proteinuria. Congenital nephrotic syndrome was sometimes included with other types of the nephrotic syndrome, sometimes excluded, sometimes the only clinical feature and frequently not mentioned in childhood studies.For the reasons given, precise numbers of these different features cannot be determined, but at least 145 series appear to have had the nephrotic syndrome as the main or only clinical feature. Other clinical features, if they were mentioned, included such things as non-nephrotic proteinuria, microscopic haematuria, renal failure, hypertension and combinations of features. Some of these were the main reason for inclusion in series.A diagnosis of FSGS was a reason for selection of cases for study in 70 series, but this was accompanied by a variety of clinical features, and was not necessarily the only reason for selection. Accordingly, there is an overlap between these 70 papers and many with the nephrotic syndrome. Thirteen papers did not specify any clinical features in those with FSGS. Ten papers selected FSGS specifically with the nephrotic syndrome but not all with the same type of nephrotic syndrome, as described above, while 34 papers selected FSGS with the nephrotic syndrome or other clinical features, eight selected either FSGS or the nephrotic syndrome and five selected FSGS with other glomerular disorders, such as IgA nephropathy or thin glomerular basement membrane disease. In some series, particularly on patients with steroid-resistant nephrotic syndrome, some were said to have FSGS even though a renal biopsy was reported to show something else , or patiWT1 mutations, such as Frasier and Denys-Drash syndromes (14 papers), various disorders associated with mitochondrial DNA abnormalities, such as MELAS syndrome and maternally inherited diabetes mellitus and deafness (10), Alport syndrome (6), Charcot-Marie-Tooth syndrome (5), Galloway-Mowat syndrome (3), Pierson syndrome (3) and Schimke immunoosseous dysplasia (3). Among the others were seven with tubular disorders, particularly Dent syndrome (3) and Bartter syndrome (2). Other reasons for inclusion in series were, as examples, that there was a known genetic mutation or polymorphism, or simply that the patients had been referred for genetic testing.In 62 papers, both series and case reports, the reason for inclusion was a variety of syndromes that included renal disease, not necessarily with the nephrotic syndrome. The most numerous syndromes were those with There were various clinical reasons for exclusion of cases from 44 series, particularly causes of secondary FSGS such as reduced renal mass or obesity  or extrarenal features (8). HIV infection was included in seven other series. The remaining series and case reports either included patients with extrarenal features (59) or did not mention whether there were any exclusions on clinical grounds (142).NPHS1 and NPHS2, a couple of the earliest genes to be described. In theory, every paper should have searched for all reported genetic causes before an apparently new abnormality was described, which may have been a coincidental and irrelevant finding. Similarly, in theory, whenever any new cause is published, authors of every previous paper should review their cases to see whether that cause was missed, and was a more likely cause or contributor to the condition. Bierzynska and Saleem [In the 252 papers, 91 excluded an abnormality in at least one relevant gene, such as mutations in d Saleem  warned tThis overview shows that genetic studies of FSGS had no consistency in the population studied. Although many patients with FSGS had steroid-resistant nephrotic syndrome, some did not. Was the FSGS reported in anyone with steroid-resistant nephrotic syndrome, for example, the same as the FSGS reported in a case of congenital nephrotic syndrome, or an adult with asymptomatic proteinuria, or a child with Alport syndrome or someone with a tubular disorder?GLA, mutated in Fabry disease, which affects podocytes [COQ2, INF2, LAMB2, LMX1B, SMARCAL1 and WT1, and so genes causing FSGS can affect podocytes and cells outside the kidney. Similarly confusing is the finding that several genetic abnormalities that cause tubular disorders [CLCN5, mutated in Dent disease 1 [SLC12A1, mutated in Bartter syndrome type 1 [The concept of phenocopies of genetic causes of FSGS or steroid-resistant nephrotic syndrome was introduced by Warejko et al. , withoutodocytes . The nonodocytes  includedodocytes , such asisorders  have beeisease 1 , and SLCe type 1 , althouge type 1 \u201339, and e type 1 , 41.WT1 mutations, and Sadowski et al. [NPHS2 mutations. Can genetic abnormalities really cause multiple glomerular disorders, or is a more likely explanation that there was inconsistency between pathologists in these studies?There seemed a readiness of many pathologists to apply the term FSGS and a readiness of those doing the genetic studies to accept the diagnosis. In only 21 papers (8% of 252) was there a mention that there was a review of light microscopic sections on every renal biopsy or every available biopsy or most biopsies by at least one pathologist. This should in theory give consistency in the cases included and excluded e.g. , 42, 43.i et al.  reportedSometimes, among conditions as well as FSGS that were reported in particular mutations, unsatisfactory terms appeared, such as \u2018minimal unspecific glomerular changes\u2019, \u2018unclear (or no) diagnosis\u2019, \u2018mild (or minor) glomerular changes\u2019, \u2018nonspecific tubulointerstitial nephropathy\u2019, \u2018not classifiable\u2019, \u2018unclassifiable glomerulopathy\u2019, \u2018nonspecific pathology\u2019 and \u2018no findings\u2019. This again emphasises how unreliable is mere copying of reports from a variety of pathologists, especially those who are not experts in renal pathology. Also, FSGS was said to occur in glomeruli with diagnoses such as IgA nephropathy , diabetiApplication of the Columbia classification would be a test of whether a paper attempted to refine the diagnosis of FSGS. Columbia appeared in 2004 , althougPhotomicrographs were common in papers on genetic causes of FSGS, and were included in 86 of 252 papers (34%). A problem is that the figures were almost always just of one glomerulus, and an illustration of one glomerulus in itself generally is of little value. For satisfactory interpretation of a segmental glomerular lesion, the ideal information should be a description of the lesion, its site within the glomerulus, which requires the landmarks of the hilum and tubular origin to be shown, the condition and size of the rest of the glomerulus, the number of glomeruli affected and the condition of the rest of the kidney . Hardly No paper mentioned that the position of segmentally sclerosed glomeruli in the cortex was analysed. Following the work of Rich , FSGS isThese findings on the selection, diagnosis, description and illustration of cases show that the literature on genetic causes of FSGS is in confusion. One reason why this is important is because the first requirement for study of any condition is to have a precise definition of that condition so that as in any scientific study, other workers can investigate and replicate or refute the original findings. At the moment, there can be little certainty that any paper on FSGS is studying the same condition as any other paper. The confused state of genetics of FSGS contrasts sharply with the relatively much more straightforward genetics of membranous nephropathy, a diagnosis that is uncontroversial compared with FSGS . The criAnother reason why this is important is related to understanding of the pathogenesis of segmental lesions. A few papers reporting mitochondrial DNA mutations have shown that the lesions in affected glomeruli are at the vascular pole or hilum and are associated with abnormalities of arterioles \u201337, 39. DGKE variants causing a glomerular microangiopathy: \u2018There were also secondary focal and segmental sclerotic glomeruli, which could be seen in the advanced stage of any glomerulopathy\u2019. Although most genes said to cause FSGS are expressed in podocytes [ACTN4 mutations, even though some biopsies did not seem to show significant loss of renal mass, nor glomerular enlargement. They speculated that podocytes with ACTN4 mutations were less mechanically robust than normal podocytes, and more sensitive to damage by normal glomerular capillary pressures. No papers analysed the distribution of segmentally sclerosed glomeruli in the cortex to see if genetic disorders showed the typical childhood pattern, with juxtamedullary glomeruli affected first [Hardly any other papers suggested a mechanism of development of segmental sclerosing lesions, other than vague ideas that the lesions were due to podocyte disorders, which would be expected to produce lesions randomly distributed in glomeruli . One papodocytes , 29, 30,odocytes , who fouodocytes , were coed first , 55.NPHS1 and NPHS2 mutations\u2019. The importance of phenotyping in genetic studies, including the renal biopsy diagnosis, has been stressed by others [A more scientific approach to all cases of FSGS may reveal more precise genotype-phenotype correlations than are currently known. Until this is done, the study of genetic causes of FSGS will remain unsatisfactory. Some workers have expressed disillusionment with reported renal biopsy findings. These include Bierzynska et al. , who wroy others , 64. They others .ESM 1(DOCX 83\u00a0kb)"}
+{"text": "Risk assessment and therapeutic options are challenges when counselling patients with an atypical ductal hyperplasia (ADH) to undergo either open surgery or follow-up only.We retrospectively analyzed a series of ADH lesions and assessed whether the morphological parameters of the biopsy materials indicated whether the patient should undergo surgery. A total of 207 breast biopsies [56 core needle biopsies (CNBs) and 151 vacuum-assisted biopsies (VABs)] histologically diagnosed as ADH were analyzed retrospectively, together with subsequently obtained surgical specimens. All histological slides were re-analyzed with regard to the presence/absence of ADH-associated calcification, other B3 lesions , extent of the lesion, and the presence of multifocality.p\u2009=\u20090.002) and presence of multifocality in VAB specimens (p\u2009=\u20090.0176) were significant risk factors for the underestimation of the disease . In the multivariate logistic regression model, the absence of calcification (p\u2009=\u20090.0252) and the presence of multifocality  in VAB specimens were significant risk factors for underestimation.The overall underestimation rate for the whole cohort was 39% . In the univariate analysis, the method of biopsy , we could not identify a subgroup that would not require an open biopsy. Atypical ductal hyperplasia (ADH) is a small, mostly unifocal, low-grade intraductal lesion in the breast, which in most cases is detected by the associated calcification seen on mammograms \u20134. The hIn our study, we collected histological slides from 207 patients diagnosed with ADH with either a preoperative diagnostic core needle biopsy (CNB) or VAB and correlated the histological findings with the biopsy method and the diagnostic outcome after a subsequent open biopsy. Our aim was to identify any histological parameters predictive of outcome in any subgroup of patients.We analyzed the clinicopathological data of 207 patients histologically diagnosed with ADH, classified as B3 , or B4 , by CNB or VAB, who had undergone subsequent open surgery in 2002\u20132015. Of the 207 diagnostic biopsies, 56 were CNBs and 151 were VABs. All biopsies were performed at the Breast Center Seefeld Zurich and were histologically processed at the Institute of Pathology and Molecular Pathology of the University Hospital Zurich, Switzerland.\u00ae , Suros Eviva\u00ae , or Mammotome\u00ae . In general, 7G or 9G needles were used for the biopsies performed under magnetic resonance imaging (MRI) and ultrasound (US), and 11G needles were used for stereotactic or tomosynthesis-guided biopsies. All procedures were performed under local anesthesia, except in two patients who required general anesthesia because multiple bilateral lesions were removed under US guidance.In 2002\u20132015, 5213 VABs were performed at the Breast Center Seefeld Zurich, using EncoreOf the 5213 VABs, 1722 were guided by US (handheld), 125 by MRI, and 3366 by stereotactic surgery . The main indication for stereotactic or tomosynthesis-guided VAB was suspicious microcalcifications, and the indications of asymmetric density and architectural distortion were observed in only a few patients. MRI-guided VAB was indicated when a suspicious MRI finding could not be confirmed by mammogram or a second look US. The indication for handheld VAB was a US finding requiring histological clarification. As well as benign findings (71.30%), DCIS (10.5%), and invasive cancers (3.95%), 13.8% of the specimens showed cellular atypia, and 191 (3.7%) of these were classified as ADH.In 2001\u20132015, 15,528 US-guided CNBs (with 14G needles) were performed, 66 of which indicated a diagnosis of ADH.The decision to perform initially either CNB or VAB depended on the imaging characteristics. CNB was the preferred method in case of a visible, mostly mass lesion on ultrasound while VAB was the preferred method in cases with mammographically detected calcification not visible at ultrasound.We included in this study only patients with ADH who had undergone subsequent open surgery and for whom it was possible to correlate the ADH diagnosis based on VAB or CNB with that based on the surgical specimen.The statistical analyses were performed with frequency tables and univariate and multivariate logistic regression models. All statistical analyses were performed by the Swiss Group for Clinical Cancer Research, Bern, Switzerland.All biopsy materials were fixed in 4% neutral buffered formalin and embedded in paraffin blocks with routine histological processing. Sections (2\u00a0\u00b5m thick) were prepared from the paraffin blocks; different levels from each block were cut separately and stained routinely with hematoxylin and eosin.For all biopsies, the diagnosis of ADH was made according to the current World Health Organization classification of breast tumors . ADH wasThe presence of calcification and multifocality (as B3 or B4 category) was noted. Findings additional to ADH, including lobular neoplasia (LN), radial scar (RS), flat epithelial atypia (FEA) and intraductal papilloma, were noted in the reports. In many cases, immunohistochemical analyses of estrogen receptors and basal cytokeratins 5/6 were also performed to confirm the ADH diagnosis. The diagnosis of ADH was confirmed if immunohistochemistry showed the diffuse upregulation of estrogen receptors and negativity for basal cytokeratins  Fig.\u00a01)1).The whole cohort underwent a retrospective review, and the histological  slides were reviewed by two pathologists who are specialists in breast pathology . The following features were assessed in the review: the histological diagnosis of ADH, the presence of multifocality in the biopsies, the presence of necrosis and associated calcification, and the extent of the lesion (in millimeters). The presence of FEA, RS, LN, and papilloma was also noted in the histological review.Additionally, an age parameter (cut-off at 50\u00a0years) was added to the criteria.\u2018Upgrade\u2019 was defined as a diagnosis of DCIS or invasive carcinoma based on the subsequently obtained surgical specimens  could be re-analyzed by reviewing the histological slides and classifying ADH according to the histological criteria mentioned above.p\u2009=\u20090.002) and the presence of multifocality in the VAB specimens  were significant risk factors (Tables\u00a0p\u2009=\u20090.0252) and the presence of multifocality  in VAB specimens as significant risk factors. The other parameters including the age parameter showed no significant association with the rate of disease upgrade based on subsequent surgical specimens. We did not identify large comedo type necrosis, however, smaller necrotic foci in association with calcification were observed. In the surrounding breast tissue, no relevant calcifications were found.The histological findings of additional B3 lesions are listed in Table\u00a0s Tables\u00a0. The mulThe upgrade rate in the cohort with the lowest risk  was 16.5% (10 DCIS and 1 invasive cancer in 63 patients). The overall upgrade rate in the whole cohort was 39% .To the best of our knowledge, this is the largest study to show that the biopsy method (CNB or VAB) influences the outcome determined based on surgical specimens, with a greater rate of disease upgrade after diagnosis by CNB than by VAB. In one previous study, Badan et al. reported similar observations in 40 patients . Our casSeveral previous studies have analyzed the relationships between pathological parameters  with the outcome determined from surgical specimens , 19, 27.Our results partly support those of previous studies regarding the importance of calcification. Our study shows that calcification predicts diagnostic upgrade only in cases of ADH diagnosed by VAB, with a significantly lower upgrade rate in patients with calcification. The upgrade rate in cases of ADH diagnosed by CNB was independent of the presence of calcification. A relationship between calcification and diagnostic upgrade has been reported in previous studies , 30. OurIt can be assumed, that ADH cases detected on VAB lacking calcifications were most likely representing a non calcified extension of DCIS, which was not detectable as such in mammography.The data reported in the literature on multifocality and the diagnostic upgrade of ADH are controversial , 20, 25.Interestingly, as in our data, the data in the literature show fairly consistently that there is no association between ADH upgrade and the presence of other B3 lesions, such as LN, papilloma, RS, or FEA , 23, 31.One earlier study by Hartman et al. reported a significant interaction between premenopausal status and histological findings. Breast cancer risk among patients <\u200945\u00a0years at the time of ADH diagnosis, was 6.99 times the expected risk, which was significantly lower (3.37) in postmenopausal patients (>\u200955\u00a0years), as was shown in Fig.\u00a02 in this paper . InteresIn conclusion, we have provided evidence that the biopsy method used (CNB or VAB) influences the diagnostic upgrade rate. Especially in cases of ADH diagnosed by VAB, the presence of calcification in a unifocal lesion is associated with a significantly lower disease upgrade rate than is the absence of calcification and/or the presence of multifocality. These factors should be considered, in addition to imaging characteristics, when deciding whether a patient should undergo open surgery or simply be managed conservatively. These data require validation in further studies."}
+{"text": "The intercalated IL-anions promoted LDH swelling in monomers and LDH delamination during the course of in-situ polycondensation, which led to the production of PBAT/LDH nanocomposites with intercalated and exfoliated morphology containing well-dispersed LDH nanoplatelets. The prepared nanocomposite films showed improved water vapor permeability and mechanical properties and slightly increased crystallization degree and therefore can be considered excellent candidates for food packaging applications.Currently, highly demanded biodegradable or bio-sourced plastics exhibit inherent drawbacks due to their limited processability and end-use properties . To overcome all of these shortcomings, the incorporation of lamellar inorganic particles, such as layered double hydroxides (LDH) seems to be appropriate. However, LDH delamination and homogenous dispersion in a polymer matrix without use of harmful solvents, remains a challenging issue, which explains why LDH-based polymer nanocomposites have not been scaled-up yet. In this work, LDH with intercalated ionic liquid (IL) anions were synthesized by a direct co-precipitation method in the presence of phosphonium IL and subsequently used as functional nanofillers for in-situ preparation of poly (butylene adipate- Currently, engineering plastics are designed as sustainable materials with an adjustable lifetime in order to minimize their impact on the environment. The challenging issue is thus to prepare advanced plastics either with (i) increased durability or (ii) easy and fast degradability by means of abiotic factors  and/or biological  attack. The first are hi-performance materials with a long service life, while the latter are often designed as biodegradable polymers for packaging with a typical life-time shorter than one year.co-terephthalate) (PBAT) is a typical representative of aliphatic-aromatic copolyesters, which provide balance between biodegradability of aliphatic segments and thermo-mechanical properties of aromatic units. PBAT is a flexible material with high elongation at break and thus suitable for coatings and film applications x+(An\u2212)x/n\u00b7mH2O, where M2+ and M3+ are divalent  and trivalent  layer cations, respectively, and An\u2212 is the exchangeable inorganic anion  in the interlayer space with x being the relative substitution rate generally ranging between 0.20 < x < 0.33 resulting in M2+/M3+ ratios of 2/1\u20134/1 . Th. Th\u22121 , (006), and (009) planes, respectively. The (003) reflection of pristine LDH corresponded to the basal spacing value of 0.77 nm, which is typical for unmodified Mg-Al LDH [The XRD measurements enabled us to indicate whether the IL-anions were intercalated into the basal spacing of LDH. The XRD pattern of pristine LDH a showed g-Al LDH ,35,36.\u03b8 range\u20143.39\u00b0, 3.05\u00b0, and 2.19\u00b0 for LDH-phosphate, LDH-decanoate, and LDH-phosphinate, respectively  reflection in the lower 2ectively , denoteser anion . Assuminer anion , then th authors  or by th32\u2212 decomposition, and dehydroxylation of the metal hydroxides proceed simultaneously.Thermogravimetric (TG) and derivative weight TG curves of all LDH samples display an initial weight loss (8\u201313 wt %) between 50 \u00b0C and 250 \u00b0C  due to t32\u2212-intercalated into Mg-Al LDH was always present as a contaminant. The PBAT nanocomposites containing LDH-phosphate  revealing that the layered particles were neither exfoliated nor intercalated during the in-situ polycondensation. On the contrary, a complete disappearance of the reflection peak at a low angle range (2\u03b8 < 4\u00b0) was observed for all PBAT nanocomposites with the modified LDH . In such conditions, the permeation, diffusion, and solubility mechanisms in the system resembled gas transport in liquids. Crystallinity of the neat PBAT film (8%) was slightly enhanced by the presence of LDH fillers (<13%\u2014see the DSC result below). However, such low overall crystallinity in the PBAT/LDH nanocomposites has a negligible effect on the evolution of gas transport properties [The permeability coefficients of all samples increase in the order of O2 << H2O . The peroperties .Since the permeability of gas and water vapor molecules was directly proportional to diffusion and solubility coefficients, the more dominating process can be determined based on the diffusion and solubility selectivities. In the cases of neat PBAT and PBAT/LDH nanocomposites, the gas and water vapor permeabilities were preferably driven by solubility in PBAT rather than by diffusion  . General2 and H2O [2 and H2O are much higher than those of O2, which further results in significantly faster permeation of these polar molecules through PBAT materials as compared to O2. Neutral oxygen interacted weakly with PBAT and therefore relatively high selectivities for CO2/O2 gases were obtained.It seems that the relatively high polarity of the PBAT backbone caused by the presence of ester groups promotes strong interactions between PBAT and polar molecules such as CO2 and O2 permeations by acting as a physical barrier, decreasing diffusion but only negligibly affecting solubility, which is in agreement with the theory of transport properties of polymeric materials filled with impermeable particles [The LDH fillers in PBAT nanocomposites were shown to slightly lower COarticles ,45. Gas articles .2 and O2 transport properties, the neat PBAT exhibited high water vapor permeabilities , where Ps and Pp are WVP of PBAT/filler (in our case PBAT/LDH) nanocomposite and neat PBAT, respectively, are depicted in Contrary to COies WVP, , which lies WVP, a. In conies WVP,  was seleOur results are promising compared to the results so far published in the literature. They show the most significant improvement in the water vapor barrier properties of PBAT/LDH-phosphate nanocomposites, especially considering the relatively low nanofiller content (1.5 wt %). The materials prepared in our last study displayed similar WVP results only when higher amounts (2 wt %) of IL-modified LDH was incorporated into the PBAT matrix . In thatOther lamellar fillers  hagT) values increased with the increasing content of the modified LDH containing IL-anions, which indicates homogenous nanofiller dispersion in the PBAT matrix and LDH delamination into individual LDH layers thus reducing mobility of PBAT chains. In contrast, the incorporation of pristine LDH showed no influence on gT due to poor nanofiller dispersion in the PBAT matrix. The increase in crystallinity (cX) and the decrease in melting temperature (mT) of PBAT with increasing content of modified LDH indicates that the particles act as heterogeneous nucleating agents promoting the PBAT crystallite growth [e growth ,9,15.dT5%) of the PBAT matrix  was used for synthesis of IL-anion intercalated LDH. Using this technique, bis (2-ethylhexyl) phosphate (IL-phosphate), decanoate (IL-decanoate), and bis  phosphinate (IL-phosphinate) anions were successfully introduced into the interlayer space of LDH compounds. The produced LDH show the increased interlayer spacing and high content of IL-anions. The results surprisingly show that LDH-IL prepared by this technique practically does not contain surface-bonded IL (in contrast to anion exchange technique used in our last study ). The wa2 and O2 permeability reduction while the water vapor permeation was significantly decreased for all PBAT/IL-modified LDH nanocomposites. From this point of view, the produced PBAT/LDH nanocomposites are considered excellent candidates for food packaging applications.In the next step, LDH with intercalated IL-anions were shown to delaminate readily in a mixture of monomers during in-situ polycondensation. The produced PBAT/LDH nanocomposites exhibited exfoliated morphology either with homogenously dispersed LDH nanoplatelets in the PBAT matrix  or formation of LDH-rich domains in the PBAT matrix . Moreover, the IL-phosphinate modifier was shown to ensure the strongest LDH-PBAT affinity resulting in optimized mechanical performances. The presence of IL-anion intercalated LDH exhibited a relatively low effect on CO"}
+{"text": "The literature investigating female and male medical students\u2019 differing career intentions is extensive. However, medical school experiences and their implications for professional identity formation and specialty choice have attracted less attention. In this study we explore the impact of medical school experiences on students\u2019 specialty preferences, investigate gender similarities and differences, and discuss how both might be related to gender segregation in specialty preference.interested and uninterested in a specialty. Utilizing a sequential mixed methods design, their responses were analyzed qualitatively to create categories that were compared quantitatively.In a questionnaire, 250 Swedish final-year medical students described experiences that made them Similar proportions of women and men became interested in a specialty based on its knowledge area, patient characteristics, and potential for work-life balance. These aspects, however, often became secondary to whether they felt included or excluded in clinical settings. More women than men had been deterred by specialties with excluding, hostile, or sexist workplace climates . In contrast, more men had been discouraged by specialties\u2019 knowledge areas .Male and female undergraduates have similar incentives and concerns regarding their career. However, the prevalence of hostility and sexism in the learning environment discourages especially women from some specialties. To reduce gender segregation in specialty choice, energy should be directed towards counteracting hostile workplace climates that explain apparent stereotypical assumptions about career preferences of men and women. Despite a preponderance of women in medical schools there remains an unequal distribution of men and women over specialties and in medical leadership , 2. A stIn parallel, there is research showing how experiences during clinical training, such as role modeling or exposure to harassment, affect students\u2019 sense of belonging and (dis)identification on different wards, thus driving them to avoid particular specialties \u201316. MostInclusion is also fundamental to professional identity formation . In ordeAttitudes towards gender in the educational environment have important implications for medical students\u2019 clinical experiences. Female students are more likely to encounter sexism such as gender related prejudice, gender discrimination, and sexual harassment, than are their male peers , 26, 27.Researchers have suggested that the cumulative effect of overt and covert forms of sexism in the learning environment create an adverse climate for female students that dampens their confidence, participation, and aspirations , 28, 29.In a questionnaire to Swedish final year medical students we included open-ended questions about concrete experiences that made them interested versus uninterested in a specialty. Our aim was to explore the impact of medical school experiences on students\u2019 specialty preferences, to investigate similarities and differences between men and women regarding the character and consequences of experiences described, and to discuss how this might be related to gender segregation in specialty preference.This study is part of the project \u2018Gender Challenges in Medical Education\u2019, aimed at investigating medical students\u2019 attitudes and thoughts about gender-related questions . A studyThe study was conducted at the medical school at Ume\u00e5 University in Northern Sweden. In Sweden, paid parental leave and subsidized childcare encourages women and men to share paid and unpaid work . At presUndergraduate medical education in Sweden comprises 5.5\u00a0years, including 3\u00a0years of clinical training. The undergraduate period is followed by an 18\u201324\u00a0month internship that is required before applying for a residency position.During the clinical phase of their program, medical students in Ume\u00e5 rotate between different wards at the university hospital, local hospitals, and health care centers in the region. The proportion of female medical students in Ume\u00e5 has been 50\u201360% in the past 10\u00a0years. According to questionnaires administered to all students in 2006\u20132009 a large majority came from a middle class background and less than 10% had parents who were born outside of Scandinavia .: \u201cCan you describe an event that made you interested in working in a certain specialty?\u201d and: \u201cCan you describe an event that made you uninterested in working in a certain specialty?\u201d. Subsequent questions and scales measuring respondents\u2019 attitudes to gender issues in medicine are not included in this analysis.Between 2011 and 2013, final-year medical students at Ume\u00e5 University were invited to respond to the anonymous questionnaire \u201cGender in medical education\u201d. After demographic queries, the survey continued with two open-ended questionsStudents were informed about the study when attending compulsory lectures, unrelated to the study, and participation was voluntary. No attempt was made to reach students who did not attend the lecture where the questionnaire was administered. The lecture, however, was mandatory and we have no reason to believe that the absent students differed in any specific way from those present.Out of 404 students  registered during the study period, 344 students were present at the lectures and therefore invited, and 305 of them stayed on to fill out the questionnaire. After exclusion of 55 students who did not answer either of the two open questions 250 remained , 104 men), giving a response rate of 73%.The Regional Ethics Committee in Ume\u00e5 approved the study: (dnr 2011\u2013262-31\u00a0M.). The return of students\u2019 completed questionnaires was considered as consent for participation.p-value <\u20090.05 was considered significant.Pearson\u2019s chi-square test was used for comparing socio-demographics for responders and non-responders. A We adopted a simple sequential mixed-method design to explore students\u2019 experiences and observe similarities and differences between men and women . It was Prior to the analysis, students\u2019 responses were \u2018blinded\u2019 from socio-demographic information, e.g. gender. However, a few participants referred to themselves as either a man or a woman in a way that was not possible to blind. The last author (JA) read one third of the answers to get an overview of the content, conducted an open coding and created a preliminary coding schedule. Answers from 100 students were then read, coded and categorized independently by the four Swedish-speaking authors . In joint sessions, the coding and labeling of categories was then compared, discussed, and reformulated. Based on this, answers from another 50 students were read, categorized, and discussed. This procedure was repeated one more time, at which point the elaborated categories seemed consistent and varied enough to capture the variety of answers. In this way a final coding schedule of eight categories was established. Thereafter, the first author (EK) reread and encoded all answers, i.e., conducted the main coding. To facilitate understanding and presentation, related categories were grouped into three themes.To check the reliability of the main coding, two of the authors  individually encoded 160 randomly chosen answers (80 respectively about experiences inducing interest and disinterest). Each answer was encoded according to the categories in the final coding schedule and the codings were then compared to the first authors coding. A total of 68 discrepancies were identified and these were spread among authors as well as answers about interest and disinterest. With three authors , 160 answers, and eight categories the percentage of discrepancies was less that 2% (68/(160\u2009\u00d7\u20098\u2009\u00d7\u20093)\u2009=\u20090,018). Thus, the inter-rater reliability test showed high consensus, suggesting that the categories were clear and consistent.p-value <\u20090.01 was considered significant. The number of words in the answers was also counted and compared between men and women (range and mean).When the main coding was completed and all answers sorted into categories, we created a chart in SPSS (version 24.0 for Mac OSX). If a category was coded in an answer it was marked with \u20181\u2019, otherwise with \u20180\u2019. The proportions of answers coded with \u20181\u2019 within each category were compared between inspiring and deterring experiences, and between men and women, and tested using the Pearson chi-square test. A Below we present students\u2019 socio-demographics, followed by the themes and categories developed in the qualitative analysis of free text answers. Finally, we present the statistical analysis of gender patterns in the categories.Men and women had similar socio-demographics, as shown in Table\u00a0The answers varied in level of detail and depth. Most responses coded for more than one category. The results comprised three themes; \u2018The character of work suits me\u2019, \u2018Inspiring and inclusive workplace\u2019, and \u2018Matches my work-life priorities\u2019. Each theme consists of two to three categories. The content and examples of all categories are outlined in Table\u00a0In our presentation of themes, more space is given to the second theme \u2018Inspiring and inclusive workplace\u2019. The reason is that this theme in particular conveyed examples of students\u2019 educational experiences and the relational settings in which professional identity formation is situated. The answers within the other two themes were often shorter, included less variation, and conveyed simple arguments and views rather than accounts of concrete experiences. Still all three themes are important and were often interconnected.In the excerpts, details about wards and staff have been modified to ensure confidentiality. For example, quotes pertaining to specific surgical specialties e.g.: \u2018general surgery\u2019, \u2018orthopedics\u2019, etc., have been changed to \u2018surgery\u2019 or \u2018surgical specialty\u2019.\u2018Knowledge area and practice\u2019: Participants recounted specific work tasks that intrigued them or matched their perceived talent, like \u2018the discussions\u2019 and the \u2018detective work\u2019 of internal medicine, or the surgical \u2018craftsmanship\u2019. Interest in specific work tasks, however, was often initiated, or maintained by invitations to hands-on participation:I was interested in surgery before I started medical school, but what kept me interested was that I got the opportunity to try a lot on my own. (woman)Correspondingly, being a passive bystander rendered the same knowledge area or work assignments being labeled as \u2018boring\u2019, \u2018monotonous\u2019, and \u2018deterring\u2019.\u2018Patient characteristics and patient contact\u2019: Rewarding patients were portrayed as grateful, or \u2018giving a lot back\u2019 \u2013 spanning from particular age groups, like children and elders to people with concrete problems that could be cured or \u2018fixed\u2019. Meeting patients with severe or incurable diseases, or trauma victims, could also make a lasting impression.When I met a patient with chronic pain, who had very little drive and who was totally uninterested in working to reach forward. For some reason, this provoked me. (man)\u2018Values and care ideology.\u2019 Some participants considered whether they shared values and care ideology (e.g. patient-centered care) with physicians in a particular specialty. Others wanted to make a difference by working in medically underserved areas of the world. Workplaces where values were at odds with those of the student were described as dissuading:I lose interest when all that matters is how fast you finish off the operation list - not how well you take care of the patients. (w)Conversely, patients suffering from \u2018multiple morbidities\u2019, diffuse symptoms, or those who were \u2018unmotivated\u2019 or \u2018ungrateful\u2019 were discouraging for some students:\u2018Workplace climate\u2019: Often participants explained that even though patients and work tasks were important, the workplace climate was paramount when opting for a specialty:The clinic and my co-workers will be of greater importance than the work itself. (w)Students were attracted by nonhierarchical wards, well functioning cooperation, and staff who seemed to enjoy their job and supported each other \u2013 factors that often were interconnected. However, workplace climates, characterized as \u2018hierarchical\u2019 and \u2018tough\u2019, and with senior physicians who treated colleagues of lower rank poorly were described as reasons for not choosing a certain specialty. Most examples concerned male-dominated and surgical wards where students also noted prevalence of \u2018abuse of power\u2019, \u2018sexist jokes\u2019, \u2018macho jargon\u2019, and \u2018macho attitudes\u2019; behaviors with which they disagreed:Chauvinistic and macho atmosphere reoccurred on morning rounds. Sexist jokes were told and I lost interest completely for that clinic. I just couldn\u2019t stand it there. (w)There were also negative accounts of competitive rather than supportive attitudes, as well as accusations and conflict rather than solidarity where \u2018not showing weakness\u2019 and \u2018not asking for help\u2019 were encouraged. Likewise, settings where residents were expected to be wholly devoted to work and have no other interests were deterring.Since I have seen what the jargon is like at several surgical clinics and how unpleasant many surgeons are, I am no longer interested in working there although I still think it is an exciting subject. (m)Overall, accounts of workplace climate did not explicitly concern gender, although \u2018macho jargon\u2019 and \u2018sexist jokes\u2019 was mentioned. Still, some women overtly discussed gender related issues. Seeing specific challenges facing women in male-dominated clinics, for example difficulty gaining respect, worried them:The only female physician expressed her disappointment over that the other physicians did not prioritize the ward. The atmosphere became very tense, and she got very little support. I do not want to work as the sole female physician in my team. (w)\u2018Supervision and participation\u2019: Participants\u2019 interest was spurred by experiences of being seen, included, and taken seriously by supervisors:I had good contact with a supervisor who gave me the \u201cright\u201d space to work independently, combined with good support. As a result I had fun during my placement and thus aroused interest in the specialty. (w)Positive attention, like constructive feedback and praise, or being offered a job, made long lasting impressions. Active participation, and getting to do practical tasks were also important. This student described his overwhelming first experience in the operating room:I went from being totally uninterested to being quite interested in surgery the first day of the clinical placement. I got to assist on a surgery and meanwhile I got fantastic tutoring from the surgeon. (m)Lack of supervision or participation on the other hand made a negative impression, as did being \u2018used as free labor\u2019, or feeling \u2018excluded\u2019 or \u2018ignored\u2019:I lost interest already at the first morning meeting when all the surgeons treated us students like we were invisible, and also ignored pretty much everything the interns and everyone else said. (m)Some respondents disclosed how they had been subjected to domination techniques, or felt humiliated by supervisors. Such treatment had the power to literally \u2018destroy\u2019 clinical placements. Experiences of neglect were also detrimental and some students, particularly women, described such incidents as gender related:I lose interest when I feel spoken to as if I'm not a potential future colleague at work - usually from a male physician. (w)\u2018Role models\u2019: Role models depicted as caring and empathic towards patients and students, who seemed enthusiastic and dedicated even after many years within the profession, were highlighted as important for specialty preferences:When I meet an experienced physician that is still excited about patients and being a supervisor. (m)Negative role models in contrast were those who demonstrated cynicism and detachment towards their work. They belittled students and staff, and tried to escape their responsibilities as physicians and supervisors. Other accounts pertained to physicians who treated patients insensitively and harshly:When a surgeon during the morning rounds in an abstruse and cold-hearted way notified a patient that he had cancer, a patient who had lost his wife in cancer a couple of years earlier. (w)Some students disclosed that experiencing a bad environment compelled them to revise previous career plans:\u2018Workload\u2019: Working office hours and being in control of one\u2019s work schedule, often related to the potential of combining paid work with family and leisure time, was generally considered preferable. Poor organization, heavy workloads, and seeing residents and consultants staying on past regular hours, and working nightshifts scared the students:It doesn\u2019t matter how interesting a specialty is, if I have to be awake at night I will not be able to appreciate it. I don\u2019t want to have to \u2018live through\u2019 a shift. I want each day to be enjoyable and rewarding. (w)\u2018Development possibilities\u2019: Some accounts pertained to the potential for professional development in a specialty, for example, \u2018platforms for improving communication skills\u2019, \u2018promising technical innovations\u2019, and research possibilities. Some also gave negative reports of \u2018scarce development possibilities\u2019 or \u2018expectations to work without pay\u2019.Most participants  responded to both of the open-ended questions, resulting in 220 answers regarding experiences inducing interest and 224 answers about experiences inducing non-interest Figures\u00a0 and non-p\u00a0=\u20090.013). Workplace climate was the third most common incentive for women. Among men, the third most prevalent incentives were \u2018Supervision and participation\u2019 and \u2018Role models\u2019 (both M\u2009=\u200919%). Compared with men, women gave longer answers including more categories. As a result, even though \u2018Supervision and participation\u2019 and \u2018Role models\u2019 did not rank among the top three incentives among women, they still occurred just as frequently as for men (at 18 and 19% respectively).Men and women showed a similar pattern with no significant differences found when comparing the relative proportions of men and women describing experiences that fostered interest in a specialty Fig . Examplep\u2009<\u20090.001). There was also a significant gender difference in the category \u2018Knowledge area and practice\u2019 which was the most common deterrent among men but the third most common deterrent for women . The category \u2018Supervision and participation\u2019 ranked second (at 28%) among women\u2019s deterring experiences, followed by \u2018Workload\u2019 (W\u2009=\u200923%). Among men, the second and third most common deterrents were \u2018Workload\u2019 (M\u2009=\u200920%), closely followed by \u2018Supervision and participation\u2019 (M\u2009=\u200919%).Gender patterns were more disparate for deterrent experiences Fig . The larWe explored how medical students\u2019 specialty preferences are shaped by educational experiences, and investigated similarities and differences between men and women regarding the character and consequences of experiences described. Male and female participants shared common inspiring experiences pertaining to knowledge area, their own talent, and patient groups. However, participants\u2019 impressions of workplace climate, supervision, and participation were often described to be of greater importance in their specialty considerations. These experiences rendered feelings of inclusion or exclusion and sometimes outweighed other experiences during clinical practice. Among women, the most common reason for avoiding a specialty was related to experiences of hierarchical, hostile, and sexist workplace climates. In contrast, perceived lack of interest in the knowledge area of a given medical field was the most common reason for men to avoid a specialty.Earlier studies have showed that it is mainly women who consider patient orientation, while men focus on technical challenges \u20138. By exEven if specialty preference at first seemed to be about students selecting it was just as much a matter of feeling selected and welcomed at a workplace. Knowledge area was the most common factor that made students, women and men alike, interested in a specialty. However, the fact that more women than men described experiences of a hostile and unwelcoming workplace climate made it more of a male privilege to choose a specialty according to interest in its knowledge base. In keeping with previous research, there were more examples of unwelcoming workplace climates from male-dominated and surgical specialties , 27. ThuA sexist jargon and difficulties for women gaining respect described by our participants, convey the message that women are less worthy and less capable than men , 29. It Publicly espoused professional values in Swedish health care include respect, empathy, and gender equality . Our resLave and Wenger  propose Another example of resignation concerned work-life priorities. Our students were dissuaded from specialties where they saw residents struggling with high workloads and expectations to prioritize work above all else. Work-life balance is often suggested to be an issue for women , 5, 8, ball students with good opportunities to pursue their career goals, and improve their professional development.Even if students can discard specialties where supervisors display unprofessional behaviors and sexism they still cannot avoid interaction with these physicians during compulsory clinical training. Although it was primarily women who raised concerns about an adverse workplace climate, quite a few men had similar complaints and changes in the climate would probably benefit the professional development of all students, even those who are not apparently troubled by the problem. By creating welcoming workplace climates, medical schools and other stakeholders in the future medical workforce can therefore provide However, this might constitute a challenge, as research shows that elusive gendered inequities are often communicated by well-intended people who are unaware of their harmful conduct , 29. TheWe used free-text answers from a questionnaire, analyzed by means of mixed methods, to explore and compare how male and female medical students experiences from clinical practice might affect their specialty preferences. The use of qualitatively evolved categories grounded in the data when performing statistical analyses helped us highlight what the students themselves chose to focus on instead of using pre-defined categories.The questionnaire enabled us to represent a large number of students and their multiple realities and to search for gender patterns. The response rate was high and it was similar for women and men, reflecting the distribution of women and men at the Ume\u00e5 medical school. Although in-depth answers from students were lacking, the answers were given spontaneously by all without having to probe. However, a limitation inherent in using a questionnaire is that it precludes researchers from asking respondents to clarify comments.In research focusing on differences between women and men there is always a risk of exaggerating gender differences. Thus, the open-ended questions were placed at the beginning of the questionnaire, in order to lower the risk of biasing responses by subsequent questions concerning gender. Furthermore, we did not search for gendered patterns until after categories were outlined, a procedure we believe limited the risk of exaggerating gender differences caused by researchers\u2019 expectations. Finally, since women\u2019s answers were, on average, longer and included more categories than did men\u2019s we considered which categories were the most common in women and men\u2019s answers respectively, rather than focusing only on the gender differences in each category separately.Our findings are confined to the perceptions of students from one Swedish medical school. Even though Sweden ranks high on international measures of gender equality , partici(Un)professional beliefs, values, norms, and behaviors among supervisors and staff shape the clinical experiences of medical students, affecting their professional identity formation and how they imagine their future careers. Although male and female undergraduates have similar incentives and concerns regarding career, they enter clinical environments that tend to be more hostile for women, resulting in them feeling less welcomed or even excluded. Irrespective of students\u2019 interests and aptitudes, feelings of inclusion or exclusion in specific workplaces, likely affect subsequent specialty choices. Thus, the understanding of gender as merely a socio-demographic factor, which primes women for \u2018family friendly\u2019 specialties and part-time work, and men for technical specialties and prestigious careers, needs to be revised. Future research and debates about gender segregated specialties should be directed towards countering problematic workplace climates that still make it more of a male privilege to choose specialties according to interests."}
+{"text": "Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors. T cells play an important role in the execution of the adaptive immune response by regulating the activity of innate and other adaptive immune cells or directly executing effector functions, such as killing by cytotoxic T cells. In general, for T cells to execute their function, they need to become activated by antigens through interaction with an antigen-presenting cell (APC). Crucial to this activation is the interaction between the T cell receptor (TCR) on the T cell surface and the peptide-Major Histocompatibility Complex (pMHC) on the APC surface. Immediately after recognition of the pMHC, the TCR, associated with the CD3 receptor complex, combines with co-stimulatory receptors CD4/CD8 and CD28 on the T cell membrane in small so-called TCR microclusters where signaling is initiated , 2. DuriCD6 is one of the cell surface co-receptors on the T cell membrane involved in T cell activation. CD6 is a transmembrane glycoprotein, part of the scavenger receptor cysteine-rich superfamily (SRCR-SF), that is expressed on thymocytes, mature T cells, a subset of B cells and NK cells, and brain parenchymal cells \u201318. On tAlready early on, it was clear that CD6 was involved in T cell activation in mature T cells, since monoclonal antibodies targeting CD6 were able to induce T cell activation, subsequent T cell proliferation and IL-2 receptor expression , 24. SinMultiple data hint at an interaction, either direct or indirect, between CD6 and the TCR. Co-precipitation studies have indicated that rat CD6 associates with protein kinases Lck, Fyn, ZAP-70, and Itk: protein kinases that also interact with and co-precipitate with the TCR or are part of the LAT signalosome , 32. ThiCD6 gene, together with the gene for its ligand ALCAM, was identified as a susceptibility locus and a potential target for treatment of multiple sclerosis  Microscopy, combined with biochemical or immunological assays, such as supported lipid bilayers , have be*105 Jurkat cells were transfected at 1325 Volt  with 3 \u03bcg of DNA in 100 \u03bcl Resuspension buffer. After transfection cells were seeded in 2 ml of 1640 RPMI with 10% FCS and 1% U-glut. Antibiotics were added after 3 h. Stable cell lines were sorted on RFP or GFP expression on a FACSAria cell sorter (BD Biosciences), and cells were maintained in complete RPMI medium as described above, additionally supplemented with 500 ng/ml geneticin .Jurkat E6.1 lymphoma T cells were maintained in 1640 RPMI (PAA) supplemented with 10% Fetal Calf Serum (Greiner Bio-one), 1 mM Ultra-glutamine  and antibiotics . Jurkat cell lines stably expressing CD6-RFP, CD6-GFP, or LifeAct-GFP were obtained by electroporation using the Neon Transfection System for Electroporation (Invitrogen) according to the manufacturer's guidelines. Shortly, 5The following primary antibodies were used: Mouse IgG2A-anti-human CD3 antibodies clone T3B and clone OKT-3 (both referred to in the text as \u03b1CD3), and Mouse IgG1 anti-human LFA-1 antibody TS2/4 were obtained from in-house hybridoma production. Mouse IgG1 anti-human phospho-tyrosine (P-Tyr-100), both unconjugated and conjugated to Alexa488, was obtained from Cell Signaling Technology; Mouse IgG1 anti-human CD6  was obtained from BD Biosciences. The following secondary antibodies were used: Goat anti-Rabbit-IgG(H+L)-Alexa647 and Goat-anti-Mouse-IgG1-Alexa488 (both from Invitrogen). Neutravidin-TexasRed was obtained from Thermo Fisher Scientific. For use in immunofluorescence staining, anti-CD3 antibody clone OKT-3 was biotinylated  at RT for 1.5 h, with a molecular ratio of IgG:Biotin at 1:15. Following the same procedure, for use in supported lipid bilayers, anti-human CD3 antibody OKT-3 was simultaneously biotinylated and conjugated to ATTO647 Carboxylic Acid, Succinimidyl ester (ATTO-TEC) at a molecular ratio of IgG:Biotin:dye at 1:15:15. In both cases, purification was performed with Zeba Desalting columns (Thermo Fisher Scientific). For preparation of supported lipid bilayers, the lipids POPC  and Biotin Capped PE , both from Avanti Polar Lipids Inc, were used, together with the fluorescent lipophilic tracer DiI . For inhibition of actin polymerization Cytochalasin D (CytoD) was used . The CD6-GFP plasmid was generated by cloning CD6 from the CD6-RFP construct into peGFP-N1 (Clontech) . LifeAct2O and dried under a N2 stream. A glass microscope slide was cleaned by rinsing consecutively with distilled H2O, 70% ethanol and 100% acetone, and was dried under a N2 stream. The stamp was then manually pushed on the cleaned glass microscope slide for 20 s and removed, after which the stamped area was back-filled with 20 \u03bcg/ml fibronectin  in PBS for 1 h at RT. The microscope slide was washed in PBS and incubated with 1% BSA for 30 min to block all uncoated glass surface. The slide was subsequently washed with PBS and dried under a N2 stream before cell seeding.PDMS stamps containing a regular pattern of 5 \u03bcm circular spots were prepared as described earlier . PDMS st2 stream. SLBs were prepared by spin coating  solution and sonified for 15 min at RT after which they were rinsed with ultra clean water and ethanol and dried under a N coating . To formImmunofluorescent staining of CD3 was performed on wildtype Jurkat T cells on microprinted antibody spots. Immunofluorescent staining of phospho-tyrosine was performed on wildtype Jurkat T cells on microprinted antibody spots and on wildtype Jurkat T cells on SLBs. LFA-1 staining was performed on wildtype Jurkat T cells on SLBs. Cells were seeded on spots or SLBs for 15\u201330 min at 37\u00b0C. Samples were washed with PBS and subsequently fixed with 4% PFA in PBS for 15 min at RT. After fixation, samples were blocked for 1 h with blocking solution (PBS/3% BSA/10 mM glycine/1% human serum) at RT. For CD3 and phospho-tyrosine staining on antibody spots and for phospho-tyrosine staining on SLBs, permeabilization was performed simultaneously with blocking by adding 0.1% saponin to the blocking solution. 0.1% saponin was added to all subsequent antibody staining solutions. For LFA-1 staining, after blocking, cells on SLBs were incubated with primary Mouse IgG1 anti-human LFA-1 antibody TS2/4 and subsequently Goat-anti-Mouse-IgG1-Alexa488. For phospho-tyrosine staining, after blocking/permeabilization, cells on antibody spots were incubated with Mouse IgG1 p-Tyr-100-Alexa488; cells on SLBs were incubated with primary Mouse IgG1 p-Tyr-100 and subsequently Goat-anti-Mouse-IgG1-Alexa488. For CD3 staining, after blocking/permeabilization, cells on antibody spots were incubated with OKT3-biotin and subsequently NeutrAvidin-Texas-Red. After immunofluorescence staining, samples on microprinted antibody spots were washed with phosphate buffer and embedded in Mowiol (Sigma-Aldrich). Immunofluorescence samples on SLBs were not embedded but imaged in PBS directly after preparation. To study the effect of inhibition of actin polymerization on IS formation, CD6-GFP Jurkat cells were taken from culture and incubated in HBSS with or without 0.5 \u03bcM Cytochalasin D for 15 min at 37\u00b0C at a concentration of 800,000 cells per ml. Next, cell suspensions were added onto \u03b1CD3-containing SLBs, reaching a final cell concentration of 400,000 cells per ml. Samples were incubated for 30 min at 37\u00b0C. After incubation, samples were fixed with 4% PFA in PBS for 15 min at RT. Finally samples were washed once and imaged in PBS directly after preparation. CD6-GFP, \u03b1CD3-ATTO647, DiI, and brightfield signals were acquired. Samples of cells seeded on microprinted antibody spots were imaged on an Olympus FV1000 confocal laser scanning microscope with a 60 \u00d7 1.35 NA oil immersion objective. Samples of cytochalasin D treated cells on SLBs were imaged using a Leica DMI6000 widefield microscope equipped with a HC PL APO 63 \u00d7 1.40 NA oil immersion objective. Samples of LFA-1 and phospho-tyrosine staining in cells on SLBs were imaged with TIRF microscopy at an Olympus IX-71 wide field fluorescence microscope equipped with a 3-line TIRF system and a Hamamatsu ImagEM EM-CCD camera equipped with a PL APO 60 \u00d7 /1.4 NA oil immersion TIRF objective.Live cell imaging in cells on SLBs was performed at 37\u00b0C with TIRF microscopy at the Olympus TIRF microscope setup described above. Prior to live cell imaging, Jurkat cells (LifeAct-GFP or CD6-GFP) were washed with PBS and resuspended in HBSS. Cells were added to the SLBs at the microscope, in a final concentration of 400,000 cells per ml HBSS. Directly after adding the cells, both cells and \u03b1CD3-ATTO647coupled to the SLBs were imaged. Images were acquired at a frame rate of 300 ms/frame or 1 s/frame with an exposure time of 10\u2013100 ms.Dynal CD3 beads coated with mouse monoclonal anti-CD3 antibody (Invitrogen) or fibronectin-coated beads, all with a diameter of 4.5 \u03bcm, were used for bead experiments. Jurkat CD6-GFP cells were seeded on fibronectin-coated coverslips in imaging medium . Subsequently, beads were added to the cells in a concentration of 5 \u03bcM. Imaging of the CD6-GFP signal and the brightfield channel of cells with beads was performed on a Zeiss LSM510 meta confocal laser scanning microscope equipped with a PL APO 63 \u00d7 /1.4 NA oil immersion objective. Cells were imaged at RT to slow down internalization of the beads.0 using a method that is known as double normalization . Photobleaching was performed at 100% laser power by scanning the bleached ROI for two iterations, yielding a total bleach time of 0.10 s and an average fluorescence loss of ~50%. Recoveries were collected with time intervals of 200 ms using 488 nm excitation. Fluorescence intensity data for the bleached ROI and a control ROI were calculated using LSM software (Zeiss). After background correction and normalization to tlization , the sinI(t) is the intensity in the bleached ROI at time t, A is the mobile fraction, and \u03c4 is the characteristic recovery time. The halftime recovery t0.5 was calculated with:where For FRAP measurements on cells in contact with beads, Jurkat CD6-GFP cells were resuspended in phenol red-free medium, incubated with \u03b1CD3-coated beads and seeded on fibronectin-coated surfaces. FRAP was performed using a 2 \u00d7 1 \u03bcm rectangular ROI. Photobleaching was performed at 100% laser power by scanning the bleached ROI for 20 iterations, yielding a total bleach time of 1 s and average fluorescence loss of ~50%. Recoveries were collected with time intervals of 100 ms using 488 nm excitation. After background correction and single normalization, FRAP curves were fitted using the Ellenberg fitting  with theI(t) is the fluorescence intensity as a function of time, Ifinal the final intensity reached after complete recovery, w the width of the rectangular ROI, and D is the one-dimensional diffusion constant. Recovery halftime t0.5 was calculated using the formula . The fraction of cells forming an immunological synapse on SLBs upon Cytochalasin D treatment was determined by manual counting. Cells having formed an immunological synapse were defined as CD6-GFP positive cells, also visible in brightfield, on top of SLB (DiI-positive area), overlaying an \u03b1CD3 positive cluster. In bead experiments, CD6 enrichment was determined as the ratio between CD6-GFP fluorescence intensity of the membrane area of cell that was in contact with the bead and the fluorescence intensity in an equal sized ROI in the membrane of the cell at the opposite side of bead contact.Image analysis was performed using Fiji Image J . To quan Image J . A ROI it-test was applied. To compare three or more groups, one-way ANOVA with post-hoc Tukey's Multiple Comparison test or Kruskal\u2013Wallis test with post-hoc Dunn's Multiple Comparison test was applied. Differences were considered statistically significant at p < 0.05.Statistical analysis was carried out with GraphPad Prism version 5.03. Data are presented as mean \u00b1 standard deviation for bar plots and median \u00b1 interquartile range for box plots. To compare two groups, a paired/unpaired Although CD6 has been recognized as a TCR co-receptor, the nature of the interaction with the TCR/CD3 complex has not been resolved. To provide a biophysical characterization of the interplay of these receptors during IS formation, we first studied CD6 and TCR/CD3 (co-)localization using antibody spots created with microcontact printing Figure  52). Wi. Wi52). To investigate the recruitment of CD6 upon cross-linking of the TCR/CD3 complex, we created Jurkat T cells stably expressing CD6-RFP or CD6-GFP. Total cell surface expression of CD6 as well as GFP and RFP expression in CD6-GFP cells, CD6-RFP cells and wildtype cells was determined with flow cytometry  Figure . To studWhen a T cells engages contact with a stimulating antigen-presenting cell, the TCR/CD3 complex is transported to the center of the immunological synapse (IS) formed at the interface between these cells. CD6 has been previously shown to co-localize with the TCR/CD3 complex in the central supramolecular activation cluster (cSMAC) of this IS , 35. HowTo assess whether Jurkat T cells formed an IS on these \u03b1CD3-containing SLBs, wildtype cells were allowed to interact with and spread on the SLBs. After fixation, the \u03b1CD3 antibody in the lipid bilayer was visualized to localize TCR/CD3 complexes. Representative brightfield images overlaying \u03b1CD3 signal are shown in Supplementary Figure Next, we investigated the dynamics of CD6-TCR/CD3 interplay during synapse formation. To this end, Jurkat cells expressing CD6-GFP were imaged during spreading on and engagement of contact with SLBs using live cell TIRF microscopy Figure . Within The actin cytoskeleton provides a dynamic mechanical framework to spatially organize the IS, and the radial transport of TCR/CD3 microclusters is dependent on retrograde actin flow , 11, 53.To better understand CD6 mobility in a cell-cell contact model, magnetic beads coated with \u03b1CD3 or with FN were added to CD6-GFP Jurkat T cells seeded on a FN-coated surface and CD6 enrichment at the cell-bead interface was determined. Brightest point reconstructions of confocal image stacks of CD6-GFP show that CD6 was a threefold more enriched to \u03b1CD3-coated beads than to fibronectin-coated beads Figures . Next, CIn this study, we applied different experimental techniques to characterize the interplay between CD6 and the TCR/CD3 complex. We show that CD6 and the TCR/CD3 complex are co-recruited to stable stimulatory clusters, both in Jurkat T cells seeded on antibody spots and in Jurkat T cells in contact with \u03b1CD3-coated beads. This association to TCR/CD3 applies to only a fraction of the CD6 population, as FRAP measurements on CD6-GFP (both in cells on \u03b1CD3 antibody spots or in cells in contact with \u03b1CD3-coated beads) indicate that more than half of the CD6-GFP population was still mobile. If the interaction was transient, a reduction in recovery time but no change in immobile fraction would have been expected. This partial association of CD6 with TCR/CD3 confirms previous reports by Gimferrer and colleagues which showed a partial association using co-precipitation . AlthougNext to recruitment to static ligands, we exploited SLBs where \u03b1CD3 could freely diffuse in the lateral plane. This setup allowed us to visualize CD6 dynamics during IS formation. We found that CD6 co-localizes with TCR microclusters on the Jurkat T cell membrane during IS formation. These CD6-TCR/CD3 microclusters were transported toward the cSMAC of the IS, which finally resulted in CD6-TCR/CD3 co-localization in the mature IS, as reported previously by us and others , 35. SinFurthermore, in all set-ups we have used antibodies directed against CD3 to induce TCR/CD3 clustering and triggering. Although this is an artificial way of inducing T cell activation, it has been shown that stimulating CD3, without presence of an MHC-antigen complex, can sufficiently induce IS formation in Jurkat T cells . FurtherThe actin cytoskeleton provides a dynamic mechanical framework to spatially organize the IS, and the radial transport of TCR microclusters depends on retrograde actin flow , 11, 53.Next to SLP-76, CD6 also interacts with the actin-binding adaptor protein syntenin-1 . SynteniDetailed investigation of the organization of CD6, TCR/CD3, SLP-76 during IS formation using super-resolution imaging, such as Sherman and colleagues showed for the TCR, LAT, ZAP-70, and SLP-76 , could pAlthough the data presented here further substantiate the interplay between CD6 and TCR/CD3 and indicate that this co-recruitment already occurs in TCR microclusters prior to stable IS formation, it still remains a subject of debate whether CD6 signaling plays a stimulatory or inhibitory role in T cell activation. On the one hand, many studies employing monoclonal antibodies or soluble CD6 to target CD6 or its interaction with ALCAM have underlined the stimulatory role of CD6 in T cell activation and proliferation \u201329. On tThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.MM, AC, and CF designed the study. MM performed microcontact printing experiments. BJ, JW, and RB assisted with microcontact printing. SM performed lipid bilayer experiments. BJ assisted in cell culture, lipid bilayer experiments and image analysis. FC and JK performed magnetic tweezer and beads experiments. MM, SM, and FC analyzed data with support of JtR. SM, MM, AC, and CF wrote manuscript, with input from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "At present, the state-of-the-art approaches of Visual Question Answering (VQA) mainly use the co-attention model to relate each visual object with text objects, which can achieve the coarse interactions between multimodalities. However, they ignore the dense self-attention within question modality. In order to solve this problem and improve the accuracy of VQA tasks, in the present paper, an effective Dense Co-Attention Networks (DCAN) is proposed. First, to better capture the relationship between words that are relatively far apart and make the extracted semantics more robust, the Bidirectional Long Short-Term Memory (Bi-LSTM) neural network is introduced to encode questions and answers; second, to realize the fine-grained interactions between the question words and image regions, a dense multimodal co-attention model is proposed. The model\u2019s basic components include the self-attention unit and the guided-attention unit, which are cascaded in depth to form a hierarchical structure. The experimental results on the VQA-v2 dataset show that DCAN has obvious performance advantages, which makes VQA applicable to a wider range of AI scenarios. Visual Question Answering (VQA) is a multimodal research task that aims to answer questions related to the given image. Compared with other multimodal learning tasks , the core of which is the Dense Co-Attention (DCA) layers stacked in depth. Each DCA layer consists of two parallel question self-attention units, an image self-attention unit, and a guided-attention unit. The self-attention unit aims to carry out intra-modal interactions, while the guided-attention unit is used to realize the inter-modal interactions between the image regions and question words. Compared with the single-layer self-attention unit in MCAN, two parallel question self-attention units can extract more fine-grained question features. When the question features are used to guide the image, more accurate image features can be obtained. Experimental results on the benchmark VQA-v2 dataset  demonstrAn improved multimodal co-attention model is proposed by stacking the self-attention unit and the guided-attention unit. It can not only describe the interactions between multimodalities in a more effective way but also take account of the dense self-attention in each modality. Compared with the existing scheme MCAN, DCAN achieves higher precision.Ablation studies on VQA-v2 are conducted to explain the effectiveness of DCAN. The qualitative evaluation results demonstrate how it generates reasonable attention to questions and images.In summary, the main contributions of this paper are as follows:The rest of this paper is organized as follows\u2014the related work is introduced in When looking at an image, the focus is necessarily on a certain part of the image. In other words, when shifting eyes to another place, attention is also shifting along with the movement of the eyes. In this sense, when people notice a target or scene, the distribution of attention within the target or at each spatial location in the scene is different. With reference to the way the human brain processes information, the attention mechanism is introduced in deep learning, which can quickly select useful information from large amounts of data. A series of methods based on the attention mechanism came into being, but these methods are not the same.Reference  had achiMultimodal feature fusion ,34 referAt present, the fusion methods include the method based on linear fusion and the method based on bilinear pooling. The former includes feature connection and element multiplication and other linear operations. The latter is expressed as the outer product of two vectors. However, the dimension of the feature obtained by the ordinary exterior product is the square of the original feature\u2019s size, making the subsequent classification model large. Therefore, the academic community has made various improvements to the bilinear pooling method, which significantly reduces the dimension of features. Kim et al.  put forwV, K, and Q, respectively. The attention function on all queries is performed simultaneously. The attended feature F is given by:The input of the scaled dot-product attention  includesh paralleled heads, and each head corresponds to a scaled dot-product attention function. On each projection of the values, keys, and queries, the attention function is executed in parallel, resulting in output values of dimension h = 8 parallel heads are applied to reduce each head\u2019s dimensionality, and the total calculation consumption is the same as that of full-dimensional single-head attention. Additionally, To further enhance the representation capacity of the attended features, multi-head attention is presented in Reference . Multi-hThe pointwise feed-forward layer is a forward neural network, which uses several weight coefficients E and P represent question features and image features respectively. The input feature i-th head concerning image features. The feed-forward layer transforms the attended image features further. The final feature is obtained as follows:Both Self-Attention (SAtt) unit  and GuidGuided-attention unit takes the question features and image features as input, which represents question-guided attention or image-guided attention. Correspondingly, the output feature represents the image features guided by the question or the question features guided by the image. As shown on the right side in This section demonstrates DCAN in detail, the main structure of which is shown in The questions and answers are encoded by Bi-LSTM. In It is assumed that M, it can be encoded as When encoding the answers, a similar method as the question encoding method is adopted. Supposing that an answer has words with the number of Inspired by bottom-up attention , Faster d represents the dimensionality of each feature in each image. Considering better performance, lower cost and computational efficiency, The output feature is As can be seen in Firstly, taking the original question features L is set to 6. SGAtt means the image self-attention is carried out firstly, then the question-guided attention is performed.Secondly, the original image features are input to a layer of the self-attention unit to model self-attention of the image. Then the obtained image features are fed to the guided-attention unit together with the question features in the above step. For each SGAtt unit, the output feature of each layer is defined as Equation (15):P is taken as an example, the final attended feature L is the number of layers stacked by DCA layers, namely After co-attention learning, the question features and image features contain abundant information about the attention weights of words and regions. Therefore, a two-layer multi-layer perceptron (MLP) is designed as an attention reduction model, which can obtain the attended features of both the question and the image. If the image feature C is the joint representation of question and image. In this paper, C is input into a non-linear layer, and the score of each candidate answer is predicted by linear mapping.s is the score of the candidate answer, After calculating the final image features The binary cross-entropy (BCE) is employed as the loss function to train the classifier of N answers.In this section, DCAN is evaluated on the VQA-v2 dataset. Firstly, the dataset is introduced, and then experimental demonstrations and results are highlighted. Finally, the qualitative analysis is presented.The VQA-v2 dataset is based on MSCOCO , which cN answers as N classes and set the number of answers to 3000. The dropout ratio in each fully connected layer is set to 0.1 to prevent overfitting. Due to GPU memory limitation, the batch size of the model is set to 64, and 13 epochs of training are performed. Finally, the best epoch is chosen in the validation set.The question feature In this section, some ablation experiments are conducted on the VQA-v2 dataset to verify the effectiveness of DCAN. For a fair comparison, all models use bottom-up object features, which are extracted from Faster R-CNN. The ablation studies are trained on the train set to save the training time, and the results are evaluated on the validation set.As shown in h is 16, accuracy is no longer improved. Considering the training time, we set h = 8 in our bes model.To explore the effect of the number of heads in multi-head attention on the accuracy, we set the number of heads As shown in To explore the effect of the depth of DCA on the accuracy, we set the number of DCA layer In this section, DCAN is compared with state-of-the-art methods under the same experimental settings. We use the train set, vg set, and validation set to train all models, where vg represents the augmented training samples from Visual Genome. First of all, the first part of It can be seen from Secondly, to further verify the effectiveness of DCAN, the second part of In this section, some results of the DCAN are visualized in This paper focuses on fine-grained interactions between multimodalities in VQA tasks. An effective Dense Co-attention Networks (DCAN) for the VQA task is developed, the core of which is a dense co-attention model. It consists of six layers of self-attention units and guided-attention units, namely, six layers of SAtt (E)-SGAtt , which achieves the fine-grained and simultaneous understanding of both images and questions. Moreover, to better capture the relationship between words that are relatively far apart and make the extracted semantics more robust, Bi-LSTM is adopted in the question encoding phase to encode the bidirectional semantic features of the question. Compared with the existing method MCAN, DCAN can make use of the complex correlation between multimodal features in a more effective way and extract more discriminative features for images and questions. This exploration of modeling dense intra- and inter-modality interactions has been applied to intelligent transportation , intelli"}
+{"text": "P\u2009=\u20090.007). A significant difference was found between different degrees of lens subluxation and the length of surgical time and complications. At follow-up, only two eyes (1.98%) were identified to have developed retinal detachments. In conclusion, better visual outcomes can be achieved when patients received an early operation with MCTR implantation.Marfan syndrome (MFS) is a hereditary disease with an incidence of 0.3%\u00a0in the general population. Approximately 60% of MFS patients with FBN1 gene mutation will suffer ectopia lentis (EL) from the age of 3. With the development of EL, severe loss of vision will accrue because of lens tilt and glaucoma. Cionni modified capsular tension rings (MCTR) has been applied in the surgery for EL in MFS patients. To evaluate visual acuity and safety of using MCTR during lens subluxation surgery in MFS patients, 66 MFS patients (110 eyes) were included in our study, with the mean duration of follow-up of 4.7\u00a0months (SD 1.76\u00a0months). The capsular bags were preserved in 101 eyes (91.81%) with MCTR implantation. There was an overall significant improvement in BCVA at 1-month follow-up which was maintained at 3\u00a0months. Multivariable linear regression revealed that older age at first visit was associated with greater postoperative BCVA at the 1-month follow-up ( The mutation of the fibrillin-1 (FBN-1) gene located on chromosome 15, which is the main component of elastic matrix microfibrils is acknowledged as the main cause of MFS with a detection rate of 97% among MFS patients4.Marfan syndrome (MFS) is an autosomal dominant disorder affecting several systemic systems, such as the cardiovascular and ophthalmologic system. The prevalence has been estimated to be 1 in 10,000 to 20,000 individuals without geographic, ethnic, or gender predilection6. Maumenee et al. 7 reported that EL was stably accrued in 12.5% of MFS children before the age of 3 and 45% of those at the age of 4 to 5, which could be the initial presenting sign of MFS. Other well-recognized ocular manifestations are myopia, early cataract, glaucoma, and retinal detachment9.Affected individuals present with typical manifestations of MFS during all phases of life, including descending aortic root aneurysm and ectopia lentis (EL). EL occurs in approximately 60% of MFS patients10. The lack of FBN-1 in the capsular bags and zonules as well as in the iris and sclera of MFS patients, which are the two ideal positions for IOL fixation, results in difficulty in the fixation of Scleral- and iris-fixated intraocular lens (IOL)12. As a consequence, intra-operative and postoperative complications have been reported13, including iris capture and lens decentration due to deficient FBN-1 in the iris and sclera. These pathological changes make EL in the setting of MFS to be one of the most challenging anterior segment surgeries.Without adequate zonular support for the lens, EL may cause a large refractive error, a partially phakic visual axis, and an anisometropia. Furthermore, the risk of developing glaucomatous damage, secondary microspherophakia, as well as endothelial compromise, can also be highly ascended by severe EL11. Moreover, intra-operatively during this procedure, the needle needs to be passed through the vascular uveal tissue that may result in bleeding and great trauma14. From a survey participated by 185 MFS patients with EL and RD, 21% with RD had prior lens surgery11. Furthermore, at 7\u201310\u00a0years after transscleral suture-fixated IOL implantation, IOL dislocation has been observed in 24% of patients due to breakage in the polypropylene suture14.The transscleral suture-fixated IOL implantation has previously been accepted as an ideal operative method for the surgical correction of EL. However, it is associated with a high risk of serious complications such as retinal detachment (RD) or glaucoma15. MCTR can provide adequate support and correct the decentration of capsular bags in the presence of progressive zonular degradation. As a simplified operation, MCTR can be implanted into capsule bags with greater ease and minimal trauma. It can also be easily fixated to the sclera with 1 or 2 sutures without compromising the integrity of the capsular bag. Other advantages of MCTR implantation, such as minimizing vitreous loss and allowing placement of posterior chamber IOL (PC-IOL) in capsular bags have also been reported16. Furthermore, the long-term risk of postoperative complications including retinal detachment, glaucoma, and IOL dislocation can also be reduced by implanting MCTR in eyes with subluxation of the lens secondary to MFS17. However, the evidence is still lacking in the practice of MCTR implantation for the subluxation of the lens with its visual outcomes in the MFS cohort.When performing anterior segment procedures, preservation of capsular bags by implanting the Cionni modified capsular tension rings (MCTR) has become a preferred practice instead of transscleral fixated IOL implantation for MFS patientsIn this study, we aimed to evaluate the visual outcomes of MFS patients following MCTR implantation in eyes with a subluxated lens. Length of surgical time, complications of the surgery, and predictive factors for postoperative best-corrected visual acuity (BCVA) were also investigated.From June 2018 to June 2020, a total of 252 MFS patients with congenital EL at the Eye and ENT Hospital of Fudan University were retrospectively reviewed and screened for suitability for this study. All of these patients had MFS confirmed by Ghent-2 criteria. Patients with microspherophakia, keratoconus, previous history of ocular surgery, uveitis, corneal disease, glaucoma, retinal detachment, or use of contact lenses in the 2\u00a0weeks prior to the examinations were excluded from this study. Finally, 66 (110 eyes) MFS patients with EL who were suitable for surgical intervention were included in this study. The family and medical histories of all patients were carefully recorded. A flow chart summarizing the selection of the study cohort was illustrated in Fig.\u00a0This study was approved by the Human Research Ethics Committee of the Eye and ENT Hospital of Fudan University that adhered to the tenets of the Declaration of Helsinki. All the participants have given their written informed consent and informed consent for the publication of the images and data, of which the participants under the age of 18\u00a0years were provided through their legal guardians. This study was an extension of our randomized controlled trial (ChiCTR2000039132).In accordance with previously published reports, data on patient demographics, preoperative ocular parameters measured by Pentacam AXL system, best-corrected visual acuity (BCVA) during the first visit and 1-, 3-, and 6-month postoperative follow-ups, degree of lens subluxation, and surgery time were collected. Intra-operative and postoperative complications were recorded, including iris dysgenesis, vitreous prolapse, a peripheral extension of the tear, retinal detachment, posterior capsular opacification (PCO), and anterior capsule opacification (ACO). The logarithm of the minimum angle of resolution (logMAR) units was used to describe BCVA.Fig.\u00a0For analyses, eyes were divided into 3 groups based on the BCVA: Group 1: logMAR\u2009<\u20090.3 ; group 2: 0.3\u2009<\u2009logMAR\u2009<\u20091 ; group 3: logMAR\u2009>\u20091 . The degrees of lens subluxation were stratified into 3 broad groups  with a rotating Scheimpflug camera was used to measure the biological characters of the eyes in MFS patients.All patients were examined by experienced ophthalmologists with data recorded as the means of three repeated measurements.All surgeries of scleral-fixated MCTR implantation with intraocular lenses (IOL) were performed by one surgeon (Dr. YX Jiang). A 2.6\u00a0mm clear corneal incision was made after general anesthesia. A continuous curvilinear capsulorrhexis was created. To stabilize the capsular bag, 2\u20134 capsular hooks  were applied. Soft nucleus and cortical aspiration were performed using an irrigation/aspiration handpiece (Alcon Laboratories Inc.) under a low flow rate with an infusion bottle height of 65\u00a0cm. After the capsular bag was refilled with DisCoVisc , MCTR was positioned in the area of maximum zonular weakness with the fixation eyelet through the main incision into the expanded capsular bag. The MCTR was sutured with 9\u20130 polypropylene to the sclera, 1.5\u00a0mm posterior to the corneal limbus. The suture needle was passed around the interlamellar sclera four times with a modified knotless Z-suture technique. The suture was tightened and cut. The end of the suture was spontaneously retracted to the interlamellar sclera. Then, an acrylic 1-piece foldable IOL  selected by the surgeon was implanted into the capsular bag. After aspirating residual OVD by I/A handpiece, the main corneal wound was sutured with 10\u20130 nylon suture while the overlying conjunctiva was closed with 8\u20130 vicryl suture.Postoperatively, Cravit Eye Drops  and Pred Forte Eye Drops  were applied three times daily for 1\u00a0month. Also, 0.1% pranoprofen  was applied three times daily for 1\u00a0month, followed by weekly tapering.t-test, one-way ANOVA, and Mann\u2013Whitney U-test were used for analyzing the data between the modes of different groups.The average standard deviation (SD) was used to describe continuous variables. Categorical variables were described by number and proportion as appropriate. To confirm the normal distribution of the variables, the Kolmogorov\u2013Smirnov test was used. The Chi-square test, Student\u2019s Univariable and multivariable line regression analyses were performed to identify the predictors of BCVA at 1-month, 3-month, and 6-month postoperatively.The cohort for this study consisted of 66 110 eyes) MFS patients with EL. However, transscleral suture-fixated IOL was performed in 9 eyes (8.18%) instead of the originally-planned MCTR implantation because of intra-operative complications. The baseline preoperative ocular characteristics of the study participants (101 eyes) who underwent MCTR implantation were shown in Table 10 eyes MAfter reviewing the surgery video of 103 EL eyes in this cohort (video data of 6 eyes with MCTR implantation was not available), a peripheral extension of the tear occurred in 7 eyes (6.8%), of which 2 developed further to a posterior extension. As a result of the peripheral extension of the tear, MCTR could not be implanted or had to be removed in 6 patients. In preoperative EL, the vitreous loss occurred in 7 eyes (6.8%) with severe zonular weakness, the breakup of the anterior vitreous membrane occurred in 5 eyes, and vitreous prolapse occurred locally in intra-operative traction of capsular bags in 2 eyes. After vitrectomy of 5 eyes, MCTR could still be maintained and IOLs were implanted into capsular bags. Transscleral suture-fixated IOL was performed on 2 eyes with vitreous loss. Iris dysgenesis appeared in 5 eyes (4.85%). Surgery time and intra-operative complications were analyzed according to the different degrees of lens subluxation, as illustrated in Table Retinal detachment (RD) occurred in 2 MFS patients (1.94%) postoperatively, first in a 10-year-old girl at 1\u00a0month after MCTR implantation, and another occurred in the left eye of a 24-year-old man on the second day after surgery. After detachment surgery and posterior capsulotomy by YAG laser, his BCVA was 20/40 at the 3-month follow-up. PCO was found in 22 eyes (21.36%) and ACO was found in 11 eyes (10.68%) at 6-month follow-ups. Dislocation of IOL-capsular bag complex was observed in 6 eyes (5.94%) with no obvious influence of visual outcome in 4 eyes while anterior capsulotomy by YAG laser was performed in 2 eyes compared with ACO. There were no cases of postoperative endophthalmitis or vitreous in the anterior chamber.P\u2009=\u20090.028, paired t-tests.). In the 28 eyes at 6-month follow-ups, there was no significant difference in the BCVA between postoperative 3-month and 6-month . MFS patients were divided into two groups by age: the \u201cchildren\u201d group with 71 eyes (age\u2009<\u200915\u00a0years) and the \u201cadults\u201d group with 30 eyes (age\u2009\u2265\u200915\u00a0years). There was no significant difference in the BCVA during the first visit and 1-, 3-, and 6-month follow-ups between the \u201cchildren\u201d group and \u201cadults\u201d group or \u201cmild\u201d, \u201cmoderate\u201d and \u201csevere\u201d group 22. Besides, the capsular tension ring (CTR) has been commonly recognized as a rewarding and effective procedure in localizing zonular weakness. However, it does not provide accurate decentration of the capsular bag in the presence of progressive zonular dialysis in MFS patients, whereby further decentration or even dislocation of the IOL-CTR-Bag Complex Subluxation may be encountered23.Patients with EL may present primarily with fluctuating vision, blurred vision, or monocular diplopiaMCTR has been recognized as an excellent device in providing good stabilization of capsular bags with zonular dialysis. As MFS represents a progressive disease with advancing age, MCTR implantation has been chosen for MFS patients in this study. Our study has shown that MCTR preserved the capsular bags safely in MFS patients and consistent surgical outcomes were observed.In our study group, over 98% of eyes showed an improvement in BCVA at 1-month while the increase of BCVA slowed down after 3-month follow-ups, and tended to be stable after 3\u00a0months as no significant difference was observed between the 3- and 6-month follow-ups. The treatment of PCO and amblyopia are essential to achieve good visual acuity. In our cohort, the treatment of amblyopia that gradually improved vision acuity was recommended one month after surgery. Conversely, PCO that developed after surgery might have negated the improved visual acuity brought by amblyopia therapy and finally contributed to the stable BCVA at 3- to 6-month follow-ups.Our analysis has found a negative correlation between age and BCVA (logMAR units) at 1-month follow-up. Univariable and multivariable line regression analyses revealed that with the additional age, BCVA (logMAR units) at 1-month follow-up would decrease, which suggested that older patients achieved a better visual outcome. Severe amblyopia is more common in MFS children with EL than the individuals who developed EL in adulthood, which may result in poorer visual prognosis in MFS children of whom with possible inherent severe phenotypes than the adults. Meanwhile, a significant difference was observed in BCVA between the first visit and 1-, 3-, and 6-month follow-ups, which indicated that better preoperative BCVA was correlated with better visual outcomes after implantation of MCTR. Although there was no significant correlation between degrees of lens subluxation and BCVA after surgery, a significant correlation was demonstrated in our analysis between degrees of lens subluxation and surgery time. These correlation results suggest that implantation of MCTR should be considered early especially when an EL has occurred.In our study, the majority  had MCTR implantation with IOL while transscleral suture-fixated IOL was performed in 9 eyes (8.18%) as a result of intra-operative complications. The most common intra-operative complications that occurred were a peripheral extension of the tear and prolapse of vitreous, which were the main reasons for not proceeding with MCTR implantation but a transscleral suture-fixated IOL was performed instead. Significant differences were observed between the varying degrees of lens subluxation and risks of prolapse of vitreous and peripheral extension of the tear, which suggested that patients with severe lens subluxation are at high risk of surgical failure. Moreover, the severity progression of EL was associated with increased surgical time, indicating the increased operative difficulty that further enhancing the risk of intraoperative complications such as a peripheral extension of the tear. Although ideal BCVA was achieved in the 9 eyes operated with transscleral suture-fixated IOL, the risk of postoperative complications also increased. Therefore, these results have further emphasized that in the EL with rapidly deteriorating BCVA, the corrective operation should be expedited to reduce the risk of potential intra-operative and postoperative complications.25 have reported a high risk of PCO of 60%\u201384% from a few months to a few years following CTR implantation in children with EL. In our study, posterior capsular opacification was observed in 22 eyes, and ACO was found in 11 eyes (10.68%) at 6-month follow-up, and 9 (93.9%) of these needed an anterior capsulotomy by YAG laser as a precautionary measure in our practice. Comparatively, the lower rates of PCO in our study might be due to the shorter length of follow-up. Retinal detachment is another potential vision-threatening complication of MFS. Without appropriate intervention, the risks of 5\u201311% in retinal detachments have been reported in MFS patients, with particularly higher risk (8\u201338%) among those with prior intraocular surgery17. In this context, MCTR minimizes pulling forces to the vitreous base especially crucial in the setting of MFS. In our study, RD was only observed in 2 MFS patients (1.94%) postoperatively, which is lower than reported in the literature.Some postoperative complications have been observed during the follow-ups in our cohort, with PCO being the most common complication observed in 21.36% of eyes. Previous studiesThere were some limitations to this study. Firstly, it was limited by a fixed, relatively small sample size and rather short and variable follow-ups due to the COVID-19 pandemic in 2020. Secondly, the retrospective methodology and the lack of a control group for comparison might have resulted in biases in our findings. Despite these, our finding of a significant association between the varying degrees of lens subluxation and intro-operative complications lays the foundation for our future study, in addition to a longitudinal study for long-term postoperative complications.In conclusion, MCTR implantation contributes to an obvious improvement in BCVA. The preoperative BCVA and age are predictors of BCVA outcome postoperatively. The severity of EL determines the length of surgical time and risks of intraoperative complications, with ACO and PCO being the main postoperative complications in the short-term follow-up. Therefore, early operation with MCTR is safe and necessary for MFS patients with EL."}
+{"text": "Bioassay functions, which are provided by the International Commission on Radiological Protection, are used to estimate the intake activity of radionuclides; however, they include considerable uncertainties in terms of the internal dosimetry for a particular individual. During a practical internal dose assessment, the uncertainty in the bioassay function is generally not introduced because of the difficulty in quantification. Therefore, to clarify the existence of uncertainty in the bioassay function and provide dosimetrists with an insight into this uncertainty, this study attempted to quantify the uncertainty in the thyroid retention function used for radioiodine exposure. The uncertainty was quantified using a probabilistic estimation of the thyroid retention function through the propagation of the distribution of biokinetic parameters by the Monte Carlo simulation technique. The uncertainties in the thyroid retention function, expressed in terms of the scattering factor, were in the ranges of 1.55\u20131.60 and 1.40\u20131.50 for within 24\u00a0h and after 24\u00a0h, respectively. In addition, the thyroid retention function within 24\u00a0h was compared with actual measurement data to confirm the uncertainty due to the use of first-order kinetics in the biokinetic model calculation. Significantly higher thyroid uptakes (by a factor of 1.9) were observed in the actual measurements. This study indicates that consideration of the uncertainty in the thyroid retention function can avoid a significant over- and under-estimation of the internal dose, particularly when a high dose is predicted. Intake estimation after internal exposure to radioiodine, which is one of the main fission products in a nuclear power plant, is typically carried out using thyroid bioassay measurements and corresponding bioassay functions . However, in addition to the measurement data, the thyroid retention function exhibits a significant uncertainty because of the lack of accurate knowledge on biokinetic models, inter-individual variability, and/or mathematical assumptions made for computational convenience . The uncIn practice, the uncertainty in the bioassay function is not introduced in general data fitting processes because of the difficulty in quantification. For this reason, dosimetrists may misinterpret the bioassay data and fitting result. For example, if a clear information of the intake time  is given but the bioassay fitting result is statistically rejected, a dosimetrist may misadjust the intake time to improve the fitting result rather than trust the given intake time information. This problem can occur if the dosimetrist is unaware that the bioassay function involves significant uncertainty in the case of a particular individual. Another example is the use of thyroid measurement data obtained within a day after exposure. After internal exposure, thyroid measurement is acquired as soon as possible; hence, dosimetrists sometimes need to evaluate the internal dose using the data measured in the early phase. However, because the thyroid activity rapidly increases until a day after exposure , the thyIn this study, to clarify the existence of uncertainty in the thyroid retention function and give dosimetrists an insight into its uncertainty, the uncertainty in the thyroid retention function was quantified through the uncertainty propagation of the biokinetic parameters. Further, the uncertainty was numerically expressed in terms of the scattering factor to practically apply it to the maximum likelihood fitting method, which is the most widely used fitting method. Within a day after exposure to radioiodine, the uncertainty in the thyroid retention function was separately analyzed and the uncertainty due to the use of first-order kinetics was additionally quantified in terms of the bias, through comparison with the available measurement data.The uncertainty quantification of the thyroid retention function was implemented through its probabilistic estimation, whereby the radioiodine activity in the thyroid is predicted not as a single value but in the form of a probability distribution. The probability distribution was generated via multiple calculations of the thyroid retention function with a set of biokinetic parameters assigned using the Monte Carlo simulation technique from the corresponding parameter distributions. The probability distributions of the thyroid retention functions were produced with a time interval of 1\u00a0h, and the scattering factors as indicators quantifying the variances of the distributions were derived from each distribution at each time step. L(I). In general, the i-th likelihood function, i(I)L, is defined as follows:i/I)PL should be expressed as in Eq. eI is the estimate of intake activity calculated using i/miM, and im is the bioassay function corresponding to iM. The probability distribution of Mi, P(Mi), represents the measurement uncertainty. The component of the measurement uncertainty can generally be divided into Type A  and Type B  errors. The Type A error is involved only in the counting statistics and is described by a Poisson distribution. The Type B error is involved in uncertainties other than the counting statistics and is generally described by a log-normal distribution; for example, the variability of the thyroid mass described by a log-normal distribution causes uncertainty in the counting efficiency, which is a Type B error. However, for simplicity, the overall uncertainty, P(Mi), can be assumed to be a log-normal distribution [i, P(mi), representing the uncertainty in the bioassay function, the type of distribution can be determined by the uncertainty propagation of the biokinetic parameters, which are used to calculate the bioassay functions. Irrespective of the type of distribution used for the biokinetic parameters, the type of distribution of the overall uncertainty, P(mi), should be inductively determined from the final distribution in which the biokinetic parameter uncertainties are combined. In this study, P(mi) will be described by the log-normal distribution (see i) and P(mi) can be described by log-normal distributions, as explained above, P(Ie/I) may also exhibit a log-normal distribution, and the scattering factor (SF) can be defined as its geometric standard deviation. Therefore, Li(I) can be written asiSF is the scattering factor as a measure of the uncertainty in eI, which can be expressed as a combination of the measurement uncertainty, MSF, and the bioassay function uncertainty, mSF, as follows.The principle of the maximum likelihood method for the bioassay has been explained in the IDEAS guidelines . In the entclass1pt{minimaribution . This aption see . If bothMSF can be derived from the experimental conditions, typical values can be used; for example, 1.2 has been suggested as the typical MSF for high-energy gamma (>200\u00a0keV) [mSF is the purpose of this study.Although laboratory-based 200\u00a0keV) . Derivinn measurement data are independent, the combined likelihood function, L(I), can be calculated by the product of the likelihood functions as in Eq. When L(I) is the maximum, the corresponding I is determined as the final intake estimate.Therefore, when 1f, for the ingestion of iodine was assumed to be 1. Although the thyroid retention functions were calculated for iodine-131, it is reasonable to apply the uncertainty quantification results to other iodine isotopes because only the biological parameters were considered as the uncertainty factors.iq is the amount of iodine in the i-th compartment, and ijr is the transfer rate from j- to i-th compartment. In this study, the thyroid retention functions were calculated only for an acute intake that was more likely to happen in emergency situations. The calculation of the thyroid retention function was conducted using the computer code developed in an earlier work [The thyroid retention function can be calculated using the biokinetic models, which can mathematically describe the behavior of radioiodine after intake. The biokinetic models were first written as a set of first-order differential equations, as in Eq. ier work , which pBiokinetic models generally include a respiratory tract model for inhalation intake, an alimentary tract model for ingestion intake, and a systemic model for describing the iodine behavior after uptake to blood. Although the uncertainty in the biokinetic model originates from both model structure and parameter, only the uncertainty in the biokinetic parameter was considered in this study. The uncertainties in the biokinetic parameters for radioiodine are explained in detail in the following with data sources.The ICRP developed a human respiratory tract model (ICRP 66) . Althoug1f, of iodine, the triangular distribution with a mode of 1.0 and a range of 0.9\u20131.0 provided by D. M. Hamby (1999) [The human alimentary tract model in ICRP 100 , which iy (1999)  was usedThe simple biokinetic model shown in The uncertainties in the transfer rates of the three-compartment model were investigated in various studies. In this study, the parameter distributions for the iodine systemic model were referenced from Hamby (1999) , as showA set of biokinetic parameters for the multiple calculations of the thyroid retention function was assigned from each corresponding distribution using a Latin hypercube sampling (LHS) method, which is the most widely used method for Monte Carlo sampling. Because the LHS method draws samples from evenly divided probability intervals of a cumulative density function, the entire range of the distribution is used for sampling . Thus, tM, obtained within a day by the bias. Thus, the corrected thyroid measurement data, correctedM, can be calculated using Eq. First-order kinetics were assumed for computational convenience. In the biokinetic model calculation with first-order kinetics, radioiodine can be removed only with constant half-life. However, the actual movement of radioiodine is more complicated, particularly in the early phase after intake. In this study, 24\u00a0h (1 d) was regarded as the time limit where the assumption of the first-order kinetics can introduce an uncertainty because after a day, most of the iodine is accumulated in the thyroid and eliminated gradually with a half-life of 80 d . The unc\u22121 and is completed within 2\u00a0h [( [) and normalized to the value at 24\u00a0h. Thereafter, the normalized %RAIUs were compared with the corresponding values calculated using the first-order kinetics. The bias was derived as the ratio of the measured values to the calculated values. Note that because the reported data used in this study were measured by well-collimated systems or were properly corrected with the background counts, the uncertainty due to the extra-thyroidal radioiodine was negligible.The bias was determined by comparing the measured and calculated radioiodine thyroid uptakes (%RAIU) within 24\u00a0h after iodine ingestion. Although the thyroid uptake data for intravenously injected iodine are required to clearly address the uncertainty related to the systemic model of iodine, the data obtained within and after 24\u00a0h in the same study were unavailable. Thus, the thyroid uptake after oral administration was used with the assumption that the mechanisms in the alimentary tract before iodine uptake to blood had little effect on the overall biokinetics of iodine. This assumption is reasonable because the absorption of iodide from the alimentary tract of humans is quite fast with a rate of approximately 5% minthin 2\u00a0h . Therefon 2\u00a0h [ and nor2\u00a0=\u00a00.98) with GM of 0.21 and GSD of 1.41. The value of ICRP 78 [The probability distributions of the thyroid retention function were generated with increasing elapsed time after exposure to quantify the time-dependent uncertainty in the thyroid retention function. Multiple calculations with 1,000 sets of parameter samples at each time step reduced the mean standard error of the distribution to below 2%; however, further calculations did not significantly improve the distribution. When the thyroid retention function at a particular time was drawn as a frequency distribution, it was log-normally distributed. For example, as shown in  ICRP 78  falls atConsidering the shape of the distribution of the thyroid retention function shown in . (1985) [The scattering factors and bias suggested in this study were applied to examples in which thyroid measurements within a day after exposure were included. First, the uncertainty was applied to the data reported by Floyd et\u00a0al. (1985) , in whicThe determination of the type of uncertainty distribution is critical for interpreting and quantifying the uncertainty. The type of distribution of the thyroid retention function was inductively determined after propagation of the biokinetic parameter uncertainties. The log-normal distribution selected for explaining the distribution of the thyroid retention function is statistically valid and corresponds with the results of the other studies; for example, the time-integrated activity of ingested iodine in the thyroid, which was calculated by integrating the thyroid retention function, was also described by the log-normal distribution . Based oAlthough measurement uncertainty has been considered in general bioassay data fitting methods, this study showed that the thyroid retention function also exhibits a significant uncertainty. Considering the typical SF of 1.2 for the measurement uncertainty of high-energy gamma, as suggested by the IDEAS guidelines, the major uncertainty can be attributed to the uncertainty in the thyroid retention function, whose SF values are higher than 1.4. In particular, the magnitude of the uncertainty was relatively large in the thyroid retention function within a day after exposure. Because the thyroid activity in the early phase after exposure is influenced by various factors, such as the amount of iodine in blood, blood-to-thyroid transfer rate, and thyroid-to-rest of the body transfer rate, it is difficult to predict the thyroid activity before the early thyroid uptake is complete. In addition, this study showed that the thyroid retention function calculated using the first-order kinetics could underestimate the actual thyroid uptake within a day after exposure. Although sufficient measured data were unavailable, obvious discrepancies between the thyroid measurement data and the calculated values were observed. There could be other reasons for the discrepancies, such as the extra-thyroidal radioiodine contribution and other factors that cannot be identified clearly. However, we judged that the influence of other factors was minor and negligible compared to the uncertainty from the use of first-order kinetics.The effects of the uncertainty in the thyroid retention function were demonstrated in the previous examples. In the first example in which the true intake was known, applying the scattering factor and bias suggested herein made a difference in the statistical explanation ability of the calculated %RAIU curve. The correction using the scattering factor and bias produced p-value higher than 0.05. However, it should be noted that despite the statistical acceptance of the fitting, the discrepancy in the early data at 4 and 5\u00a0h indicated that the thyroid retention function in the early phase after intake would still involve a significant uncertainty. In the second example in which artificial measurement data were used, it was shown that the intake estimate could significantly vary depending on whether the uncertainty in the thyroid retention function was considered or not. In the example, the intake estimates changed by approximately 17%. In particular, the overestimation of the intake could be avoided by scaling down the data at 2 and 4\u00a0h, and the fitting curve became closer to the data after 24\u00a0h. From the results, it can be concluded that applying the uncertainty in the thyroid retention function reduces the importance of the unstable thyroid measurement data obtained within a day, but strengthens the relative reliability of the data obtained after a day. Depending on the situation, considering the uncertainty can also reduce the possibility of underestimation. If the exact time of intake is unknown, applying the bias can estimate an intake time later than that without the bias and thus result in a higher intake estimate.The best way to avoid the uncertainty from the unstable measurement data in the early phase is by measuring the thyroid after 1 d of exposure. If only data measured after 1 d are used, the uncertainty is relatively low, and the result of intake estimation using the bioassay data fitting may not change because equal weightings are induced to all the measurement data regardless of the time. In this case, introducing the uncertainty in the thyroid retention function to the data fitting can only affect the good of fit, and thus the determination of acceptance or rejection of fit. Moreover, the thyroid measurement after 1 d is preferred because it can reduce the background effect of extra-thyroidal iodine on the thyroid measurement. Until radioiodine is absorbed into the thyroid or excreted via urine, significant amounts of radioiodine distributed to organs or tissues other than the thyroid can influence the thyroid measurement counting. Nevertheless, in actual situations, dosimetrists often need to perform intake estimation using only the data obtained within a day or data obtained both before and after a day. The first dose assessment should not be delayed after internal exposure. Therefore, the time-dependent uncertainty is vital for practical dose assessments and should be considered particularly important when early measurement data are used.It is also important to clarify the exposure categories and situations where the uncertainty investigated in this study could be applied in practice. ICRP publication 103  has clasThis study attempted to quantify the uncertainty in the thyroid retention function for practical applications. More importantly, the objective was to provide insights to dosimetrists into the uncertainty in the thyroid retention function rather than giving the exact numerical values. Although the uncertainty is not considered numerically, the dosimetrist should understand the extent of uncertainty in the thyroid retention function and thus be able to practically judge which data are more important for intake estimation. Because the internal dose calculation depends on the judgment of the dosimetrist, a firm understanding of the uncertainty is essential to derive more accurate dose estimates.However, it should be noted that this study does not guarantee that applying the uncertainties suggested herein would always yield a more accurate dose estimate. Because the dose assessment needs to be based on a comprehensive understanding of the given data and conditions, dosimetrists should focus on the use of uncertainties depending on the situation.This study revealed the uncertainty in the thyroid retention function based on the Monte Carlo method and performed a comparison with actual measurement data. Moreover, the study provides new insights to dosimetrists into the uncertainty in the thyroid retention function and methods of applying it to practical dose assessments. The thyroid retention functions were assumed to be independent of one another; the accuracy of the uncertainty quantification can be improved by better understanding the correlation between the thyroid retention functions and through the development of a more extensive database. In addition, the use of recently developed biokinetic models for iodine can improve the accuracy of the thyroid retention function. This study lays a foundation for the uncertainty quantification of bioassay functions. Future studies should be directed toward quantifying the uncertainty in various bioassay functions for chronic intakes and for other radionuclides.The authors have no conflicts of interest to declare.This research was supported by a grant of the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by Ministry of Science and ICT (MSIT), Republic of Korea. (No.50445-2020)."}
+{"text": "This study aimed to evaluate the association between optical coherence tomography (OCT)-measured retinal layer thickness parameters with clinical and patient-centred visual outcomes in healthy eyes. Participants aged 40 and above were recruited from the Singapore Epidemiology of Eye Diseases Study, a multi-ethnic population-based study. Average macular, ganglion cell-inner plexiform layer (GCIPL), and outer retinal thickness parameters were obtained using the Cirrus High\u00a0Definition-OCT. Measurements of best-corrected visual acuity (BCVA) and 11-item visual functioning questionnaire (VF-11) were performed. Associations between macular thickness parameters, with BCVA and Rasch-transformed VF-11 scores (in logits) were assessed using multivariable linear regression models with generalized estimating equations, adjusted for relevant confounders. 4,540 subjects  with a mean age of 58.8\u2009\u00b1\u20098.6 years were included. The mean BCVA (LogMAR) was 0.10\u2009\u00b1\u20090.11 and mean VF-11 score was 5.20\u2009\u00b1\u20091.29. In multivariable regression analysis, thicker macula  and GCIPL  were associated with better BCVA , while thicker macula  and GCIPL  were significantly associated with higher VF-11 scores . In conclusion, among healthy Asian eyes, thicker macula and GCIPL were associated with better vision and self-reported visual functioning. These findings provide further understanding on the potential influence of macular thickness on visual function. Using this technique, recent studies have shed insights on variations of macular thickness across gender, age, ethnicity, axial length and refractive error profiles8.Optical coherence tomography (OCT) is a non-invasive imaging technique and an essential clinical assessment tool in retinal diseases14, and macular thickness has thus been proposed as a potential surrogate for visual function. Previous studies also reported poorer vision in cases who presented with signs of disrupted retinal layers such as the retinal inner layers, external limiting membrane or the ellipsoid zone17. The association between macular thickness and self-reported visual functioning has only been evaluated in diseased eyes such as age-related macular degeneration18. However, the associations between macular thickness with visual acuity and visual functioning have yet been evaluated in healthy eyes. It is postulated that a thicker retina is correlated with better vision in non-pathological eyes, but this has not been evaluated comprehensively.A series of past studies has shown that changes in the retinal or its sublayer\u2019s thickness in diseased eyes were associated with visual acuity and functionHence, the purpose of our study was to evaluate the association between macular thickness and visual acuity and visual functioning, in a multi-ethnic Asian population. The findings from this study may provide further understanding on the relationship between retinal anatomical structure and visual function.21. Briefly, participants aged 40\u201380+ years residing in the Southwestern part of Singapore were recruited and underwent standardised ocular and systemic examinations. Our study population is made up of 3,353 Chinese from the baseline visit in year 2009\u20132011 (response rate 72.8%), 1,901 Malays from the 6-year follow up visit in 2011\u20132013 (response rate 72.1%), and 2,200 Indians from the 6-year follow up visit in year 2013\u20132015 (response rate 75.5%). The baseline visits in the Malay and Indian population did not undergo OCT assessments and hence were not included in this study. This study follows the principles of the Declaration of Helsinki with ethical approval obtained from SingHealth Centralised Institutional Review Board. Written informed consent was obtained from all participants.The Singapore Epidemiology of Eye Diseases (SEED) Study is a cross-sectional population-based study in Singapore and comprises of adults from three major ethnicities: Chinese, Malay, and Indian. The methodology of the SEED study has been previously describedAll participants who underwent an OCT scan were included in our study. Exclusion criteria included poor fundus photo quality e.g. dense media opacity and artefacts, low OCT signal strength of <6, history of glaucoma or high cup-disc ratio\u00a0of\u00a0>0.75, previous retinal procedures such as laser photocoagulation, retinal surgery, intravitreal injection, and pre-existing retinal diseases such as diabetic retinopathy, age-related macular degeneration, epiretinal membrane and macular hole.8. The presenting visual acuity (VA) was recorded and subjective refraction subsequently performed to ascertain best-corrected VA (BCVA) of each participant. Both parameters were measured in the units of logarithm of the minimum angle of resolution (LogMAR), using number chart  at 4 meters. High myopia was defined as spherical equivalent of \u22125 dioptres or more.A standardised examination protocol was used across all three ethnic groupsA detailed interviewer-administered questionnaire was used to collect information including demographics, ocular history, medical and surgical history and medication use, height, weight, blood pressure and pulse rate. Body mass index (BMI) between 18.5 to 25 was defined as normal, <18.5 as underweight, 25 to 30 as overweight, and> 30 as obese. A non-fasting venous blood sample was collected for serum lipids, glycosylated haemoglobin A1c (HbA1c), creatinine, and random glucose. Diabetes was defined as random glucose \u2265 11.1\u2009mmol/L, HbA1c\u2009\u2265\u20096.5%, use of diabetic medications and/or self-reported history. Hypertension was defined as systolic blood pressure \u2265 140\u2009mmHg, diastolic blood pressure \u2265 90\u2009mmHg, use of anti-hypertensive medications and/or self-reported history. Hyperlipidaemia was defined as total cholesterol \u2265 6.2\u2009mmol/L, use of lipid-lowering medications and/or self-reported history. Cardiovascular disease (CVD) history was defined as self-reported history of angina, heart attack and/or stroke. Lastly, low socioeconomic status was defined as fulfilling all three criteria of primary education or below, monthly income <2,000 SGD and residing in 1 to 2-room public housing flat.22. The VF-11 is a modified version of VF-14 which has been validated to suit the local Singapore cultural context. The questionnaire was administered by interviewers fluent in English, Chinese, Malay and Tamil. The VF-11 assesses participants\u2019 ability to perform activities of daily living, such as reading the newspaper, reading street signs, recognising friends, seeing stairs, watching television, cooking, and driving during the day or night. Items 1\u20139 were given a numerical grading from 0 to 4, where 0 represents no difficulty in performing such activity, and 4 represents inability to perform that activity, while items 10 and 11 were rated from 0 to 2. A non-applicable option for each item was available if participants did not do the activity for reasons other than their vision, and these data were excluded from the analyses.The VF-11 questionnaire was used to assess the impact of retinal thickness parameters on subjects\u2019 vision functioning23. The scores were reversed so that higher scores represented better visual functioning. We used the overall VF Rasch-transformed score derived from items 1\u20139 of VF-11 questionnaire excluding item 10 and item 11 (\u2018driving during the day\u2019 and \u2018driving at night\u2019) as Rasch analysis of the VF-11 revealed these two items to have high level of misfit.Rasch analysis, a form of Item Response Theory, was applied to assess the psychometric properties of the VF-11, in which raw VF-11 scores are transformed to estimates of interval level Rasch measures, expressed in log of the odds units or logits2 area centred on the fovea was acquired based on the 512 \u00d7 128 protocol and automatically analysed by the Cirrus HD-OCT software V6.5. The macula is subdivided into nine macular subfields as defined by the Early Treatment Diabetic Retinopathy Study (ETDRS) with the foveal central subfield defined as central 1\u2009mm diameter, and the inner ring and outer ring with diameters of 3\u2009mm and 6\u2009mm, respectively. Both inner and outer rings are further divided into superior, inferior, nasal and temporal quadrants. The average overall macular thickness was measured from the internal limiting membrane to the retinal pigment epithelium (RPE) of the macular cube. The average ganglion cell-inner plexiform layer (GCIPL) thickness and outer retinal thickness were measured across an elliptical annulus within the 6 \u00d7 6 mm2 area. GCIPL was segmented from the outer boundaries of retinal nerve fibre layer to the inner plexiform layer, and outer retinal layer was segmented from the outer plexiform layer to the RPE layer  was used to capture images of the macula following pupil dilation. In each study eye, a macular cube scan of 6 \u00d7 6 mmAll statistical analyses were performed using Stata 14.0 . Eye-specific analysis data were used. Mean and standard deviation were reported for continuous variables, while frequency and percentage were reported for categorical variables. Univariate linear regression analysis was performed to examine the associations between demographic, systemic and ocular factors with the two outcomes of interest, BCVA and VF-11 Rasch-transformed scores. Variables significantly associated with the two outcomes in univariate analysis  and older age groups (\u226560 years old) . Despite this close relationship, association between the respective macular thickness parameters with VF-11 scores remained significant after adjusting for presenting VA. This further substantiates the observed associations. Furthermore, as age and axial length were major determinants of macular and GCIPL thicknesses, we performed subgroup analyses by age and axial length profiles27. For example, a thicker OCT-measured macular thickness in eyes with vein occlusion or uveitis, as shown in the Standard Care vs. Corticosteroid for Retinal Vein Occlusion (SCORE) study and Multicentre Uveitis Steroid Treatment (MUST) trial, was associated with poorer baseline visual acuity. In comparison, our study evaluated this association in healthy eyes, whereby the differences in macular thickness represent a larger physiological variation among normative population, rather than as a sequela to a macular disease. Our finding of a positive correlation between thicker macula with vision and patient-centred visual function suggests that macular thickness could also be one of the determinants for visual outcomes. Our observed finding may be explained by the notion that thicker macula reflects a more abundant cell number especially of the ganglion cell type which functions to transmit crucial visual information from the retina to the brain28.To the best of our knowledge, this is a novel finding in healthy eyes. This contrasts with most other studies which looked at pathological eyes where increased macular thickness was generally associated with poorer visual acuityOur analysis showed that within the inner retinal layer, average GCIPL thickness was also significantly associated with better BCVA and visual functioning scores, but the outer retinal thickness was not. This may be because the majority of the ganglion cells are located in the inner retinal layer of the macular and have greater influence on visual function. Overall, our study supports the existing literature that GCIPL thickness is a determinant of visual outcomes, even in healthy eyes.18. They reported an inverted U-shaped correlation between central foveal thickness and visual function, assessed using National Eye Institute Visual Function Questionnaire (VFQ) scores, with best visual function peaking at 220\u2009\u00b5m of macular thickness. In contrast, our study evaluated healthy eyes and further took into account a range of potential confounders including individual\u2019s presenting vision status. This shows that, other than clinically measured vision status, macular and GCIPL thickness also potentially contributes to good visual functioning. In healthy eyes, macular thickness can possibly aid in other areas crucial for visual functioning such as depth perception, contrast sensitivity, stereo-acuity, and visual fields, further supporting the role of macular thickness as a factor associated with better visual functioning29. Overall, despite the statistically significant associations observed between macular parameters and visual functions, it should also be noted that the observed effect estimates were generally small. Hence, the clinical impact of these observed associations remained to be evaluated in future studies.We also presented a novel finding in which macular and GCIPL thickness were positively associated with self-reported outcomes in healthy eyes, independent of individual\u2019s vision status. One study looked at the correlation between foveal morphology and visual function in participants with neovascular age-related macular degenerationStrengths of our study include a large, multi-ethnic population. Furthermore, measurements were conducted comprehensively and according to a standardized protocol, allowing us to account for a range of potential confounders. Our study is limited by the lack of other parameters that measure quality of vision (e.g. contrast sensitivity) and information on temporality, which limits inference on causality between retinal layer thickness and visual outcome. Thus, future longitudinal studies in this aspect are still warranted. Our study was also limited by some missing responses on VF-11 questionnaire, which ranged from 0.4 to 36.4% by individual question. However, only three items related to difficulty in cooking, playing games and filling out lottery forms had missing responses of\u00a0>20%. The higher missing rates in these 3 items were unlikely to have impacted our original findings substantially. Lastly, although the GCIPL thickness was measured using an elliptical annulus area at the macula, we acknowledge that the effect of retinal ganglion cell displacement at the macula was still not fully accounted for in our measurements, and might impact inner retina-related evaluation.In conclusion, our study demonstrates that in an adult Asian population with healthy eyes, thicker macula and GCIPL were associated with better VA and better visual functioning. Our findings suggest that macular and GCIPL thickness parameters may be useful determinants for visual functions.Supplementary Tables."}
+{"text": "The antioxidant system in islets of Langerhans is weak, which can lead to diabetes. Meanwhile, the main component of cloves that produce antioxidant effects is eugenol. Accordingly, the present study was conducted to investigate the antioxidant effect of eugenol on oxidative stress induced by hydrogen peroxide . Finally, the islet's lipid peroxidation and antioxidants levels were measured by the ELISA assay method.  In this experimental study, adult Naval Medical Research Institute (NMRI) mice (20-25\u2009g) were prepared. The collagenase digestion method was used for dissecting the islets of Langerhans. HP < 0.05). MDA diminished in H2O2+eugenol 50, 100, and 200\u2009\u03bcM (P < 0.01) groups versus the H2O2. TAC was elevated when eugenol 50, 100, and 200\u2009\u03bcM was administered in oxidative stress-induced islets (P < 0.001). Also, CAT increased in the H2O2+eugenol 50 (P < 0.05) group in comparison with the H2O2 group.  Malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD), and catalase (CAT) increased in all groups when compared to the control (2O2 induced oxidative stress and lipid peroxidation in the islets, and administration of eugenol recovered these alterations by raising the level of TAC and CAT, while reducing MDA as a lipid peroxidation biomarker. In conclusion, H NumerouIn this experimental study, adult Naval Medical Research Institute (NMRI) mice (20-25\u2009g) were kept at a 12\u2009:\u200912 hour light\u2009:\u2009dark cycle. The animals were treated in accordance with the principles and guidelines on animal care of Dezful University of Medical Sciences as reviewed by an ethics committee (IR.DUMS.REC.1398.022), as well as free access to tap water and commercial chow ad libitum.The islets of Langerhans were isolated by the protocol for the isolation of islets from rodent pancreas using collagenase digestion. Briefly, the animals were anesthetized with ketamine/xylazine (70/10\u2009mg/kg), and their pancreas removed and transferred to a Petri dish containing Krebs-bicarbonate buffer solution . Then, the separated pancreas was cut into 1\u2009mm pieces and centrifuged at 100 \u00d7g for 5\u2009min. Krebs-bicarbonate buffer solution plus collagenase type P (1-2\u2009mg/pancreas)  were added to the sediment of the centrifuged conical tube to separate the islets from exocrine tissue. Next, the conical tube was transferred to an incubator at 37\u00b0C with 800 oscillations for 5-10\u2009min. Then, a cold Krebs-bicarbonate buffer was added to the conical tube to stop collagenase digestion and centrifuged at 500 \u00d7g for 5\u2009min. Finally, the isolated islets were transferred to a Petri dish and separated manually under a stereomicroscope .2O2 (50\u2009\u03bcM) was added to each islet's sample and incubated for 30\u2009min. Then, the samples were centrifuged at 400 \u00d7g for 10\u2009min. In order to reduce the oxidative stress in islets, 50, 100, and 200\u2009\u03bcM of eugenol were added to each islet's sample and incubated for 2 hours at 37\u00b0C; after the incubation period, the oxidative-induced protocol with H2O2 (50\u2009\u03bcM) was repeated. Finally, the supernatant of each microtube was stored at -70\u00b0C until the experimental measurement. Each microtube contained 7 islets, and the number of samples is repeated 6 times for each group [To create oxidative stress in the isolated islets of Langerhans, Hch group , 19.Group 1: controlGroup 2: H2O2 (it received 50\u2009\u03bcM of H2O2 for 30\u2009min)Group 3: H2O2+eugenol (50\u2009\u03bcM) (it received 50\u2009\u03bcM of eugenol for 2 hours after which H2O2 (50\u2009\u03bcM) was added to each sample for 30\u2009min)Group 4: H2O2+eugenol (100\u2009\u03bcM) (it received 100\u2009\u03bcM of eugenol for 2 hours after which H2O2 (50\u2009\u03bcM) was added to each sample for 30\u2009min)Group 5: H2O2+eugenol (200\u2009\u03bcM) (it received 200\u2009\u03bcM of eugenol for 2 hours after which H2O2 (50\u2009\u03bcM) was added to each sample for 30\u2009min)To measure the level of MDA, total antioxidant capacity (TAC), SOD, and CAT in the isolated islets of Langerhans, the ELISA method as well as specific commercial kits  was used.P was considered significant at less than 0.05 in all experiments.The results were statistically analyzed using the Statistical Package for Social Sciences (SPSS) software with one-way analysis of variance (ANOVA), followed by post hoc least significant difference (LSD) tests. All results were represented as mean \u00b1 standard\u2009error\u2009(SE) where P < 0.001). This lipid peroxidation variable diminished in H2O2 plus eugenol 50 (P < 0.001), 100, and 200\u2009\u03bcM (P < 0.01) administrated groups versus the H2O2 group , as well as H2O2 plus eugenol 50, 100, and 200\u2009\u03bcM (P < 0.001) groups compared with the control. This variable revealed a significant increase following eugenol 50, 100, and 200\u2009\u03bcM (P < 0.001) administration under oxidative stress islets compared to the H2O2 group (The islets' level of TAC increased in HO2 group .P < 0.01). However, this antioxidant enzyme did not show any significant alteration across the H2O2-administered groups  and H2O2 plus eugenol 50 (P < 0.001), 100, and 200\u2009\u03bcM (P < 0.01) groups compared to the control. Also, this enzyme was elevated in the H2O2 plus eugenol 50\u2009\u03bcM (P < 0.05) group in comparison with the H2O2 group (CAT level increases in HO2 group .2O2 would lead to higher MDA levels in the isolated islets of Langerhans. However, H2O2 could increase TAC, SOD, and CAT, but this increasing effect could not improve the damage induced by oxidative stress and lipid peroxidation in the islets. The administration of eugenol decreased MDA along with increase TAC and CAT in pancreatic islets under oxidative stress condition induced by H2O2. Also, the effect of low doses of eugenol on improving the damage induced by oxidative stress was greater than that of other doses. This effect can be attributed to the fact that a low dose of eugenol resulted in a significant increase in catalase as well as in further reduction in MDA in the isolated islets.The results of the present study showed that the induction of oxidative stress by H\u03b2 cells are sensitive to oxidative stress where ROS may play a central role in \u03b2 cell death causing T1DM. Also, this disorder has a secondary pathogenic role in the development of T2DM. On the other hand, it was demonstrated that antioxidants such as phenolic compounds reduced this complication. H2O2 has multiple effects on \u03b2 cells including metabolic inhibition and enhancing plasma membrane permeability, impairing glucose metabolism, and reducing insulin secretion [\u03b2 cell, causing type 1 and type 2 diabetes [2O2-induced proliferation inhibition in \u03b2 cell, and it was suggested that these antioxidants can be used in T2DM to increase \u03b2 cell proliferation [Pancreatic ecretion . Oxidatidiabetes . In addiferation . Diabeteferation . Overproferation . Accordi2O2 at higher concentrations [It was revealed that antioxidant agents such as polyphenols or flavonoids scavenge free radicals depending on their concentration . Furthertrations . So, in 2O2 induced oxidative stress and lipid peroxidation in the isolated islet of Langerhans, and administration of eugenol recovered these alterations by raising the level of TAC and CAT and reducing MDA as a lipid peroxidation biomarker. Also, among the administered concentrations of eugenol, a low dose of this phenolic compound was more potent to produce a therapeutic effect in the function of antioxidant enzymes and reducing the oxidative stress of the pancreatic islet. Hence, this dose of eugenol could be used to treat diabetes by improving the function of \u03b2 cells in the islets of Langerhans, but a future clinical study is required to clarify this finding.In conclusion, H"}
+{"text": "The present study suggested that early mobilization after cardiac surgery may improve physical function at discharge.The objective effects of early mobilization on physical function in patients after cardiac surgery remain unknown. The purpose of the present study was to clarify the effects of early mobilization on physical function in patients after cardiac surgery through meta-analysis. Four electronic databases were searched on 2 August 2019. We used search keywords related to \u201cearly mobilization\u201d, \u201ccardiac surgery\u201d, and \u201crandomized controlled trials\u201d. All randomized controlled trials conducting early mobilization after cardiac surgery were included. We defined early mobilization as the application of physical activity within the first five postoperative days. Citations and data extraction were independently screened in duplicate by two authors. The meta-analysis was conducted using random-effects modeling with EZR software. The primary outcome was the distance walked during the six-minute walking test at hospital discharge. Six randomized controlled trials comprising 391 patients were included following screening of 591 studies. All studies included coronary artery bypass grafting as the cardiac surgery conducted. Early mobilization started on postoperative days 1\u20132 and was conducting twice daily. Early mobilization showed a trend of being combined with respiratory exercise or psychoeducation. The meta-analysis showed that the distance walked during the 6-min walking test improved by 54 m (95% confidence interval, 31.1\u201376.9; I Cardiovascular diseases (CVD) such as coronary artery disease (CAD) are a major cause of mortality worldwide . With raEarly mobilization is defined as the application of physical activity within the first two to five days of critical illness or injury ,8. In crA decline of physical function (gait speed) with bed rest was seen in 17% of patients after surgery . FurtherRamos Dos Santos et al. found that three quarters of the studies related to cardiac surgery that they analyzed reported early mobilization to have significant effects on improving physical function such as that assessed in the 6MWT . HoweverThis systematic review and meta-analysis was conducted according to the PRISMA statement . We inclStudies were identified by searching four electronic databases . We searched for keywords related to \u201cearly mobilization\u201d, \u201ccardiac surgery\u201d, and \u201crandomized controlled trials\u201d . The lasEligibility assessment was performed independently in a standardized unblinded manner by two independent reviewers (Y.K. and T.S.). First, we screened the titles and abstracts of each study. After the results of this screening were integrated, we screened the full text of the manuscripts. Disagreements regarding inclusion of a study were reconciled via consensus.We used datasheets based on the Cochrane Consumers and Communication Review Group\u2019s data extraction template. Two reviewers independently extracted the data listed below from the included studies. After that, each result was integrated, and disagreements were resolved by discussion between the two independent reviewers (Y.K. and T.S.). If agreement could not be reached, we requested intermediation by a third party. We contacted seven authors directly to collect additional data. After that, three full texts and the details of the results of two studies were collected and included in the present analysis. The following information was extracted from the included studies: patient characteristics , interventions , and outcomes .To assess the validity of the included studies, two reviewers independently used the \u201cCochrane risk of bias tool\u201d, which consists of seven items that were evaluated as \u201clow risk\u201d, \u201chigh risk\u201d, or \u201cunclear risk\u201d and are summarized in p-value was < 0.05 in the Cochrane Q test or >50% in the I2 test as a measure of inconsistency. No other additional analyses were conducted.The distance walked during the 6MWT at discharge was the primary outcome of the intervention effect. In the meta-analysis, we calculated the mean distance walked during the 6MWT in each group at discharge using the weighted average \u201crandom-effect model\u201d included in the EZR analysis software . We judgWe searched 591 studies through four database searches along with four additional studies obtained from the Ramos Dos Santos et al. study . After dEarly mobilization started on postoperative days 1\u20132 and was conducted twice daily by physical therapists or nurses. The exercise protocol was performed via a progressive approach, and it consisted of such activities as active upper and lower limb exercise training, ambulation, cycle ergometer, and ascent/descent of stairs. Ambulation was started by postoperative day 1 or 2 in almost all studies. Interventions in four studies were continued until discharge, although in two studies, they were continued until 4 months after surgery. Respiratory physiotherapy  and psychoeducation were implemented as additional interventions with early mobilization. The aim of psychoeducation was to improve disease coping skills  by applying a patient-centered approach. These interventions in two of the studies were conducted in the same institution. In addition, the psychoeducation was conducted four times per patient by trained nurses and was based on the Human Becoming Practice Methodologies . Herdy e2 test was 52%, and the P-value for heterogeneity was 0.06, and although these results indicated moderate heterogeneity in the six studies, early mobilization significantly increased the distance walked during the 6MWT at hospital discharge.In the meta-analysis of all included studies, the distance walked during the 6MWT at discharge was set as the objective variable. In total, 391 patients were included in the analyzed studies . The results of the meta-analysis are shown in In the present meta-analysis, early mobilization was shown to clinically significantly improve physical function at discharge. To our best knowledge, this is the first meta-analysis to examine the effectiveness of early mobilization on physical function in patients after cardiac surgery.In a previous systematic review, early mobilization was shown to have a positive effect on improving physical function . The outAccording to previous studies, the minimum important clinical difference in the 6MWT is an increase in distance walked of 25 m for patients with CAD and 14.0\u201330.5 m for patients with other diseases ,32. The Bed rest induces changes in skeletal muscle atrophy and inflammatory markers . CardiacThe present analysis showed that not only early mobilization but also respiratory exercise and psychoeducation were conducted as interventions after cardiac surgery. Respiratory dysfunction and depression are reported likely to occur after cardiac surgery ,39. In tNone of the analyzed studies reported adverse events during early mobilization. Serious adverse events are uncommon during early mobilization when safety considerations are addressed prior to mobilization ,30. TakeThere are several limitations in the present meta-analysis. First, only six studies were included, and these studies were conducted only in Brazil and Denmark. In addition, two of the six studies were from 10 years ago. As the technology and context of cardiac surgery have continued to advance in the last decade, such as in the reduction of its invasiveness, the background of these older studies is likely to be different. Although the present analysis could not clarify this, the combination of current improvements in cardiac surgery and earlier mobilization may have resulted in a better postoperative prognosis compared to the results reported here. Moreover, this meta-analysis included only 391 patients after cardiac surgery, and the female ratio was 13\u201345%. In the present meta-analysis, about 60% of the patients were from the studies of Hojskov et al., and thus, this could influence the results of the meta-analysis, which included only six studies. Based on the above results, this meta-analysis shows heterogeneity, and the possibility of overestimation may be present. Second, only patients diagnosed as having CAD or ischemic heart disease and underwent CABG only were included. In addition, the present study could not distinguish whether on-pump or off-pump CABG was performed. If off-pump CABG is conducted, patients benefit from early discharge and recover their physical function better at hospital discharge. On the other hand, to the best of our knowledge, there are few RCTs examining the effects of early mobilization after other types of cardiac surgery, such as aortic valve replacement and mitral valve plasty, on physical function. The type of cardiac surgery is a determinant of distance walked during the 6MWT , so furtThird, the effect of early mobilization alone was not considered because respiratory exercise and psychoeducation were also conducted as interventions along with early mobilization in the included studies. On the other hand, early mobilization suggests a comprehensive intervention that includes respiratory exercise and psychoeducation. These interventions will be performed in almost 100% of postcardiac surgery patients in the clinical setting. However, the novelty of the present meta-analysis was to clarify the effect of these interventions on improving physical function at hospital discharge. In addition, in almost all included studies, the difficulty of blinding the participants and personnel to early mobilization itself indicated a high risk of bias. Fourth, only the 6MWT was included in the present assessment of physical function. Other assessments of physical function  were included in the analyzed studies, and thus, these indexes should also be studied. Finally, the primary outcome was assessed only at discharge from hospital, and the effects of early mobilization on long-term factors of prognosis such as mortality and hospital readmission were not considered. In fact, one of the included studies showing significant differences in the 6MWT between the intervention and control groups reported that the significant difference in the 6MWT was lost at four weeks after discharge . As a neBy integrating the data of RCTs of early mobilization for physical function in patients after cardiac surgery, the findings of the present meta-analysis underscore the fact that early mobilization after cardiac surgery may improve physical function  at hospital discharge. Five of the six included studies showed a significantly positive effect of early mobilization, and no adverse events occurred during early mobilization after cardiac surgery. Early mobilization after cardiac surgery also tended to be combined with respiratory exercise and psychoeducation. Further study is required to examine the effectiveness of early mobilization with increased numbers of studies and patients and for other types of cardiac surgery."}
+{"text": "Vigna unguiculata) plays a key role in family farming systems in Senegal. It makes an essential contribution to economic, nutritional and food security. Although it is crucial, little is known about how farmers classify the diversity of local varieties or about the social practices associated with them. The aim of this study is to characterize the farming practices associated with growing cowpea in Senegal. Surveys were conducted involving 335 rural farmers living in 37 villages, spread across seven regions that produce cowpea. An average of ten farmers were randomly selected in each village. The results reveal that cowpea is a key feature of cropping systems in the studied area. Our findings highlight the high diversity of local cowpea varieties with 59 local names inventoried. In 75% of cases, the name refers to the seed\u2019s morphology or color. Cowpea production is more diverse in Diourbel and Louga and less diverse in the south. More than half the farmers (57%) acquired their cowpea seeds  outside their village, either from markets, seed suppliers or NGOs. This new understanding of farmers\u2019 expertize in the management of cowpea and its local variability will help to valorize local diversity in breeding programs.Cowpea (The online version contains supplementary material available at 10.1186/s13002-022-00506-y. Vigna unguiculata (L.) Walp.) is one of the most important leguminous plant grown in tropical savannah zones in Africa [Cowpea  and valorization of this legume. It is particularly relevant for breeding programs, which require the availability of a wide genetic diversity . In thisBased on new collections and specific more exhaustive surveys, this study aims to characterize the farming practices associated with growing cowpea in Senegal for the first time. In particular, it aims to: (i) identify the role that cowpea has in the cropping system, by describing the range of species that it is associated with; (ii) survey and characterize its diversity based on the local nomenclature and the date to reach maturity and (iii) identify the farmers\u2019 seed supply.The surveys were conducted between September 2015 and March 2016 in the main cowpea producing regions in Senegal . The K\u00e9dougou region was also surveyed in order to identify the characteristics of the cowpea varieties grown in this area. The villages surveyed were chosen in consultation with agents from the services of Regional Rural Development Division to facilitate access to villages that grow cowpea. To optimize the coverage of the main cowpea producing zones, three departments were visited in each region  in Senegal for conservation.The villages\u2019 geographic coordinates were recorded on a tablet with the aid of the software Sygic: GPS Navigation 17.3.27 Android.The age, ethnic group and profession were used to characterize the farmers interviewed. The frequency, the average salience and Smith\u2019s index for each species and variety were calculated with the R AnthroTools package . The freThe number of cowpea morphotypes that farmers identified and named was used to estimate the varietal richness . To furtStats and FactoMinR were used for exploratory statistical analyses and to test the hypothesis.The farmers\u2019 responses regarding the sowing and harvest dates allowed to propose a classification system according to the phenology of the cowpea varieties. The association between the variety types and the regions was checked using a Chi-square test. The maps showing the village locations and the spatial distribution of the accessions were compiled using the software R (version 3.6.0 for Windows). The software packages The panel of interviewees comprised 156 women and 179 men, for a total of 335 people. In the different regions, on average, about ten farmers were randomly selected per village\u2014except in the K\u00e9dougou Region, where it was only possible to interview four farmers per village . Among those interviewed, 50.8% spoke Wolof, which is the language mainly spoken in the regions of Thi\u00e8s, Louga, Diourbel and Saint-Louis. The Serer, which represented 17.9% of the interviewees, are found in the Fatick, Thi\u00e8s and Diourbel regions. Lastly, the Toucouleur (10.5%) and Moors (3.3%) occupy the Louga and Saint-Louis regions, while the Mandinka, Jola, Bainuk, Bedick and Manjak live in the Fatick, K\u00e9dougou and S\u00e9dhiou regions Table .Table 2CTwenty-four (24) different species grown with cowpea were identified in the seven regions which were studied. The most frequently cited species grown with cowpea were groundnut and millet, which on average are grown, respectively, by 85% and 71% of the farmers interviewed Table . Howeverp-value\u2009=\u20090.008).The correspondence analysis shows that the regions of Thi\u00e8s, S\u00e9dhiou, Louga, Fatick and Diourbel have similar cropping profiles: red sorrel, sesame and sorghum, in addition to cowpea, groundnut and pearl millet. The Saint-Louis region differs, with watermelon (grown by 62.9% of interviewees) and onion (22.6%), melon, cucumber and tomato, whereas the K\u00e9dougou region is characterized by fonio, pearl millet and cotton, rarely grown elsewhere , \u201cSosso\u201d\u00a0is used by the Mandinka, \u201cNiao\u201d\u00a0by the Serer, \u201cDeulleugane\u201d by the Moors and \u201cOufithion\u201d\u00a0by the Manjak.During the survey, 702 cowpea accessions were collected in Thi\u00e8s (84), Louga (155), Diourbel (158), Fatick (85), Saint-Louis (122), K\u00e9dougou (19) and S\u00e9dhiou (79) . One to A wide range of reasons is used by the farmers to identify their cowpea varieties. Indeed, 75% of names make reference to morphology (seed color and size or vegetative cycle), 14% are named after a person  and 1% refer to the geographic origin (the zone they came from). Lastly, 9% have names that refer to a specific event (details not provided here) or are arbitrary Table .Table 6PMost of the time, the names of varieties are composed of a generic name for cowpea in the local language plus a second term, which either refers to simple morphological characteristics (seed color), people\u2019s names or zone of origin. Among the Mandinka, for example, cowpea is known by the generic name \u201cSosso.\u201d In order to identify red cowpea, farmers add the suffix \u201cwoul\u00e9roung\u201d (red) to the name \u201cSosso.\u201d In all the regions visited, the names generally referred to morphology, particularly seed color . Sometimes seed size is added . Some cowpea names are associated with the seeds\u2019 geographic origin (Fouta cowpea) or a person . In Senegal, the GOANA agriculture program, launched in 2008 by the former President of the Republic, Abdoulaye Wade, coincided with the introduction of a cowpea variety that is now called after the program. The Goana variety is sometimes called \u201cpea\u201d (because the shape of the seed is quite round or full) or \u201cnenou naat,\u201d which means \u201cguinea fowl\u2019s egg,\u201d in reference to the marks on the seed\u2019s integument Table .Table 7LAfter standardizing the spelling and identifying the synonyms, 36 names of varieties were kept. Irrespective of the ethnic group, the cowpea varieties called white cowpea , red cowpea (25%) and black cowpea (15%), and Baye Ngagne (9%) are the most commonly grown in Senegal.The zone in the north and center of the groundnut producing area has the greatest diversity (Louga and Diourbel), whereas K\u00e9dougou has the fewest varieties. Cowpea production is more diversified in the regions of Diourbel and Louga, followed by Thi\u00e8s, Saint-Louis and S\u00e9dhiou, respectively Fig.\u00a0.Fig. 5AvThe average number of cowpea varieties per farmer ranged from 1 (K\u00e9dougou) to 3 (Diourbel and Louga) Table . The DioThe majority of the farmers interviewed grow cowpea as a single crop (65%). This method of cultivating cowpea is far more frequent in four regions in Senegal, namely Louga, Diourbel, K\u00e9dougou and Saint-Louis. Groundnut is the species most commonly associated with cowpea. This association was described for 28% of the farmers surveyed, especially in the regions of Thi\u00e8s, Fatick, S\u00e9dhiou and Diourbel. Cowpea is also associated with maize (3%) in the Saint-Louis region, millet (0.3%) in the K\u00e9dougou region and market gardening (0.85%) in the regions of Louga, Saint-Louis and K\u00e9dougou Table .Table 9CIn the regions of Thi\u00e8s, Fatick, Diourbel, K\u00e9dougou and S\u00e9dhiou, cowpea is grown in the rainy season. In general, sowing is in June and July (53.42%) and harvesting is in September and October (93.44%). In the Louga region and part of the Saint-Louis and Diourbel regions, sowing is in August and September (42.60%) and harvesting is in November. Floodplain cultivation of cowpea is only found in the Saint-Louis region (3.99%). For this type of production, sowing occurs between November and January and harvesting is between February and March.There are three groups of cowpea varieties grown in Senegal that can be distinguished according to their development cycle: early (number of days\u2009<\u200970), semi-early (between 70 and 90\u00a0days) or late (number of days\u2009\u2265\u200990). The early maturity varieties represent 81.34% of the varieties grown. They are found in all regions, except K\u00e9dougou. Semi-early varieties (3.84%) are grown in Louga and Diourbel. Lastly, late maturity varieties (14.67%) are generally grown in the regions of K\u00e9dougou, Thi\u00e8s and Saint-Louis Table .Table 10Most of the interviewees (57%) stated that they obtained their first cowpea seeds at markets or from seed suppliers, NGOs, cooperatives or farmer organizations outside the village. Forty-two percent (42%) obtained them from relatives or neighbors in the village. How seeds are acquired varies depending on the region Table . Eighty-Drawing on the new collections and the recent surveys, which were more exhaustive than earlier surveys, the aim of this study was to characterize the farming practices associated with growing cowpea in Senegal. It focused particularly on the range of species grown in association with cowpea. The richness and variability of cowpea varieties were established in reference to the farmers\u2019 nomenclature. We also identified where farmers obtained their seeds.In all the zones surveyed, cowpea producers also grow groundnut and millet. In Senegalese farming systems, these three species are complementary. Along with sorghum, cassava, watermelon and red sorrel, they are the main cash crops grown in the center and north of the groundnut growing area, which is ideal for growing cowpea. Our findings on the regional distribution of species diversity are similar to those obtained when the FAO conducted inventories of the agricultural species in rural areas , in whicThe majority of farmers surveyed grow cowpea as a single crop. This cropping system is found in the regions of Louga, Diourbel and K\u00e9dougou. In the groundnut growing area, which includes the regions of Diourbel and Louga, there has been a rainfall deficit for decades. However, cowpea is adapted to these conditions. More and more land is being used to grow cowpea. Between 2012\u20132017, cowpea was grown on 165\u00a0452\u00a0ha, on average. This increased to 257\u00a0219\u00a0ha in 2019 . In thesIn the regions of Thi\u00e8s and Louga, young people grow cowpea, which could help reduce immigration. In fact, in this part of the country, the legume is grown as a cash crop on large areas of land. In the S\u00e9dhiou region, young people also grow cowpea, although it is often neglected in favor of other crops. This could be explained by the fact that varieties from other crops are better adapted to the groundnut producing zone, such as the S\u00e9dhiou region. In S\u00e9dhiou, cowpea is traditionally valorized by women. In the regions of Diourbel, Fatick and Saint-Louis, cowpea is grown by aged farmers, who probably know more about traditional accessions and their cropping practices.This study helped to confirm the area of distribution of cowpea production in Senegal. In fact, in the regions of Diourbel, Louga, Thi\u00e8s and Saint-Louis, collecting several varieties from one farmer is common, whereas in the S\u00e9dhiou and K\u00e9dougou regions, cowpea is less common and, on average, there is seldom more than one variety per farmer. The cultivation of this legume is more diversified in Diourbel and Louga. This reveals the importance and richness of the species in the central north and north, the main cowpea growing areas in Senegal . The depThe analysis of diversity based on the local names for cowpea allowed us to identify six appellations for the cowpea species. On a varietal level, 59 different names were identified. Varieties whose seeds have the same morphological features may have different names depending on the ethnic group. These names essentially refer to seed color, size or people\u2019s names. Thus, the farmer manages diversity by recognizing perceptible characteristics, especially morphological features . By stud. (2007), on a local level, cowpea diversity is generally due to its phenological adaptability to environmental constraints. The abundance of early maturing accessions may be due to the adoption of improved varieties that are early. Late varieties are no longer grown in the main cowpea producing areas because rainfall has been irregular or insufficient for four decades. This may also explain the high number of early varieties. The earliest varieties were collected in S\u00e9dhiou, which has the longest rainy season. However, in this region, very small areas were cultivated for home-consumption. The variability of rainfall in the different regions could explain the phenological diversity observed. In fact, more late accessions are grown in the K\u00e9dougou region, where rainfall is higher, and in the Saint-Louis region, where floodplain cropping plays an important role. These types of varieties are valuable because they are dual purpose and can be used as seed and fodder. In fact, under favorable conditions, they produce a large amount of seeds and fodder [Cowpea is mainly grown during the rainy season in all the zones surveyed, except the Saint-Louis region, where cowpea is also grown on the floodplain. Three groups  were identified according to the varieties\u2019 development cycle. According to Kouakou et ald fodder . The latd fodder , late mad fodder .In the last two decades, most of the seeds in the farmers\u2019 possession were purchased at the market or obtained from agricultural services, NGOs, farmers\u2019 organizations and cooperatives. These types of structure are common to several villages. Consequently, the same variety can be found in different villages or regions, even if it has different names. Thus, the pleasing appearance of seeds of one cowpea variety can encourage people to buy it at a market, even if they are unaware of its germination performance and agricultural value.Many of the people surveyed obtained their first seeds in the village, either through donations or by trading with relatives, friends or neighbors. Similarly, married women obtain their first seeds from their husband or parents-in-law, along with plots of land, after leaving their place of birth to go to their husband\u2019s place of residence. Thus, women rarely take seeds from their home or continue to obtain seeds from their relatives, especially if they live in different villages.The majority of seeds from the season preceding this study were home-grown. In fact, farmers keep a share of their previous harvest for seed. Consequently, farmers only purchase or obtain seeds at the market or from relatives or neighbors the year after a poor harvest or a food shortage.Identifying the nomenclature for the local cowpea varieties and their seed management system is essential for optimizing local diversity. This study revealed the considerable diversity of local names. This diversity is an indicator of the importance of cowpea in Senegalese farming systems. The names primarily refer to the seed morphology or color, a feature that facilitates identification. The named diversity of cowpea is greater in regions where the crop systems are less diversified. In the studied area, more than half the cowpea seeds grown by farmers are obtained from markets, NGOs, agricultural services and projects and then farmers produce and conserve their own seeds. Cowpea is generally grown as a single crop or associated with groundnut or maize. The length of the growing cycle is rarely used by farmers to identify their varieties. However, we classified varieties in terms of development cycles because of the difference observed between sowing and harvesting dates. This study made it possible to characterize the diversity of cowpea grown in Senegal. Undoubtedly, the diversity of farming practices and cowpea cropping systems is closely linked to the diversity of the biological types grown in the country and vice versa.Additional file 1: Collection areas and socio-cultural characteristics of surveyed farmers.Additional file 2: The various species cultivated with cowpea in the areas visited.Additional file 3: List of the 702 cowpeas accessions, their local names and signification, regions of collection, acquisition and cycle."}
+{"text": "Polytrichum have been shown to contain rare benzonaphthoxanthenones compounds, and many of these have been reported to have important biological activities. In this study, extracts from Polytrichum formosum were analyzed in vitro for their inhibitory properties on collagenase and tyrosinase activity, two important cosmetic target enzymes involved respectively in skin aging and pigmentation. The 70% ethanol extract showed a dose-dependent inhibitory effect against collagenase (IC50 = 4.65 mg/mL). The methanol extract showed a mild inhibitory effect of 44% against tyrosinase at 5.33 mg/mL. Both extracts were investigated to find the constituents having a specific affinity to the enzyme targets collagenase and tyrosinase. The known compounds ohioensin A (1), ohioensin C (3), and communin B (4), together with nor-ohioensin D (2), a new benzonaphthoxanthenone, were isolated from P. formosum. Their structures were determined by mass spectrometry and NMR spectroscopy. Compounds 1 (IC50 = 71.99 \u00b5M) and 2 (IC50 = 167.33 \u00b5M) showed inhibitory activity against collagenase. Compound 1 also exhibited inhibition of 30% against tyrosinase activity at 200 \u00b5M. The binding mode of the active compounds was theoretically generated by an in-silico approach against the 3D structures of collagenase and tyrosinase. These current results present the potential application from the moss P. formosum as a new natural source of collagenase and tyrosinase inhibitors.Mosses from the genus  Polytrichum formosum Hedw. is a moss that belongs to the genus Polytrichum (Polytrichaceae). Polytrichum species are known to have ethnobotanical applications as a hair growth stimulant, burn and wound healing and anti-inflammatory agents, diuretic, antipyretic, antidotal, and also for the treatment of pneumonia  + and 387.0880 [M \u2212 H] \u2212) with UV\u03bbmax at 268 and 345 nm  was collected in the Black Forest, Germany , in April 2018 and taxonomically identified by Professor Dr. Nils Cronberg . The specimen is identical to the voucher specimen with ID no MT20211 sent for deposition at the Lund University Botanical Museum (LD). In this study, the whole plant was used for analysis.P. formosum was dried at room temperature and ground to a fine powder using a bead mill. The dried powder was homogenized in 70% ethanol (v/v) in water, methanol and ethyl acetate solvents for the extraction of small molecules. The solution (1:10 g/mL of dry weight to solvent ratio) was macerated for 30 min by rotating mixer at room temperature. After centrifugation, the supernatant was collected and used for analysis. Then, 70% ethanol and methanol extracts were diluted to the final concentrations as indicated in each experiment. The ethyl acetate extract was evaporated and the dry extract was dissolved in ethanol absolute (1:10 g/mL of dry plant weight to solvent ratio).Clostridium histolyticum  from Sigma-Aldrich was determined by the procedure previously described by Chajra et al. [50 values were calculated from the equation generated by a logarithm fit of the experimental data.Collagenase inhibition activity was measured by following the enzymatic conversion of the synthetic substrate FALGPA (N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala) purchased from Bachem (ref. 4006713.0025) to FAL (N-(3[2-Furyl]acryloyl)-Leu) + Gly-Pro-Ala (GPA). The collagenase activity from a et al. . EthylenThe mushroom tyrosinase inhibitory activity was determined by a spectrophotometric method using a microplate reader, Synergy HT (Biotek), based on Kamkaen et al.  with modTyrosinase-driven conversion of L-Tyr to dopachrome was followed by an increase in the absorbance of the samples at 475 nm for 25 minutes. All tested samples were incubated at 25 \u00b0C during the process of data acquisition. Kojic acid  was used as a positive control. The points in the linear range of the absorbance versus time plots were applied to calculate the slopes, directly proportional to tyrosinase activity. Then, the values of tyrosinase inhibition, expressed as the percent of the activity of the test samples versus the control experiment (pure solvent), were calculated for all samples according to the following equation:P. formosum to collagenase and tyrosinase was investigated by the Target Binding\u00ae technology [Clostridium histolyticum (type IA) was prepared at 1.5 mg/mL in 50 mM phosphate buffer (pH 7.5). Moreover, mushroom tyrosinase was prepared at 1.25 mg/mL in 50 mM ammonium acetate buffer. Collagenase and tyrosinase solutions were mixed with the corresponding plant extract and incubated at room temperature for 10 min and 5 min, respectively. After the incubation step, the mixtures were filtered using a 10 kDa cut-off centrifugal filter. After a series of washing steps to eliminate the unbound compounds, the target\u2013ligand complexes were solubilized in water and the recovery of bound compounds was obtained by the addition of acetonitrile. All experiments were performed in duplicate.The affinity of the constituents from chnology , a methochnology  with few\u00ae for collagenase was performed using the analytical method containing water and 0.1% vol. of formic acid (A) and pure acetonitrile (B) in 0.5 mL/min with the gradient mobile phase of B phase as follows: 5\u201345% (0\u201312.50 min); 45\u201395% (12.50\u201317.50 min); hold at 95% (17.50\u201320.49 min); 95\u20135% (20.49\u201320.50); hold at 5% (20.50\u201322.50 min) in the Kinetex EVO C18 reverse-phase column , maintained at 40 \u00b0C. Target Binding\u00ae for tyrosinase was performed using the analytical method containing water and 0.1% vol. of formic acid (A) and pure acetonitrile (B) in 0.5 mL/min with the gradient mobile phase of B phase as follows: 5\u201325% (0\u20136 min); 25\u201390% (6\u201315.45 min); 90\u201395% (15.45\u201315.50 min) hold at 95% (15.50\u201318.90 min); 95\u20135% (18.90\u201319 min); hold at 5% (19\u201321.50 min) in the Kinetex Biphenyl reverse-phase column , maintained at 40 \u00b0C.Finally, the ligands and raw extracts were analyzed by UHPLC  with a photodiode array detector coupled to the LCMS2020 mass spectrometer (electrospray ionization in negative and positive ion mode). Target Bindingv/v) in water, three times, at 40 \u00b0C for 30 min in the ultrasound bath followed by 24 h in an agitation mixer. The combined extracts were concentrated under a vacuum at 40 \u00b0C. The dry crude extract (1.00 g) was partitioned in distilled water and ethyl acetate to concentrate and remove more polar compounds. The ethyl acetate phase was evaporated and 160 mg of dry extract was then dissolved in 2.4 mL of absolute ethanol. The solution was used to separate the compounds 1\u20134 by preparative liquid chromatography (LC) Armen Spot Prep II (Armen) with a C18 column . The fractions were purified using water containing 0.1% vol. of formic acid (A) and pure acetonitrile (B) with the gradient mobile phase of B of 45% (0\u201312.5 min), 45\u201370% (12.5\u201318 min), 70\u201395% (18\u201320 min), 95% (20\u201325 min) at a flow rate of 120 mL/min and an UV detection at 250 and 270 nm. The fractions containing the purified compounds 1\u20134 were evaporated under vacuum which provided the corresponding quantities: 1  >95%); 2  82%); 3  >95%) and 4  73%). All isolated compounds were analyzed using the HPLC Agilent 1200 system (Agilent) with an Agilent 1260 Infinity Diode array Detector (applied range: 210\u2013600 nm) coupled to a mass spectrometer Agilent 6120 Quadrupole LC/MS , using a Vydac Denali C18 reverse-phase column  maintained at 25 \u00b0C during all analyses. The mobile phase was composed of water containing 0.1% vol. of formic acid (A) and pure acetonitrile (B), delivered at 1.5 mL/min with the gradient of B phase as follows: 45% (0\u201312.5 min), 45\u201370% (12.5\u201318 min), 70\u201395% (18\u201320 min), 95% (20\u201325 min).The dried and powdered plant (1:10 g/mL of dry weight to solvent ratio) was extracted with 70% ethanol , using water/acetonitrile mobile phase, both containing formic acid at 20 mM (phase A/B respectively). Phase B increased from 10% to 100% in 10 min, then held at 100% B for 2 min, returned to 10% in 0.1 min, and equilibrated for 2 min at a flow rate of 350 \u00b5L/min, and column temperature of 40 \u00b0C.Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was realized on Agilent 1290 Infinity II UHPLC (Agilent Technologies) with diode array detector (DAD) coupled to an Agilent 6545 QTOF with an electrospray ionization source. Analyses were performed in negative and positive ion mode. Compound The raw data were processed by MassHunter workstation software (Agilent Technologies), Qualitative Analysis (version B.07.00).1H and 13C chemical shifts are reported relative to TMS (\u03b4(1H) = 0.0 ppm, (\u03b4(13C) = 0.0 ppm) using the solvent signals as secondary reference (DMSO: \u03b4(1H) = 2.49 ppm and \u03b4(13C) = 39.5 ppm; acetone: \u03b4(1H) = 2.05 ppm and \u03b4(13C) =29.9 ppm). The HSQC spectra were acquired using a data matrix of 4096 \u00d7 1024 complex points with acquisition times of 200 and 15 ms in F2 and F1, respectively. Adiabatic bilevel 1H decoupling was employed during acquisition. The HMBC spectra were acquired using a data matrix of 4096 \u00d7 512 complex points with acquisition times of 220 and 6 ms in F2 and F1, respectively. The DQF-COSY spectra were acquired using a data matrix of 4096 \u00d7 1024 complex points with acquisition times of 220 and 53 ms in F2 and F1, respectively. NMR spectroscopic data are provided in the The presented NMR spectra were recorded on either a 600 MHz Avance III HD spectrometer equipped with a BBFO SmartProbe or an 800 MHz Avance III HD spectrometer equipped with a 5 mm TCI CryoProbe (Bruker Biospin). All compounds were designed by ChemBioDraw Ultra 14.0 while the protein file was downloaded from the protein data bank . P. formosum extracts and isolated ohioensins. Molecular docking was utilized to suggest the binding modes of the compounds inside the tested enzymes. Additional biological assays should be performed to verify the efficacy and safety of application for human use. The obtained results encourage further investigation of the active compounds from bryophytes to the cosmetic and medicinal fields. Moreover, bryophyte species are being increasingly used in biotechnological applications, especially for their advantage to grow in bioreactor-based cultivation systems [Polytrichum species, a protonema suspension culture of the species Polytrichum juniperinum was already established as a potential platform to produce bioactive compounds of interest [In conclusion, this study reports the first in vitro analysis on collagenase and tyrosinase inhibitory activities of  systems . In the interest . Therefo"}
+{"text": "The overall pelvic floor dysfunction rate in the WPHR group was lower when compared with both control groups (95% CI: 2.15\u20133.62 vs. 2.34\u20133.54 in women from PAW group and vs. 3.0\u20134.56 in women from WNPA group). Based on this study, it can be concluded that all of the pelvic floor related symptoms, their frequency, and severity levels do not qualify recreational horseback riding as being a risk factor for developing pelvic floor dysfunction in women.The aim of this study was to compare the condition of the pelvic floor in women who are involved in regular recreational horseback riding, with both physically active women as well as women not undertaking any recreational physical activity. Taking into account horseback riding and physical activity, 140 healthy women aged 17 to 61 were divided into three groups: women practicing horseback riding (WPHR) (46 persons), physically active women (PAW) (47 persons) and women not physically active (WNPA) (47 persons). The Australian Pelvic Floor Questionnaire (APFQ) was used to measure the extent of pelvic floor dysfunctions in women from all three groups. The lowest average values were found in the group of women practicing recreational horseback riding, and the highest in the group of women not physically active . Statistically significant intergroup differences were recorded only for the bowel function rate ( In recent years, there has been an increasing interest in horseback riding as a form of leisure and recreational activity for women in Poland. According to the data by the Polish Equestrian Federation , the numHorseback riding is a physical activity of a moderate effort at 3\u20136 MET, with the aerobic cost increasing as the horse speed accelerates . PositivPositive effects of horseback riding on body posture and motor skills are connected with movements of the back of the horse that forces rider\u2019s pelvis to move. Depending on the speed of riding, these movements may almost accurately imitate pelvis movements when walking. During the slowest horse gait, the walk, the rider\u2019s body is subjected to a rocking motion of up-down, forward-back, and right-left movements. Hence, horse movement is described as three-dimensional. When the saddle is raised, the pelvis is subjected to a retroversion, and when it falls the pelvis undergoes an anteversion, which is opposite to the direction of the saddle movement. The forward-back movement of the pelvis is superimposed by up-down and right-left movements, which in practice looks as if the ilia were moving along two ellipses, separate for the right side and the left side . During Thus, horseback riding is a form of physical activity that, more than any other physical activity, directly affects the female pelvis. This effect is both, movement-related (connected with the pelvic movements adjusted to the speed of riding), as well as sensation-related (resulting from a direct contact between the lower part of the pelvis and the saddle). Therefore, by engaging the pelvis to such an extent, horse-back riding has a significant impact on the female pelvic floor. During the vaginal electrode EMG tests, it was demonstrated that the tension of the female pelvic floor increased with the increase in the horse\u2019s speed rate. The highest tension was recorded in the canter in a two-point position, slightly lower in the sit-position canter, and it returned to the resting state when the horse was stationary . On the A cross-sectional study investigated the occurrence of symptoms of pelvic floor dysfunctions among 140 potentially healthy women aged 17 to 61. Taking into account the horseback riding and physical activity, the analyzed women were divided into three groups: women practicing horseback riding (WPHR), physically active women (PAW) and women not physically active (WNPA). The criterion for inclusion in the group of women engaged in recreational equestrian activities was to practice horseback riding for at least one year for a minimum of 1 h per week. The criterion for inclusion in the group of physically active women was to do other forms of recreational physical activity also for at least one year for a minimum of 1 h per week. Finally, the criterion for inclusion in the group of women not physically active was lack of doing any recreational activities. The exclusion criteria for all of the three groups were as follows: poor health, chronic cardiovascular and respiratory diseases, metabolic disorders, renal diseases and mobility impairment. Women who did not know the Polish language to the extent allowing them to understand the questionnaire and answer the questions in an informed manner were also excluded from the study. In the group of women practicing horseback riding, the additional criterion for exclusion was regular recreational exercise of additional physical activity and in the group of women physically active . The enrolment process of the surveyed women is presented in Finally, 46 women aged between 17 and 61 were enrolled in the group of women systematically involved in a recreational horseback riding. In this group of women, the actual number of years of equestrian experience varied between 6 and 32 (16.1 \u00b1 6.2) and the number of hours they spent on horseback riding was between 1 and 6 h a week (3.3 \u00b1 1.9). A total of 47 women aged 21\u201360 were included in the group of physically active women, most of whom attended fitness activities (42.6%) for 1 h twice a week, did jogging (19.2% of the group) between 1 and 7 h a week or attended the gym (14.9%) one to three times a week. The group of women not physically active comprised of women aged 20\u201360 who declared a lack of any recreational form of physical activity taken regularly. The study was conducted using anonymous surveys. A group of women involved in recreational horseback riding was selected from all over Poland. The questionnaires were distributed during nationwide training on horseback riding, at the national conference of hippotherapists in Pozna\u0144, and among women who use the services of boarded horses in Silesia. Printed copies of anonymous surveys in the form of a stapled set comprising of a cover letter explaining the circumstances and purpose of the stilt; a survey with socio-demographic data; a questionnaire on the pelvic floor ailments; and a pen were distributed either in a white envelope marked with a stamp and a return address or in a white unmarked envelope when it was possible to return the surveys to the survey collection container. Physically active women were tested in the same way in fitness centers, gyms, swimming pools or recreational areas in parks in Upper Silesia. Women not physically active were selected from the civil servants in the voivodeship of Silesia. The survey did not specify the time required to complete the questionnaire; it was possible to return the surveys back on the same day or the next day or send them back by post.The Australian Pelvic Floor Questionnaire (APFQ)  was usedThe surveyed women completed the questionnaire anonymously and on their own. The study was authorized by the Bioethics Committee for Scientific Studies at the Jerzy Kukuczka Academy of Physical Education of Katowice. All of the study procedures were performed according to the Helsinki Declaration of Human Rights of 1975 (modified in 1983).2 test. The level of significance was set at p < 0.05.The characteristics of the participants were described by mean and standard deviation. The occurrence of pelvic floor dysfunction symptoms is presented as a percentage within the groups. Differences in the demographic parameters  and the APFQ scores were analyzed by a one-way analysis of variance (ANOVA) with the \u201cgroup\u201d as a between-subjects factor. For significant results, the post hoc Tukey\u2019s analysis was performed. Differences in the number of childbirths and the frequency of the symptoms were analyzed by the Chip = 0.025 and PAW group p = 0.016) did not have significant impact on the values of the individual indicators of the pelvic floor dysfunction symptoms . p = 0.021). The post-hoc analysis (Tukey\u2019s test) confirmed significantly lower values of this rate in the WPHR group (p = 0.033) and in the PAW group (p = 0.05) as compared to the WNPA group. 2 test did not reveal any statistically significant differences . (Most of the surveyed women claimed that they did not have any negative symptoms of urinary bladder or lower urinary tract function (except for the urgency symptom in the WNPA group). The surveyed women who complained of such symptoms, usually reported that these symptoms were present occasionally (score\u20141 point). In individual cases, some women claimed that the symptom occurred always/daily or more than 15 times (for urinary frequency) and more than 3 times (for nocturia symptoms), which were rated at 3 points. In the WPHR group, this was the case for urinary frequency (in three women), in the case of urgency (one woman), in the case of a weak stream (one woman), in the sense of incomplete bladder emptying (one woman), and in the case of dysuria (one woman). In addition, in individual cases, the highest severity of the symptoms (3 points) was reported by women in the PAW group . A similar situation was observed by the members of WNPA group, where one person reported the maximum severity with regard to the following symptoms: nocturia, stress incontinence, incomplete bladder emptying, and dysuria. For most of the symptoms rated in this part of the questionnaire, the women practicing horseback riding recreationally reported the symptoms of urinary bladder and lower urinary tract dysfunctions the least frequently. On the contrary, the women not physically active usually indicated the occurrence of such symptoms the most often. However, the Chi 0.061). .2 test showed that problems with bowel consistency were statistically significant more frequently in the women in the WNPA group as compared to the women in the WHR and PAW groups (p = 0.0045). At a level close to the statistical significance (p = 0.052), there were differences in the occurrence of obstructed defecation between the members of the PAW group and the WPHR group.  occurrence of this symptom . Only two women not physically active reported a daily (3 points) occurrence of this symptom. With regard to other bowel symptoms, it was only among the women in the WNPA group where the majority reported having such problems. These symptoms were related to the bowel consistency symptom (72.34%) and the occurrence of fecal urgency (51.06%). No women in the WPHR group reported the maximum severity of the bowel symptoms (score\u20143 points). Among the women in the PAW group, this applied to one case (incomplete bowel movement). Most often, the highest degree of severity was recorded among the members of the WNPA group . The ChiR group. .Amongst all of the symptoms related to the pelvic floor dysfunction, surveyed women indicated that the occurrence of prolapse symptoms was the least frequent. In both, the WPHR and PAW groups, symptoms of this type occurred in five cases , and in each case the symptoms were sporadic (APFQ score\u20141 point). In the WNPA group, these types of symptoms occurred in six women , and were occasional (<1/week). .2 test showed statistically significant differences (p = 0.0059). (The same number of women (5) in each of the groups were not sexually active. A similar percentage of women was sexually active\u00a0most days or daily . Sexual function disorders were found in a small proportion of the respondents, with the exception of dyspareunia symptoms in the WNPA group being present in 40.42% of the women. Nobody in the WPHR or PAW groups reported a high severity of the symptoms (APFQ score \u20143). On the other hand, in the WNPA group, this was indicated by six women . Only for occasional coital incontinence symptoms , the Chi0.0059). .Horse riding is a physical activity that significantly engages not only the pelvis but also pelvic floor itself. However, the survey results show that recreational horseback riding does not affect the subjective sensation of the pelvic floor adversely.The assessment of the pelvic floor was applied to bladder function, bowel function, prolapse of reproductive organs, and sexual function.The movement of a horse in each gait vast majority of the time is carried out in the vertical axis (up and down), with this movement being the most clearly seen in the canter. In the trot the movement amplitude is lower, but the frequency is higher, which makes it difficult for the rider\u2019s joints to absorb the moves in a smooth way. Jumps, as a part of physical activity, are identified by the researchers as a risk factor that may contribute to urinary incontinence in women ,23. The Bowel function of women practicing horseback riding and physically active women is very similar, whereas a greater degree of constipation is observed in the group of women not physically active. According to the studies on constipation in the group of 62,032 women aged 36\u201361, the occurrence of constipation depends on the level of physical activity and the diet maintained, in particular on the fiber content in the diet . StudiesThe results of the studies showed that the prolapse of pelvic organs was not a problem in any of the surveyed groups.In regard to sexual function, the women practicing horseback riding rated this function as slightly better than the women from the reference groups. However, this was not confirmed statistically. These results confirm the observations indicating that horseback riding does not have a negative impact on this area of life for female recreational horseback riders. This is in line with the results obtained by Alanee et al. .The results of this cross-sectional study indicate that recreational horseback riding performed by women does not result in increased symptoms of pelvic floor dysfunction compared with control groups. It is unknown whether such a risk could occur in women who undertake this type of activity later in life . Therefore, further study should be undertaken to assess the influence of horseback riding on the pelvic floor in the period around and after the menopause, which is critical for the appearance of dysfunction in this part of the body. It is also unknown what effect different riding techniques exert on the activity of the pelvic floor. In future studies, the pre-activity and reflex activity of pelvic floor muscles during horseback riding should therefore be analyzed, both in women with a properly functioning pelvic floor and in women with symptoms of pelvic floor dysfunction. Such study would allow to assess the possible therapeutic potential of horseback riding in relation to pelvic floor dysfunction. The results of the study show that women practicing horseback riding are not suffering from severe lower urinary tract problems and that there is no negative impact of recreational equestrianism on bowel function. Moreover, this study has demonstrated that there are no severe symptoms related to the prolapse of pelvic organs and no adverse effects on sexual function among women practicing recreational horseback riding were found. Therefore, it can be concluded that compared to women practicing other types of physical activity and physically inactive women, horseback riding does not contribute to the appearance of symptoms of pelvic floor dysfunction in women."}
+{"text": "Virtual reality (VR) has gained popularity in daily life, and VR food cues seem to elicit food cravings, similar to real food cues. However, little is known about the impact of VR food cues on actual food intake.In real life (RL), exposure to food cues in a situation in which the desire to eat food interferes with the completion of a food-related task reduces the subsequent food intake . In this study, we examine, on the one hand, whether the pre-exposure effect could be replicated in RL and, on the other hand, whether this effect could be extended to VR contexts.The study used a 2 (stimulus type: food vs nonfood) \u00d7 2 (mode: VR vs RL) between-subject design (n=175). Participants were randomly assigned to 1 of the 4 conditions.P=.02). In addition, among female participants, we found that exposure to both food cues  resulted in lower food intake than exposure to both nonfood cues (P=.05). In contrast, this effect was not observed among male participants (P=.34). Additionally, VR and RL cues generated similar emotional and behavioral responses .We found the main effect of mode on food intake, with a higher food intake after both VR conditions than after RL conditions  reduces food intake, indicating that a VR pre-exposure procedure may effectively be applied exclusively for women.ClinicalTrials.gov NCT05169996; https://clinicaltrials.gov/ct2/show/NCT05169996 It is teir eyes . Howevereir eyes . This mens et al  and has ns et al  and for ns et al . These dns et al . Given tRecently, technological advances, such as virtual reality (VR), have made it possible to run the pre-exposure procedure in a cost-efficient and flexible way. VR can simulate a virtual environment with tempting food stimuli by delivering multisensory cues , which lThe pre-exposure procedure is a 2-phase paradigm. In the original study that identified this procedure, participants in the experimental condition performed a consumer knowledge task in which they were asked to associate various wrappers of candies with the corresponding flavors . In contIn sum, the first aim of this paper is to replicate the pre-exposure effect with real food temptations among adults. We posit that exposure to real tempting food cues decreases subsequent intake of a similar tempting food as compared to exposure to nonfood cues. Moreover, the aim of the research is not only to replicate the pre-exposure effect with real foods but more importantly to better understand the potential of VR in the pre-exposure procedure. The next sections will focus on the extension of the pre-exposure effect to VR contexts.There is a large body of research on how consumers respond to food cues in VR. Prior research has shown that VR food cues can produce emotional  or behavioral  responses similar to those observed in real-life (RL) contexts ,11,18,19Overall, the prior literature suggests that consumers\u2019 emotional and behavioral responses in food-related VR contexts are similar  to those in the RL ,22. In aIn sum, although there is a burgeoning body of research that focuses on how VR affects food cravings, marketers and researchers are struggling to fully understand the impact of VR on eating behaviors . It is important to note that our research focuses on the actual food intake (instead of food cravings) after interacting with food or nonfood cues in both VR and RL contexts. For the application in the VR context, we designed a task context where people could interact with the food in VR. To validate that this procedure could be used to trigger the pre-exposure effect, we tested it both in an RL and in a VR context. We assume that participants\u2019 behavior in the VR context will be similar to their behavior after exposure to physical food temptations. In addition, prior research has shown that passive exposure to VR food cues induces high levels of food cravings as compared to VR nonfood cues . TherefoThis study used a 2 (stimulus type: food vs nonfood) \u00d7 2 (mode: VR vs RL) between-subject design. Participants were randomly assigned to 1 of the 4 conditions. A total of 218 participants (18-30 years old) were recruited with flyers and posters from a large Western European university. Participants received course credits or \u20ac7.50 for participation. This study was approved by the university ethical committee of the institution (file no. 2018-PC-9033) where the corresponding author was employed at the time of data collection. All participants provided written informed consent. In addition to the 4 conditions reported in this paper (n=175), an additional condition  was collected with the aim to investigate the effect of brands presented in VR on brand memory and purchase intention, and the results are reported elsewhere ,25.Participants were asked to refrain from eating 2 hours before the study. After entering the laboratory, participants were told that they were participating in 2 unrelated studies: a puzzle game and a taste test. First, they were asked to finish a tangram (puzzle game) with either food products  or nonfood products , either in VR or in RL (depending on their condition). Following that, they were asked to participate in a taste test of chocolate candies. Given the taste test, it was required that the participants were not allergic to peanuts (self-reported). Finally, they completed a questionnaire measuring game experiences  and emotional responses . We also measured the attractiveness of chocolate and the desire to eat chocolate for participants in the food cues condition (both VR and RL contexts). Participants also reported their demographic data  and data on height and weight. In addition, participants\u2019 hunger levels and the completion time of the puzzle game were measured as covariates. After all measurements, the participants were thanked for their participation and debriefed on the fact that both studies were related. Note that the groups that were in the RL conditions also got an opportunity to play the VR game at the end of the procedure after all measurements (as the study was advertised as a study involving VR and the participants were told in the factsheet that they would engage in a VR game during participation).An immersive VR game was developed with a gameplay based on the pre-exposure effect. The game is played by wearing a head-mounted display VR  instrument and players can interact in the virtual environment with handheld controllers in the lab. The task in the game is to finish a tangram puzzle. Two versions of the game were developed, one in which the tangram pieces were tempting food products  and the other in which the tangram pieces were plain pieces see . PlayersIn the taste test, participants were presented with 2 bowls of chocolate, one with chocolate-covered peanuts from the brand M&M\u2019s and the other with private label chocolate-covered peanuts. Participants were instructed to taste at least 1 of each bowl and were allowed to eat as much of the chocolate as needed to evaluate the products on several dimensions . Food intake was measured by weighing the bowls before and after the test. The participants were left alone in the lab for 5 minutes during the taste test to avoid socially desirable behavior. The distribution of food intake was skewed, so we transformed the food intake with a logarithmic format.We measured the perceived entertainment and difficulty of puzzle games as indicators of game experiences. Specifically, the perceived entertainment was measured with 4 items  on a 7-point scale . The perTo assess whether emotional responses induced in VR contexts were similar to those generated in RL contexts, we measured arousal and valence using the Self-Assessment Manikin (SAM) scale . The SAMr=0.75, P<.001).In the food cues condition, we also measured the appeal of food. Specifically, the attractiveness was measured with a single item  on a visual analog scale (VAS) ranging from 0  to 100 (\u201ca whole lot\u201d). Similarly, the desire to eat chocolate was measured with a single item  on a 7-point scale ranging from 1  to 7 (\u201ca whole lot\u201d). The 2 measures were correlated with each other (2 (SD 2.7). Data from 13 participants were excluded from analysis because of nonconforming to the study tasks , impossible values , reporting a low preference for chocolate , or spontaneous mention of not liking the peanut M&M\u2019s (n=1). The final sample consisted of 162 adults  with a mean age of 22.4 years (SD 4.1) and a mean body mass index (BMI) of 21.9 kg/mF3,158=0.092, P=.97), gender , BMI , weight concerns , chocolate preference , hunger , and time since last intake . These results indicate that our randomization was successful.We conducted a randomization check to test whether the sample was distributed equally across conditions see . We compF1,158=2.234, P=.14, partial \u03b72=0.014). A main effect of mode was found . Food intake (g) was higher in the VR condition  than in the RL condition . The interaction between stimulus type and mode was not significant . R2 of the complete model was 0.047 (adjusted R2=0.029); we also performed an analysis controlling for the hunger level, liking of chocolate candies, and time since the last intake as covariates, and the significant level of the main effects was not substantially different.ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and food intake  as the dependent variable was performed. No significant main effect of stimulus type was found . Food intake (g) was higher in the nonfood condition  compared to the food condition . A main effect of mode was found . Food intake (g) was higher in the VR condition  than in the RL condition . The interaction between stimulus type and mode was not significant . R2 of the complete model was 0.070 (adjusted R2=0.045).ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and food intake  as the dependent variable was performed. A significant main effect of stimulus type was found . No significant main effect of mode was found . The interaction between stimulus type and mode was not significant . R2 of the complete model was 0.038 (adjusted R2=\u20130.034).ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and food intake  as the dependent variable was performed. No significant main effect of stimulus type was found . No main effect of mode was found . The interaction between stimulus type and mode was not significant . R2 of the complete model was <0.001 (adjusted R2=0.019).To test whether the game experience produced by VR cues was similar to that produced by RL cues, ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and entertainment as the dependent variable was performed. No significant main effect of stimulus type was found . No main effect of mode was found . The interaction between stimulus type and mode was not significant . R2 of the complete model was 0.031 (adjusted R2=0.012).ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and difficulty as the dependent variable was performed. No significant main effect of stimulus type was found . No main effect of mode was found . The interaction between stimulus type and mode was not significant . R2 of the complete model was 0.012 (adjusted R2=\u20130.007).To test whether emotional responses produced by VR cues are similar to those produced by RL cues, ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and valence as the dependent variable was performed. No significant main effect of stimulus type was found . No main effect of mode was found . The interaction between stimulus type and mode was not significant . R2 of the complete model was 0.007 (adjusted R2=\u20130.012).ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and arousal as the dependent variable was performed. No significant main effect of stimulus type was found . R2 of the model was 0.014 (adjusted R2=0.002).To test whether food cravings produced by VR food cues are similar to those produced by real food, ANOVA with mode (VR vs RL) as the independent variable and chocolate attractiveness  as the dependent variable was performed. Note that we only used half of the data set for subsequent analyses because only half of the participants were exposed to food cues. No main effect of mode was found . R2 of the model was 0.033 (adjusted R2=0.020).ANOVA with mode (VR vs RL) as the independent variable and the desire to eat chocolate (answer to \u201cHow much did you feel like eating the chocolate?\u201d) as the dependent variable was performed. No main effect of mode was found . The completion time was longer in the nonfood condition  compared to the food condition . A main effect of mode was found . The completion time (seconds) was longer in the RL condition  than in the VR condition . The interaction between stimulus type and mode was also significant . Simple contrasts revealed that when in RL mode, the completion time was longer in the nonfood condition  than in the food condition . However, in VR mode, there was no significant difference in the completion time between the 2 conditions . Additionally, simple contrasts revealed that when the stimulus type was nonfood, the completion time was longer in the RL condition  than in the VR condition . However, when the stimulus type was food, there was no significant difference in the completion time between the 2 conditions . R2 of the complete model was 0.120 (adjusted R2=0.104).In this study, we also measured the completion time of the puzzle game. To test whether participants spent a similar amount of time in completing the puzzle game between VR and RL contexts, ANOVA with stimulus type (food vs nonfood) and mode (VR vs RL) as independent variables and completion time as the dependent variable was performed. As shown in Due to the rising popularity of VR in our daily life, it is necessary to better understand how VR food cues affect consumers\u2019 eating behavior . However, prior research has mainly focused on the impact of VR food cues on food cravings ,12,20,21Unexpectedly, we found that the main effect of mode (VR vs RL) on food intake is significant. Specifically, we found a higher food intake after both VR conditions (puzzle game in VR with either food or nonfood cues) than after RL conditions. A possible explanation could be that playing a game in VR is arousing and that arousal leads to increased food intake. There is ample evidence that arousal may lead to increased food intake . Howeverfemale=6.000 (SD 1.147) versus Mmale=5.364 (SD 1.382), with F1,160=8.792 and P=.003. Consequently, given that tempting chocolates may induce a behavioral conflict between the desire to eat and the completion of a food-related task among females, we observed the pre-exposure effect. In contrast, chocolates may not have been sufficiently tempting for males, therefore resulting in a lack of behavioral conflict and activation of control processes in males.We did not replicate the pre-exposure effect in the full sample. In addition, there was no interaction effect between stimulus type (food vs nonfood) and mode (VR vs RL) on food intake in the full sample. Prior studies have shown that pre-exposure effects are in some instances specific to males  or femalMoreover, we found that emotional and behavioral responses  induced by VR cues are similar to those generated by RL cues. This suggests that there were no additional confounders between the conditions. In addition, this study showed that exposure to VR food cues elicits similar food evaluations  compared to exposure to RL food cues. Given that the appeal of food (measured in this study) was similar to food cravings (measured in prior studies), this study does not provide evidence that VR food cues induce weaker food cravings compared to RL food cues ,22. In tAlthough this study provides useful insights into the impact of VR cues on food intake, 3 limitations should be considered. First, in this research, we found that manipulation affects the completion (exposure) time of puzzle games. Specifically, participants spent more time completing puzzle games in nonfood or RL conditions as compared to food or VR conditions. We tried to replicate the pre-exposure effect in RL contexts; however, the completion time was longer in the nonfood condition (around 5.5 minutes) than in the food condition (around 3.5 minutes). Prior research on the pre-exposure effect used a consumer knowledge task , a puzzlSecond, this research used the pre-exposure paradigm  to examine the effect of VR cues on food intake. Both tasks are more specific to laboratory contexts. Until now, we are not clear whether the pre-exposure procedure still works well outside laboratory contexts. Given the unlimited possibility of creating various eating environments with HMD-VR, future research could examine how VR affects food intake in different situations. Prior research on VR food cues focused on some daily environments, such as living rooms, kitchens, and restaurants. For instance, there was no significant difference in food intake between the restaurant scene and the blank scene in VR environments . HoweverThird, in this study, we exclusively used confectionery food products when manipulating pre-exposure  and when measuring food intake . Both products belong to the same food category, which warrants caution with generalizing our results across all food products. Concretely, individual differences in perceptions of how appealing the stimulus food was, as well as strong preferences in particular brands of confectionery food products, could have affected food intake (at least) on the individual level. To rule out any potential product-specific bias, future studies could consider incorporating different or various food categories to manipulate pre-exposure as well as to measure food intake.Our findings contribute to 2 streams of literature: the pre-exposure procedure and the responses to VR food cues. Although prior research on the pre-exposure procedure has studied how food cues versus nonfood cues affect the subsequent intake of tempting foods in RL contexts ,4,15, ouFurthermore, our research extends the VR food cues literature by examining the impact of VR on actual food intake, instead of food cravings, considering that actual food intake is critical to understanding whether VR food cues increase or decrease subsequent intake of tempting foods in more naturalistic settings. In addition, in line with prior research on the effects of VR food cues on eating behavior\u2013related outcomes ,10, the Overall, in this study, we were unable to replicate the exposure effect in our complete sample. Subgroup analyses, however, showed that for women, exposure to food cues (either in VR or in RL) does reduce food intake, indicating that a VR pre-exposure procedure may effectively be applied exclusively for women. Moreover, we found that exposure to cues in VR (either food or nonfood) results in a higher overall food intake as compared to exposure to cues in a similar RL setting. Finally, we demonstrated that VR and RL cues elicit similar emotional responses ."}
+{"text": "The as-prepared fully exposed Pt3 cluster catalyst shows higher conversion (35.4%) and remarkable alkene selectivity (99.0%) for n-butane direct DDH reaction at 450\u2009\u00b0C, compared to typical Pt NP and Pt SA catalysts supported on ND@G. Density functional theory calculation (DFT) reveal that the fully exposed Pt3 clusters possess favorable dehydrogenation activation barrier of n-butane and reasonable desorption barrier of butene in the DDH reaction.Metal nanoparticle (NP), cluster and isolated metal atom  exhibit different catalytic performance in heterogeneous catalysis originating from their distinct nanostructures. To maximize atom efficiency and boost activity for catalysis, the construction of structure\u2013performance relationship provides an effective way at the atomic level. Here, we successfully fabricate fully exposed Pt 3 clusters and correlate dehydrogenation activity with average coordination number of Pt-Pt bond.Direct dehydrogenation has been a thematic research area resulting from the energy shortage and petroleum gas upgrading scenario. Here, the authors fabricate fully exposed Pt For a typical supported metal catalyst, many factors including the crystallographic surface, chemical composition, particle size, and metal\u2013support interaction can affect catalytic performances2. Recently, it has been found that well-dispersed nanoparticles (NPs), clusters, and isolated metal atoms  exhibited surprisingly different catalytic performances from bulk materials with the development of the advanced characterization tools and well-controlled synthesis technology, leading to the establishment of the structure\u2013performance relationship at the atomic level11.Heterogeneous catalysis plays an indispensable role in chemical production12, low adsorption energy of reactants/intermediate13 and maximal atom utilization15. Nevertheless, it is worth noting that clusters, though with inactive bulk components, were more active than SAs in some catalytic reactions. Anderson et al.16 deposited size-selected Aun+  on TiO2 support to study the relationship between Au size and CO oxidation activity. Owing to adverse CO-Au binding, Au or Au2 was inactive for CO oxidation. The activity was found in the order of Au7\u2009>\u2009Au3\u2009>\u2009Au4\u2009>\u2009Au2\u2009\u2248\u2009Au1. Corma et al.18 studied the size effect of different types of Pt species  supported on various oxides. In the low-temperature NO reduction reaction with CO, the surface of Pt clusters favored NO dissociation and CO oxidation. The moderate adsorption of CO on Pt clusters could suppress catalyst poisoning, leading to higher activity than that of Pt SAs. Szanyi et al.19 found that Ru clusters favored CH4 formation in contrast to Ru single atoms. Due to the limited ability to activate hydrogen, single Ru atoms can only allow CO formation but cannot further hydrogenate it to CH4. However, we find later that the modulation of the chemical state of metal species by strong metal\u2013support interaction is more important for observed selectivity regulation  in CO2 hydrogenation reaction over Ir/CeO2 catalyst with different size of Ir species20. Nevertheless, the highly dispersed subnanometer-sized metal clusters (<1\u2009nm) achieve maximum atomic exposure and utilization and possess various chemical coordination environments and chemical states that eventually affect the activity, selectivity, and stability of catalysts. It is the cluster with certain size, instead of single atoms, that was linked to high stability, selectivity, and activity in the heterogeneous catalysis, where the catalysts showed remarkable performance on structure-sensitive catalytic reactions22. But the small clusters always suffered from aggregating into metal NPs at high temperature, limiting their applications into high-temperature reactions26. Therefore, considering industrial application, designing thermodynamically stable metal cluster with low metal loading  still remains highly desired.In several previous works, SAs catalysts exhibit advantages such as unique reaction pathway28. The primary catalyst designated for DDH is a Pt-based catalyst30. The side reactions of DDH including coke deposition, hydrogenolysis, and cracking are usually structure-sensitive, which prefer to occur on Pt NPs with large average Pt\u2013Pt coordination numbers (CN)31. An ideal solution to suppress side reactions and promote catalytic performance is to downsize Pt NPs and regulate CN of Pt to a moderate level. In general, the addition of promoter metals to Pt afforded bimetallic and alloying systems, which can improve both the dispersion and stability for Pt species. For instance, as to n-butane dehydrogenation, Pt particle sizes decreased with increasing Sn loading in the PtSn/\u03b8-Al2O3 catalyst. Higher atomic efficiency and the formation of PtSn alloying phases were correlated with higher activity and n-C42\u2212 selectivity. But the conversion rate remained low at each active site in such a large size PtSn alloying system32. Considering maximize atomic utilization, Pt/Cu single atom alloy catalyst is proposed in propane dehydrogenation. Compared with Pt NPs, isolated Pt atoms dispersed on copper nanoparticles dramatically enhance the propylene selectivity and stability33. Several bimetallic cluster catalysts have also been reported, including small raft-like PtSn clusters34, PtSn/ND@G35, Pt/Sn-Beta catalysts36, PtSn cluster38, and PtZn clusters defined in zeolites40, which displayed high activity for DDH reaction with excellent durability. But it also remains major challenges to synthesize clusters with precisely controlled metal-metal coordination numbers, and fundamentally understand the structure effects  on dehydrogenation performance at atomic level.Direct dehydrogenation (DDH) for light olefins production has been a thematic research area resulting from the energy shortage and petroleum gas upgrading scenario3 clusters stabilized on the defective graphene through Pt\u2013C bond, with the geometric partitioning by the atomically dispersed Sn promoter, which can precisely tune the CN of supported Pt clusters based our previous reported methods35. The obtained Pt3 clusters with 0.5\u2009wt% Pt loading showed higher conversion for n-butane DDH than Pt NPs and Pt SAs supported on ND@G. At a relatively low temperature (450\u2009\u00b0C), n-butane rate of Pt3 clusters has achieved 1.138\u2009mol\u00b7gPt\u22121\u00b7h\u22121. Moreover, we systemically established the structure\u2013performance relationship by correlating the DDH activity with the average CN of Pt\u2013Pt bond on ND@G supported Pt NP, Pt cluster, and Pt SA catalysts. By density functional theory (DFT) calculations, we found that the Pt3 cluster catalyst\u2019s unique structure facilitates the activation of C\u2013H bond and the desorption of butene. Such a structure\u2013performance relationship may provide an insight for rationally designing highly active heterogeneous DDH catalysts in atomic scale.In this paper, we fabricated fully exposed Pt3 cluster catalysts, a serial of PtSn/ND@G catalysts were prepared by the co-impregnation method, denoted as Pt0.8Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20090.85), Pt1.7Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20091.7), Pt3.4Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20093.4), and Pt6.8Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20096.8). The Pt and Sn precursors anchored on the ND@G support composed of a diamond core and a defect-rich graphene shell  was carried out. Due to the difference in Z-contrast, the supported Pt species can be easily distinguished from Sn atoms, as shown in Fig.\u00a00.8Sn/ND@G  and Pt6.8Sn/ND@G (the dispersion of Pt was 5.6%). In other words, the excess Sn species did not further promote the Pt dispersion but covered up the Pt clusters, preventing Pt atoms from being exposed to adsorbed reactant molecules.Besides, from the XRD profiles , Pt foil, and PtO2 are shown in Fig.\u00a00.8Sn/ND@G catalyst, it exhibited a distinct peak at 1.7\u2009\u00c5 and a weak peak at 2.6\u2009\u00c5, which matched up with the first coordination shell of Pt\u2212C/O and Pt\u2013Pt, respectively, indicating that Pt NPs were located on ND@G support through Pt\u2013C bonds. The average CN of Pt\u2212C/O was 3.1, and the average CN of Pt\u2212Pt was 3.2. Notably, for the Pt1.7Sn/ND@G, Pt3.4Sn/ND@G and Pt6.8Sn/ND@G samples, they all showed a strong signal of Pt\u2212C/O and a relatively weak signal of Pt\u2013Pt. For the Pt1.7Sn/ND@G catalyst, the average CN of Pt\u2212Pt is ~2 (2.3), verifying the presence of Pt3 clusters. Due to the limitation of the characterization methods ), the coordination property of Pt atoms is statistically averaged. The realistic Pt clusters on current catalyst could have distribution in both atomicity and configuration42, but the majority of Pt clusters compose of around three Pt atoms. Moreover, all the Pt atoms in the Pt3 clusters (dispersion of Pt was 99.1%) are fully exposed. Significantly, both the features of high dispersion and fully exposure allow all the Pt atoms accessible for adsorbing reactant molecules. For the Pt3.4Sn/ND@G and Pt6.8Sn/ND@G catalyst, the CN of Pt\u2013Pt is 2.0 and 2.1, respectively, similar to that of Pt1.7Sn/ND@G, suggesting that Pt atoms in Pt3.4Sn/ND@G and Pt6.8Sn/ND@G mostly existed in the form of Pt3 clusters. In contrast, for the Pt/ND@G catalyst, the average CN of Pt\u2212Pt bond was 6, indicating the formation of large Pt NPs, agreeing well with the STEM results. The wavelet transformation (WT) of Pt L3-edge EXAFS oscillations visually displayed the structure of Pt species in both the k and R spaces  measurement was employed to provide detailed information of the structure and the local environment of Pt and Sn species. From the X-ray absorption near edge structure (XANES) spectroscopy, the intensity of the white line for as-prepared catalysts situated above that of Pt foil, indicating the existence of slightly positively charged Ptort Fig.\u00a0. The EXAces Fig.\u00a0. Figure\u00a00.8Sn/ND@G, Pt1.7Sn/ND@G, Pt3.4Sn/ND@G, and Pt6.8Sn/ND@G to understand the role of Sn species on catalytic performance under the atmospheric condition and at 450\u2009\u00b0C. The conversion of n-butane and the selectivity to C4 olefin over those catalysts are shown in Fig.\u00a0Pt\u22121\u00b7h\u22121. The value of Kd  and the initial selectivity at 10\u2009h were 0.0421\u2009h\u22121 and 96.0%, respectively. In general, the deactivation of DDH reaction was attributed to the sintering of Pt NPs with large Pt\u2013Pt CN, resulting in structure-sensitive side reactions and coke formation to block Pt active sites on the catalyst surface43. Notably, benefited from the mono-dispersed Sn, the catalytic performance of Pt0.8Sn/ND@G was dramatically enhanced. The butane conversion reached 25.9% and then dropped to 20.8% in 10\u2009h test. The selectivity towards C4 olefin reached 98.9% at the initial stage. The value of Kd was 0.0313\u2009h\u22121, showing that the lifetime was extended with decreasing CN of Pt\u2013Pt. Significantly, the Pt1.7Sn/ND@G catalyst possessed excellent catalytic activity and remarkably high selectivity for n-butane DDH. Figure\u00a04 olefin was as high as 99.0% at the initial stage, and the value for Kd was only 0.0223\u2009h\u22121. When further adding Sn species, the conversion and n-butane rate dramatically decreased over the Pt3.4Sn/ND@G and Pt6.8Sn/ND@G catalysts as shown in Fig.\u00a04 olefin selectivity over Pt3.4Sn/ND@G and Pt6.8Sn/ND@G were both close to that of Pt1.7Sn/ND@G. For Pt3.4Sn/ND@G and Pt6.8Sn/ND@G, the structure of atomically dispersed Pt3 was almost intact, but the excess Sn species covered up the Pt3 clusters, causing Pt dispersion decrease from 99.1% to 5.6%, as shown in Fig.\u00a03 clusters can significantly reduce the conversion and n-butane rate. As shown in Fig.\u00a00.8Sn/ND@G and Pt1.7Sn/ND@G. When Sn/Pt\u2009 =\u20091.7, the atomically dispersed Pt3 cluster was thought to be the optimum catalyst, as all the Pt atoms are full exposed for n-butane DDH. Moreover, with the increase of reaction time, the butane conversion and the selectivity for Pt1.7Sn/ND@G catalyst remained constant at 24% even after 50-h of reaction , and Pt1/ND@G (Pt SA). Firstly, aberration-corrected HAADF-STEM was employed to investigate the morphology of Pt1/ND@G. As shown in Supplementary Fig.\u00a01/ND@G showed that no Pt crystal or Pt-oxide phase was observed , Pt1.7Sn/ND@G (Pt3 Cluster), and Pt1/ND@G (Pt SA), respectively. The fully exposed Pt3 clusters showed the best catalytic performance for n-butane DDH reaction than Pt/ND@G and Pt1/ND@G. Clearly, the n-butane DDH rate of Pt1.7Sn/ND@G (1.138\u2009mol\u00b7gPt\u22121\u00b7h\u22121) was higher than those of Pt/ND@G (0.373\u2009mol\u00b7gPt\u22121\u00b7h\u22121) and Pt1/ND@G (0.193\u2009mol\u00b7gPt\u22121\u00b7h\u22121). Consistently, the initial selectivity towards butene on Pt1/ND@G was only 97.1%, followed by Pt1.7Sn/ND@G (96.6%) and Pt/ND@G (93.1%). Figure\u00a031. But interestingly, the n-butane conversion rate did not increase proportionally with a decrease of the Pt\u2013Pt CN in Fig.\u00a01.7Sn/ND@G, but Pt1/ND@G.Figure\u00a01.7Sn/ND@G, and Pt1/ND@G. In the DFT calculations, Pt (111) surface, Pt SA on single-lay graphene (Pt1-Gr), and a triangle Pt3 cluster doped into single-vacancy graphene (Pt3-Gr) were used to model Pt/ND@G, Pt1/ND@G, and Pt1.7Sn/ND@G, respectively. The dehydrogenation process follows four steps: (i) the adsorption of n-butane; (ii) the dehydrogenation of n-butane to surface adsorbed 2-C4H9 species (2-C4H9*); (iii) the surface adsorbed 2-C4H8 (2-C4H8*) is generated via the further dehydrogenation of 2-C4H9*; (iv) the desorption of 2-C4H8* to form the product 2-butene gas. Supplementary Tables\u00a01-Gr, Pt3-Gr, and Pt (111). The Gibbs free energy profile of butane dehydrogenation to 2-butene on these three models is shown in Fig.\u00a01-Gr), 1.19\u2009eV (Pt3-Gr), and 2.00\u2009eV (Pt (111)), respectively, suggesting that the Pt3-Gr is the most active for butane dehydrogenation. These data provide a rational interpretation for the high catalytic activity of Pt3-Gr and low activity of Pt1-Gr in our experimental observations. Deep hydrogenation  is known to be the origin of coke and hydrogenolysis44. The difference between the energy barriers of deep dehydrogenation (EDH) and the desorption (EDP) of 2-butene (\u0394ES\u2009=\u2009EDH\u2009\u2212\u2009EDP) can be used to evaluate the selectivity of dehydrogenation from alkanes to alkenes44. The more positive of the value \u0394ES indicates the better selectivity of the catalyst. Supplementary Fig.\u00a0S for Pt1-Gr (0.45\u2009eV), Pt3-Gr (0.11\u2009eV), Pt (111) (\u22120.04\u2009eV), respectively. Due to insufficient metal active sites to the further dehydrogenation and weak adsorption of 2-butene, Pt1-Gr exhibits the best selectivity compared to Pt3-Gr and Pt (111). Based on the calculated results, Pt3-Gr is predicted to have high activity and selectivity for the butane dehydrogenation to butene, while Pt1-Gr is expected to have high selectivity and relatively low activity, which agrees well with the aforementioned experimental observations.To better understand the relationship between metal size and its catalytic performance, DFT calculations were conducted to unveil the difference in dehydrogenation among Pt/ND@G, Pt3 clusters where the atomically dispersed Sn atoms play the role of geometric partitioning. We constructed the structure\u2013performance relationship between Pt NP, fully exposed Pt3 cluster, and Pt SA for n-butane DDH reaction. The fully exposed Pt3 clusters showed the highest n-butane conversion and remarkable alkene selectivity, compared to Pt NPs and Pt SAs, resulting from the facilitated activation of C\u2013H bond and the desorption of butene. Such relationship between Pt CN and n-butane DDH activity provides a valuable insight in the structure effect on catalytic performance and thus a new avenue to design DDH catalysts with high activity, selectivity, and stability.By optimizing the loading amount of Sn promoter, we fabricated fully exposed Pt2PtCl6\u2009\u2219\u20096H2O) and Tin (II) chloride dehydrate (SnCl2\u2009\u2219\u20092H2O) as metal precursors were purchased from Sinopharm Co. Ltd.Nanodiamond (ND) powders with the average diameter of 30\u2009nm were purchased from Beijing Grish Hitech Co., China. Analytical grade chloroplatinic acid (H\u22121) for 4\u2009h under flowing Ar gas (80\u2009mL\u00b7min\u22121). When it finished and cooled to room temperature, the final powder, ND@G, was collected for further use. A series of PtSn/ND@G catalysts were prepared by co-impregnation method using a fixed weight loading of Pt (0.5%) with varying weight loading of Sn: Pt0.8Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20090.85), Pt1.7Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20091.7), Pt3.4Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20093.4), Pt6.8Sn/ND@G (Sn/Pt atomic ratio\u2009=\u20096.8). First, certain amount of H2PtCl6 (20\u2009g/L) and SnCl2\u2009\u2219\u20092H2O (6.0\u2009g/L) were dissolved in 1\u2009ml ethanol and generated a clear solution. Then, 100\u2009mg ND@G powder was added and impregnated in the liquid solution. After that, the samples were dried in air at 80\u2009\u00b0C for another 6\u2009h. Finally, the solid sample were calcined in Ar (80\u2009mL\u2009\u22c5\u2009min\u22121) at 500\u2009\u00b0C for 4\u2009h and subsequently reduced in H2 gas (80\u2009mL\u2009\u22c5\u2009min\u22121) at 500\u2009\u00b0C for 1\u2009h. For reference, the Pt/ND@G (0.5\u2009wt% Pt) was prepared with the same process without the addition of Sn. Pt1/ND@G (0.1\u2009wt% Pt) was also prepared with the similar process without the addition of Sn and reduced by thermal treatment in H2 gas at 200\u2009\u00b0C for 1\u2009h.The nanodiamond@graphene (ND@G) hybrid carbon support was prepared by annealing fresh nanodiamond powders at 1100\u2009\u00b0C (heating rate 5\u2009\u00b0C\u00b7min\u03bb\u2009=\u20091.54\u2009\u00c5). HAADF-STEM measurements were conducted with a JEOL JEM ARM 200CF aberration-corrected scanning transmission electron microscope at 200\u2009kV accelerating voltage. XAFS measurements of the samples were carried out in Shanghai Synchroton Radiation Facility (SSRF). The H2-O2 titration measurements were performed on a Micromeritics AutoChem II 2920 equipped with a thermal conductive detector.The series of samples were characterized by X-ray diffraction (XRD) on a D/MAX-2500 PC X-ray diffractometer with monochromated Cu K radiation  of 18,000\u2009mL\u2219gcat\u22121\u2009\u2219\u2009h\u22121 on the basis of the whole feed gas .The catalytic test for the DDH reaction of n-butane was tested using a fixed-bed stainless-steel micro-reactor with a quartz lining under atmosphere pressure at 450\u2009\u00b0C, equipped with an online gas chromatography instrument (Agilent 7890 with an FID and a TCD detector). First, 50\u2009mg of sample was loaded into the stainless-steel reactor. The reaction was carried out in a feed gas with a composition of 2% H4 olefin  were calculated by the following formula:The rate and conversion of n-butane and the selectivity of total Ci is initial conversion after reaction 30\u2009min; Cf is final conversion value; t represents the reaction time (h); and kd is the deactivation rate constant (h\u22121) that is used to evaluate the catalyst stability .The catalyst stability was described by a first-order deactivation model:46 was used to perform spin-polarized DFT computations with the projector augmented wave (PAW) method48. The generalized gradient approximation in the form of the Perdew-Burke-Ernzerhof functional (PBE)49 was chosen for electron exchange and correlation. An energy cutoff of 400\u2009eV was employed for the plane wave expansion. The ground-state structure of bulk and surfaces were obtained by minimizing forces with the conjugate-gradient algorithm until the force on each ion is below 0.02\u2009eV/\u00c5, and the convergence criteria for electronic self-consistent interaction is 10\u22125.The Vienna ab initio simulation package (VASP) code3 cluster and Pt single atom embedded into a monovacancy at 5\u2009\u00d7\u20095 supercell of graphene was adopted to simulate the active site of butane dehydrogenation (Pt3-Gr) through comparative investigation between potential Pt cluster models and EXAFS data. The vacuum layer was set to 20\u2009\u00c5 to avoid interaction from adjacent cells. The Monkhorst\u2013Pack k-point set to 3\u2009\u00d7\u20093\u2009\u00d7\u20091 in the reciprocal lattice, and the electronic occupancies were determined according to the Gaussian smearing method with \u03c3\u2009=\u20090.1\u2009eV. Spin-polarized calculations were performed. For Pt (111) surface, a four-layer slab with a (3\u2009\u00d7\u20093) supercell  was employed. The successive slabs were separated by a vacuum region as thick as 20\u2009\u00c5 to eliminate periodic interactions. The Brillouin zone is sampled with a 3\u2009\u00d7\u20093\u2009\u00d7\u20091 k-points mesh by the Monkhorst\u2013Pack algorithm. The electronic occupancies were determined according to the Methfessel-Paxton scheme with \u03c3\u2009=\u20090.2\u2009eV. The bottom two layers of the slab were kept fixed to their crystal lattice positions. Spin-polarization is not considered in Pt (111) calculation. We have calculated zero-point energies (ZPE) of reaction species and transition states.A model with Pt3-Gr, Pt1-Gr and Pt (111) surface were obtained by the standard minimization of density functional theory (DFT). These configurations were used as the initial states, from which the constrained optimization method as described by Plessow. P. N50. was used to search the transition states (TS). The TS optimization convergence was regarded to be achieved when the force on each atom was less than 0.05\u2009eV/\u00c5. All transition states have been verified to include only one imaginary harmonic frequency corresponding to the transition vector of the reaction. Furthermore, small distortions along the transition vector followed by optimization toward the minima verified the connectivity of the transition states. The entropy contributions of butane, 2-butene and hydrogen gas were included in the free energy calculations. The most important contributions arise from the translational entropy51, which can be calculated using the following equation:2 gas gained 1.34\u2009eV and 1.02\u2009eV. It should be noted that the partial calculation work on the Pt3-Gr model is based on the theoretical part of our previous work35, and has been further improved in the Gibbs free energy calculations according to the main contribution of translational entropy.The most stable configurations of the reactant and intermediates on PtSupplementary InformationPeer Review File"}
+{"text": "Volumes of produced hydrogen were up to 25 and 6 times larger than those obtained with the corresponding free and cucurbit[7]uril-bound platinum monomer, respectively, at equal Pt concentration. The thermodynamics of the proton-coupled electron transfer from the Pt( The cucurbit[8]uril macrocycle can secure a platinum terpyridyl complex into a particularly reactive dimer that catalyzes the photoreduction of water. Sakai showed that Platinum(ii) terpyridyl (tpy) chloride 1a can be used both as a photosensitizer and H2-evolving catalyst in the presence of ethylenediaminetetraacetic acid (EDTA) as the sacrificial electron donor.3 A turnover number of approximately 3  was measured after 7 h in the presence of 0.4 mM of Pt complex 1a in a 2-(N-morpholino)ethanesulfonate (MES) buffer at pH 5.The design of artificial photosynthetic systems that split water into molecular hydrogen and oxygen remains a major challenge of our times.z2\u2013dz2 orbital overlap of the Pt centers,15\u201317 Sakai questioned whether sensitization is triggered by complex 1a as a monomer or dimer. A dimerization constant of 4 (\u00b12) \u00d7 103 M\u22121 and the quadratic dependence of the initial rate of H2 production as a function of the concentration of complex 1a (below 0.4 mM) indicate that either (a) photosensitization is mostly achieved by the 1a2 dimer \u2013 via a proton-coupled electron transfer (PCET) to the corresponding Pt(ii)\u2013Pt(iii)\u2013H hydride ruthenium(ii) cation as photosensitizer and methylviologen as electron relay. Furthermore, Yamashita and coworkers showed that grafting complex 1a onto solid supports  improves its catalytic activity by packing the complexes into arrangements with short Pt\u2013Pt distances. This led us to consider the cucurbit[8]uril (CB[8])-secured Pt tpy dimers prepared recently in our group as a possible new family of single component catalysts for water reduction.23\u201326 In the past few years, we have shown that Pt tpy complexes substituted at the 4\u2032-position with CB[8]-binding aryl units form 2\u2009:\u20091 head-to-head \u201cstacked\u201d assemblies, with both Pt tpy units sitting on top of each other at the same CB[8] portal, i.e. with two positively charged guests interacting with the same CB[8] portal while leaving the opposite one void of any host/guest interaction , a weakly bonding p\u03c3 LUMO and a strongly anti-bonding p\u03c3*. The stabilization of the d\u03c3* HOMO confers a net energy gain to the interaction.27,28Sakai also showed that amidate-bridged Pt dimers . Hydrogen nuclei H7, H8 and H9 located at the CB[8]-binding tolyl unit experience a typical upfield shift upon encapsulation , and the Caryl\u2013Cpyridyl torsional angles \u03d51 and \u03d52 between the CB[8]-binding tolyl group and the tpy scaffolds. In one assembly, both set of angles rotate in the same direction , and in the other, the directions are opposite  \u2192 \u03c0*(tpy)] transitions (the \u03c0*(tpy) orbital being better described as a 6pz(Pt)/\u03c0*(tpy) hybrid). Absorption bands above 350 nm are broad with lower extinction coefficients; they correspond to 1[5d(Pt) \u2192 \u03c0*(tpy)] metal-to-ligand charge transfers (1MLCT).The X-ray crystal structure of assembly CB[8]\u00b71bcell see . This ar\u03bb = 447 \u00b1 10 nm; 250 mW/LED). Aliquots were removed from the headspace and analyzed by gas chromatography as a function of time  in the presence of EDTA as sacrificial electron donor (30 mM). Solutions  were irradiated with 10 royal blue LEDs . CB[7] encapsulation of the tolyl tpy substituent does not significantly enhance performance, with up to 0.20 mL H2/10 mL solution at 1.0 mM Pt. Increasing the catalyst load above 0.50 mM and 1.0 mM is detrimental to H2 production, in the case of complexes 1b and CB[7]\u00b71b, respectively . To the contrary, H2 production is increased by 25- and 6-fold when the CB[8]-secured Pt dimer is used as catalyst, compared to complexes 1b and CB[7]\u00b71b . A pronounced increase in hydrogen production is observed as the catalytic load increases  formation using X-ray absorption fine-structure spectroscopy (XFAS),44 after irradiation of complex 1a (4.0 mM) and EDTA (30 mM) in a sodium acetate buffer at pH 5.2 solution was also monitored by electrospray ionization mass spectrometry, with 1.5 mM EDTA as sacrificial electron donor in water. Before irradiation, assembly CB[8]\u00b71b2, its binary complex CB[8]\u00b71b fragment, its free guest fragment, and an EDTA bridged Pt(tpy) dimer were detected  hydride cation  and the Pt concentration might have been detected when complex CB[8]\u00b71b2 is used as the photocatalyst . However, the more soluble Pt thiolate 1c obtained after exchange of the chloride ligand with l-cysteine afforded a dimerization constant KPt\u2013Pt of 4.0 (\u00b10.4) \u00d7 104 by ITC in water, in very good agreement with the 7-fold lower constant measured for complex 1b in the high ionic strength buffer environment. A ternary binding constant \u03b2 for complex 1c towards CB[8] of 1.9 (\u00b10.6) \u00d7 1013 M\u22122 (see equilibrium 1) was extracted. In other terms, dimer 1c2 (and most likely dimer 1b2) can be considered as a standalone guest forming a very tight 1\u2009:\u20091 complex with CB[8], with a binding affinity K\u2032 of 5 (\u00b12) \u00d7 108 M\u22121, obtained from 26 on a parent system.A linear correlation between the initial rate of Hlyst see . This re low see . As ment\u22121, respectively, in excellent agreement with the typical strength of Pt\u2013Pt interactions.45 They also indicate that approximately 52, 66, 74 and 81% of Pt complex 1b is already present as a dimer at concentrations of 0.2, 0.5, 1.0 and 2.0 mM, respectively, even in the absence of CB[8], in the MES/EDTA buffer. While a simple dimerization process is sufficient to fit enthalpograms and changes in UV-Vis absorption, further aggregation into trimers or larger oligomers cannot be ruled out even if, in our case, considering those processes leads to over-parametrization.The dimerization constants of complexes 1b and 1c return free energy terms of \u22125.1 (\u00b10.1) and \u22126.3 (\u00b10.1) kcal moliii)\u2013H hydride formation from the corresponding Pt(ii) complex  to calculate the free energy of Pt.11 Here, we can report that optimization in the gas phase with Grimme's newly developed B97-3c functional50 and def2-mTZVP basis sets51,52 returns accurate Pt\u2013Pt distances and geometries highly similar to those measured by X-ray crystallography. The two CB[8]\u00b71b2 arrangements  and 3.35 \u00c5 (vs. 3.44 \u00c5), respectively. Cl\u2013Pt\u2013Pt\u2013Cl dihedral angles are +3\u00b0 and \u221221\u00b0, respectively, in perfect agreement with the X-ray crystal structure. Grimme also showed that for a set of reactions involving transition metals, the B97-3c functional not only returns realistic geometries, but also more accurate free energies compared to the B3LYP functional, even when the latter is supplemented with a dispersive term.50 The enthalpic and entropic contributions at 25 \u00b0C, as well as the free energies of solvation, were calculated using the tight-binding semi-empirical method GFN2-xTB currently developed by Grimme and coworkers53\u201355 , thereby indicating an even competition between the Coulombic repulsion of the positively charged Pt centres and the dz2\u2013dz2 orbital overlap. This is also consistent with the presence of both arrangements in the unit cell of the X-ray crystal structure.Sakai notes that Pt\u2013Pt distances obtained by DFT optimization (B3LYP/LANL2DZ)ents see  were reo2 were optimized at the same level of theory. The Cl\u2013Pt\u2013Pt\u2013Cl dihedral angle in the most stable conformation of dimer 1b2 is 124\u00b0, and the Pt\u2013Pt distance 4.99 \u00c5 \u2013H hydrides at the same level of theory. As noted by Sakai, the Pt\u2013Pt bond is reinforced compared to Pt(ii)\u2013Pt(ii) interactions, and can be seen as a three-center two-electon Pt(ii)\u2013Pt(iii)\u2013H system11,56\u201358 with a Pt\u2013Pt bond order of 0.5 (further oxidation to a d7\u2013d7 Pt(iii)\u2013Pt(iii) system would afford a \u03c3 bond between both metals, as the dz2\u2013dz2 anti-bonding orbital would be empty). A unique CB[8]\u00b71b2\u2013H assembly is obtained with a pronounced shortening of the Pt\u2013Pt bond  complexes into mixed-valence Pt(ii)2Pt(iii)2 complexes.59 The Pt\u2013Pt axis also crosses the tpy planes almost perpendicularly -dz2(Pt)-s(H) hybrid  was also obtained (see ESIWe then optimized the corresponding open-shell Pt(brid see . A simild see ESI section.E) and free energies (\u0394G) of the PCET processes . (2) CB[n] encapsulation does not provide any electrostatic contribution to the PCET process, as it is separated from the Pt centers by the tpy ligands . (3) Aggregation of free Pt complex 1b must severely hamper the catalytic process, as calculations indicate a more favorable PCET process for dimer 1b2 compared to complex CB[7]\u00b71b , whereas hydrogen production is even poorer. (4) Most importantly, in addition to efficiently deaggregating Pt complex 1b (and likely preventing the formation of higher order oligomers), CB[8] secures the Pt dimer into a conformation that facilitates the PCET process. If dimerization of Pt complex 1b were quantitative in the presence and absence of CB[8], a 2.2 kcal mol\u22121 gain for the PCET process would be obtained upon addition of the macrocycle . The energy gain upon addition of CB[8] therefore ranges from 1.8 to 2.1 kcal mol\u22121 depending on Pt concentration. Also, removing CB[8] from complexes CB[8]\u00b71b2 and CB[8]\u00b71b2\u2013H and carrying out single point energy calculations returns an electronic contribution \u0394E of \u221231.4 kcal mol\u22121 for the PCET process, a 4.6 kcal mol\u22121 improvement compared to dimer 1b2 in its most stable conformation in the absence of CB[8] . In addition to being computed using adequate functionals and semi-empirical methods, we note that the quality of these calculated energy gains must be further enhanced by error compensation, as they represent relative stabilities of very similar assemblies. One can thus conclude that CB[8] encapsulation destabilizes dimer 1b2 by at least 1.8 kcal mol\u22121, enhances its reactivity, and promotes hydrogen production.The following observations can be made from the electronic contributions (\u0394sses see : (1) the60\u201362 we showed here, as a proof of concept with arguably modest TONs, that a macrocycle like CB[8] can be used as a tool to enhance the catalytic activity of a well-established family of multifunctional photocatalysts, namely Pt complexes. CB[8] effectively secures a pair of Pt complexes 1b on top of one another into reactive dimer CB[8]\u00b71b2, which upon irradiation yields volumes of hydrogen up to 25 and 6 times larger than those obtained with the corresponding free monomer 1b and the CB[7]-bound control CB[7]\u00b71b, respectively. A combination of semi-empirical and DFT calculations suggest that the kinetics and activity of the photocatalysts are governed by the thermodynamics of the PCET reaction from the Pt(ii) complexes to the corresponding Pt(iii)\u2013H hydride intermediates. CB[8]-promoted dimerization not only renders the process more exergonic , as the PCET process is more favourable starting from a Pt(ii) dimer compared to a monomer, but also by forcing the dimer into a more reactive conformation for Pt(ii)\u2013Pt(iii)\u2013H hydride formation .While noble metal-free catalysts and chromophores can promote water reduction efficiently,All analytical data is provided in the ESI.\u20202. RR, SS, HB and MN conducted all other experiments. EM, RR, SS, HB and MN wrote the manuscript.EM conceived the project, and mentored RR, MN and HB; TAW mentored SS; TAW and SS designed the photolysis experiments. NK determined the single-crystal X-ray diffraction structure of assembly CB[8]\u00b71bThere are no conflicts to declare.SC-012-D1SC03743A-s001SC-012-D1SC03743A-s002"}
+{"text": "The catalysts demonstrated excellent durability compared to a commercial Pt/C in load cycling, experiencing less than 50% changes in the mass-specific activity (MA) and surface area-specific activity (SA). In stop-start durability cycling, the new materials demonstrated high stability with more than 50% retention of electrochemical active surface areas (ECSAs). The results can be rationalised by the high BET surface areas coupled with an array of meso and micropores that led to Pt confinement. Further, pair distribution function (PDF) analysis of the catalysts confirmed that the nitrogen and oxygen functional groups, as well as the shell curvature/roughness provided defects and nucleation sites for the deposition of the small Pt nanoparticles. The balance between graphitic and diamond-like carbon was critical for the electronic conductivity and to provide strong Pt-support anchoring.The durability and long-term applicability of catalysts are critical parameters for the commercialization and adoption of fuel cells. Even though a few studies have been conducted on hollow carbon spheres (HCSs) as supports for Pt in oxygen reduction reactions (ORR) catalysis, in-depth durability studies have not been conducted thus far. In this study, Pt/HCSs and Pt/nitrogen-doped HCSs (Pt/NHCSs) were prepared using a reflux deposition technique. Small Pt particles were formed with deposition on the outside of the shell and inside the pores of the shell. The new catalysts demonstrated high activity (>380\u00a0\u03bcA\u00a0cm The global dependence on fossil fuels for everyday energy needs is detrimental to the long-term sustainability of the earth. Over the years, scientists have shown that the combustion of fossil fuels for energy is the driver of rising global temperatures, recurring droughts and other adverse weather and climatic conditions . TherefoThe use of hydrogen fuel cells, commonly referred to as proton exchange membrane fuel cells (PEMFCs), could provide one solution to environmental and energy problems. These PEMFCs use platinum or its composites as the cathodes and anodes for the generation of electric currents usable in power machinery and equipment. The critical hydrogen oxidation reaction  or the oxygen reduction reaction  takes place on the Pt . Current2 and Pt is known to dissolve and redeposit on larger clusters facilitating the debilitating processes of dissolution and agglomeration (2\u00a0g\u22121), large pore volume (2.8\u00a0cm3\u00a0g\u22121) and mesoporous structure and they attributed the distribution and dispersion of small-sized Pt particles to these textual properties. In electrocatalytic reactions, they observed that the mass current density on Pt/HCS electrocatalyst was 1.7 times as high as that of commercial Pt/C in ORR. The stability of the electrocatalysts was attributed to the mesopores that provided a physical interaction force between the Pt and the HCSs. The researchers acknowledged both the superiority of Pt nanoparticles in electrocatalysis and the role of the mesoporous hollow carbon spheres. Also in other studies  of other supports like metal oxides. In fuel cells, degradation occurs during load cycling or during the stop-start events where cell potentials can surpass 1.5\u00a0V vs. RHE. Above these potentials, carbon is electrochemically oxidized to COmeration . These dmeration . Though meration . Even thmeration . Separatmeration . Yan and studies , the mes studies .Therefore, herein we report on the electrochemical activity and load cycling and stop-start durability of Pt nanoparticles supported on HCSs and NHCSs (ca. 40\u00a0wt.% loading) and compare the data to a commercial benchmark Pt/C catalyst . The worPair distribution functional analysis (combined with other techniques) provided key information on the Pt-C and Pt-NC interactions and allowed for explanation of the data in terms of surface interactions.2, 97%], tetraethyl orthosilicate , resorcinol, formaldehyde (37%), Nafion perfluorinated resin solution  and ethylene glycol were all purchased from Sigma-Aldrich and used without further purification. Nochromix crystals (Gordax laboratories), ammonia solution , absolute ethanol , perchloric acid , ultrapure water , alumina polish , argon gas , oxygen gas , nitrogen gas , were obtained and used without further purification.Hydrofluoric acid (48%), cetyltrimethylammonium bromide (CTAB), ethylene glycol, absolute ethanol, melamine, methanol (99%), sulfuric acid (98%), platinum acetylacetonate [Pt (acac)The method described by Stober and colleagues was used, with minor changes, to prepare spherical silica templates . In the 2) template. In the procedure, the silica powder (1.5\u00a0g) was dispersed by sonication in a solution of absolute ethanol (105\u00a0ml) and deionized water (25\u00a0ml). The surfactant and porogen, CTAB (2\u00a0g), was added to a premixed solution of 37% formaldehyde solution (0.3\u00a0ml) and resorcinol (0.3\u00a0g) and 25% NH4OH solution (3\u00a0ml). The procedure proceeded with magnetic stirring for 24\u00a0h leading to the formation of SiO2@RF as the solution turned deep brown with vigorous stirring. After 24\u00a0h the products were filtered and washed with 500\u00a0ml of deionized water, followed by drying in an oven overnight at 100\u00b0C  was deposited on the silica (SiOat 100\u00b0C .2@RF) was transformed into a carbonaceous material using a tubular horizontal furnace saturated with argon at 900\u00b0C. Typically, the SiO2@RF (50\u00a0mg) was loaded into a quartz boat and placed in the centre of the furnace, under Ar flowing at 50\u00a0ml\u00a0min\u22121. The furnace was ramped up to 900\u00b0C at a heating rate of 10\u00b0C min\u22121 and kept isothermal for 2\u00a0h. The quartz boat was cooled using a fast flow of compressed air for 30\u00a0min. The black soot (SiO2@C) was etched using a 10% HF solution in water (100\u00a0ml) for 24\u00a0h to remove the SiO2 template. To prevent the room temperature evaporation of the HF solution, the etching was conducted in a sealed Teflon container placed in a fume hood. The solution was vacuum filtered, washed with copious amounts of DI water and dried in an oven at 100\u00b0C overnight. The formed HCSs were annealed in a tubular horizontal furnace at 900\u00b0C for 2\u00a0h at a heating rate of 10\u00b0C min\u22121 under an argon flow rate of 50 ml\u00a0min\u22121. The formed HCSs (600\u00a0mg) gave a yield of 88% based on the starting resorcinol and formaldehyde used.The dried brown product  followed by sonication for 30\u00a0min. The products were dried in an oven at 70\u00b0C for 2\u00a0h. The SiO2@RF-melamine was transformed to SiO2@NHCSs using the same procedure used for the SiO2@HCSs, after etching with HF to give NHCSs (The NHCSs were prepared from SiOve NHCSs .2 (27\u00a0mg) in a solution of absolute ethanol (100\u00a0ml), deionized water (10\u00a0ml) and ethylene glycol (10\u00a0ml) and the mixture sonicated for 30\u00a0min to allow for thorough mixing. The composite, placed in a round bottom flask (attached to a reflux condenser), was then placed in an oil bath heater. The bath was heated to 200\u00b0C at a slow heating rate of 2.5\u00b0C\u00a0min\u22121 and kept isothermal for 2\u00a0h while the reaction continued under reflux. The reactor was allowed to cool naturally to room temperature, and the products were filtered under vacuum and washed twice with 300\u00a0ml deionized water followed by drying at 100\u00b0C  and Pt (acac)at 100\u00b0C .2 as the purge gas (20 ml\u00a0min\u22121) and air for combustion (10 ml\u00a0min\u22121) and a heating rate of 10\u00b0C\u00a0min\u22121. Nitrogen adsorption/desorption was measured using a Micromeritics Tristar 3000 surface area and porosity analyser set at \u2212195\u00b0C, with sample degassing conducted at 150\u00b0C overnight. Raman spectroscopy measurements were performed on a Horiba Jobin-Yvon Raman spectrometer with a laser wavelength of  ca. 10\u00a0mg of each catalyst in 1\u00a0ml ethanol for 5\u00a0min. About 3 drops of the material rich ink were deposited onto a lacey carbon copper grid, and analysis by TEM was done after drying the samples in air. Particle size distributions of the Pt particles, HCSs and NHCSs, were obtained by measuring at least 100 particles using ImageJ. The powder X-ray diffraction measurements were carried out on a Bruker D2 phaser diffractometer with a Cu K\u03b1 radiation source operating at 40\u00a0kV to determine the crystalline phases present in the catalyst with 2\u03f4 between 10\u00b0 and 90\u00b0. Indexing the compounds detected by the PXRD technique was achieved using the EVA software. Particle sizes of platinum particles were calculated using the EVA software embedded Scherrer equation Thermal stability and metal loading of the catalysts was performed with a Perkin-Elmer STA6000 (TGA) analyser using N GSAS-II . The Qma4 purged with nitrogen for cyclic voltammetry (CV) or oxygen for the oxygen reduction reaction (ORR) experiments. The counter electrode was a high surface area Pt wire and the reference electrode was Ag/AgCl (3.0\u00a0M KCl). All Ag/AgCl potentials were converted to RHE by calibrating the potential between the reference electrode and an in-situ RHE prepared by saturating a clean Pt wire immersed 0.1\u00a0M HClO4 with hydrogen gas. Arbitrarily, no iR drop correction was conducted. For Pt and support durability studies, the high surface area Pt wire was replaced with a high surface area gold counter electrode. A catalyst thin film coated glassy carbon (GCE) electrode with a working area of 0.196\u00a0cm2 was used as the working electrode (WE). The catalyst inks were prepared by dispersing about 5\u00a0mg of the catalyst in a solution of ultrapure water , isopropyl alcohol  and Nafion perfluorinated resin solution  followed by a low-temperature 30\u00a0min sonication. The electrochemical cell, electrolyte volumetric flasks, purge tubes and reference electrode bridge tubes were all thoroughly cleaned with a solution of Nochromix and concentrated sulfuric acid followed by multiple rinses in Merck-millipore ultra pure water. A Biologic SP300/VMP300 potentiostat coupled to a Gamry RDE (rotating disk electrode) 710 Rotator was used for CV and RDE measurements.The electrochemical experiments were performed in a three-electrode cell at room temperature (approx. 25\u00b0C) in a solution of 0.1\u00a0M HClO\u22121 until a reproducible CV was obtained. The scan rate was then reduced to 50\u00a0mVs\u22121 and the third cycle was used for the calculation of the electrochemical active surface area (ECSA) assuming a monolayer charge associated with hydrogen adsorption of 210\u00a0\u00b5Ccm\u22122. The area under the CV curve, representing the charge associated with the underpotential deposition of hydrogen (QDES), was integrated and used for the calculation of ECSA according to the equation:The potential of the WE was cycled between 0.0 and 1.2\u00a0V vs. RHE for 100 cycles at 100\u00a0mVsPt is the loaded amount of Pt particles on the 0.196\u00a0cm2 surface area of the working electrode. Oxygen reduction reaction (ORR) I (current)-V (voltage) polarization curves were obtained at 1,600\u00a0rpm on the electro-catalyst coated working electrode. The WE was cycled at 10\u00a0mV\u00a0s-1 between 0.0\u20131.2\u00a0V vs. RHE in the cathodic direction. To correct for non-ORR background current, the LSV obtained in the nitrogen saturated electrolyte without rotation was subtracted from that obtained from the oxygen saturated electrolyte. The kinetic ORR currents (Ik) were extracted from the measured ORR currents (I) and the limiting currents (Ilim) determined at 0.4\u00a0V vs. RHE using the Koutecky-Levich equation.The LFinally, kinetic currents were normalised with the ECSA and the initial Pt mass loading to obtain the surface area-specific activity (SA), \u22121 to clean the catalysts of any impurities and contaminants and to produce a reproducible CV. Load cycling for catalyst durability measurements was carried out in a 0.1\u00a0M HClO4 solution at 25\u00b0C, in a nitrogen saturated electrolyte. The WE was cycled at 50\u00a0mV\u00a0s\u22121 between 0.6\u20131.0\u00a0V vs. RHE. Load cycling was carried out in units of 10, 100, and 1,000 cycles until when the 6,000th cycle was reached after 27\u00a0h of continuous cycling. As Pt durability was under investigation, in all durability experiments, the Pt counter electrode was replaced with a gold counter electrode of high surface area (Durability load cycling was carried out after the measurement of the beginning of life (BOL) CV and ORR activity. Initially, the WE was cycled between 0.0\u20131.2\u00a0V vs. RHE at 100\u00a0mV\u00a0sace area .\u22121 in an N2 saturated 0.1\u00a0M HClO4 electrolyte at 25\u00b0C. Cycling was carried out for a total of 6,000 cycles with ECSA CVs sampled after 10, 100 and 1,000 cycles until the 6,000th cycle was reached after 27\u00a0h of continuous cycling and argon saturation (Start-stop durability cycling was carried out after obtaining the beginning of life (BOL) ECSAs of the three catalysts. The WE electrode was cycled between 1.0\u20131.6\u00a0V vs. RHE at a scan rate of 50\u00a0mV\u00a0sturation .2 hybridized carbons while the D band is attributed to the Raman vibration of sp3 hybridized carbons. The extent of graphitization was measured by the ratio of the D and the G band areas (ID/IG ratio). As expected, the HCSs with an ID/IG ratio of 0.98 were more graphitic when compared to the NHCSs, with an ID/IG ratio of 1.22. The nitrogen groups in the NHCSs introduced carbon vacancies in the structure of the NHCSs resulting in defects from displacement of carbon atoms by the nitrogen , amorphous diamond like carbon with sp3 hybridization, C\u2013C (284.6\u00a0eV), C=N/C-O (285.8\u00a0eV), C\u2013N/C=O (287.6\u00a0eV) and finally O\u2013C=O (289.9\u00a0eV). The C=N and C-N bonds indicate the successful incorporation of the nitrogen functionalities into the carbon matrix of the NHCSs (7/2 (71.1\u00a0eV) and Pt4f5/2 (74.9\u00a0eV) and the +2 oxidation state of the Pt, Pt4f7/2 (72.1\u00a0eV) and Pt4f5/2 (76.2\u00a0eV), respectively,  . The Pt 2 bonding. The second peak (B) at 2.44\u00a0\u00c5 represents the distance between the three atoms coordinating a central carbon or the shortest diagonal in the hexagon. The third peak (C) at 2.86\u00a0\u00c5, which is twice the first C-C distance, is the second, long diagonal in the hexagon. As can be seen from the simulation of graphite C-C bond distances in To further explore the effect of nitrogen on the HCSs structure, total scattering data were obtained and are plotted in 3 bonds originating from a R-3m rhombohedral diamond-like structures, albeit in lesser amounts relative to the graphite/ene phase  is not exactly half that of the second coordination sphere (B) . The NHC3 respectively which is just below the atomic density of graphite at 0.11 atoms/A3  from the scatterer is noted along the graphene sheet. This is likely due to the curvature of the sheets to form spheres, and the distribution of varying degrees of curvature in the sample, rather than from termination of sheet fragments. The effective atomic density of the scattering volume can be estimated from the slope of the measured G(r). For the HCSs and NHCSs samples the atomic density is 0.095 and 0.105 atoms/Aatoms/A3 . No inteatoms/A3 . All peaatoms/A3 .Atomic correlations are more significantly damped beyond 13\u00a0\u00c5 for HCS than NHCS. This is the range corresponding to the lateral coherence of the structures and also where stacking differences are important. This behaviour of G(r) indicates that short-range two-dimensional atomic order is similar to graphite but stacking along the c-axis is strongly disordered . HoweverThe effect of the addition of Pt to the HCSs and N/HCSs was also evaluated from total scattering data. The PDFs of HCSs and Pt/HCSs are shown in Fitting of the PDFs revealed mean Pt particle sizes of 2.6 and 2.4\u00a0nm for the Pt/HCSs and Pt/NHCSs, respectively. It is important to contrast the Pt particle sizes determined from XRD, TEM and PDF measurements. Considering XRD, Bragg diffraction has limitations in accurately determining particle sizes below 5\u00a0nm while the resolution and magnification of the TEM images likely result in an underrepresentation of very small particles smaller than 2.5\u00a0nm. PDF is highly sensitive to small particles as both Bragg and diffuse scattering are detected resulting in a more accurate particle size determination. The interaction of the Pt particles with the carbon surface has been shown to result in a local rearrangement of carbon atoms, with the stronger the adsorption energy, the larger the C\u2013C bond elongation . As seenvia DFT calculations, this adsorption energy is proportional to the Pt particle sizes and has been shown to have profound effects on the Pt cluster properties  and kinetic current than the Pt/HCSs. The least active was the commercial benchmark Pt/C catalyst. The ORR onset, ECSAs, kinetic current and E1/2 data are summarized in \u22121 compared to 96 \u00b1 6.5\u00a0mVdec\u22121 (Pt/HCSs) and 102 \u00b1 mVdec\u22121 , thereby confirming that ORR kinetics are more favourable on the nitrogen-doped catalyst, showing superiority to the commercial benchmark Pt/C  and ORR activity. The materials demonstrated a standard Pt/C cyclic voltammogram , with tyark Pt/C .\u22121, the Pt/HCSs a value of 387 \u00b1 8\u00a0A\u00a0g\u22121, both of which are ca. 10% larger than the commercial Pt/C (350 \u00b1 10\u00a0A\u00a0g\u22121) catalyst (\u22122) > Pt/HCSs (246 \u00b1 17\u00a0\u00b5A\u00a0cm\u22122) >Pt/C (203 \u00b1 7\u00a0\u00b5A\u00a0cm\u22122).The area-specific activities (SA) of the catalysts were obtained from the normalization of the kinetic current with the ECSAs of the catalysts. The Pt/NHCSs had a SA of 391 \u00b1 16\u00a0A\u00a0gcatalyst . The kincatalyst . The obsThe variation of catalysts activity was attributed to the physicochemical properties of the supports and N doping which affected the electronic properties of the catalysts. The interaction of the Pt and the N groups altered the electronic properties of the Pt-support resulting in better activity and a better Pt-support interaction. From the Pt surface, electron density is transferred to the \u03c0-conjugated system on the surface of the nitrogen groups due to differences in electronegativity creating a sea of electron density between the Pt particles and the N-functionalized surface. The same is less true on a pristine carbon surface. Here charge transfer occurs but the higher electronegativity of oxygen terminal functional groups means more of the electron density from the Pt is transferred to the oxygen functional groups. Thus, less of the charge is transferred to the carbon surface. As a result, the sea of charge on the pristine support is smaller than that on the N doped surface. Thus, electron flow is much easier on the N-doped surface than on the pristine surface, resulting in better activity in the former .2g\u22121) and Pt/NHCSs (309\u00a0m2g\u22121) and an array of micro and mesopores promotes better mass transport of oxygen to the Pt active sites compared to the less porous Pt/C benchmark catalyst with a surface area <200\u00a0m2g\u22121. The larger pores in the Pt/HCSs  and Pt/NHCSs  facilitates better \u201cin-pore\u201d confinement of the Pt inside the pores and thereby promotes a better Pt-support contact  for 6,000 cycles at 50\u00a0mV\u00a0s vs. RHE . The deg vs. RHE . Also ob vs. RHE . The ini vs. RHE .The start-stop durability LSVs of the catalysts before and after the tests are shown in Or4+ in PtO2, undergo dissolution faster than lower-order Pt species according to the equation.The presence of a facial layer of oxide on the surface due to the presence of terminal oxygen functional groups as shown in the O1s spectra of the HCSs is responsible for part of the degradation of the Pt. The formation of the Pt \u2013O bonds weakens the pre-existing Pt\u2013Pt bonds. As the PtO can be further oxidised, higher-order Pt oxidation states such as Pt.2+and subsequently Pt4+ as further Pt oxidation occurred. Notwithstanding the quantifiable oxygen functionalities on the HCSs and NHCSs supports, significant confinement of the Pt particles inside the pores reduced the rate of migration of dissolved and undissolved Pt particles to form larger Pt clusters.The oxidation state of the Pt particles is closely related to Pt dissolution and Ostwald ripening processes. The cathodic dissolution of the Pt particles is a thermodynamically feasible process that occurs below 0.837\u00a0V according to The shift in the LSVs is also observed in the changes in the MA and SA of the catalysts after durability tests . There ivia processes that include Pt dissolution, Ostwald ripening, agglomeration, particle detachment as well as support corrosion (DES), and as shown by The decline in MA was also coupled with a decline in SA. For the benchmark Pt/C, a 40% decline was observed compared to a 22.4% (Pt/HCSs) and 20.7% (Pt/NHCSs) decline for the two new catalysts. Degradation in fuel cells is known to occur orrosion . These pThe losses in ECSA, MA and SA were attributed to the agglomeration of Pt resulting in the formation of particles that are bigger and with less surface area. The growth in Pt particles can be seen from the comparison of the TEM images before and after stress testing . As show\u22121 from 1.0\u20131.6\u00a0V vs. RHE  for 6,000 cycles at 50\u00a0mV\u00a0s vs. RHE . Under lEven though the thermodynamic potential for this process is lower , at highThe load cycling durability CVs shows a comparison between the before and after AST tests . The CVs2  as confirmed in the XPS C1s and O1s spectra. This presence results in the ease of interfacial oxidation of the thin layers of carbon. The low formation of the Q-HQ couple on the commercial benchmark could be attributed to the low BET surface area as a result of the carbon degradation to COEven though the double-layer capacitance and the features associated with the ECSA of the catalysts are reduced, due to degradation of the Pt/HCSs and the Pt/NHCSs catalysts, more degradation is observed for the commercial Pt/C catalyst . The AST2/g) and NHCSs (604\u00a0m2/g) supports both have a higher BET surface area and an array of mesopores and micropores which promotes the confinement of Pt particles, resulting in better support to metal contact. Also, confined Pt particles are more resistant to migration and agglomeration. The higher loss in activity of the benchmark Pt/C is ascribed to low pore confinement compared to the new materials. Pore confinement of the Pt in the structure of the HCSs based supports was confirmed in a similar study involving broken hemispherical hollow carbon spheres  analysis of the catalysts was studied. Analysis of the data revealed that the NHCSs showed increased buckling of the carbon plane compared to the pristine sample due to the nitrogen doping. Addition of Pt to the spheres also gave rise to a Pt-C interaction that could be detected by PDF analysis and the strength of the interaction of the Pt particles with the carbon surface is such that the larger the interaction the larger the C\u2013C bond elongation. The stronger Pt-C interaction was responsible for part of the observed long-term durability of the Pt/HCSs and Pt/NHCSs catalysts.The PDF method is highly sensitive to small particles resulting in an accurate particle size determination. The higher proportion of smaller average Pt sizes as determined by the PDF technique compared to PXRD, could partly explain the higher activity in ORR observed for the Pt/HCSs and Pt/NHCSs.\u22121 from 0.6 to 1.0\u00a0V vs. RHE. The data showed that after 6,000 cycles, the ECSA retentions were Pt/C (47.6%) < Pt/NHCSs (62.1%) < Pt/HCSs (65.4%). In other experiments the start-stop durability LSVs of the catalysts, before and after the tests, was studied and the data revealed LSV shifts in the order Pt/HCSs (18\u00a0mV) < Pt/NHCSs (22\u00a0mV) < Pt/C (30\u00a0mV). A shift in LSVs is attributed to the loss of activity due to the degradation of the Pt catalysts. The losses in ECSA, MA (and SA) were attributed to the agglomeration of Pt and this agglomeration was detected by post analysis of the samples from TEM images.The Pt catalysts prepared by the ethanol reflux method demonstrated higher activity and durability than the commercial benchmark Pt. The durability the Pt catalysts were subjected to accelerated stress tests (AST) for 6,000 cycles at 50\u00a0mV\u00a0sThe two effects of pore confinement and the presence of defects/nucleation sites is proposed to be responsible for the enhanced durability of the catalysts. Based on these findings, the Pt/HCSs and Pt/NHCSs are good candidates for PEMFC catalysts and strategies to further enhance the durability of the catalysts and reduce Pt use can be further initiated from the above studies."}
+{"text": "However, it is still not completely understood how c-Myc functions in GC. Here, we generated a stomach-specific c-Myc transgenic mouse model to investigate its role in GC. We found that overexpression of c-Myc in +Atp4b gastric parietal cells could induce gastric adenoma in mice. Mechanistically, c-Myc promoted tumorigenesis through the AKT/ mammalian target of rapamycin (mTOR) pathway. Furthermore, AKT inhibitor (MK-2206) or mTOR inhibitor (rapamycin) inhibited the proliferation of c-Myc overexpressing GC cell lines and the initiation of gastric tumorigenesis in c-Myc transgenic mice. Thus, our findings highlight that gastric tumorigenesis can be induced by c-Myc overexpression through activation of the AKT/mTOR pathway.Gastric cancer (GC) is one of the most common malignant cancers in the world.  Gastric cancer (GC) is one of the most common malignant cancers in the world. It was reported that in 2018, the incidence of GC ranked the fifth in the world, while the mortality rate ranked the third , GC can Mouse model is commonly used to investigate the pathogenesis of various cancers since the protein-coding genes of mice and human share high similarity . Establic-Myc is a well-known oncogene involved in various cancers, including GC. Amplification of c-Myc in GC has been reported in several studies [c-Myc copies (\u22653) is linked with late on-set, intestinal-type, advanced tumor stage, and distant metastasis, while c-Myc hypomethylation is associated with diffuse-type GC [c-Myc overexpression is more frequently observed in GC than gene amplification [c-Myc overexpression was described in over 40% of GC [c-Myc mRNA expression, which was associated with deeper tumor extension and metastasis [c-Myc is more frequently observed in intestinal-type GC than diffuse-type GC [c-Myc is sufficient to cause GC remains unclear.The  studies -11. Gain-type GC . It is rfication ,14. c-My0% of GC . De Souztastasis ,16. Nota-type GC ,17,18 an-type GC . MYC pro-type GC ,22. Howec-Myc plays a crucial role in several cellular functions, such as cell proliferation, differentiation, and cell cycle progression [Myc transcriptionally regulates the expression of TRAP1, which controls primary and metastatic tumor growth [Myc induces RCC in a glutamine-addicted way [c-MYC proteins, which is resulted from alterations of the Wnt and Ras pathways, is often seen in 70% colorectal cancer [BRD4 could promote GC progression through positive regulation of c-Myc in transcription and epigenetic levels [c-Myc could inhibit the growth and proliferation of GC cell lines [c-Myc/NAMPT/SIRT1-dependent FOXO1 and YAP signaling pathways [in vitro and in vivo [Lats1 and Lats2 [Myc-driven mouse models of cancer, including prostate cancer [c-Myc on GC would be of great interest to uncover new therapies for GC.The gression . It was r growth . In renacted way . Signifil cancer -28. Whilc levels  and knocll lines ,31. Liu pathways . Xu et a in vivo . Choi etnd Lats2 . There he cancer  and renae cancer , but note cancer . Thus, ic-Myc is highly expressed in gastric parietal cells to investigate the definite role of c-Myc in GC. We present data indicating that these mice developed the phenotypic features of the gastric adenoma, with a step-wise tumorigenic progression from hyperplasia to metaplasia, dysplasia, and finally adenoma in gastric mucosa. Importantly, our findings highlight a mechanism by which gastric adenoma can be induced by stomach-specific c-Myc overexpression through activation of the AKT/mammalian target of rapamycin (mTOR) pathway.In this study, we generated a novel gastric tumor model in which human Atp4b-cre mice were gifted from Dr. Xiao Yang and described previously [fl/flMyc mice were purchased from the Jackson Laboratory and were also described previously [fl/+Atp4b-cre; Myc, referred as OEAtp4b-cre; Myc mice were generated by crossing Atp4b-cre mice with fl/flMyc mice. fl/flMyc mice were used as control. Both male and female mice were used for experiments since no difference of sex has been observed. All the mouse strains were generated in a C57BL/6 background and were born and maintained in a specific-pathogen-free (SPF) facility and all experimental procedures were approved by the Animal Ethics Committee of School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University. All institutional and national guidelines for the care and use of laboratory animals were followed. Primers for genotyping are shown in eviously . Mycfl/feviously . Atp4b-c2O2. Blocking was performed with 5% BSA for 1 hour at room temperature. The primary antibodies used here were anti-c-MYC , anti-Ki67 , anti-MUC2 , and anti-MUC5AC . Periodic Acid-Schiff/Alcian Blue (PAS/AB) staining was performed using AB/PAS stain kit . Staining intensities were calculated using Image J software.Mice were sacrificed at different ages . Mouse stomachs were then harvested, cut open through the greater curvature, and washed 3 times in phosphate-buffered saline by vigorous shaking. Tissues were fixed in 4% poly-formaldehyde for 24 hours at 4\u00b0C, then dehydrated and embedded in paraffin. Sections (5 \u03bcm) were cut and stained with H&E. Tumor grades (0-4) were scored according to the previous report . For IHCTissue and cell lysates were prepared by strong radioimmunoprecipitation assay buffer  containing protease inhibitors and supplemented with protein phosphatase inhibitors . The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 6% skim milk in Tris-Buffered Saline Tween-20 for 1.5 h at room temperature and subsequently incubated with specific primary antibodies overnight at 4\u00b0C followed by incubation with secondary antibodies for 1 h. The primary antibodies used in this study were as follows: Anti-flag-tag , anti-c-MYC , anti-\u03b2-Actin , anti-PI3K , anti-p-PI3K , anti-AKT , anti-p-AKT , anti-mTOR , anti-p-mTOR , and anti-GAPDH .p value was calculated using the Student\u2019s t-test. The primers used in qPCR were listed in Total RNA was extracted from tissues using RNA extraction kit (Bioteke) following the manufacturer\u2019s protocol. RNA was then reverse-transcribed with RT reagent kit . The cDNAs were subsequently subjected to SYBR Green-based real-time PCR analysis. GAPDH was used for normalization. Data were shown as average values \u00b1 standard error of the mean (SEM). The OEAtp4b-cre; Myc and wild type (WT) mice. Differential gene expression was analyzed using the DESeq2 package. The list of significance was determined by setting a false discovery rate (FDR) threshold at a level of <0.05 and |log2FC| of more than 0.585. All differentially expressed genes were subsequently analyzed for gene ontology (GO) and pathway analysis.Gastric mRNA was obtained from 12-week-old 2 atmosphere.AGS cell line was obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Thermo), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Thermo) at 37\u00b0C in a humidified 5% COc-Myc cDNA was generated by PCR and cloned into pCMV6-Entry vector with Myc-tag and flag-tag. The constructs generated were confirmed by DNA sequencing. For transient transfection, AGS cells were transfected with the jetPRIME\u00ae transfection reagent (Polyplus) according to the manufacturer\u2019s instruction. Primers used for amplification of human c-Myc cDNA were as follows: Sense: 5\u2019-AGTAAAGCTTATGGATTTTTTTCGGGTAGTGGAA-3\u2019 and antisense: 5\u2019-ATATACGCGTCGCACAAGAGTTCCGTAG-3\u2019.Human 4 cells/well. After cultured for 4 hours, cells were treated with MK-2206 and rapamycin at a concentration of 10 uM and 25 uM, respectively. After cultured for 12 hours, 24 hours, 36 hours, and 48 hours, cells were incubated with CCK8 for 2 hours at 37\u00b0C. Cell proliferation was determined by measuring the optical density value at 450 nm using a microplate reader (BioTek).Cell counting kit-8 , being non-radioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. Cells that transfected for 24 hours were seeded in 96-well plates at a density of 1 \u00d7 10OEAtp4b-cre; Myc mice, at 7 weeks of age, were treated with two inhibitors, MK-2206 and rapamycin, 3 mice in each group. MK-2206  was prepared in 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG300), 5% Tween-80, and 45% saline and administered to c-Myc transgenic mice by oral gavage at a dose of 100 mg/kg every other day for 2 weeks. Rapamycin  was prepared in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline and administered to c-Myc transgenic mice by intraperitoneal injection at a dose of 5 mg/kg daily for 3 weeks. Mice were then sacrificed and stomachs were harvested for further HE staining.p < 0.05 was considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.All experiments were repeated at least three times. Unless otherwise indicated, data were presented as mean \u00b1 SEM and analyzed for statistical significance by Kruskal\u2013Wallis or Mann\u2013Whitney using GraphPad Prism 6 software or SPSS 19.0 software. RNA-Seq raw data have been deposited in the Gene Expression Omnibus (GEO) under accession number GEO: GSE145583.c-Myc in GC, we first generated a mouse model with Cre-dependent targeted overexpression of c-Myc. c-Myc-floxed mice were crossed with Atp4b-cre to obtain OEAtp4b-cre; Myc mice  in OEAtp4b-cre; Myc mice and PAS+ cells  in WT mice  were upregulated compared with WT mice. Notably, the RNA expression of Smad2/3/4, which is related to regulation of transcription, was significantly downregulated in c-Myc transgenic mice compared with WT mice. Furthermore, the RNA expression of Mcm2/Mcm5/E2f2, associated with cell cycle, was significantly upregulated in OEAtp4b-cre; Myc mice.To explore the potential mechanism underlying ed genes  and B. Aigenesis . RT-qPCRigenesis . As expec-Myc overexpression enriched genes correlated with the PI3K-Akt pathway and mTOR signaling  compared with WT mice  or an mTOR inhibitor (rapamycin). As shown by CCK8 assays, overexpression of c-Myc in AGS cells  were associated with malignant characteristics [c-Myc transgenic mice, and inhibition of AKT and mTOR can significantly decrease cell proliferation in AGS cells overexpressing c-Myc and inhibit or postpone the onset of gastric tumors in vivo. Importantly, our experiments demonstrate not only that c-Myc can be a driver for gastric adenoma but also that the AKT/mTOR pathway could be the underlying mechanism of gastric tumorigenesis caused by c-Myc overexpression.By the establishment of a conditional transgenic mouse model, we show that gastric adenoma induced by eristics . Activateristics , skin caeristics ,49, and eristics . It has eristics ,52. Our c-Myc gene can prompt the gastric tumorigenesis of transgenic mice toward a faster and more severe way. Based on our observation, 14-week-old fl/flAtp4b-cre; Myc mice exhibit submucosal invasion, while OEAtp4b-cre; Myc  mice do not at the same age. Similarly, in human GC, it is reported that increased Myc copy number is associated with a late-onset, intestinal-type cancer and the presence of distant metastasis [Atp4b-cre; fl/flMyc mice exhibit distant metastasis needs further investigation.It is worth pointing out that our study also shows an increased copy number of tastasis . Whetherc-Myc affects the development and progression of GC. More importantly, it will aid the clinical detection and therapeutic strategies for intervention at precancerous stages of GC so to improve patient survival.Taken together, we generated a novel autochthonous transgenic mouse model of gastric adenoma that is generally useful for studying the initiation and progression of GC. It provides a new platform to further study the roles of more genes involved in GC through combining with mutations in other genes. It will facilitate our better understanding of the development of early GC and shed light on the molecular mechanisms by which"}
+{"text": "Deceased donor kidneys with acute kidney injury (AKI) are often discarded because of concerns about inferior transplant outcomes. A means of grading the quality of such kidneys is the performance of procurement biopsies.This is a retrospective study of 221 brain death donors with marginal kidneys transplanted in 223 recipients in Germany. Marginal kidneys were defined as kidneys with procurement biopsies done exceptionally to assess suitability for transplantation in otherwise potentially discarded organs. The impact of deceased donor AKI on patient survival and death-censored graft survival at 1, 3 and 5\u00a0years and graft function at 1 and 3\u00a0years after transplantation was investigated.Stage 1: 1.435 (95% CI 0.438\u20130.702), ORStage 2: 2.463 (95% CI 0.656\u20139.245), ORStage 3: 4.784 (95% CI 1.421\u201316.101)] but a similar graft and patient survival compared to recipients of donors without AKI and with AKI stage 1 and 2 as well. The coexistence of recipient DGF and donor AKI was associated with the lowest graft survival and function rates.Recipients of kidneys with stage 3 AKI had a greater incidence of delayed graft function [DGF; ORThe transplantation of deceased donor marginal kidneys with AKI confers a higher risk for DGF but is associated with acceptable graft and patient outcomes, which do not differ in comparison with marginal donor kidneys without AKI. Graft prognosis is especially poor if donor AKI and recipient DGF concur. Donor AKI was a risk factor independent of the histological lesions of procurement biopsies.The online version contains supplementary material available at 10.1007/s11255-022-03277-3. Kidney transplantation (KT) continues to remain the best available renal replacement therapy for most patients with end-stage renal disease. The shortage of organs and the continuously increasing number of patients on the waiting list led to the increased usage of organs from marginal donors . The chaAcute kidney injury (AKI) is very common in kidney donors and is strongly correlated with DGF \u20138. PreviGiven these open questions, we aimed to investigate AKI in kidneys from marginal donors with post-explantation biopsies. We evaluated the impact of clinical donor characteristics and histological findings of their biopsies on short-term patient and graft survival and short-term graft function.We extracted data from the Deutsche Stiftung Organtransplantation (DSO) Region Nord and from the German transplant centers of kidneys allocated between January 2003 and March 2012. We included adult recipients of deceased-donor kidney-only transplants of marginal organ quality in Germany. Recipients were excluded if they were\u2009<\u200918\u00a0years old at the time of transplantation, if they received multiple types of organs, or if their donors were from outside of Germany. According to German regulations, only brain-dead donors were included in the study. For the same reason, normothermic ex vivo kidney perfusion systems for organ preservation were also not used.Donor variables, procurement biopsy results, recipient variables and transplant factors included in the analysis are listed in Tables Marginal kidneys were defined according to the current clinical practice in Germany, i.e., kidneys with procurement biopsies done exceptionally to assess suitability for transplantation in otherwise potentially discarded organs. Such kidneys had, for example, proteinuria or presumed chronic kidney disease, were of poor macroscopic or perfusion quality, had heavy aortic patch and/or renal artery atherosclerosis, had multiple accessory renal arteries or were recovered from donors with long ICU stay, diabetes and multiorgan failure. Macroscopic grading of the external aspect of the donor kidney was provided by the explanting surgical team as good, medium, or poor; likewise, atherosclerosis was characterized as no, mild or severe and perfusion quality as good, medium or poor. Extended criteria donors (ECD) were classified as brain death donors 60\u00a0years of age or donors 50 to 59\u00a0years of age with at least 2 of the following features: history of hypertension, terminal serum creatinine\u2009>\u20091.5\u00a0mg/dl or cerebrovascular cause of death .The original Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to calculate eGFR . AKI wasOverall graft loss was defined as time from transplantation to return to dialysis, or death with a functioning graft. Death-censored graft failure was the same apart from censoring those who died with a functioning graft. Patient death was defined as the time from date of transplantation to patient death, not censored at graft failure. All survival times were censored at the end of follow-up or loss to follow-up.Four different outcomes were analyzed: (1) primary non-function (PNF), (2) DGF, (3) recipient eGFR/creatinine at 3, 12 and 36\u00a0months, and (4) graft loss, death-censored graft failure and patient survival at 1 and 3\u00a0years.All biopsies were processed in paraffin according to the routine protocol at the Institute of Pathology, Hannover Medical School, which involves multiple level sections stained with hematoxylin and eosin, periodic-acid Schiff, Jones silver, trichrome elastica. Histopathological parameters were retrospectively determined by an experienced nephropathologist and included type of biopsy, total number of glomeruli and ratio of globally sclerosed glomeruli, focal and segmental glomerulosclerosis (FSGS), number of arteries (media\u2009\u2265\u20092 smooth muscle cell layers), presence of FSGS, Banff Lesion Scores i, t, v, g, ptc, ci, ct, cv, cg, ah according to Banff 2011 \u201318, arteP\u2009<\u20090.05. Variables were also considered confounders if they changed the coefficient of the explanatory variable by\u2009>\u200910%. The different exposure variables were inserted into the model. We compared the models using F test, adjusted R2 and the Hosmer\u2013Lemeshow goodness of fit and the C statistic.The baseline characteristics of the study cohort were expressed as mean (SD). We created logistic regression models for the outcome of DGF, adjusting for covariates. The linearity assumption was assessed through categorization of continuous variables. We checked for interaction terms using forward elimination. Nonsignificant variables were removed from the model using backward elimination with a cutoff of F test and adjusted R2.Three multilinear regression models for the outcomes of recipient eGFR at 3, 12 and 36\u00a0months were created, adjusting for covariates. Collinearity of different variables was assessed using the variance inflation factor. The linearity assumption was assessed using scatter plots of residual values for each continuous variable. Effect modification was assessed for using the forward elimination method. Nonsignificant variables were removed from the model using backward elimination. The different exposure variables were then assessed in the different models. The Wald test was used to assess the significance of exposure variable plus any interaction terms. We then compared the variables of interest for the different models using the C statistic and Akaike Information Criterion (AIC).Three separate multivariable Cox proportional hazards models were created to assess the outcomes of death-censored graft failure, and patient death. Nonlinear continuous variables were made categorical. The nonsignificant variables were removed from the model using backward elimination. Wald statistics were used to assess the significance of exposure variables. The models were assessed using the Harrell The variables for which the various models were adjusted for in the multivariate analyses are summarized in Supplementary File 1.P value below of 0.05 was considered significant in all two-sided tests. Statistical analysis was performed with SPSS software, v24  and IBM SPSS Statistics Essentials for R.A The study protocol was conducted in accordance with the Declarations of Helsinki and Istanbul on organ trafficking and transplant tourism and approved by the Ethics Committee of Hannover Medical School (No. 1519-2012).From 442 kidneys of marginal quality considered for transplantation and with procurement biopsies, 149 were not transplanted. For the remaining 293 transplanted kidneys, follow-up data were available for 223 organs . Furthermore, the KDRI was 1.553\u2009\u00b1\u20090.506 and 1.426\u2009\u00b1\u20090.516 in the non-AKI and AKI donor groups. The mean donor BMI and the prevalence of diabetes were higher, whereas less numbers of ECDs were observed in the AKI group. No differences in donors\u2019 gender, hypertension, smoking rate, Hepatitis B and C and CMV serology, causes of brain death and distribution of KDPI were observed between the non-AKI and AKI donor groups. Traumatic brain injury was more common in donors without AKI, but duration of brain death was comparable between groups. Donors with AKI remained for a longer time in the ICU and received less often volume expanders and steroids.Donors\u2019 characteristics are summarized in Table In the AKI donor group serum creatinine at admission, peak serum creatinine and creatinine and recovery were higher. Furthermore, AKI donors had more proteinuria and reduced diuresis in the last 24\u00a0h before cross-clamp.Regarding perfusion and organ quality, there were no differences between kidneys with and without AKI Table . HoweverTable P\u2009=\u20090.002), patient and death-censored graft survival at 1, 3 and 5\u00a0years graft function at 3\u00a0months, 1 and 3\u00a0years, proteinuria and number of rejections were similar in both groups.Clinical outcomes are presented in Tables C statistics: AKI\u2009=\u20090.772 (CI 0.703\u20130.842), AKIN classification\u2009=\u20090.784 (CI 0.714\u20130.853))]. Increasing stage of donor AKI was associated with a higher rate of DGF  showed that there was no significant association between AKI stage and patient survival, death-censored and non-death-censored graft failure Table . SimilarR2 values were worse .Interestingly, there was weak evidence of an association between 3- and 12-month recipient eGFR and increasing stage of donor AKI Table . The modResults on graft survival are illustrated in Fig.\u00a0The deceased donor pool is limited and living kidney donation does not suffice to close the gap in organ shortage. For donors with AKI, several aspects have to be taken into account, such as surgical issues, hemodynamic compromise, immunological issues and ischemia reperfusion injury . UnfortuShort-term patient and allograft survival and graft function appear acceptable after transplantation of marginal kidneys with and without AKI to expand the donor pool.The incidence of DGF was significantly higher only in recipients of kidneys from marginal donor kidneys with AKIN stage 3, but not in recipients of donor kidneys with AKIN stage 1 and 2.Donor AKI and recipient DGF in combination were considerably associated with graft loss.Histopathological assessment of donor kidneys was not helpful in predicting outcomes;The rate of cumulative rejections or the level of proteinuria at follow-up was not higher in recipients of marginal donor kidneys with AKI.The main findings of our study were:Similar to others, we confirmed classical risk factors for AKI, such as diabetes mellitus, BMI and preexisting chronic kidney disease \u201323. MoreThe discrepancy between macroscopic and microscopic findings in the no-AKI group  implicates that macroscopic assessment of a recovered organ from transplant team is a subjective and probably not accurate parameter of organ quality. Furthermore, it underpins the pitfalls of procurement biopsies in marginal kidneys and corroborates the allocation policy of the European senior program (ESP) of Euro Transplant (ET), where biopsy is not a prerequisite and indeed is not performed in the great majority of the recovered organs. Regarding histopathology, we suppose that the opposite as expected patterns in donor kidneys with AKI are due to selection bias, since only post-explantation biopsies of marginal kidneys were assessed. In that cases, histological findings are unfavorable in general and differences between marginal donor kidneys with and without AKI would not be anticipated. This was also probably the reason why, except for DGF, histological findings failed to predict clinical outcomes, in the multivariate analysis. Perhaps, the statistical analysis did not confirm the significance of these lesions, although their clinical relevance was evident, i.e., that AKI kidneys could be transplanted with satisfactory outcomes. Hence, our results do not support the routinely performance of procurement biopsies in deceased donor kidneys with AKI.The occurrence of DGF in presence of donor AKI is plausible but the extremely poor outcomes after concurrence of both unfavorable conditions are probably due to the superimposed damage in the transplanted organ before recovery from AKI. The development and severity of AKI are known risk factors for the transition to chronic- or end-stage renal disease . What\u2019s The clinical implication of our findings is that patients at high risk of developing DGF should be cautiously selected if kidneys with AKI are offered and in the case of transplantation, careful observation is required during the first three months of follow-up. Such a time frame is of relevance for the allocation policy of ET since patients can be relisted without losing previous accrued waiting-list points in case of graft failure. Insisting on rescuing a graft deemed to get lost could finally result in much longer waiting times for a second transplant. Giving-up the graft and re-initiating dialysis timely is therefore crucial for those patients.Strengths of the study are the analysis of detailed donor items, concerning the treatment during the ICU stay and the explantation procedure, with data on kidney function from admission to the ICU until recovery, elaborate hemodynamic parameters, concomitant medications and histopathological scoring of the procurement biopsies in a center with experienced nephropathology.Limitations of our study were the small sample size, the retrospective design, the amount of missing data and the bias toward marginal organs. Lastly, quality of life was not investigated. This is a fundamental item considering the significantly inferior outcomes in graft survival.In conclusion, our results suggest that transplantation of marginal kidneys with AKI puts recipients in disadvantage only regarding transplanted organ survival and function but not patient survival and may be recommended, always considering an unfavorable risk-to-benefit ratio. Since functioning grafts in the long run outperform dialysis, an individualized approach in the context of a personalized medicine strategy is essential. The non-acceptance of an organ leads in the end to an increase in patients\u2019 mortality due to their longer time on the waiting list .Supplementary file1 (DOCX 18 kb)Supplementary file2 (DOCX 25 kb)Below is the link to the electronic supplementary material."}
+{"text": "E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human \u03b2-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalismprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while \u0394mprF2 and \u0394mprF1 \u0394mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the \u0394mprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in \u0394mprF2 and \u0394mprF1 \u0394mprF2. In the mprF mutants, particularly \u0394mprF1 \u0394mprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the \u0394mprF1 \u0394mprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both \u0394mprF2 and \u0394mprF1 \u0394mprF2, compared to the wild type and \u0394mprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF.The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While  Enterococcus faecalis is a Gram-positive commensal bacterium that naturally inhabits the harsh environment of the human gastrointestinal tract. The enterococci are among the most clinically significant nosocomial pathogens and cause a variety of opportunistic infections in susceptible individuals, including endocarditis, urinary tract infections, bacteremia, and wound infections , amphipathic molecules that are part of the innate immune response against microbes during infection . CAMPs aE. faecalis, CAMPs are thought to target phosphatidylglycerol (PG), an anionic phospholipid which constitutes a major portion of the cell membrane , and a flippase domain responsible for transferring modified PG from the inner leaflet to the outer leaflet of the cytoplasmic membrane  were deleted, exogenous fatty acid supplementation resulted in tolerance to the cationic antibiotic daptomycin and changes in lipid composition  in in OG1RF . Our finymyxin B . In OG1Rposition . Mutatiofaecalis . Such adfaecalis  and phosfaecalis .mprF genetic perturbation with cationic antimicrobial resistance and lipidomic alterations suggest a gap in knowledge about how mprF brings about such adaptive lipid remodeling. Thus, we sought to more thoroughly investigate the mechanism by which mprF affects lipid homeostasis in E. faecalis. Using lipidomic methods that we had previously established , and assays for membrane fluidity, secretion, biofilm formation, and CAMP susceptibility, we have discovered a previously unappreciated role for mprF in global lipid regulation and cellular physiology.Our previous work and the above literature linking ablished , we quanE. faecalis more strongly at foci situated at or near the division septum in the \u0394mprF2 mutant than in the wild type, and that only MprF2, and not MprF1, protects against hBD-2-mediated killing  at 50\u2009\u03bcg/mL  to quantify phosphatidylglycerol (PG) and lysyl-PG (L-PG) molecules in this strain and in two daptomycin-resistant strains  studies which lacked the sensitivity to discriminate between individual lipid molecules and quantify their levels, using LC-MS/MS, we were able to observe that MprF1 also contributes to L-PG synthesis in E. faecalis OG1RF under these laboratory conditions.We previously observed that human \u03b2-defensin 2 (hBD-2) binds  killing . The \u0394mp strains . In the ble L-PG , as prevble L-PG , whereas10.1128/mbio.03073-22.1FIG\u00a0S1mprF mutants. Deletion of mprF paralogs increases susceptibility to CAMPs to different degrees. CFU enumerated after 2-hour exposure to increasing concentrations of (A) hBD2 or (B) LL-37. Error bars represent the standard error of mean from 2 biological replicates averaged from 6 technical replicates each. *, 0.05 > P \u2265 0.01; ****, P\u2009<\u20090.0001; Fisher\u2019s least significant difference (LSD) test for ANOVA. Loss of mprF contributes to changes in individual species of L-PG and PG. Normalized quantities of (C) 8 native lysyl-PG species, (D) 18 native PG species. Error bars represent the standard error of mean from 5 biological replicates. *, P\u2009<\u20090.05; **, P\u2009<\u20090.01; ***, P\u2009<\u20090.001; ****, P\u2009<\u20090.0001; Dunnett\u2019s test for ANOVA. Inactive MprF2 does not complement L-PG and PG levels. Normalized amounts of total lysyl-phosphatidylglycerol (L-PG) (E) and total phosphatidylglycerol (PG) quantities (F) in E. faecalis wild type and mprF mutants are shown. Each bar represents the mean \u00b1 standard error of measurement calculated from 5 biological replicates, each represented by an open circle. **, P\u2009<\u20090.001; ****, P\u2009<\u20090.0001; Fisher\u2019s least significant difference (LSD) test for ANOVA. Inactive MprF2  does not complement L-PG and PG levels at the individual species level. Normalized quantities of (G) 8 native lysyl-PG (L-PG) species, and (H) 18 native phosphatidylglycerol (PG) species. Each bar represents the mean \u00b1 standard error of measurement calculated from 5 biological replicates, each represented by an open circle. No difference in lipoteichoic acid (LTA) levels between wild type and mprF mutants. (I) Western blots for LTA in whole cell lysates and supernatants. SecA used as a loading control. Loss of mprF contributes to changes in individual species of DGDAG. Semi-quantitative analysis of (J) 15 native DGDAG species in the mprF mutants. Error bars represent the standard error of the mean from 5 biological replicates. *, P\u2009<\u20090.05; **, P\u2009<\u20090.01; ***, P\u2009<\u20090.001; ****, P\u2009<\u20090.0001; Dunnett\u2019s test for ANOVA. Download FIG\u00a0S1, PDF file, 1.0 MB.CAMP susceptibility assessment and analysis of individual species of lysyl-PG (L-PG), phosphatidylglycerol (PG) and, diglucosyl-diacylglycerol (DGDAG) and lipoteichoic acid (LTA) in the Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mprF gene expression, we performed quantitative reverse transcription PCR (RT-qPCR) of the mprF paralogs in the wild type. RT-qPCR revealed that mprF2 expression is 200-fold higher than mprF1 expression  Absolute quantitation of mprF1 and mprF2 transcripts using reverse transcription quantitative PCR (RT-qPCR) of mid-log phase cultures of the wild type displaying fold change of mprF2 relative to mprF1 copy number. (B) Relative quantification of mprF1 and mprF2 transcripts using RT-qPCR of mid-log phase cultures of WT, \u0394mprF1, \u0394mprF2 and \u0394mprF1 \u0394mprF2. Data displays fold change of transcripts relative to the wild type. Each bar represents the mean \u00b1 standard error of measurement calculated from 3 biological replicates averaged from 3 technical replicates each. ***, P\u2009<\u20090.001; Unpaired t-test for (A), Fisher\u2019s least significant difference (LSD) test for ANOVA for (B). No difference in growth rates or cell viability between wild type and mprF mutants. (C) Optical density measured at 600 nm for the wild type and mprF mutants across 9 time points is shown. Error bars represent the standard error of the mean from 2 biological replicates averaged from 2 technical replicates each. (D) CFU enumeration results of the wild type and mprF mutants across 9 time points are shown. Error bars represent the standard error of the mean from 2 biological replicates averaged from 5 technical replicates each. (E) Percentage of live and dead cells for OG1RF WT and mprF mutants across 4 bacterial growth phases. Enumerated from at least 100 cells per strain. Download FIG\u00a0S2, PDF file, 0.2 MB.Expression of the Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mprF2 and \u0394mprF1 \u0394mprF2 mutants had significantly less PG than wild type. Moreover, like our observation for L-PG, \u0394mprF1 also had an intermediate amount of PG compared to the wild type and the other two mprF mutants (pgsA) is thought to be essential in E. faecalis, based on the fact that this gene is essential in many other bacterial species , as previously described in B. licheniformis  reveals that full length MprF is required for restoration of PG and L-PG levels in \u0394mprF1\u0394mprF2. Normalized quantities of PG and L-PG from the \u0394mprF1\u0394mprF2 with plasmid-based expression using the PsrtA promoter of full length mprF1, mprF2 or the N-terminal, C-terminal domains of either mprF. Total normalized quantities of (A) L-PG and (B) PG in the strains tested as well as individual normalized quantities of (C) L-PG and (D) PG. Error bars represent the standard error of mean from 5 biological replicate. Plasmid backbone used here for all strains are pGCP123-PsrtA, except for pEmpty which are pGCP123 instead. Download FIG\u00a0S3, PDF file, 0.5 MB.Expression of individual Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mprF2 is required for L-PG production  Text detailing how the identities of the TLC spots in Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03073-22.4FIG\u00a0S4mprF mutants. (A) Iodine staining, (B) ninhydrin staining and (C) 32P-radiolabeling staining of 1D-TLCs of empty vector controls of WT, \u0394mprF1, \u0394mprF2, \u0394mprF1 \u0394mprF2 and pmprF2-HA complemented \u0394mprF2, \u0394mprF1\u0394mprF2 together with PG, CL and lysyl-PG standards under chloroform: methanol: water (65:25:4) solvent system. Iodine stains most lipids while ninhydrin stains amino modified lipids. 6 major spots were observed. (D) These spots were scraped off iodine strained TLC plates of WT and \u0394mprF1 \u0394mprF2 and placed through lipid extraction before analysis using LC-MS. Spots were identified and labelled accordingly. (E) 1D- and 2D-TLCs of 14C and 32P labelled lipid extracts of wild type and \u0394mprF1 \u0394mprF2. chloroform: methanol: water (65:25:4) solvent system was used in the first dimension while chloroform: hexane, methanol, acetic acid (50:30:10:5) was used in the second dimension. Comparing positions of the spots in the first and second dimension, we can resolve the positions of PG and CL. In the solvent system used in the second dimension, CL migrates ahead of PG. Download FIG\u00a0S4, PDF file, 0.4 MB.Identification of lipid spots in 1D- and 2D-TLCs of lipid extracts from WT and the Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03073-22.8TABLE\u00a0S1Table\u00a0S1, DOCX file, 0.05 MB.Supplementary tables for methods. (A) Bacteria strains and culture conditions. (B) Lipid standards used. Procured from Avanti Polar Lipids. (C) Primers used for RT-qPCR. (D) Components in chemically defined media (CDM). (E) Antibody and developing solution concentrations for Western blot. (F) PCR Primers. (G) Antimicrobial peptides used. Download Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03073-22.10TABLE S2E. faecalis from untargeted MS. (B) MRM transition list for DGDAG. (C) Lipid species observed from untargeted MS analysis of TLC spots. (D) GC-FAME analysis of BHI. (E) Fatty acids detected in wild type and \u0394mprF1 \u0394mprF2 grown in BHI or CDM. (F) RNAseq analysis of \u0394mprF1 vs wild type. (G) RNAseq analysis of \u0394mprF2 vs wild type. (H) RNAseq analysis of \u0394mprF1 \u0394mprF2 vs wild type. Download Table\u00a0S2, XLSX file, 0.2 MB.(A) DGDAG species detected in Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 14C radiolabeled and iodine-stained TLC plates indicate that L-PG is lower only in \u0394mprF2 and \u0394mprF1 \u0394mprF2 and that reduction was restored by complementation with pmprF2-HA (mprF2 and \u0394mprF1 \u0394mprF2 (32P radiolabeling) but no amino groups (negative ninhydrin staining)  that were consistent across all species, suggesting that these species belong to the same class   to E. Bame class . Compleme levels .14C-radiolabeled TLC revealed faint spots for GPDGDAG. Since it was not possible to discern any differences in GPDGDAG levels via TLC  (which is polyglycerophosphate polymerized onto a GPDGDAG membrane anchor) might be affected as well  surrogate standard . \u0394mprF1 pe level . The sampe level .mprF1 \u0394mprF2 while CL appeared unchanged   , F. All mprF deletion resulted in alterations in phospholipid and glycolipid levels, we hypothesized that these altered levels might be due to altered expression of genes involved in lipid metabolism. To test this hypothesis, we performed RNA sequencing to compare the gene expression profiles of the wild type and the mprF mutants. In total, 301 genes (14% of the genome) were differentially expressed between the wild type and \u0394mprF1 \u0394mprF2  were downregulated in the double mutant  Global gene expression profile comparing E. faecalis OG1RF with \u0394mprF1 \u0394mprF2 . Gene functional categories are based on KEGG pathway membership and manually curated for category assignment. The majority of the differentially expressed genes were categorized under metabolism and membrane transport. A vertical black line represents the median value for each category. (B) Principal component analysis of global gene expression profile comparing E. faecalis wild type (orange circle), \u0394mprF1 (green triangle), \u0394mprF2 (blue square), and \u0394mprF1 \u0394mprF2 (purple cross). Smaller symbols represent the spread of each biological replicate and larger symbols represent the centroid of their respective clusters. The two dimensions measured account for 70.6% of the variability of all differentially expressed genes. (C) Venn diagram represents differentially expressed E. faecalis genes in the mprF mutants relative to wild type. The number of genes (FDR\u2009<\u20090.05) in each section was calculated from mprF1 \u0394mprF2 showing a slight increase in pgsA and decrease in fatty acid biosynthesis gene expression levels. Each bar represents the mean \u00b1 standard error of measurement calculated from 4 biological replicates averaged from 3 technical replicates each. **, P\u2009<\u20090.01; ****, P\u2009<\u20090.0001; Fisher\u2019s least significant difference (LSD) test for ANOVA. Download FIG\u00a0S5, PDF file, 0.2 MB.\u0394Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the de novo fatty acid biosynthesis is downregulated in \u0394mprF1 \u0394mprF2, we speculated that this mutant might require exogenous fatty acids for growth and survival. E. faecalis can incorporate exogenous fatty acids into its membrane when grown in bovine heart infusion (BHI) culture medium (16:0] and stearic acid [C18:0])  lacking fatty acids, while growth of the single mutants was less severely impaired  . We thusimpaired . Supplemimpaired  or stearimpaired  promotedimpaired  promotedimpaired  and D. Uimpaired  and F. Oimpaired , H and I10.1128/mbio.03073-22.6FIG\u00a0S6mprF mutants cultured in chemically defined media (CDM) while high concentrations of unsaturated fatty acids, myristic acid, and lauric acid fail to promote growth. Growth curves of wild type and \u0394mprF1 \u0394mprF2 grown under fatty acid supplementation of (A) palmitic acid (C16:0) or (B) stearic acid (C18:0) at lower concentrations of 62.5, 31.25 or 15.63 ng/mL.  Growth curves of combinations of palmitic and stearic acid in a 3:1 ratio at varying concentrations. Growth curves of wild type and \u0394mprF1 \u0394mprF2 grown under higher concentrations of unsaturated fatty acid supplementation of (E) palmitoleic acid (C16:1 cis-9) or oleic acid (C18:1 cis-9) and (F) cis-vaccenic acid (C18:1 cis-7) or linoleic acid , as well as (G) lauric acid (C12:0), myristic acid (C14:0) or palmitic acid (C16:0) and (H) stearic acid (C18:0) or arachidic acid (C18:0). Fatty acids were supplemented at 5\u2009\u03bcg/mL. (I) Growth curves of 1, 5, and 10\u2009\u03bcg/mL of arachidic acid. Equal volumes of ethanol (EtOH) were used as the solvent control for comparison. Total fatty acid concentrations are shown. Data points for all graphs represents the mean \u00b1 standard error of measurement calculated from 3 biological replicates averaged from 3 technical replicates each. Fatty acid abbreviations:  and . For curves that overlap the blank, arrows corresponding to their respective colors are shown next to the blank curve. Download FIG\u00a0S6, PDF file, 0.8 MB.Saturated fatty acid supplementation at low concentrations promotes growth of Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03073-22.7FIG\u00a0S7mprF1 \u0394mprF2. (A) Proportion of acyl-ACPs within WT and \u0394mprF1 \u0394mprF1. Values obtained through mass spectrometry were normalized against internal standards and total protein concentration before conversion to proportions. Error bars represent the standard error of mean from 4 biological replicates. *, P\u2009<\u20090.05; **, P\u2009<\u20090.01; ****, P\u2009<\u20090.0001; Fisher\u2019s LSD test for ANOVA. Control experiments show that the Laurdan assay can sensitively and accurately measure differences in membrane fluidity. Wild type cells grown at a range of temperatures that result in different membrane fluidities (B) and exposure to the membrane fluidizer, benzyl alcohol (C). Benzyl alcohol was spiked in just after the initial reading at time, t\u2009=\u20090 minutes. Experiments were done in a microtiter plate set-up with measurements taken using a plate reader instead due to the technical constraints in the microscope set up in FIG\u00a0S7, PDF file, 0.1 MB.Acyl-ACP analysis and control experiments testing sensitivity of the Laurdan dye. Analysis of individual species of acyl-ACPs in WT and \u0394Copyright \u00a9 2023 Rashid et al.2023Rashid et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Lactococcus lactis uses the multiple antibiotic resistance repressor (MarR) family repressor FabT to regulate expression of the fab gene operon  species that have long-chain acyl moieties  amino acid sequence present at the acyl attachment site, leaving behind a DSL peptide connected to 4\u2019-phosphopantetheine and an acyl-group, which was then detected using mass spectrometry. The conserved nature of the Asp-N cleavage site and consistent structure of the digestion products makes quantification of acyl-ACP species possible  possesses the conserved DSLD sequence, while AcpB (the ACP involved in uptake of exogenous fatty acids) does not . Gas chromatography analysis of total fatty acid methyl esters (GC-FAME) was performed on lyophilized cell pellets of the wild type and \u0394mprF1 \u0394mprF2 grown in either BHI or CDM. The fatty acid profiles of the wild type and \u0394mprF1 \u0394mprF2 grown in BHI were very similar , ~11% less cis-vaccenic acid (C18:1 \u03c97 cis), and ~2.5% less C19 cyclo \u03c97 acid relative to the wild type, whereas the wild type showed a decrease of 2% and 3% in palmitic acid (C16:0) and stearic acid (C18:0), respectively, and an ~6% increase in cis-vaccenic acid (C18:1 \u03c97 cis) compared to growth in BHI , an ~7% decrease in cis-vaccenic acid (C18:1 \u03c97 cis), and an ~3.5% decrease in C19 cyclo \u03c97 relative to growth in BHI  Secretion. Anionic membrane phospholipids promote efficient secretion via the generalized Sec pathway  Membrane fluidity. The fluidity of the cell membrane is influenced by the amount of unsaturated lipids present, where a higher degree of unsaturation results in more fluid membranes (mprF mutants (o) or disordered (Ld) the local lipid environment is, its emission spectra will blue-shift (in Lo regions) or red-shift (in Ld regions), respectively  Biofilm formation. In E. faecalis strain 12030, the loss of mprF enhances biofilm formation  across all daily time points over a 5-day period  leads to unexpected lipidomic, transcriptomic, and functional changes in E. faecalis OG1RF, \u0394mprF1, \u0394mprF2, and \u0394mprF1 \u0394mprF2. One advantage of such methods is that they are much more sensitive than traditional thin-layer chromatography (TLC), enabling a more comprehensive understanding of lipid homeostatic shifts that are dependent upon mprF  than \u0394mprF1 , L-PG is completely absent in \u0394mprF2 (mprF1 present). The unexpected observation that MprF1 alone does not synthesize L-PG suggests that MprF2 expression is necessary for MprF1 to function. This possibility warrants further investigation.In the current study, we used previously established mass spectrometry-based methods  to analypon mprF . When wen \u0394mprF1 . Unlike mprF2 and \u0394mprF1\u0394mprF2 have significantly lower PG levels than wild type and \u0394mprF1. This is a novel and unexpected finding because mprF acts downstream of pgsA, one of two genes  involved in PG synthesis . Thus, only a fraction of the lipidome was involved in adaptive remodeling. Different sets of lipidomic features  were involved in responses to changes in temperature, high salt concentrations, and stationary growth phase. One of the more striking observations was that varying the degree of acyl chain saturation in PE, PG, or phosphatidylcholine (PC) had varying effects on lipid packing, a property that is correlated with other physical properties, including viscosity , as previously described . For metE. faecalis were quantified by LC-MS/MS using multiple reaction monitoring (MRM) using a previously described methodology  . The QToF instrument was set to positive ion mode, at an electrospray voltage of \u22123,500 V (Vcap), a temperature of 200\u00b0C, a drying gas rate of 14 L/min. Spectra were acquired in auto-MS2 mode with MS1 acquisition rate at 4 spectra/s and the MS2 acquisition rate at 20 spectra/s with fixed collision energy at 40\u2009eV. The list of detected species of DGDAG in the samples can be found in To determine the species of DGDAG present in m/z, 1,071.6; MS2 m/z, 153.0. Integrated peak areas were normalized against cell weight.Due to the absence of suitable internal standards, semiquantitative analysis of DGDAG was carried out instead. Lipid extraction was performed as described above without addition of internal standards. Analysis of DGDAG lipid species was performed by LC-MS/MS via MRMs using monoglucosyl-diacylglycerol (MGDAG) 34:1 as a surrogate standard  for extemprF1, \u0394mprF2, and \u0394mprF1 \u0394mprF2 strains. Detailed methods are described in the supplementary information file.Sequencing of RNA was done from OG1RF, \u039415N acyl-ACP standards  were then spiked into the sample at 5-\u03bcM equimolar concentrations. Proteins were precipitated from standard spiked lysates via TCA-precipitation and resuspended in 50\u2009mM MOPS buffer, pH 7.5. Resuspended proteins were then treated with the Asp-N protease at 20:1 ratio (protein:enzyme) at 37\u00b0C overnight. Reaction was then stopped by addition of methanol to a final concentration of 50%. These samples were then analyzed using LC-MS with MRMs using previously described solvent gradients and MRM parameters  or CDM (72\u2009h) were lyophilized and sent together with powdered BHI for GC-FAME analysis at the Identification Service of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. Cellular fatty acids were converted into fatty acid methyl esters (FAME) and analyzed by GC-MS using used C21:0 FAME in a defined amount per biomass as internal standard for normalization.Late stationary phase cultures of wild type and \u039414C]-acetate or [32P]-disodium phosphate  was added into 5\u2009mL of media at 0.2 \u03bcCi/mL or 1 \u03bcCi/mL, respectively, before culturing strains overnight at 37\u00b0C for 16 to 18\u2009h at static conditions. Lipids were then extracted as previously described and resuspended in 50\u2009\u03bcL of chloroform-methanol solution (1:1 vol/vol) (Radiolabeling of lipids was performed as previously described with the following modifications : [14C]-avol/vol) . Next, 1SDS-PAGE and Western blot were performed as described in a previous study . 4\u201312% o600) readings of the cultures were measured. Cell-free supernatants were obtained by centrifugation at 6,000 rcf for 5 min at 4\u00b0C and filtering the supernatants into fresh Eppendorf tubes using 0.2-\u03bcm syringe filters. Next, 1.6\u2009mL of filtered supernatant was mixed with 400\u2009\u03bcL of 100% wt/vol tricholoroacetic acid (TCA) solution (1:4 ratio of TCA to sample) and incubated at 4\u00b0C for 10 min. Tubes were centrifuged at 20,000 rcf for 15 min at 4\u00b0C. The precipitated protein pellet was washed once with 2\u2009mL of 100% ice-cold acetone and placed on a 98\u00b0C heat block to evaporate residual acetone. The pellets were resuspended in 500\u2009\u03bcL of PBS. Twenty-five \u03bcL of these protein solutions were used for estimation of protein content using the Pierce BCA protein assay kit  in a microtiter plate format according to the manufacturer\u2019s protocol. Protein concentrations of the samples were then normalized to OD600 of 1.0 based on the respective OD600 readings of the individual cultures.Late stationary phase cultures were prepared by growing cultures in BHI broth overnight for 16 to 18\u2009h at 37\u00b0C, static conditions. Optical density at 600 nm . PhoZF secretion was monitored by its ability to convert para-nitrophenyl phosphate (pNPP) into a colored product that can be measured by absorbance at 405\u2009nm. Mid-log phase cultures of strains harboring the pABG5 plasmid containing the chimeric alkaline phosphatase enzyme (PhoZF) were normalized to an OD600 of 0.5. Cell-free supernatants were obtained by centrifuging samples at 6,000 rcf for 5 min at 4\u00b0C and filtering the supernatant through 0.2-\u03bcm syringe filters. Next, 25\u2009\u03bcL of supernatant was added to 200\u2009\u03bcL of 1M Tris-HCl, pH 8.0, in a 96-well microtiter plate. Then, 25\u2009\u03bcL of 4\u2009mg/mL para-nitrophenyl phosphate (pNPP)  was added to each well to start the reaction. The plate was placed into a Tecan Infinite M200 Pro spectrophotometer and incubated at 37\u00b0C with the absorbance read at 405\u2009nm every 10 min for 18\u2009h.Secretion of the strains were monitored by their ability to secrete a chimeric alkaline phosphatase, PhoZF (600 of 0.7 in PBS and incubated with 100 \u03bcM Laurdan for 10 min at 37\u00b0C. Cells were washed twice with PBS and 10\u2009\u03bcL of the cell suspension was spotted onto PBS-agarose pads (1% wt/vol) mounted on glass slides. Coverslips were placed over the agarose pads and sealed using paraffin wax.Late stationary and mid-log phase cultures were normalized to an ODSlides were imaged using a Zeiss LSM 880 laser scanning microscope with Airyscan, using a Plan-Apochromat 63x/1.4 Oil DIC objective with an incubation chamber set to 37\u00b0C. The slides were equilibrated for 10 min within the chamber before imaging and excited using a 405-nm laser with emission collected between 419 and 455 nm (blue) and between 480 and 520 nm (green) simultaneously. Digital images were acquired using the Zen (Zeiss) software and analyzed using ImageJ. Using ImageJ, regions of interests (ROIs) of individual cells or cell clusters were selected and mean fluorescence intensities (MFIs) of each ROI for each channel were measured and tabulated in Microsoft Excel. Using the following formula, the average GP values for each ROI were calculated and then plotted using GraphPad Prism software:Laurdan was validated to be responsive to changes in membrane fluidity via control experiments subjecting stained cells to a gradient of temperatures, and membrane fluidizer, benzyl alcohol  and C.Mid-log phase cultures of the strains were tested for their daptomycin MIC using the microplate broth dilution methods, as previously described .mprF1, \u0394mprF2, and \u0394mprF1 \u0394mprF2 across a 5-day period . These bacterial cultures were then normalized to an OD600 of 0.5 and harvested by centrifugation at 6,000 rcf for 5 min at 4\u00b0C. The supernatant was discarded, and the remaining cell pellet was washed and resuspended in 1\u2009mL of 0.01 M low-salt phosphate buffer (PB). Bacterial resuspensions were then serially diluted 500-fold and 25\u2009\u03bcL of each sample was added twice into a 96-well microtiter plate, for a total of 2 technical replicates. An equal volume of human \u03b2-defensin 2 (hBD2) or LL-37  was added to the samples and incubated statically for 2\u2009h at 37\u00b0C . Then, 5\u2009\u03bcL of bacterial suspension from each well was then spotted 3 times onto a BHI agar plate, for a total of 6 technical replicates per sample. The spotted agar plates were then incubated statically overnight at 37\u00b0C and surviving bacteria were determined by CFU enumeration.Overnight OG1RF cultures were subcultured in BHI liquid broth at a 1:10 dilution and grown to mid-log phase .RNAseq files are available on NCBI, Sequence Read Archive (SRA accession no."}
+{"text": "Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events  than the R group . The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder ( Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae [Nidovirales and the family Coronaviridae. The SARS-CoV-2 virus triggered the breakout of the coronavirus disease 2019 (COVID-19) pandemic, which is yet ongoing. There have been few reports of nidoviruses in aquatic species, which are mostly confined to the subfamily Piscanivirinae and the family Tobaniviridae [Piscanivirinae subfamily is comprised of two genera, namely Bafinivirus and Oncotshavirus. White bream virus isolate DF24/00 (WBV DF24/00), the first fish nidovirus, was isolated in 2001 from white bream (Blicca bjoerkna) in Germany [Piscanivirinae include fathead minnow nidovirus (FHMNV) [The International Committee on Taxonomy of Viruses (ICTV) formally recognized the order Nidovirales in 1996, with four families: iviridae , there aiviridae . The Pis (FHMNV) , chinook (FHMNV) , crucian (FHMNV) , Atlanti (FHMNV) , and chi (FHMNV) . These nStudies on virus\u2013host interactions at various levels are critical for gaining a full understanding of virus properties. To understand the molecular processes of virus infection and possible locations of intervention, a thorough investigation of gene expression following virus infection at the entire genome scale is required. High-throughput sequencing is a precise method for investigating transcriptome data on fish immune to infection with bacteria, viruses, or parasites ,10,11. HParalichthys olivaceus) is widely distributed along the Korean Peninsula, Japan, and China. In China, it is a commercially significant marine culture fish. The recent expansion of the fish culture business has resulted in several illnesses that have resulted in significant losses in the Japanese flounder population. The diseases induced by viral infection are becoming a major threat to the Japanese flounder culture [Japanese flounder , Chinook salmon (Oncorhynchus tshawytscha) embryonic tissue (CHSE), or Japanese flounder brain (JFB) cells in 6 well plates at 1:10 dilutions and was incubated at 17 \u00b0C in L15 with 5% fetal bovine serum for 20 days. The infected JFB cells and supernatant mixture were lysed for 3 cycles of freeze-thaw at \u221280 \u00b0C. Cell debris and contaminants were removed by centrifuging at 1807\u00d7 g for 20 min at 4 \u00b0C. The purification of virions was performed using ultracentrifuge (himac CP70MX) according to the protocol described by Gao et al. [The pooled tissues were homogenized in sterile PBS (1:10 weight\u2013volume ratio) and centrifuged at 1000\u00d7 GenBank Virus RefSeq database using Bowtie software and then assembled into contigs by the SPAdes and Velvet software. After assembled contigs, annotation was conducted with five databases .To narrow down the range of candidate viruses, the RNA of virus-infected JFB cells was extracted using RNA simple total RNA kit , and cDNA was synthesized by PrimeScript RT reagent Kit with gDNA Eraser . The RNA was then used for small RNA library construction, siRNA sequencing, and analysis according to the protocol described by Wu et al. . In partTo obtain the whole genome sequence of the isolated virus, the RNA of purified virions was extracted, and cDNA was synthesized. After quality checking, the sequencing library was constructed and clustered following the manufacturer\u2019s recommended protocols. The library was sequenced on an Illumina NovaSeq 6000 platform and 150 bp paired-end reads were generated. The raw reads were trimmed using Trimmomatic , and theThe full genome sequence of the isolated virus in this work, as well as additional ten nidovirus sequences retrieved from NCBI, were utilized for phylogenetic analysis. The sequences were aligned, and phylogenetic relationships were inferred using the neighbor-joining method. The phylogenetic tree was constructed with 1000 bootstrap replications of the Tamura\u2013Nei model using MEGA X .2O. The cycling conditions were as follows: 98 \u00b0C for 3 min; followed by 35 cycles of 10 s at 98 \u00b0C, 5 s at 55 \u00b0C, and 5 s at 72 \u00b0C on a GeneAmp PCR system 9700 . The primer sequences were listed in For the detection of CSBV Bces-Po19, each reaction volume (50 \u03bcL) contained 200 ng of cDNA, 25 \u03bcL of 2\u00d7PrimeSTAR Max Premix , 300 nM of each primer, and ddHThe gills, liver, spleen, kidney, heart, and stomach of diseased fish were sampled and fixed in Bouin\u2019s solution for 24 h before being rinsed with 70% alcohol and kept at room temperature. The samples were dehydrated in ethanol serial dilutions, sectioned (5 \u03bcm thickness), and then stained with Harris\u2019s hematoxylin and eosin. The liver, kidney, ovary, and spleen of diseased fish as well as inoculated JFB cells containing 70% cytopathic effects (CPEs) were processed for TEM  using the previously reported methodology ; the fixWe randomly selected 3 moribund fish that displayed symptoms (Sensitive (S)) and 3 fish that had no symptoms (Resistant (R)). Each fish\u2019s head kidney was immediately frozen in liquid nitrogen for 48 h and kept at \u221280 \u00b0C until use. The TRIZOL Kit was used to extract RNA . To remove genomic DNA from extracted RNA, RNase-free DNase I was used. The quality of the extracted RNA was evaluated using an Agilent 2100 Bioanalyzer System . The presence of virus in all S and R samples was confirmed by PCR using specifically designed primers  in the iThe sequence data obtained were processed with the SMRTlink 5.0 software. Circular Consensus Sequence (CCS) was created from subread BAM files with the following parameters: min_length 200, max_drop_fraction 0.8, no_polish TRUE, min_zscore \u22129999, min_passes 1, min_predicted_accuracy 0.8, and max_length 18,000. The output of CCS BAM files output was acquired as full-length reads and non-full-length reads using pbclassify.py script, ignore polyA false, and minSeqLength 200. The non-full-length and full-length fasta files were sent into the cluster step, which involved isoform-level clustering (ICE), followed by final arrow polishing, hq_quiver_min_accuracy 0.99; bin_by_primer, false; bin_size_kb, 1; qv_trim_5p, 100; and qv_trim_3p, 30. Using the Illumina RNA-Seq data and the LoRDEC program, further nucleotide mistakes in consensus reads were repaired . FollowiUnmapped transcripts and novel gene transcripts obtained from gene structure analysis were annotated as NT using BLAST ; the software Diamond BLASTX  was used to annotate to NR, KOG/COG, Swiss-Prot and KEGG; and the software of Hmmscan  was used in Pfam database analysis. p-value of 0.05 were set as the threshold for significantly differential alternative splice. The full-length transcript that was mapped on two or more loci in the reference genome with more than 99% alignment coverage and having the locus 100 kb apart from each other on the reference genome and a 10% alignment of each gene locus to the corresponding transcript was predicted as a fusion transcript [TAPIS was used to analyze the gene structure of polished isoforms. The exon-intron structure of each transcript was predicted. Newly found loci and isoforms were identified by comparing them to the reference genome annotation, using the same criteria as for loci and isoform identification . Alternaanscript . The animal TFDB 2.0 database was used for transcript factor (TF) analysis . The trahttps://bigd.big.ac.cn/gsa (Unpublished Data).Total RNA from S1 to S3 and R1 to R3 were prepared separately for RNA-Seq. Briefly, the sequencing libraries were created with the NEBNext UltraTM RNA Library Prep Kit for Illumina  and then tested for quality with an Agilent Bioanalyzer 2100. Following library preparation, index-coded samples were clustered, and libraries were sequenced using an Illumina Hiseq XTen platform, yielding paired-end reads. The raw sequencing data presented in this paper were deposited in the Genome Sequence Archive in National Genomics Data Center, Beijing Institute of Genomics , Chinese Academy of Sciences, under accession number CRA003368 that are publicly accessible at p < 0.05 were differentially expressed. Raw reads were cleaned before being mapped to the Japanese flounder reference genome  using Hip < 0.05 were deemed significantly enriched. The statistical enrichment of DEGs in KEGG pathways was tested using KOBAS (v3.0) [GOseq (v1.10.0) was used to perform a gene ontology (GO) enrichment study of differentially expressed genes (DEGs) . GO keywS (v3.0) , which w\u2212\u0394\u0394Ct method was used to analyze gene expression levels. qRT-PCR was used to confirm the RNA-Seq data. Ten DEGs with high levels of significance were selected for qRT-PCR analysis, with the 18S gene serving as a control . The qRTThe moribund fish exhibited bleeding on their fins, muscles, liver, gonads, etc. . The infThe infected EPC, CHSE, and JFB cells all demonstrated severe CPEs that resulted in large plaques of detached cells and the loss of the monolayer . The infThe isolated virus was named as CSBV Bces-Po19, and its full-length genome sequence (NCBI accession no.: OM830952) was 26,597 nt in length, including a 5\u2032 UTR (959 nt) and five open reading frames (ORFs) that encode ORF1a , ORF1b , spike glycoprotein , membrane protein , nucleocapsid protein , and a 3\u2032 UTR (215 nt) A. Oncotshavirus, both nucleotide and amino acid homology analysis were performed. Phylogenetic analysis of the viral genome placed the CSBV Bces-Po19 in the Oncotshavirus genus, within the Tobaniviridae family of Nidovirales, forming a clade with CSBV WHQSR4345 (NCBI accession No. MG600027) and CSBV HB93 (NCBI accession No. MH171482) (To identify the genetic relationship between CSBV Bces-Po19 and other viruses belonging to the genus H171482) B. The comparison of complete genome sequences in GenBank Blast searches showed that the CSBV Bces-Po19 isolate had a similarity of 93.17\u201398.62% (in nucleotides) to other aquatic nidoviruses isolates, with the highest percent identity to CSBV WHQSR4345. Nucleotide and amino acid sequence analyses of five ORFs are shown in Using the designed CSBV Bces-Po19-specific primers, bands with 757 bp were detected in CSBV Bces-Po19-infected JFB cells, while no band was detected for the control cells. However, the internal reference gene \u03b2-actin was found in all samples in both control and infected cells . This reThe primers used in this experiment were designed to amplify ORF1a of CSBV Bces-Po19. However, due to the close species relatedness, CSBV Bces-Po19 primer pairs could cross-react with CSBV WHQSR4345, CSBV HB93, and CSBV Cefas-W054. As such, the viral species should be initially screened by PCR and then further validated through whole-genome sequencing.The ultrastructural examination was performed on the liver, kidney, ovary, and spleen of Japanese flounder infected with CSBV Bces-Po19. Cell necrosis was the most common pathogenic alteration in all four tissues. The spherical virions were visible, measuring 55\u2013110 nm in diameter, and featured surface spikes that protruded 14\u201321 nm from the virion wrap A,B. The Pathological alterations in CSBV Bces-Po19-infected JFB cells included necrosis and extensively disrupted spherical and bacilliform virions A\u2013C. In tFrom the head kidney samples of CSBV Bces-Po19-sensitive and -resistant Japanese flounder, Illumina sequencing yielded 41,175,958 to 55,892,426 clean reads. The Japanese flounder reference genome was uniquely mapped to an average of 90.13% of clean reads . FPKM vaThe Iso-Seq on PacBio Sequel machine produced 23.31 and 24.30 G polymerase read bases for CSBV Bces-Po19 R and S samples, respectively . After eThe polished consensus reads were adjusted with Illumina RNA-Seq data to reduce further nucleotide mistakes in full-length sequencing reads. After correction, 463,495,705 (R) and 535,777,939 (S) nucleotides were retrieved, representing 99.86% and 99.84% of the total nucleotides before correction, respectively . The N50The polished consensus reads, after correction, were mapped to the reference genome of Japanese flounder, yielding a mapping rate of 86.84% (R) and 84.60% (S), respectively . There wDEG analysis was used to analyze the transcriptome profile of Japanese flounder in response to CSBV Bces-Po19. A comparison of the CSBV Bces-Po19 S and R transcriptomes revealed 1444 DEGs, 931 of which were highly up-regulated and 513 of which were significantly down-regulated A. A heatA KEGG pathway analysis found that 129 pathways were implicated in the Japanese flounder\u2019s reaction to CSBV Bces-Po19. Ribosome biogenesis in eukaryotes, metabolic pathways, RNA transport, spliceosomes, and pyrimidine metabolism are the top five enriched pathways B. IntereSUPPA software was used to identify seven types of AS events: skipped exon (SE), mutually exclusive exon (MX), alternative 5\u2032 splice site (A5), alternative 3\u2032 splice site (A3), retained intron (RI), alternative first exon (AF), and alternative last exon (AF) (AL). There were 12,352 AS events in 10,743 of the total S group genes and 11,452 AS events in 12,989 of the total R group genes . This sutead3 gene had the most AS in the R group, with 52 AS, which is 25 higher than the S group. The highest number of AS was found in the S group in the slco4a1 gene, which contained 57 AS, which is 21 more than the R group  were found in three immune-related pathways, namely \u201cphagosome\u201d, \u201cp53 signaling pathway\u201d, and \u201cNOD-like receptor signaling pathway\u201d. Two AS isoforms were found in both the \u201cphagosome\u201d and \u201cp53 signaling pathways\u201d. Six AS isoforms were discovered in the RNA-related pathways: \u201cRNA transport\u201d, \u201cRNA degradation\u201d, \u201cribosome biogenesis in eukaryotes\u201d, \u201cspliceosome\u201d, and \u201cmRNA surveillance pathway\u201d were found to have six AS isoforms . The AS isoforms eif4g1_novel05, rmb8a_novel01, eif2s2_novel01, and ran_novel05 were involved in the \u201cRNA transport\u201d pathway. All the AS isoforms had similar expression patterns with their related genes. A total of twelve isoforms were up-regulated, and four isoforms were down-regulated in the S group in comparison with the R group  splicing, resulting in multiple mature mRNAs from a single pre-mRNA. The AS increases transcriptome complexity and thereby the proteome diversity of a cell, and it also modulates the abundance of functional mRNAs . It is ccdk4/6, necessary for cells to enter the G1 phase from the G0 phase [A variety of viruses have been reported to cause G0/G1, S, or G2/M arrest in the host cells, hence promoting replication of progeny viruses after viral infection . InfectiG0 phase , was sig"}
+{"text": "Classification and phenotype identification of lettuce leaves urgently require fine quantification of their multi-semantic traits. Different components of lettuce leaves undertake specific physiological functions and can be quantitatively described and interpreted using their observable properties. In particular, petiole and veins determine mechanical support and material transport performance of leaves, while other components may be closely related to photosynthesis. Currently, lettuce leaf phenotyping does not accurately differentiate leaf components, and there is no comparative evaluation for positive-back of the same lettuce leaf. In addition, a few traits of leaf components can be measured manually, but it is time-consuming, laborious, and inaccurate. Although several studies have been on image-based phenotyping of leaves, there is still a lack of robust methods to extract and validate multi-semantic traits of large-scale lettuce leaves automatically.In this study, we developed an automated phenotyping pipeline to recognize the components of detached lettuce leaves and calculate multi-semantic traits for phenotype identification. Six semantic segmentation models were constructed to extract leaf components from visible images of lettuce leaves. And then, the leaf normalization technique was used to rotate and scale different leaf sizes to the \u201csize-free\u201d space for consistent leaf phenotyping. A novel lamina-based approach was also utilized to determine the petiole, first-order vein, and second-order veins. The proposed pipeline contributed 30 geometry-, 20 venation-, and 216 color-based traits to characterize each lettuce leaf. Eleven manually measured traits were evaluated and demonstrated high correlations with computation results. Further, positive-back images of leaves were used to verify the accuracy of the proposed method and evaluate the trait differences.The proposed method lays an effective strategy for quantitative analysis of detached lettuce leaves' fine structure and components. Geometry, color, and vein traits of lettuce leaf and its components can be comprehensively utilized for phenotype identification and breeding of lettuce. This study provides valuable perspectives for developing automated high-throughput phenotyping application of lettuce leaves and the improvement of agronomic traits such as effective photosynthetic area and vein configuration.The online version contains supplementary material available at 10.1186/s13007-022-00890-2. The morphogenesis and growth of lettuce leaves have complex regulation mechanisms. In addition to genetics, habitat can also influence the variation in their leaf structure, color, and venation. Explainable and highly distinguishable features for large-scale lettuce varieties are very important for the applications of phenotype identification and phenotype\u2013genotype association analysis. It is impossible to find two identical leaves worldwide , but theThe basic structure of leaves is established in the early stage of leaf development. Leaf morphogenesis includes the formation of the petiole, mid-rid, lamina, and marginal structures . These iFrom an image processing perspective, data-driven image techniques such as convolutional neural networks (CNNs) and their derived models can be used for the segmentation and classification of vegetable plants/leaves , 16. CNNThis study aimed to develop an automated phenotyping pipeline to quantify detached lettuce leaves and evaluate the multi-semantic traits for large-scale lettuce cultivars. The study highlights the efficacy of an automated phenotyping pipeline in recognizing multiple semantic components of lettuce leaves and evaluating traits differences not only for leaf components but between positive and back leaves.More than 400 lettuce varieties were grown in a terraced greenhouse at the Beijing Academy of Agriculture and Forestry Sciences (BAAFS) from January 1 to February 25, 2020. Lettuce varieties included Butterhead, Celtuce love, Italian, Red Coral, Red Lettuce, Red Oakleaf, and Salad Grand Rapid (SGR) . A studiAs shown in Fig.\u00a0The classical U-NET  was usedLettuce leaves could be represented using size-related and size-free traits. The size-related traits of lettuce leaf consist of common geometry indices, such as leaf area, perimeter, length, and width. These traits were calculated based on the resulted semantic components from the end-to-end semantic segmentation. However, leaves of different lettuce varieties had significant differences in size and leaf area at the same growth period. Thus, these leaves were rotated to the same orientation and scaled to the same size (width) to eliminate the effect of the size difference and improve the efficiency of the phenotyping process.Leaf normalization was performed to obtain standard \u201csize-free\u201d images of lettuce leaves via rotation and scaling procedures. The leaf was rotated to an upright position according to its mid-rid  Fig.\u00a0A. It couFour semantic components  of positive and back leaves could be skeletonized Fig.\u00a0A\u2013D. The Geometry and color traits of each semantic component could be calculated directly using semantic segmentation results. However, more valuable descriptions could be extracted from these semantic components, such as the petiole and vein architecture. The phenotyping pipeline of lettuce leaves was proposed to calculate the petiole, vein architecture, and related statistical traits, as shown in Fig.\u00a0The petiole was defined as the region along the mid-rid from the leaf base to the first lamina; thus, the mid-rid could be divided into the petiole and first-order veins. The laminas and mid-rid could be combined into a new object by a dilate operation of the mid-rid region. The max contour of the new object could contain all adjacent lamina regions, and the lamina closest to the leaf base was used to determine the petiole. Moreover, the mid-rid could divide the whole leaf into the left and right parts. Thus, the skeleton line of the mid-rid was utilized to divide semantic components  into two parts (the left and right parts).The vein architecture mainly contained the angles and distribution of the second-order veins. In this study, the lamina-based approach was used to analyze vein architecture Fig.\u00a0A. All laVein angles could be calculated based on the first-order laminas (LM_L and LM_R). The contour of each lamina was simplified as a polygonal chain using fewer points based on the Douglas\u2013Peucker algorithm. The approximated polygon retained the original contour shape (controlled by the specified tolerance), and fewer nodes were used to eliminate the ambiguity in angle calculation. The simplified polygon had a subset of the points of the original contour. Due to the normalized leaves, a default tolerance, such as five, could be applied to almost all the laminas. Among the polygon nodes, the closest point to the leaf base and mid-rid was selected as the appropriate attachment point. The corresponding vein angle of each attachment point was calculated using its two adjacent line segments of the polygon.The complex shape and structure of the lettuce leaves, such as leaf folds, twisted veins, multiple main veins, etc., presented challenges to the leaf phenotyping. For these particular leaf cases, it was difficult to extract leaf semantic components accurately by image techniques. The phenotyping pipeline could only be applied for the leaf types that conformed to data annotation specification Fig.\u00a0D, not foSince data annotation was highly time-consuming and labor-intensive, only 41 representative lettuce leaves were annotated based on the specification in Fig.\u00a0Leaf normalization operation was an important procedure to ensure the consistency of phenotyping analysis. Each leaf image was rotated in an angle sequence  to construct test image sets which were used to evaluate the robustness and reliability of the proposed leaf normalization method. The data normalization results for the positive and back leaves are shown in Fig.\u00a0The rotation invariance of data normalization could be assessed by the scaling factors. The mean and std of scaling factors were 1.949 and eaf Fig.\u00a0. The Smaeaf Fig.\u00a0A. The noeaf Fig.\u00a0B\u2013G. ThisLeaf area rapidly increased during leaf vegetative growth, while the vein architecture (the mid-rid and second-order veins) slightly increased. Scaling factors could be used as an individual trait of lettuce leaves since they directly reflected leaf growth. Data normalization was valuable to extract size-related traits for evaluating lettuce leaves with large size differences. Moreover, data normalization greatly reduced the image size, thus improving the computation performance of the time-consuming techniques.Eleven traits of 112 images from 56 lettuce leaves (each leaf contained positive and back images) were manually measured to validate the computation accuracy of the phenotyping pipeline. Manually measured traits are listed in Table The comparison analysis of the 11 traits was conducted to identify the relationship between manual and computation measurements. MR_L had a higher correlation coefficient than BD_L. PE_L was determined by the position judgment of the first lamina in manual measurement. PE_L had the least correlation between manual and computation methods  were utilized to verify the accuracy of the proposed method and evaluate the trait differences between positive and back images.The common geometry traits of lettuce leaves, such as length, width, pixel area, convex hull area, were calculated based on each semantic component after data normalization  were used to represent the color traits of each semantic component of leaves. Each color space had three channels (18 channels). The mean and standard deviation of each channel was calculated. A feature set was constructed for each semantic component (each feature vector contained 18 mean color features) to identify the most important color feature of each semantic component. The orientation type (positive or back leaves) was used as a classification variable.A classification decision tree was used to determine the most important color features based on the classification variable Fig.\u00a0. All colColor features of six semantic components were integrated into a feature set containing 216 color features to quantify the whole leaf color. The orientation (positive-back leaf) and semantic components were respectively used as classification variables to investigate the important scores of color features Fig.\u00a0A and B. These semantic components of lettuce leaves contributed lots of accurate and quantitative traits that were difficult to be measured manually. Some traits had clear physiological and ecological significance, or are related to lettuce quality and yield, such as color differences of positive-back leaves, and area ratio of leaf veins, etc. Thus, these image-based traits were not only valuable for gene function analysis and mapping of lettuce varieties, but also could be used for classification and identification of lettuce leaves. The selection of important features were valuable for refining the description of lettuce leaf and its components. Principal component analysis (PCA) could be used for dimensionality reduction via fusion to reduce the number of features greatly. Both the single important feature or principal components could be used for leaf classification and grading. Herein, the feature sets were classified into four types , then used for PCA analysis (400 lettuce varieties). The top 10 principal components of each feature set could explain 98.415%, 97.493%, 95.138%, and 89.004% of the overall variance, respectively  of 0.77. CLR_PC4 was negatively correlated with GEO_PC1 and VEN_PC2 . The first three PCs of COM and CLR were highly positively correlated. The detailed correlation coefficients among different PCs are shown in Fig.\u00a0Lettuce leaves could be clustered into different categories based on their feature set by minimizing the intra-class gap and maximizing the inter-class gap. Each type of lettuce leaves was assigned an individual label for further analysis. The hierarchical clustering based on Euclidean distance was conducted using four types of PCs Fig.\u00a0. For posHere we developed a phenotyping pipeline to extract and quantify multiple semantic components of leaves for different lettuce varieties. By fine definition and image annotation, semantic segmentation models were employed to automatically decompose lettuce leaves into six types of semantic components. Through the normalization operations, leaves of great differences in size and orientation were rotated and scaled to the same level for trait quantification and comparison. The positive-back lettuce leaves were also for the first time used to evaluate the accuracy of semantic segmentation and their essential difference from the geometry, color, and venation perspectives. These semantic components contribute a large number of traits that are difficult to be measured manually and are helpful to discover the statistical differences of lettuce leaves for leaf classification and identification. The proposed method can be improved in segmentation accuracy by supplementing richer annotated leaves and be extended for leaf phenotyping of other crops.Additional file 1:Table S1. Model evaluation for six semantic components of lettuce leaves. Table S2. Geometry traits of lettuce leave. Table S3. Vein architecture traits of lettuce leaves."}
+{"text": "The application of dendrimeric constructs in medical diagnostics and therapeutics is increasing. Dendrimers have attracted attention due to their compact, spherical three-dimensional structures with surfaces that can be modified by the attachment of various drugs, hydrophilic or hydrophobic groups, or reporter molecules. In the literature, many modified dendrimer systems with various applications have been reported, including drug and gene delivery systems, biosensors, bioimaging contrast agents, tissue engineering, and therapeutic agents. Dendrimers are used for the delivery of macromolecules, miRNAs, siRNAs, and many other various biomedical applications, and they are ideal carriers for bioactive molecules. In addition, the conjugation of dendrimers with antibodies, proteins, and peptides allows for the design of vaccines with highly specific and predictable properties, and the role of dendrimers as carrier systems for vaccine antigens is increasing. In this work, we will focus on a review of the use of dendrimers in cancer diagnostics and therapy. Dendrimer-based nanosystems for drug delivery are commonly based on polyamidoamine dendrimers (PAMAM) that can be modified with drugs and contrast agents. Moreover, dendrimers can be successfully used as conjugates that deliver several substances simultaneously. The potential to develop dendrimers with multifunctional abilities has served as an impetus for the design of new molecular platforms for medical diagnostics and therapeutics. It showed great relaxation, which confirms that Mn (II) is a potential alternative to Gd-based measures [Contrast agents used in magnetic resonance imaging (MRI) allow for the obtention of more sensitive images. The two common types of images are hyperintense images  and the MDA2 antibody targeting malondialdehyde (MDA) -lysine epitopes to significantly enhance atherosclerotic lesions . In theiThe development of nanotechnology is an innovative approach to replace the classic regimens of anti-cancer therapies and classic diagnostics. The modification of drugs and contrast agents that already exist, as well as the search for new ones, is the future and a challenge of modern medicine. The use of dendrimer-coated magnetic nanoparticles allows for the development of a drug delivery system and targeted therapy that improves the effectiveness of therapy and reduces the risk of toxicity by using a lower drug dose. In addition, implementing treatment regimens using pH-sensitive pharmaceuticals would help to reduce drug resistance during anti-cancer therapy. Therefore, it is essential to characterize dendrimers and pay attention to analytical techniques in the process of their manufacture. Depending on the dendrimer group and the surface group, it is possible to create conjugates of interest. The use of nanoparticles allows for the minimization of side effects and the adjustment of the appropriate dose of the drug, which will bring the desired therapeutic effect. Due to their properties and the ability to adjust the size of molecules, PAMAM dendrimers, used as drug carriers, can be successfully used in anti-cancer therapy. It is possible to attach specific ligands to the surface of the dendrimer, which will be recognized by receptors overexpressing cancer cells. In addition, research into new MRI contrast agents is proving to be important. The currently used contrast agents cause a number of side effects due to the high dose administered. The research focuses on the development of contrast agents based on functionalized Gd dendrimers that could be administered in clinical trials at a lower dose while maintaining the same sensitivity and diagnostics in clinical trials as traditional contrast agents. This review presents advancements in dendrimer-based research in various fields. It should be emphasized that the spectrum of the use of dendrimers is very wide. The applications of dendrimers include, but are not limited to, chemotherapy, radiation therapy, photothermia, and photodynamic therapy. In addition, they are also used in gene therapies because they allow for the condensation of nucleic acid materials. Due to the unknown metabolism of the therapies used, there is a need for further research into the use of dendrimers, despite the fact that they are characterized by higher biocompatibility and the improved effectiveness of the therapies used. Despite numerous studies, there is a need for continued research and an attempt to transfer them to in vivo research. There is a need to improve the effectiveness of the applied therapies and to understand metabolic mechanisms. In summary, the use of dendrimers in oncology is a modern approach that can overcome the limitations of standard diagnostics and therapy by improving the time required to detect neoplastic lesions and monitoring the effectiveness of the therapy. The use of dendrimers for the synthesis of new drugs or the modifications of existing drugs is a challenge of modern pharmacology. In addition, the development of effective drug delivery methods to cancer cells would allow for better therapy outcomes. The development of targeted therapies would make it possible to improve the effectiveness of treatment and minimize the negative side effects of the used therapies. It will also make it possible to bypass the disadvantages of classical diagnostic and therapeutic methods and improve the effectiveness of the used therapies."}
+{"text": "The prevalence of chronic kidney disease (CKD) risk factors such as diabetes mellitus, hypertension, and obesity among the young Malaysians are increasing. Understanding the factors associated with CKD knowledge could assists healthcare providers to design health education programmes. There are scarce local studies on CKD knowledge and its associated factors among university students. This subpopulation comprises of young people with diverse background and characteristics. This study was aimed to assess the CKD knowledge and its associated factors among university students. A cross-sectional study was conducted among Universiti Kebangsaan Malaysia students from July 2020 to August 2020. A convenience sampling method was applied. All students were invited to complete an online survey using Google Forms that were sent to their email. The survey consisted of questions related to their sociodemographic, socioeconomics, university programme enrolled, medical history, lifestyle characteristics and CKD knowledge. The data were analysed using SPSS Statistics 26.0. Multiple logistic regression analysis was performed to identify the final associated factors after controlling for confounders. A total of 3074 students participated and 32.6% of them had below average CKD knowledge. Students of male gender, enrolment in undergraduate programmes and non-health-related faculties/institutes were more likely to have below average CKD knowledge. Students who are Chinese, from high monthly household income family and with family history of CKD were less likely to have below average CKD knowledge. Almost one-third of the students had below average CKD knowledge. The six associated factors with CKD knowledge were non-modifiable. Of the six factors, three were associated with students being more likely to have below average CKD knowledge; another three were associated with students being less likely to have below average CKD knowledge. Future health education programmes to enhance CKD knowledge should be designed focusing on students who are more likely to have below average CKD knowledge. CKD is an important global health problem as its prevalence and global burden are rising rapidly. Worldwide, approximately 200 million people have CKD [Chronic kidney disease (CKD) is defined as abnormalities of kidney structure or function, that are present for > 3 months, and present health implications. CKD is classified based on the cause, estimated glomerular filtration rate (eGFR), and albuminuria category . CKD canIn Malaysia, CKD prevalence increased from 9.1% in 2011  to 15.5%A person who is knowledgeable and aware of CKD would tend to adopt a healthy lifestyle, which could lower the risk of CKD . Early-sCKD can affect any age group, including the young. The prevalence of CKD risk factors, especially diabetes mellitus and hypertension among young adults in Malaysia has increased throughout the years . Among tUniversity students are a segment of the general population wherein the majority are young adults. This subpopulation comprises people with diverse sociodemographic, and socioeconomic characteristics and university programme enrolment. Some university students join health- or non-health related programmes that might or might not provide detail information on CKD. Regardless, university students need to be equipped with such knowledge, to understand the seriousness of CKD and its implications to not only for themselves but also their family members. The adaptation of a healthy lifestyle in early years would likely be maintained throughout a person\u2019s lifetime  and woulUniversity students in Rwanda, East Africa, showed poor knowledge on CKD risk factors and preventive measures . FurtherMore studies should be conducted as Malaysia is a multi-ethnic country with highly diverse sociocultural characteristics. Such studies could aid recognition of the knowledge gap in certain sub-population, who could be disproportionately affected by CKD, based on factors such as age and ethnicity . TherefoA cross-sectional study was conducted among undergraduate and postgraduate students of Universiti Kebangsaan Malaysia (UKM) from July to August 2020 to assess CKD knowledge and its associated factors. UKM is a public university which was established in 1970 and has since become a reputable research public university . The maiA convenience sampling method was applied in which students were invited to participate in the study via email; their email addresses were provided by the UKM Centre of Academic Management. The current study adhered to the tenets of the Declaration of Helsinki. Study approval was obtained from the UKM Research Ethics Committee (research code: FF-2020-271). The inclusion criteria were Malaysian citizens, aged \u226518 year and full-time students. The exclusion criterion was self-reported history of CKD. The sample size was calculated using PS software  at 5% siThe students were invited to participate in July\u2013August 2020. A set of questionnaire prepared in Google Forms was distributed via the students\u2019 email addresses. Informed consent was provided in the first section of the online survey form where they are fully informed as to the intent and purpose of the study. The written informed consent has been waived by the UKM Research Ethics Committee. No minors were involved in this study. Students who agree to have their data used for the study, can click an \u201cI Agree\u201d button and have their data submitted online. Students who do not agree to have their data used in the study, can click an \u201cI Do Not Agree\u201d button and their data is not submitted and collected online. We opted to use this method because the study was conducted when Malaysia was under a Movement Control Order following the coronavirus disease 2019 (COVID-19) pandemic outbreak. Students were prohibited from being on-campus to curb the pandemic, and all teaching and learning sessions were conducted online. The questionnaire consisted of two sections: Section A, which assessed the student\u2019s sociodemographic, socioeconomics, university programme enrolled, medical history and smoking status.Section B consisted of a validated CKD knowledge questionnaire from a study from Singapore . ApprovaAnatomy: How many healthy kidney(s) does a person need to lead a normal life? (correct answer: one)Physiology: What is the function of a kidney in the human body? (correct answer: to filter waste products in the blood)Aetiology: What can cause kidney disease? Presentation: What are the symptoms of early kidney disease that might progress to kidney failure? (correct answer: can present without any symptoms/ complaints)Progression: Which of the following statement(s) about kidney disease is incorrect? (correct answer: identified that CKD cannot be cured with medication)Resources available: Where can dialysis treatment be carried out? Treatment: What is the best medical treatment for end-stage kidney failure? (correct answer: kidney transplant)Each correct answer was awarded 1 point; incorrect answers or \u2018I don\u2019t know\u2019 were awarded 0 points, yielding a maximum score of 7 points and a minimum score of 0 points.The study outcome was below average knowledge, which was defined as a score for <4/7 correct answers. Average CKD knowledge was considered a score for \u22654/7 correct answers . The socUniversity programme enrolled comprised programme level (undergraduate or postgraduate), faculty/institute  and years of study. Medical history included personal medical history  and family history of chronic illness . Smoking status was assessed by asking \u2018Have you ever smoked \u2019 and was categorized as yes or no.n) and percentage (%). Normally distributed data were described using the mean and standard deviation (SD). The crude odds ratio (COR) and its corresponding 95% confidence interval (CI) were determined using simple logistic regression. Adjusted analysis of multiple logistic regression analysis (forward likelihood ratio) was performed to identify the adjusted OR (AOR) of the final factors associated with below average CKD knowledge after controlling for possible confounders. The final model was tested for all possible two-way interactions (multiplicative interaction) between the independent variables, and its fitness was assessed. Statistical significance was set at p < 0.05.The data were analysed using SPSS Statistics 26.0 . Categorical data were described as the frequency  years. The majority of the students were young adults . The majority were female (76.4%) and Malay (72.5%). Most of the respondents (70.5%) were not working and 55.2% were in the B40 monthly household income group. A total of 80.6% and 85.3% were single and did not have children, respectively. Almost two-thirds of the students (62.5%) were undergraduates at non-health-related faculties/institutes (69.9%). Most of the students (86.7%) did not have any personal medical history, family members with chronic illnesses (73.8%) and non-smokers (91.5%). Out of the 3074 students, 32.6% (95% CI: 31%-34.3%) had below average CKD knowledge. Approximately half of the respondents answered correctly for knowledge on anatomy of the kidney (43.5%). Most of the students answered correctly for knowledge on kidney physiology or function (96.5%) and CKD aetiology (67.5%). However, approximately one-third of the students answered correctly for CKD symptom presentation (38.4%) and knowledge on CKD progression (34.7%). More than half of the students (53.8%) answered correctly for knowledge of resources of dialysis treatment centres. Most of the students correctly answer the best treatment for end-stage renal failure (81.7%). Despite the low response rate in this study, the sample size was relatively larger than that of a previous local study  involvinCKD is a common and growing public health problem that requires everyone to have adequate knowledge to reduce its development and progression. Our results indicate that almost one-third of UKM students have below average knowledge of CKD. This is lower than that of a local study that showed that 43.5% of respondents had below average knowledge . In thatOur study reported a high proportion of correct answers for physiology as compared to a Greek study involving primary school pupils . In thatA greater proportion of students in our study correctly identified the risk factors (aetiology) and best medical treatment for end-stage kidney failure in our student\u2019s population as compared to the general populations , 35. ThiThe low knowledge could be due to health education on disease progression commonly being designed for high-risk groups or patients with CKD . It is cThe present study showed that male students were more likely to have below average CKD knowledge. The below average CKD knowledge among male students is possibly due to sex differences in health information-seeking behaviour, which requires further evaluation. A study among college students in China suggested that male students are less likely than female students to seek health information on self-care, disease prevention and self-medication . Female Here, we found that Chinese students and students from high monthly household income families were less likely to have below average CKD knowledge. The association of ethnicity with CKD knowledge is inconsistent with that from a local study  and fromThere is limited information on the association of household family income and CKD knowledge among students for further comparison. However, a review has suggested that a higher household income enables parents to acquire better goods such as healthy diet, books and learning materials for their children to thrive better, including cognitive development and health . MoreoveThe present study indicates that undergraduate students and students from non-health-related faculties/institutes were more likely to have below average CKD knowledge compared to postgraduate students and those at health-related faculties/institutes. A descriptive study of university students in the United States showed that CKD knowledge was low in this population, particularly among the younger students . We postApart from the level of educational attainment, the students\u2019 health literacy might have been influenced by their younger age. A Malaysian Health Literacy Survey showed that the Malaysian aged 18\u201324 years had a higher proportion of limited health literacy compared to Malaysian aged 25\u201339 years . A GermaThere is a scarcity of information measuring the association between faculty/institute type with CKD knowledge. Most recent studies have involved students either in health-related , 46 or nThe strength of our study included the participation of Malaysian students of varying ethnicities and backgrounds. To our knowledge, this is among the first local studies on CKD knowledge and its associated factors that include both undergraduate and postgraduate students across all courses and programmes. The findings presented were also adjusted for confounders. Furthermore, our findings can be used for formulating future studies. Our study has several limitations. This was a single-centre, cross-sectional study that used non-probability sampling. Causal inferences may not be drawn, and the findings cannot be inferred to the whole student population. Selection bias may be present, where the participating students could be more health conscious and more inclined to answer the questions. There is the possibility of information bias, as the students could have sought the correct answers prior to answering the survey, resulting in overestimation of CKD knowledge in this study.Despite the involvement of content experts (nephrologists and primary care physicians) in the questionnaire development, the authors suggested that further studies should be performed with a more refined questionnaire . We postFurthermore, the questionnaire validation was not clearly explained. A questionnaire is one of the most common techniques used to measure the knowledge level in a community . A validFactors that we hypothesized earlier that would influence CKD knowledge, such as health information-seeking behaviour and health literacy, should be examined further. More studies are needed to explore the types of knowledge , which influence motivational factors (perceived health threat, attitude towards health action, attitude towards health outcome and subjective norms that lead to decisions .Stratified analysis based on programme level and of faculty/institute type should also be investigated as the students have different roles and obligations that might influence their knowledge level. Our study could provide a foundation for intervention strategies for improving the CKD knowledge of this young population. Although CKD prevalence in childhood is uncommon and children were not our focus study population, we hope that our findings highlight the need to design a school education programme on kidney function and the CKD risk factors as early as the primary school level. Such a programme may ensure a healthy and health-literate population starting from a young age . In addiAlmost one-third of the students in this study had below average CKD knowledge. The associated factors were non-modifiable and included male gender, Chinese ethnicity, high monthly household income family, undergraduate students, enrolment in non-health-related faculties, and family history of CKD. Future health education programmes or training to enhance CKD knowledge should be designed focusing on students who are likely to have below average CKD knowledge."}
+{"text": "We aimed to investigate the association between a new definition of metabolic health (MH) and subclinical atherosclerosis in a cohort of patients without previous cardiovascular disease (CVD). In total, 7824 community-dwelling adults were categorized as normal weight, overweight, or obese. Metabolically healthy obesity (MHO) was defined as obesity accompanied by all of the following criteria: systolic blood pressure (BP) < 130 mmHg, no use of BP-lowering medication, waist-hip ratio <0.832 (women) and <0.887 (men), and no prevalent diabetes. Carotid atherosclerosis was defined as carotid plaque or mean carotid intima-media thickness \u2265 1.1 mm. The prevalence of carotid atherosclerosis was 8.3% and 1113 (14.2%) patients were classified as having MHO. All individuals classified as metabolically unhealthy were at an increased risk of carotid atherosclerosis independent of body mass index categories. Conversely, the risk of carotid atherosclerosis in individuals with MHO was not significantly increased compared to that in metabolically healthy normal weight participants . This new definition of MH was able to identify people with MHO without an increased risk of CVD in an Asian community cohort. The prevalence of obesity continues to rise exponentially worldwide and is extensively associated with multiple comorbidities, such as cardiovascular diseases (CVD), diabetes, hypertension, and several cancers ,2,3. ObeStudies on MHO and CVD have shown inconsistent results, as the diagnostic criteria varied among the studies ,10,11,12The Cardiovascular and Metabolic Diseases Etiology Research Center (CMERC) cohort consisted of community-dwelling Korean adults aged 30\u201364 years who were free from myocardial infarction, heart failure, or stroke. All participants completed health questionnaires and examinations. The sampling and measurement procedures in the CMERC cohort have been described in detail previously ,18. BrieEvery participant\u2019s height and body weight were measured while they wore light clothing and without shoes and were measured to the nearest 0.1 cm or 0.1 kg. BMI was calculated as body weight in kilograms divided by the square of standing height in meters. Waist circumference (WC) was measured at the midpoint between the lower point of the rib cage and the upper point of the iliac crest. Hip circumference was measured in the widest region of the hip.Blood samples were collected from the antecubital vein after overnight fasting. All analyses were performed at a single laboratory center . Fasting blood glucose and creatinine levels were determined using a colorimetry method . Hemoglobin A1c (HbA1c) levels were determined using high-performance liquid chromatography with a Variant II Turbo . Fasting insulin levels were determined using a radioimmunoassay with an SR-300 apparatus . Total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and high-sensitivity C-reactive protein (hs-CRP) levels were assayed by enzymatic methods . To evaluate insulin resistance, the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: [fasting glucose (mg/dL) \u00d7 fasting insulin (\u03bcIU/mL)]/405 . The ChrParticipants were classified as \u201cmetabolically healthy\u201d or \u201cmetabolically unhealthy\u201d according to the recently proposed definition of MH by Zembic et al., which consists of three cardiometabolic components , with mo2), overweight (OW) (23 to <25 kg/m2), OB class 1 (25 to <30 kg/m2) or OB class 2 (\u226530 kg/m2) [BMI was classified as normal weight (NW) (<23 kg/m) 23 to < kg/m2, OCarotid artery ultrasonography was performed using high-resolution ultrasonographic systems . All operators were trained according to a predefined protocol. Carotid intima-media thickness (CIMT) was scanned at the 1-cm segment of the common carotid arteries proximal to the bulb region. The mean and maximum CIMT values were measured on both the right and left sides. A carotid artery plaque was considered present if the thickness was greater by 50% or more than the surrounding intima-media thickness (IMT), or if its size was \u22651.5 mm . CarotidThe hepatic steatosis index (HSI) was used to define NAFLD, and those with HSI > 36 were considered to have NAFLD: HSI = 8 \u00d7 (ALT/AST ratio) + BMI  .p-value less than 0.05.Data are presented as mean \u00b1 standard deviation or median (interquartile range) for continuous variables and as numbers (percentages) for categorical variables. Comparisons among the three groups according to obesity status  were performed by using one-way analysis of variance with Bonferroni-corrected post hoc comparisons  or Kruskal-Wallis tests with post hoc pairwise comparisons . Due to the right-skewed distribution, HOMA-IR, TG, and hs-CRP levels were entered into the models as log-transformed units. Multivariate logistic regression analyses were performed to examine the independent association of obesity phenotypes with carotid atherosclerosis or NAFLD after adjustment for age, sex, TG, HDL-C, LDL-C, hsCRP, HbA1c, HOMA-IR, eGFR, smoking status, alcohol consumption, physical activity, and the other definition of MH according to the NCEP-ATP III criteria. We repeated the same analysis using MH definition by NCEP-ATP III. All statistical analyses were performed using SPSS software . Statistical significance was defined as a two-sided There were, in total, 7824 participants with a mean age of 51.5 years and mean BMI of 24.2, and 65.1% were female . The prep for trend = 0.003) even after adjustment for age, sex, TG, HDL-C, LDL-C, hsCRP, HbA1c, eGFR, HOMA-IR, smoking status, alcohol consumption, physical activity, and the NCEP definition. When MH was defined by the NCEP-ATP III criteria, similar associations were observed only in OW and OB subjects. Moreover, none of the groups displayed increased risks when adjusted for the new definition  who had adiponectin levels measured at baseline. Consistent with these animal studies, MH individuals who are OW or with obesity had higher levels of adiponectin than metabolically unhealthy individuals who are OW or with obesity . Although it is beyond the scope of this study, it is plausible that metabolically healthy groups of individuals may have a favorable body fat distribution with higher circulating levels of adiponectin, thus protecting them from CVD. Interestingly, this protection was limited to subclinical atherosclerosis but not fatty liver, which is consistent with a previous study [The underlying mechanism involved in the MHO phenotype is not fully understood; however, accumulating evidence suggests that less visceral adiposity and less insulin resistance are involved in the MHO phenotype . Adiponeus study .There was a marked difference in the prevalence of NAFLD between the groups. The prevalence of NAFLD increased substantially with increasing BMI, regardless of MH status. The prevalence of NAFLD in NW, OW and obese participants were 1.3%, 9.0% and 55.0% respectively. These findings are further supported by previous large-scale cohort studies, which found that obesity was strongly associated with incident NAFLD and worsening of liver fibrosis in NAFLD independent of MH ,40,41. AWe found that the CVD risk among those with the MHO phenotype did not increase compared to that of the MHNW. To date, recommendations for obesity treatment do not consider differences between healthy and unhealthy OW or OB phenotypes. However, the stratification of OB individuals based on their cardiometabolic phenotype may be important for identifying those who are to be prioritized for early pharmacological treatment in addition to lifestyle intervention. Future longitudinal studies are needed to evaluate the impact of lifestyle and treatment interventions on changes in body phenotypes and their effect on the delay or acceleration of subclinical atherosclerosis.However, this study has several limitations. First, as in all cross-sectional studies, we cannot conclude that there is a causal effect of body phenotype and subclinical atherosclerosis. Although we tried to adjust for known risk factors using multivariate modeling, there may have been residual confounding by unmeasured and measured variables. However, similar results were observed when the same definition of MH was applied to an independent prospective cohort . SecondlThis study had several strengths. This study included a large, community-based cohort study of middle-aged Asian participants with a broad spectrum of clinical variables as clinical risk factors. To our knowledge, this is the first study to validate the utility of this new, revised definition of MH by evaluating associations with carotid atherosclerosis, in comparison with other definitions in an Asian population without clinical CVD.In conclusion, with a new and simple definition of MH, we were able to classify people with MHO who are not at an increased risk for subclinical atherosclerosis. This new definition was also helpful in detecting lean people at risk. Routine screening of CVD risk among the metabolically unhealthy, regardless of BMI category, may provide an opportunity to institute intensive pharmacological therapy to prevent subsequent CVD."}
+{"text": "Objective\u2003The present study aims to compare the maternal and fetal outcomes of parturients with and without a gestational diabetes diagnosis.Methods\u2003A case-control study including parturients with (cases) and without (control) a gestational diabetes diagnosis, who delivered at a teaching hospital in Southern Brazil, between May and August 2018. Primary and secondary data were used. Bivariate analysis and a backward conditional multivariate logistic regression were used to make comparisons between cases and controls, which were expressed by odds ratio (OR), with a 95% confidence interval (95%CI) and a statistical significance level of 5%.Results\u2003The cases (n\u2009=\u200947) were more likely to be 35 years old or older compared with the controls (n\u2009=\u200993) (p\u2009<\u20090.001). The cases had 2.56 times greater chance of being overweight (p\u2009=\u20090.014), and a 2.57 times greater chance of having a positive family history of diabetes mellitus (p\u2009=\u20090.01). There was no significant difference regarding weight gain, presence of a previous history of gestational diabetes, height, or delivery route. The mean weight at birth was significantly higher in the infants of mothers diagnosed with diabetes (p\u2009=\u20090.01). There was a 4.7 times greater chance of macrosomia (p\u2009<\u20090.001) and a 5.4 times greater chance of neonatal hypoglycemia (p\u2009=\u20090.01) in the infants of mothers with gestational diabetes.Conclusion\u2003Therefore, maternal age, family history of type 2 diabetes, obesity and pregestational overweightness are important associated factors for a higher chance of developing gestational diabetes. Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from insulin deficiency. Its incidence has been increasing over the years. The number of adults with DM worldwide increased from 30 million in 1985 to 135 million in 1995, and to 173 million in 2002, and currently this number is \u223c 415 million. In women, when detected for the first time during pregnancy, this condition is classified as gestational diabetes (GD), which is considered an important risk factor for the future development of type 2 DM (T2DM), and has a prevalence of 1\u201337.7%, with a worldwide average of 18%.The risk factors for the development of GD should be evaluated in each pregnant woman so that the diagnosis can be established prematurely, enabling an adequate and early treatment. Some of the factors associated with the development of GD are overweightness, obesity or excessive weight gain during pregnancy, family history of T2DM in a first-degree relative, previous history of GD, hypertension, or preeclampsia in the current pregnancy.During pregnancy, insulin resistance increases due to the release of diabetogenic placental hormones. Pregnant women with GD have higher insulin resistance than pregnant women without GD; therefore, the postprandial glycemic values in these pregnant women are even higher. The complications of GD include fetal macrosomia, birth injury, increased rates of cesarean sections, neonatal hypoglycemia, neonatal respiratory distress syndrome (RDS), prematurity, and fetal death. Pregnant women with GD have an increased chance of developing T2DM after delivery. They also have an increased risk of developing hypertensive disorders during pregnancy, characterizing the pregnancy as high-risk, a fact that demands greater care and follow-up to prevent possible complications and death.Considering the increasing presence of the main risk factors for the development of GD in women of childbearing age and the fact that pregnancies associated with GD are characterized as high-risk, therefore requiring more caution and attention, the present research aimed at comparing the maternal and fetal outcomes of parturients with and without a diagnosis of GD.This was a case-control study including parturients with and without a GD diagnosis, who delivered between May and August 2018 at Hospital Nossa Senhora da Concei\u00e7\u00e3o (HNSC), located in the city of Tubar\u00e3o, in the state of Santa Catarina, Southern Brazil.The HNSC has an obstetric center that is a reference for high-risk management in the South of Santa Catarina, and \u223c 200 deliveries are performed there monthly. Using the Statcalc function of the Epi Info 3.5.4  software, and with the purpose of conducting a case-control study in the ratio of cases: 1:2 controls, we assumed a prevalence of \u223c 19% of obesity as a risk factor for the development of GD in the general population, considering that the incidence of gestational diabetes mellitus (GDM) in obese pregnant women is three times higher than in the general population. The sample size was calculated as 140 parturients (47 cases and 93 controls).The present study included parturients with and without a GD diagnosis, who delivered at the HNSC between May and August of 2018; after being informed, they accepted to participate in the study and signed an Informed Consent Form (ICF). Parturients with type 1 DM (DM1) and T2DM were excluded. The diagnostic criteria of GD followed the Consensus on Gestational Diabetes: 2017 Update.Data collection was performed from May to August 2018. The data source used was primary, in which the researchers contacted the parturients admitted to the HNSC, who, through the ICF, agreed to participate; and secondary, in which the researchers sought information related to the newborn (NB) in electronic medical records. The controls were selected among the puerperae without GD in the same period.The researchers visited the hospital daily and contacted all of the parturients admitted to explain the research and to apply the questionnaire to those who accepted to participate.2); method of diagnosis; GA at diagnosis (in weeks); family history of DM; previous history of GD; presence of gestational hypertension; delivery route ; GA at delivery (weeks); fetal weight at birth (kg); Apgar score at 1 and 5\u2009minutes; hypoglycemia at birth; RDS; fetal death; and the NB gender . The pregestational and current (time of delivery) weight and height values were mentioned by the participants, considering the impossibility of the researchers to be present full time to carry out the measurements. The BMI was calculated by the researchers (weight/height2), overweightness (BMI 25\u201329.99\u2009kg/m2), and obesity (BMI\u2009\u2265\u200930\u2009kg/m2).The instrument used was a self-administered questionnaire and a protocol developed by the researchers containing the variables of interest, such as: age (years); weight (kg); weight gain ; height (cm); previous BMI kg/m; method In the present study, excess weight was characterized as overweightness and obesity, and gestational hypertension was characterized by decompensated chronic hypertension during pregnancy or hypertension developed during pregnancy. The GA at delivery was defined as premature when the delivery occurred before 37 weeks. Macrosomia was defined as birth weight \u2265 4\u2009kg in term deliveries or\u2009>\u2009the 90th percentile for the GA. Hypoglycemia at birth was defined by the pediatrician. The researchers had no access to the absolute blood glucose levels in cases of NBs with neonatal hypoglycemia.t test for quantitative variables and the Chi-squared or Fisher exact tests for categorical variables, according to data suitability. Comparisons between cases and controls were expressed using odds ratio (OR), with a 95% confidence interval (95%CI) and a statistical significance level of 5%. The variables that presented p-values\u2009<\u20090.20 in the bivariate analysis were submitted to backward conditional multivariate logistic regression to evaluate the independent association with the outcome.The collected data were inserted and stored in a database created with the help of the software Epi Info, version 3.5.4. The data analysis was performed using this software and complemented with the Statistical Package for the Social Sciences  software, version 20.0. Descriptive statistics were used as absolute numbers and percentages, measures of central tendency and dispersion. The bivariate analysis was performed using the Student The present research followed Resolution 466/2012 of the Brazilian National Health Council, and was approved by the Ethics in Research Committee of Universidade do Sul de Santa Catarina (UNISUL) on March 28, 2018, under opinion number 2.569.614.In the 140 women included in the present study (47 cases and 93 controls), the minimum age was 18 years old, and the maximum age was 44 years old. The mean age of the cases was 32.8\u2009\u00b1\u20096.4 years, which was statistically higher than that of the controls: 27.2\u2009\u00b1\u20096.9 years. The cases were more likely to be 35 years old or older (42.6%) compared with the controls (14%). Regarding the previous BMI, the cases presented more pregestational overweightness (72.3%) than the controls (50.5%). There was also a greater chance of obesity in the cases than in the controls .There was no significant difference in mean weight gain, previous history of GD, and height between cases and controls. The cases had a greater chance of having a positive family history of DM than the controls. The risk factors associated with the development of GD analyzed in the present study are described in p\u2009=\u20090.032). Furthermore, women older than 35 years of age were more obese . Considering the previous BMI and age as continuous variables, both were associated with the occurrence of GD; but, when combined in the model, age prevailed as an associated factor. However, the adequacy measure of the model (R2\u2009\u2264\u20090.21) indicated little explanation for the relationship between the surveyed variables and GD, suggesting that there were other factors influencing the outcome that were not evaluated in the present study.In the conditional logistic regression, age emerged as an independent variable associated with the occurrence of GD in all models . We veriThe mean GA at diagnosis was of 25.2\u2009\u00b1\u20098.51 weeks, with a minimum GA of 7 weeks, and a maximum GA of 38 weeks. The most prevalent method of diagnosis of GD was the OGTT, which established the diagnosis in 76.6% (36) of the cases, while 23.4% (11) were diagnosed by fasting glucose (FG).The mean birth weight was significantly higher in the infants of mothers diagnosed with GD. Regarding the delivery route, no statistically significant difference was found between mothers with and without GD, and those who had GD also had no greater chance of having gestational hypertension than the controls.There were no significant differences in the mean GA at delivery, and 22 out of the 140 (15.7%) parturients had preterm births. The mean Apgar score at 1\u2009minute ranged from 1 to 10, while at 5\u2009minutes it ranged from 7 to 10. One infant had RDS, and no deaths were observed in the overall cohort. The outcomes related to the development of GDs are described in Out of the 47 women diagnosed with GD, 87.2% (41) underrwent some type of treatment, including diet alone (27/57.4%), insulin alone (04/8.5%), diet plus oral medication (08/17%), and diet plus insulin (02/4.3%). A total of 12.8% (06) of the cases underwent no treatment despite medical advice to do so. None of the participants used only oral medication. There were no significant differences regarding submission or not to treatment and the occurrence of maternal and fetal outcomes. A comparison between submission or not to treatment in relation to the outcomes is shown in Observing the type of treatment to which the women with GD were submitted and the related factors, we found no significant differences regarding the use of insulin in relation to maternal age, previous BMI, and overweightness. Regarding pregnancy outcomes, the use of insulin was not associated with the occurrence of preterm delivery, and although cesarean sections were performed more frequently in women taking insulin, this finding was not significant. The evaluation of the other types of treatment in regard to the related factors was limited by the small sample of women with GD who underwent treatment. The relationship between the use of insulin for treatment in women with GD, the related factors, and pregnancy outcomes is shown in p\u2009=\u20090.0003). Regardless of the presence of GD, macrosomia was a more common event in males (19/72 [26.4%]) than in females (8/68[11.8%]); p\u2009=\u20090.028. In the same way, hypoglycemia was an outcome more frequent among infants of mothers with GD (5/47 [10.6%]) than among those of mothers without the disease (2/93\u2009=\u20092.2%); p\u2009=\u20090.042.Out of the NBs with macrosomia (27), 17 (63%) were born to mothers with GD, and 10 (10.8%) were infants of women without GD (n\u2009=\u2009430 women) of the complications found. In a case-control studyn\u2009=\u2009206 cases and n\u2009=\u2009286 controls) in China, older maternal age was also associated with risk of developing GD. Findings in Southern Brazil of two cohortsn\u2009=\u2009375) and 30\u2009\u00b1\u20096 years (n\u2009=\u2009216). This relationship between age and GD was evident in the present study, in which the mean age of the cases was higher than that of the controls.The literature shows that age\u2009>\u200935 years is a risk factor for the development of GD. Alves et al2 . Miao et al,n\u2009=\u2009832), showed that, in general, 21.4% (n\u2009=\u2009178) of the women were obese or overweight, and 35.2% (n\u2009=\u2009298) presented excessive weight gain during pregnancy. In the present study, there was no significant difference in the mean weight gain between cases and controls. This is probably due to the fact that, in the present study, weight values were referred by the parturients, and were not measured.The cases had a greater chance of being overweight and obese than the controls. Feleken\u2009=\u2009996) and without GD (n\u2009=\u2009996) showed that a positive family history of DM was significantly more frequent in the cases than in the controls (15.6% versus 2.4% respectively). In relation to the presence of previous history of GD, differently from what was found in the present study, Bhat et aln\u2009=\u2009300) were 5.3 times more likely to have a previous history of GD than the controls (n\u2009=\u2009300). This difference is, perhaps, due to the fact that in their Study, the number of cases was at least one-sixth times lower.Corroborating the data obtained in the present research, a retrospective study in ChinaIn patients with GD, the mean height of the cases was above the one considered as a predisposing factor for GD (150\u2009cm). However, in a cohort studyn\u2009=\u20094,053) of the sample had a GD diagnosis, with a mean GA at diagnosis of 21 weeks, varying from 15 to 27 weeks. Furthermore, in a prospective studyn\u2009=\u200935; controls: n\u2009=\u2009465), 11.4% (n\u2009=\u20094) of the sample had their diagnosis in the initial 16 weeks, and 88.6% (n\u2009=\u200931), in 24\u201328 weeks.The mean GA at diagnosis is within the period of highest physiological insulin resistance in pregnancy, and the one recommended for GD screening (24\u201328 weeks). The most prevalent method of diagnosis of GD was the OGTT, probably due to the fact that most diagnoses were established in the period of higher hyperglycemia risk, in which the investigation is performed through the OGTT and not the FG. In a studyn\u2009=\u2009201; controls: n\u2009=\u2009201), found hypoglycemia at birth to be the most frequent complication of GD, and identified hypoglycemia and RDS as important causes of neonatal morbidity; in relation to the birth weight of the NBs, there were no statistical differences. In the present study, the mean birth weight and rate of neonatal hypoglycemia were significantly higher in infants of mothers with GD, and, when considering RDS, the same did not occur in the study by Miranda et al.Miranda et al,n\u2009=\u2009703) in a hospital in the city of Joinville, Southern Brazil: 52.2% (n\u2009=\u2009367) had cesarean sections as the delivery route.The proportion of cesarean sections in the present study was similar to that found in a retrospective cohort studyn\u2009=\u200986) conducted in the city of Canoas, Southern Brazil, suggested a greater frequency of prematurity in women with GD; 3 deaths were recorded in the study. Likewise, in a retrospective cohortn\u2009=\u2009255; controls: n\u2009=\u2009267), women with GD showed twice the risk of delivering preterm infants. In addition, the study showed that the association between GD and a low Apgar score at 1 and 5\u2009minutes was not significant. In the present study, there were no significant differences regarding the Apgar score and the mean GA at birth in infants of mothers with and without GD. The absence of neonatal deaths in our study is probably related to the current greater care dedicated to pregnant women with GD and also to therapy individualization, which reduces unwanted GD outcomes.Zanrosso et al,n\u2009=\u2009159 pregnant women) conducted in the city of Macei\u00f3, Northeastern Brazil, in which the outcomes of interest were GD and gestational hypertensive syndrome (GHS), observed no association between GD and gestational hypertension, since none of the diseases occurred concomitantly.As in the present study, a transversal studyn\u2009=\u2009799; controls: n\u2009=\u20092,843) in 3 maternity hospitals in Chennai, India, showed similar results: most women underwent a diet as the only treatment 42.2% (n\u2009=\u2009338) or diet associated with insulin 57.1% (n\u2009=\u2009457), while only 0.5% (n\u2009=\u20094) used oral medication.According to the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study,n\u2009=\u2009705 women with GD) performed in the city of Joinville, Southern Brazil, in which all women underwent some kind of treatment, 10.22% (n\u2009=\u200972) of the treated pregnant women developed hypertensive pregnancy disease, and 4.8% (n\u2009=\u200934) of the infants were premature.Regarding the relationship between outcomes and treatment in GD patients, lack of treatment is known to increase the risk of unwanted outcomes. In the present study, undergoing treatment did not interfere in the occurrence of macrosomia, hypertension, or prematurity. This differs from the observation of a systematic review,n\u2009=\u200950), and the type of treatment performed did not interfere with the delivery route, reflecting the same finding observed in the present study, except that the study in question found a higher rate of cesarean sections among parturients taking insulin. Watanabe et aln\u2009=\u20094) of them were overweight, and 10% (n\u2009=\u20091) had a BMI 30\u2009kg/m2 before pregnancy, which was not significant.Also in the aforementioned study,n\u2009=\u2009612 GD cases) showed that the mean age of the women who used insulin was 31.4 years versus 30.9 years for those who did not use it. Furthermore, women who took insulin had a higher BMI than those who did not require it (28.3\u2009\u00b1\u20097.00\u2009kg/m2). In the present study, overweight parturients had more chance of using insulin than those who were not overweight, a finding with no statistical significance.Regarding the maternal age and insulin use, no significant differences were found either. Differently, a retrospective studyn\u2009=\u2009149; controls: n\u2009=\u2009711), the prevalence of NBs with macrosomia ranged from 5 to 20%. In addition, the study showed that male NBs had 3.33 times more chance of being macrosomic when compared with female NBs. This finding was also observed in the present study. The predominance of macrosomia in males may be associated with the fact that, during the third trimester, male fetuses tend to gain more weight than female ones.According to Ribeiro et alA limitation of the present study was the reduced number of cases, which hinders the generalization of the findings to other populations. Furthermore, specific information, like treatment adherence and body weight, could not be measured, and the self\u2013reported data are subject to errors.The present study found maternal age, family history of DM2, obesity and pregestational overweightness as important factors related to a higher chance of developing GD. Regarding the neonatal outcomes, we found that the children of women diagnosed with GD had higher prevalence of fetal macrosomia and hypoglycemia at birth than the children of women who did not have GD. There was no significant difference between cases and controls regarding gestational hypertension, prematurity, cesarean section, neonatal RDS and neonatal death."}
+{"text": "However, the effect of an elevated BMI (\u2265\u200925 kg m\u22122) on humoral immunity to SARS\u2010CoV\u20102 infection and COVID\u201019 vaccination remains unclear.Class III obesity . We also collected blood samples from people approximately 5 months of post\u2010second dose COVID\u201019 vaccination (the majority of whom did not have a prior SARS\u2010CoV\u20102 infection). We measured their humoral responses to SARS\u2010CoV\u20102, grouping individuals based on a BMI greater or less than 25 kg m\u22122), when accounting for age and sex differences, is associated with reduced antibody responses after SARS\u2010CoV\u20102 infection. At 3 months of post\u2010infection, an elevated BMI was associated with reduced antibody titres. At 13 months of post\u2010infection, an elevated BMI was associated with reduced antibody avidity and a reduced percentage of spike\u2010positive B cells. In contrast, no significant association was noted between a BMI \u2265\u200925 kg m\u22122 and humoral immunity to SARS\u2010CoV\u20102 at 5 months of post\u2010secondary vaccination.Here, we show that an increased BMI . In contrast, there was no association between increased BMI and the humoral immune response to SARS\u2010CoV\u20102 vaccination. SARS\u2010CoV\u20102 has resulted in >\u2009765 million infections globally (May 2023).\u22122, the antibody response to SARS\u2010CoV\u20102 infection partially declines within 6\u2009months post infection,\u22122). In our study, we established a cohort of people from Brisbane, Australia who had recovered from a single SARS\u2010CoV\u20102 infection in March 2020\u00a0and were not exposed to SARS\u2010CoV\u20102 or vaccinated in the subsequent 13\u2009months. We also recruited individuals from Melbourne, Australia who had been vaccinated against SARS\u2010CoV\u20102. Using these cohorts, we sought to determine whether an elevated BMI (BMI\u2009\u2265\u200925\u2009kg\u2009m\u22122) affected the humoral response to SARS\u2010CoV\u20102 infection and\u00a0vaccination.There is a very little evidence to identify the host factors that are associated with the susceptibility to SARS\u2010CoV\u20102 reinfection and the impairment of an effective humoral response. Previous studies have suggested that host co\u2010morbidities,\u22122, in order to assess the effects of both overweight and obesity on the humoral response, as per previous studies.Participants who tested positive for SARS\u2010CoV\u20102 in early 2020 were recruited for the study. To note, the SARS\u2010CoV\u20102 infection cohort employed in the present study was relatively unique in so far as the early nature of their infection (March 2020) and the fact that vaccination did not begin in Australia until February 2021. In addition, the cohort represents humoral immune responses after a single infection, as Queensland, Australia did not have SARS\u2010CoV\u20102 circulating in the community until state borders opened in December 2021. Blood samples from these recruited unvaccinated patients were taken from these participants at two\u00a0different time points: approximately 3\u2009months post\u2010infection (Visit 1) and 13\u2009months post\u2010infection (Visit 2). Participants were subsequently grouped according to BMI from Visit 1 with the threshold set at 25.0\u2009kg\u2009mTo assess the effects of BMI on the longevity of humoral responses after infection, the total spike\u2010specific binding antibodies , neutralisation capacity , cross\u2010strain neutralisation, avidity and ADCC were assessed between both visits using a paired analysis of matched samples Figure\u00a0. A signiTogether with paired participant sampling at both time points, additional individual samples were collected at single time points Tables\u00a0 & 2. To \u22122) and humoral responses to vaccination. Forty\u2010seven vaccinated participants without any clinical history of positive SARS\u2010CoV\u20102 tests, and with a limited percentage with N antibodies, were recruited for the study  deleteriously affects the humoral response to SARS\u2010CoV\u20102 infection.Here, we provide evidence that an elevated BMI  at the time of writing, only 20.7% of people in low\u2010income countries have\u00a0received at least one dose of a COVID\u201019 vaccine.\u22122 did not affect the humoral immune response to COVID\u201019 vaccination at approximately 5\u2009months post\u2010second dose of vaccination. At present, there are conflicting reports as to whether infection\u22122) and a healthy BMI 6\u2009months after the second SARS\u2010CoV\u20102 vaccine dose. However, in contrast to the findings of the present study, van der Klaauw and colleagues found that the function of these antibodies, as measured SARS\u2010CoV\u20102 neutralisation, was reduced in individuals with severe obesity. This discrepancy may reflect the different BMI threshold of the present study (\u2265\u200925\u2009kg\u2009m\u22122) and that of van der Klaauw and colleagues (>\u200940\u2009kg\u2009m\u22122).\u22122). This range applies to a larger number of individuals in society and therefore, we believe that any associated immune defects have far reaching implications. With the caveat that different findings may have been observed in the present study if different timepoints post\u2010infection/vaccination were sampled or different BMI cut\u2010offs were used, our data provide an insight into the comparisons between infection\u2010induced and vaccine\u2010induced SARS\u2010CoV\u20102 humoral responses.Interestingly, BMI\u2009\u2265\u200925.0\u2009kg\u2009mIn addition to BMI, age is known to affect the humoral response to SARS\u2010CoV\u20102 vaccination.The present study has several limitations. Firstly, the number of donor samples employed herein were limited compared to other studies. This reflects the fact that there was only a limited number of individuals in Queensland, Australia who were infected with SARS\u2010CoV\u20102 in March 2020. This population cohort was important to recruit and could not be supplemented with individuals infected later in the pandemic, as it would then not be possible to assess immunity at 13\u2009months post\u2010infection in the absence of intervening reinfections or vaccinations. Indeed, several participants were lost from the Visit 2 cohort of the present study as they were amongst the earlier adopters of COVID\u201019 vaccination in Australia. Accordingly, it is not possible to state whether the fewer statistically significant relationships observed at Visit 2 is indicative of a biological phenomenon or a function of the reduced sample size. Secondly, it is possible that the improved immunity associated with hybrid immunity,Recruitment of human donors was approved by the human ethics boards of Mater Research (HREC/MML/62705), the QIMR Berghofer Medical Research Institute (Ethics: P3618), the David Serisiser Research Biobank (DSRB) (HREC/14/QPAH/275) at Mater Misericordiae Ltd and La Trobe University (Ethics: HEC21097). All methods were performed in accordance with institutional guidelines and regulations. Written consent was obtained from all study participants. To study the impact of BMI on the humoral response to SARS\u2010CoV\u20102 infection, individuals with a history of PCR\u2010confirmed SARS\u2010CoV\u20102 infection were recruited to the study from Brisbane, Australia. To study the impact of BMI on the humoral response to vaccination, individuals with a history of ChAdOx1\u2010S or Pfizer\u2010BioNTech vaccination were recruited to the study 5\u2009months after the second dose of vaccine was\u00a0administered from Melbourne, Australia. At point of recruitment, patient details including BMI were collected.g for 10\u2009min. Serum and plasma were heat treated at 56\u00b0C for 1\u2009h, aliquoted accordingly and stored in \u221220\u00b0C prior to analysis, or in \u221280\u00b0C for long term storage.Blood samples were collected from patients recovered from PCR\u2010diagnosed SARS\u2010CoV\u20102 infection at different timepoints by qualified phlebotomists. Blood was either collected in EDTA or heparin tubes  or BD SST\u2122 Vacutainers\u00ae (BD Biosciences). All blood samples were processed within 24\u2009h of blood collection. To isolate serum and plasma, tubes containing blood samples were spun down at 2000\u2009A volume of 10 mL of blood was collected in BD Vacutainer\u00ae EDTA tubes (BD Biosciences). Peripheral blood mononuclear cells (PBMC) were isolated with Lymphoprep  according to manufacturer's instructions. Isolated PBMCs were subsequently frozen down in foetal calf serum  containing 10% DMSO  at \u221280\u00b0C until analysis. For long\u2010term storage, PBMCs were stored in liquid nitrogen.2 incubator in DMEM (Dulbecco's Modified Eagle Medium) media containing 10% foetal bovine serum  and 1% Penicillin\u2013Streptomycin\u2010Glutamine . Purified DNA was incubated for 20\u2009min with Polyethylenimine (PEI)  at a ratio of 1:3. Following incubation, the DNA:PEI mix was added to the cell culture flasks in DMEM containing 1% FBS, 5% cocktail of supplements referred to as Mastermix , and placed back in the CO2 incubator for 7\u2009days at 37\u00b0C. The supernatant was harvested and purified over affinity and size exclusion column . The protein was run on an SDS\u2010page gel to confirm purity and then biotinylated and stored at \u221220\u00b0C.The design of a prefusion\u2010stabilised SARS\u2010CoV\u20102 spike, called HexaPro, was performed essentially as described previously.\u22121 SARS\u2010CoV\u20102 spike proteins were coated on ELISA plates (Thermo Fisher Scientific) overnight at 4\u00b0C. Plates were blocked and serially diluted sera or plasma samples were added to the plates and incubated at 37\u00b0C for 1\u2009h. Spike\u2010specific antibodies were detected with mouse anti\u2010human IgG HRP  for 1\u2009h at 37\u00b0C. Plates were visualised with 2,2\u2032\u2010Azino\u2010bis  in the presence of hydrogen peroxide  at 405\u2009nm on SpectraMAX 190. EC50 values were background subtracted and calculated on Graphpad Prism 9.0.2  using \u2018log(agonist) versus response; variable slope (4 parameters)\u2019 function.Anti\u2010spike IgG antibodies in patient samples were assessed with SARS\u2010CoV\u20102 spike ELISAs essentially as described previously.m sodium thiocyanate (Sigma Aldrich) for 15\u2009min at room temperature before incubating with secondary antibodies. An avidity index (%) was derived from EC50 values from treated samples over EC50 values from untreated samples.Avidity assays were conducted similarly to spike ELISAs described above, except samples were treated with 1\u2009m sulphuric acid and the optical density at 450\u2009nm (OD450) was read using the SpectraMAX M5. The final OD450 values were obtained after the background subtraction of mean\u2009+\u20093\u2009\u00d7\u2009standard deviation OD450 of four healthy individuals who were neither SARS\u2010CoV\u20102 infected nor vaccinated.Anti\u2010Nucleocapsid IgG antibody in sera was quantified using modified ELISA as previously described.2 in Dulbecco modified Eagle medium  supplemented with 10% foetal calf serum  and 1% Pen Strep Glutamine . The cells were maintained in DMEM with 2% FCS and 1% PSG during viral infection to generate stocks.Vero E6 cells and Vero E6\u2010hTMPRSS2 cells were maintained at 37\u00b0C and 5% COSARS\u2010CoV\u20102 isolates hCoV\u201019/Australia/QLD02/2020  and hCoV\u201019/Australia/QLD1893C/2021  were initially isolated from patient nasopharyngeal aspirates and inoculated on Vero E6 cells. Subsequently, passage 2 of these samples were kindly provided by Queensland Health Forensic and Scientific Services, Queensland Department of Health and stocks were generated in Vero E6\u2010hTMPRSS2 cells as described previously.50 readings were calculated on Graphpad Prism 9.0.2 (Dotmatics) using the \u2018log(inhibitor) versus normalised response\u2019 function.Microneutralisation assays were performed essentially as previously described.10 dilution curve was performed to calculate the area under the curve using GraphPad Prism v9.Antibody\u2010dependent cellular cytotoxicity function of recovered patient samples were analysed with ADCC reporter assay  accordingly to manufacturer's instructions.The Decoy tetramer was prepared as previously described.Six higher BMI and six lower BMI samples were randomly chosen for B cell analysis. Staining for flow cytometry analysis was performed using cryo\u2010preserved PBMCs. PBMCs were stained with Fixable Viability Stain 780  diluted in PBS 2% FCS for 15\u2009min at room temperature. Cells were washed with PBS 2% FCS and then stained with CD20\u2010AF700 , CD27\u2010V450 , CD38\u2010FITC , IgD\u2010BV510 , CD10\u2010PE\u2010Cy7 , CD21\u2010FITC , FcRL5\u2010BV510 , CD19\u2010BV650 , CD14\u2010PerCP\u2010Cy5.5  CD16\u2010PerCP\u2010Cy5.5 , HexaPro spike protein Tetramer\u2010PE (1:50) and HLA\u2010A2\u2010M1 Decoy Tetramer\u2010PE*DL650  diluted in PBS 2% FCS for 20\u2009min on ice. Cells were washed with PBS 2% FCS and fixed for 1\u2009h using Cytofix (BD Biosciences), then washed with PBS 2% FCS and resuspended in PBS 2% FCS. Cells were analysed using an LSRFortessa X\u201020 (BD Biosciences) and flow cytometry data was analysed using FlowJo\u2122 v10.8 software (BD Biosciences). A representative gating strategy is shown in Supplementary figure\u00a0U\u2010test whilst the difference between categorical values was determined on Graphpad Prism 9.0.2 (Dotmatics) using a Chi\u2010squared test and paired analysis was performed with two\u2010way ANOVAs. Heteroscedasticity was diagnosed by inspection of the residual for a non\u2010weighted Multiple Linear Regression (MLR) model with Age, BMI and Gender as predictor variables for all categories of response variable . For non\u2010weighted MLR we first fitted a non\u2010weighted MLR to the squared\u2010residuals themselves to estimate the change in variance that is occurring in the response variable. That is,wi=1/ri=1/\u03c3i2 for our weighted Multiple Linear Regression (MLR) model. The outputs from our variance\u2010weighted multiple linear regression model were the coefficients b1 (Intercept), b2 (Age), b3 (BMI) and b4 (Gender). We performed a t\u2010test on each coefficient in the model with null hypothesis that the coefficient is not significantly different from zero. A P\u2010value of less than 0.05 (95% confidence limit) was set to determine which variables in the variance\u2010weighted MLR model have a coefficient that is not significantly different from zero. All analysis was performed in MATLAB.Differences between continuous variables was analysed on Graphpad v9.0.2 (Dotmatics) using a Mann\u2013Whitney Marcus ZW Tong: Data curation; investigation; methodology; writing \u2013 original draft; writing \u2013 review and editing. Julian D. J. Sng: Data curation; methodology. Meagan Carney: Formal analysis; software; visualization. Lucy Cooper: Formal analysis; methodology. Samuel Brown: Data curation; methodology. Katie E Lineburg: Data curation; investigation; methodology. Keng Yih Chew: Data curation; funding acquisition; investigation; methodology; project administration. Neve Collins: Data curation; methodology. Kirsten Ignacio: Data curation; methodology. Megan Airey: Data curation; methodology. Lucy Burr: Data curation; investigation; project administration; resources; writing \u2013 review and editing. Briony A Joyce: Methodology. Dhilshan Jayasinghe: Data curation; methodology. Christopher LD McMillan: Methodology; resources. David A Muller: Methodology; resources. Anurag Adhikari: Investigation; methodology; resources. Linda A Gallo: Investigation; supervision. Emily S Dorey: Data curation; investigation; project administration; resources. Helen L Barrett: Project administration; investigation; conceptualization; supervision; writing \u2013 review and editting. Stephanie Gras: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; supervision; validation; writing \u2013 review and editing. Corey Smith: Conceptualization; data curation; funding acquisition; investigation; methodology; project administration; resources; supervision; writing \u2013 review and editing. Kim Good\u2010Jacobson: Conceptualization; data curation; funding acquisition; investigation; methodology; project administration; writing \u2013 review and editing. Kirsty R Short: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; supervision; writing \u2013 original draft; writing \u2013 review and editing.KRS is a consultant for Sanofi, Roche and NovoNordisk. The opinions and data presented in this manuscript are of the authors and are independent of these relationships. Other authors declare no competing interests.Supporting informationClick here for additional data file."}
+{"text": "KwaZulu-Natal ranked second highest among South African provinces for the number of laboratory-confirmed cases during the second wave of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The seroprevalence of SARS-CoV-2 among certain vulnerable groups, such as people living with HIV in KwaZulu-Natal, is unknown.The study aimed to determine the prevalence of SARS-CoV-2 immunoglobulin G (IgG) in HIV-positive versus HIV-negative patients.This was a retrospective analysis of residual clinical blood specimens unrelated to coronavirus disease 2019 (COVID-19) submitted for diagnostic testing at Inkosi Albert Luthuli Central Hospital, Durban, from 10 November 2020 to 09 February 2021. Specimens were tested for SARS-CoV-2 immunoglobulin G on the Abbott Architect analyser.p < 0.0001) and increased with increasing age, with a statistically significant difference between the farthest age groups . The seroprevalence increased from 17% on 10 November 2020 to 43% on 09 February 2021 during the second wave.A total of 1977/8829 (22.4%) specimens were positive for SARS-CoV-2 antibodies. Seroprevalence varied between health districts from 16.4% to 37.3%, and was 19% in HIV-positive and 35.3% in HIV-negative specimens. Seroprevalence was higher among female patients (23.6% vs 19.8%; Our results highlight that during the second COVID-19 wave in KwaZulu-Natal a large proportion of people living with HIV were still immunologically susceptible. The reduced seropositivity in people with virological failure further emphasises the importance of targeted vaccination and vaccine response monitoring in these individuals.This study contributes to data on SARS-CoV-2 seroprevalence before and during the second wave in KwaZulu-Natal, South Africa, which has the highest HIV prevalence globally. Reduced seropositivity was found among people living with HIV with virological failure, highlighting the importance of targeted booster vaccination and vaccine response monitoring. KwaZulu-Natal province ranked second-highest nationally for the number of laboratory-confirmed cases during the second wave of the SARS-CoV-2 epidemic in South Africa.1Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is one of the most significant pandemics in history. In Africa, South Africa has the highest number of confirmed cases  is most accurate for the diagnosis of acute SARS-CoV-2 infection.7Even though South Africa has passed the third, fourth and fifth waves of the SARS-CoV-2 epidemic and is currently experiencing a transition phase, serological surveillance remains a vital tool to understand the true extent of SARS-CoV-2 exposure and inform public health responses.10 A population-based seroepidemiological survey in Gauteng province in South Africa, that started 8 weeks into the first wave and ended at the peak of the second wave, estimated that there were 2.89 million SARS-CoV-2 infections, which is 7\u20138 times higher than the reported 332 000 PCR cases.10 Local SARS-CoV-2 seroprevalence rates were over 60% in black South African blood donors11 and 30% \u2013 40% among patients accessing care during the first wave in the Cape Town Metropolitan sub-districts.12 Similarly, seroprevalence studies in Spain, Geneva and New York reported seroprevalence higher than the number of reported cases.15Seroprevalence in 2020 was estimated to be 5\u201310 times higher than reported cases identified by PCR.16 However, serosurveys require extensive resources, and involve active recruitment and prospective follow-up of individuals. Using residual clinical specimens submitted for either routine screening or diagnostic testing could provide a snapshot17 and is a convenient way to conduct passive surveillance. In South Africa, seroprevalence studies using convenience specimens have been conducted by the South African Blood Bank Services (SANBS) in the Western Cape and Gauteng.18Ideally, population-based serosurveys should be used to estimate seroprevalence nationally.19 and has the second highest number of reported SARS-CoV-2 infections nationally. Second-wave infections in South Africa caused by the beta variant were associated with increased disease severity, especially in people living with HIV20 when no vaccines were available. Thus, an accurate estimate of SARS-COV-2 seroprevalence among people living with HIV was important in the early phase of the SARS-CoV-2 epidemic for outbreak surveillance, implementation of vaccination strategies, and to assess the effectiveness of non-pharmaceutical interventions in KwaZulu-Natal. We therefore determined the SARS-CoV-2 immunoglobulin G (IgG) seroprevalence among people with HIV infection in KwaZulu-Natal.KwaZulu-Natal has the world\u2019s highest HIV prevalenceThis study was approved by University of KwaZulu-Natal Biomedical Research Ethics Committee (Ref. BCA256/010). Informed patient consent was not required as this was a retrospective study using residual diagnostic specimens. No additional specimens were collected. All residual clinical specimens were de-identified and labelled with a unique study number; patient privacy and confidentiality data were protected in accordance with the Declaration of Helsinki. Data were collected in a password-protected Excel spreadsheet  on a dedicated computer behind firewall-protected servers, which was only accessible to the primary investigator.The authors retrospectively tested residual clinical sera and plasma specimens submitted to the Department of Virology at Inkosi Albert Luthuli Central Hospital, Durban, for diagnostic testing unrelated to COVID-19 from 10 November 2020 to 09 February 2021, representing the period before and during the second wave (22 November 2020 to 27 March 2021) of the SARS-CoV-2 epidemic in KwaZulu-Natal.http://www.openepi.com/)21 based on a SARS-CoV-2 prevalence of 5% to 10% before the second wave peak with a maximum 1% margin error and using published seroprevalence data from studies done globally , the assumed point prevalence was calculated.A minimum sample size of 7294 was estimated using OpenEpi  sampsi function, the sample size was calculated to compare SARS-CoV-2 seropositivity in HIV-positive and HIV-negative individuals with a power of 80%, two-sided alpha of 0.05 and equal size in each group. A sample size of 3682 would be sufficient to detect a 1% difference if the prevalence is low (1%) and a 3% difference if the prevalence is high (13%).22Using the STATASera and plasma specimens with sufficient volumes from the viral serology  were selected for SARS-CoV-2 IgG testing. Specimens with inadequate volumes (< 100 \u00b5L) were excluded. The selected specimen results were linked to demographic information extracted from the laboratory information system. Specimens from the same patient were tested once only.23 Sera and plasma specimens were stored at 2 \u00b0C \u2013 8 \u00b0C for not more than 7 days and \u221220 \u00b0C for long-term (> 7 days) storage. The assay is an automated two-step chemiluminescent microparticle immunoassay for qualitative detection of SARS-CoV-2 IgG antibodies against the SARS-CoV-2 nucleocapsid protein. The assay was performed and interpreted according to the manufacturer\u2019s instructions.24Testing for SARS-CoV-2 IgG was performed on the Abbott Architect using the Abbott Architect SARS-CoV-2 IgG assay .\u00ae version 15 . We performed a logistic regression analysis to determine whether age, gender, HIV seropositivity, and HIV viral load were associated with SARS-CoV-2 seropositivity, with significance level set to 0.05.Statistical analysis was done using STATAn = 248) or mismatched laboratory information (n = 6). A total of 8829  (p < 0.0001)  while male patients had lower odds of seropositivity ( 0.0001) . Seropos 0.0001) . Seropos 0.0001)  was high25We assessed the SARS-CoV-2 IgG seropositivity in convenience specimens submitted to the Department of Virology at Inkosi Albert Luthuli Central Hospital, Durban, before and during the peak of the second COVID-19 wave caused by the beta variant. We observed an increase in seropositivity from 17% before to 43% during the second wave in KwaZulu-Natal, reflective of the increase in the number of infected individuals during the second wave, with a mean SARS-CoV-2 seropositivity of 22.4% across the study period. There were statistically significant associations between SARS-CoV-2 seropositivity, age, gender, and HIV status. Our results indicate that the true extent of the SARS-CoV-2 epidemic in KwaZulu-Natal may have been underestimated due to reporting of PCR results alone. This is similar to other local seroprevalence studies that report higher seroprevalence compared to the number of reported PCR cases.18 The lower prevalence may be because the specimens were not collected immediately after the first wave in KwaZulu-Natal; they were collected 4 months later, thus affecting the detection of IgG, which wanes over time. The sentinel surveillance studies were started directly after the first wave in both Cape Town and Gauteng.The seropositivity in our study was lower than the seroprevalence reported by surveillance studies in Cape Town (40%) and Gauteng (27.8%).26 However, in individuals on antiretroviral therapy, immune responses to SARS-CoV-2 are reported to be similar to that in HIV-negative individuals.28 People on antiretroviral therapy may display immune reconstitution;26 however, chronic immune activation and impaired B-cell responses in individuals on antiretroviral therapy with virologic failure may result in reduced antibody responses28 and lower IgG concentrations,29 accounting for the lower seropositivity in those with HIV infection.Furthermore, the majority (63%) of the specimens in our study were from HIV-positive individuals; 10% of these specimens had an HIV viral load greater than 1000 copies/mL and reduced SARS-CoV-2 seropositivity. HIV viral load greater than 1000 copies/mL had a statistically significant association with lower odds of SARS-CoV-2 IgG positivity. HIV infection is associated with a reduced immune response to infectious pathogens.30 which may be lower than the detectable limit of the assay. Based on a local evaluation of the performance of the Abbott Architect SARS-CoV-2 IgG assay, sensitivity is highest 30\u201341 days after a positive PCR, following moderate to severe infection.23 However in our study reduced seropositivity was most likely due to reduced immunity in people with uncontrolled HIV and the timing of sampling in relation to the epidemic waves.23Lower SARS-CoV-2 IgG concentrations may affect the detectability of IgG,25 and internationally.15We have demonstrated that SARS-CoV-2 seropositivity increased with age, with children under 10 years having the lowest seropositivity (11.1%). Similar patterns have been described locally31 suggesting that PCR testing alone may have underestimated infection rates or that these children were asymptomatic. However, a seroprevalence in children under 9 years during the first wave in Gauteng was reported to be about 20%,18 most likely due to Gauteng having the highest infection rate nationally during the entire epidemic.1Our reported seropositivity (10%) in children under 10 years during the second wave in KwaZulu-Natal indicated that these children may have been more vulnerable to SARS-CoV-2 infection. However, the rate of infections in South Africa among these children was 1.4%,1We found no discernible pattern in the district seroprevalence during the second wave. However, the rate of infections in the Ethekwini, iLembe, Ugu, and Uthukela districts were subsequently lower in the third wave.Firstly, the specimens tested were from individuals accessing public healthcare and thus may not represent the general population. However, it potentially represents people living with HIV accessing antiretroviral therapy in our province.32 Thirdly, seropositivity may be underestimated since SARS-CoV-2 IgG detection and durability is directly proportional to disease severity.34 Although antibody positivity was still high in another study at 8 months after mild and asymptomatic infection,35 overall detectability and durability may be reduced in HIV-positive persons.36Secondly, surveillance using residual clinical specimens without exposure time data, as done in this study, may underestimate seropositivity.37Fourthly, the Abbott Architect SARS-CoV-2 IgG assay detects IgG to nucleocapsid antigens only and at the time was the only approved commercial serological assay by the South African Health Products Regulatory Authority.32 Nucleocapsid IgG declines more rapidly than spike IgG. However, nucleocapsid IgG is more sensitive within the first 14 days of SARS-CoV-2 infection as compared to spike IgG.34 Lastly, antibody cross-reactivity with other Betacoronaviruses may lead to false positives.38 However, the Abbott Architect SARS-CoV-2 IgG has a specificity of 99%.38The use of nucleocapsid IgG solely to determine seroprevalence may underestimate the true rate of past infections.40Our results highlight that after the second wave in KwaZulu-Natal, many people accessing public healthcare were immunologically susceptible. Furthermore, reduced seropositivity in HIV-positive individuals with HIV viral loads greater than 1000 copies/mL further emphasised the importance of targeted vaccination of people living with HIV and monitoring response to vaccination in these individuals."}
+{"text": "Although similar to trunk and limb skeletal muscles, masticatory muscles are believed as unique in both developmental origins and myogenesis. G\u03b1i2 has been demonstrated to promote muscle hypertrophy and muscle satellite cell differentiation in limb muscles. However, the effect of G\u03b1i2 on masticatory muscles is still unexplored. This study aimed to identify the role of G\u03b1i2 in the proliferation and differentiation of masticatory muscle satellite cells, further exploring the metabolic mechanism of masticatory muscles. The proliferation rate, myotube size, fusion index of masticatory muscle satellite cells and Pax7, Myf5, MyoD, Tcf21 and Musculin expressions were significantly decreased by G\u03b1i2 knockdown, while in cells infected with AdV4\u2010G\u03b1i2, the proliferation rate, myotube size, fusion index and Tbx1 expression were significantly increased. Masticatory muscle satellite cells also displayed phenotype transformation as G\u03b1i2 changed. In addition, G\u03b1i2 altered myosin heavy chain (MyHC) isoforms of myotubes with less MyHC\u20102A expression in siG\u03b1i2 group and more MyHC\u2010slow expression in AdV4\u2010G\u03b1i2 group. In conclusion, G\u03b1i2 could positively affect the adult myogenesis of masticatory muscle satellite cells and maintain the superiority of MyHC\u2010slow. Masticatory muscle satellite cells may have their unique G\u03b1i2\u2010regulated myogenic transcriptional networks, although they may share some common characteristics with trunk and limb muscles. The mice were all healthy and had no trauma, being provided with free access to normal chow and water. Animal studies were approved by the Ethics Committee of Wuhan University School and Hospital of stomatology. All procedures were proceeded according to the institutional guidelines on the use of animals in research.2 incubator at 37\u00b0C throughout all of the experimental procedures.Primary masticatory muscle satellite cells were isolated from masseters of C57BL10 mice as previously reported.2.2siG\u03b1i2 and siRNA negative control (siCtrl) construct were purchased from Genepharma. Masticatory muscle satellite cells cultured in GM2 were transfected with siG\u03b1i2 and siCtrl construct using Lipofectamine\u00ae 2000 Reagent (Invitrogin).Adenoviral constructs AdV4\u2010G\u03b1i2 and control empty vector AdV4\u2010NC were generated, amplified and purified by Genepharma. Masticatory muscle satellite cells cultured in GM2 were infected for 24\u2009h with AdV4\u2010G\u03b1i2 or AdV4\u2010NC (multiplicity of infection\u00a0=\u00a0200). After infection, the cells were cultured in GM2 or DM for another 48\u2009h.2.3The cell proliferation after G\u03b1i2 knockdown or overexpression was evaluated by Cell\u2010Light\u2122 EdU Apollo\u00ae488 In Vitro Imaging Kit (RiboBio) according to the manufacturer's instructions. Briefly, cells were labelled with 5\u2010ethynyl\u20102\u2032\u2010deoxyuridine  at a final concentration of 50\u2009\u03bcM for 2\u00a0h. Then, the absorbance was determined at a wavelength of 488\u2009nm.2.42, PEG 35000  in distilled water with a pH of 7.4. Cells were then fixed with 4% paraformaldehyde (PFA) in CSB for 15\u2009min and permeabilized with 0.2% Triton in CSB at room temperature. After the nonspecific binding was blocked with 10% goat serum (Gibco), myotubes were incubated with monoclonal antibody MF20 which recognized all MyHC isoforms  at 4\u00b0C overnight. Subsequently, cells were stained with Alexa488 and 4,6\u2010diami\u2010dino\u20102\u2010phenylindole (DAPI) (Aspen), and mounted in Vectashield mounting medium (biosharp).To determine changes in myotube size and fusion index, myotubes were washed with cytoskeleton stabilising buffer (CSB) containing 80\u2009mM PIPES, 5\u2009mM EGTA, 1\u2009mM MgClFor the measurement of myotube diameter, three pictures were taken in each well. Thirty largest myotubes from each picture were selected and measured by Image\u2010Pro Plus 6.0. After dividing myotubes into thirds, the myotube diameter was calculated as the mean of distances between the midpoints of each portion. To analyse fusion index, the nuclei of 60\u201390 myotubes in each well were counted to obtain the average number of nuclei per myotube.2.5For immunofluorescent detection of different phenotypes of muscle satellite cells under different conditions, the following antibodies were used: rabbit polyclonal anti\u2010MyoD  and mouse monoclonal anti\u2010Pax7 . After fixation with 4% PFA, cells were incubated with primary antibodies at 4\u00b0C overnight, nonspecific binding was blocked with 10% goat serum (Gibco) and the primary antibodies were visualized using appropriate species\u2010specific 488 and 549 fluoro\u2010chrome\u2010conjugated secondary antibodies , stained with DAPI (Aspen), and mounted in Vectashield mounting medium (biosharp).2.6\u2212\u0394\u0394CT method.RNA was isolated with Total RNA kit I (OMEGA) and cDNA was prepared from 200\u2009ng RNA according to the manufacturer's instructions (Thermo Fisher Scientific). The relative level of gene expression was determined by quantitative real\u2010time polymerase chain reaction (qRT\u2010PCR) using a 7900 HT Fast Real\u2010Time PCR System (Applied Biosystems). Primers used for detection are listed in Table\u00a02.7t\u2010test. p\u2010value\u2009<\u20090.05 was considered as statistically significant. Statistical analysis was conducted with SPSS 13.0 statistical software (IBM Corp).All the experiments in this study were independently performed three times. Data was expressed as mean\u2009\u00b1\u2009standard deviation. Differences between two groups were evaluated using paired Student's 3Primary masticatory muscle satellite cells were successfully isolated, cultured and differentiated into long and multinuclear myotubes Figure\u00a0.3.1Fluorescence\u2010labelled siRNA was used to suppress the expression of G\u03b1i2 in masticatory muscle satellite cells Figure\u00a0. Knockdo3.2Adenovirus was employed to overexpress G\u03b1i2 in masticatory muscle satellite cells under growth and differentiation conditions Figure\u00a0. Under g3.3Masticatory muscle satellite cells were immunostained for Pax7/MyoD after knocking down or overexpressing G\u03b1i2 to explore the role of G\u03b1i2 in the functional status of cells Figure\u00a0. Muscle In experiments of knocking down of G\u03b1i2, the percentage of different phenotypes of masticatory muscle satellite cells was calculated and the percentage of Pax7+/MyoD+ cells was found to be higher in the siG\u03b1i2 group (Pax7+/MyoD+ 62.37\u2009\u00b1\u20094.00%) compared with the siCtrl group (Pax7+/MyoD+ 51.78\u2009\u00b1\u20093.31%). By contrast, the percentage of both Pax7+/MyoD\u2212 and Pax7\u2212/MyoD+ cells were obviously lower in the siG\u03b1i2 group  than the siCtrl group (Pax7+/MyoD\u2212 16.09\u2009\u00b1\u20092.09% Pax7\u2212/MyoD+ 32.13\u2009\u00b1\u20091.80%) Figure\u00a0.In experiments of over\u2010expression of G\u03b1i2, there were less cells with Pax7+/MyoD\u2212 and Pax7\u2212/MyoD+ in AdV4\u2010G\u03b1i2 group  compared with AdV4\u2010NC group . Additionally, a greater number of Pax7+/MyoD+ cells were found in the AdV4\u2010G\u03b1i2 group (Pax7+/MyoD+ 74.30\u2009\u00b1\u20092.80%) compared with the AdV4\u2010NC group (Pax7+/MyoD+ 57.52\u2009\u00b1\u20092.11%) Figure\u00a0.3.4The most accepted methods to classify muscle fibre types are based on specific myosin profiles, especially the MyHC isoform complement.Under differentiation condition, when G\u03b1i2 was suppressed in masticatory muscle satellite cells, the mRNA expression of MyHC\u20102A was significantly decreased but other types of MyHC had no significant changes Figure\u00a0. In cont4Masticatory muscle mass and function, similar to other skeletal muscles, are maintained by signalling networks that control the proliferation and differentiation of cells as well as the synthesis and degradation of proteins. There is a strong correlation between masticatory muscles and dentofacial deformities. Masticatory muscle hyperfunction could increase sutural growth and affect the growth rotation pattern of the mandible, influencing the transversal and vertical dimensions of craniofacial bones.G\u03b1i2, as a required component of various GPCR actions, plays an important part in regulating many biological processes including development and differentiation. For example, \u03b22\u2010adrenoceptor agonists have been indicated in regulating skeletal muscle mass.In this study, we explored the possible role of G\u03b1i2 in the proliferation and differentiation of masticatory muscle satellite cells by knocking down and overexpressing G\u03b1i2. Our results showed that G\u03b1i2 can positively affect the proliferation rate, myotube diameter and fusion index of masticatory muscle satellite cells. The mRNA expression level of some transcription factors, cell phenotypes and muscle fibre types also displayed alteration as G\u03b1i2 changed. Since proteins we extracted from primary culture of masticatory muscle satellite cells were insufficient to support a reliable Western blot analysis and significant heterogeneity may arise when culturing primary cells from different mice together, we did not obtain a reliable result of Western blot analysis and the protein level of transcription factors and MyHC isoforms was not displayed in this study.First, we focused on three crucial transcription factors associated with adult myogenesis of skeletal muscle satellite cells, including Pax7, MyoD and Myf5. Pax7 regulates the expansion and differentiation of muscle satellite cells during both neonatal and adult myogenesis.Skeletal muscle satellite cells may at different functional status undergoing divergent cellular fates: Pax7+/MyoD+ cells in an activated state, Pax7+/MyoD\u2212 cells retaining self\u2010renewal and Pax7\u2212/MyoD+ cells with myogenic differentiation capacity.From the perspective of physiology, skeletal muscle fibres could change their structural and functional properties in reaction to different circumstances. The expression of MyHC isoforms is regarded as the most acceptable criterion to classify skeletal muscle fibre types and the transition between different types of MyHC represents the remodelling of muscles.In summary, our study demonstrated that G\u03b1i2 could positively affect the adult myogenesis of masticatory muscle satellite cells and maintain the superiority of MyHC\u2010slow. Masticatory muscle satellite cells may have their unique G\u03b1i2\u2010regulated myogenic transcriptional networks, although they may share some common characteristics with trunk and limb muscles.Lin Kong: Conceptualization (supporting); data curation ; formal analysis ; investigation ; writing \u2013 original draft . Yi Fang: Conceptualization (supporting); data curation ; formal analysis ; investigation ; writing \u2013 original draft . Mingyuan Du: Funding acquisition (supporting); methodology ; resources ; writing \u2013 review and editing (supporting). Yunlong Wang: Methodology ; resources ; writing \u2013 review and editing (supporting). Hong He: Conceptualization (supporting); supervision ; writing \u2013 review and editing (supporting). Zhijian Liu: Conceptualization (lead); funding acquisition (lead); supervision ; writing \u2013 review and editing (lead).The authors declare no conflict of interest.Figure S1.Click here for additional data file."}
+{"text": "Background: Healthcare labor market shortages due to migration, inadequate investments, and lack of continuous training are essential concerns in the Eastern European region. This article aims to describe and reflect on the experience with the implementation of continuous medical education among mother-and-child healthcare providers in Ukraine, including achievements, challenges, and barriers. We analyze this case based on two international collaboration initiatives: the Swiss\u2013Ukrainian program in mother-and-child health that ran from 2000 to 2015, supplemented by the recent Ukrainian\u2013Swiss project \u201cMedical education development\u201d in 2018\u20132023. Methods: We use a case study approach as the methodology for our study. We collected data from documents (project reports reviews) and in-depth interviews with stakeholders. We apply the method of directed qualitative content analysis. Results: As a result of the Swiss\u2013Ukrainian collaborations, the knowledge and awareness of medical personnel were greatly improved. Modern clinical concepts not well understood at the outset became commonplace and were incorporated into clinical activities. Nevertheless, obstacles to the implementation and rapid uptake of changes were found in the lack of knowledge of the English language among medical doctors, the fear of changes, and the lack of openness and readiness for novel evidence-based clinical practices. However, primary healthcare practitioners in this new project seem to be more inclined to change. Conclusions: A modernized continuous medical education which is based on the values of openness, respect, dialogue, and professionalism can be implemented with the input of an international assistance program despite the resistance of the system towards change. Healthcare labor markets are experiencing growing shortages in terms of an insufficient number of healthcare professionals and a lack of specific skills to tackle emerging health and environmental risks. The increased use of technology is not sufficient to fill the healthcare labor gap. This creates a mismatch between supply and demand in healthcare. The problem has manifested itself in all geographical regions, though to a different extent within and between countries . There iSuch labor market challenges have been reported for the maternal healthcare sector as well. The shortage of skilled maternal healthcare providers results in overburdened and fatigued staff, which affects their well-being as well as the quality of services they offer . InsuffiIn the Eastern European region, the shortages in the healthcare labor markets described above, including insufficient staff and lack of skills in the maternal healthcare sector, are even more pronounced than in other parts of Europe . As in WThis article focuses on modernized approaches to continuous medical education (CME) for medical personnel of maternity wards and hospitals in Ukraine. In this paper, continuous medical education refers to the life-long professional education of medical personnel who have completed post-graduate schools; it covers a variety of activities, including different types of training, simulation courses, conferences, self-education, distance learning, and e-learning . SimilarThe aim of this article is to describe and reflect on the experience with the implementation of CME among mother-and-child healthcare providers in Ukraine. In particular, we explore achievements, challenges, and barriers in implementing modernized complex CME approaches  in the Ukrainian healthcare context. We define CME as \u201cthe set of training activities that contribute to keeping physicians\u2019 competencies and skills up-to-date\u201d , and we The rest of the article is organized as follows. The background information and case description are presented in the following subsections, which provide details on the peculiarities of the Ukrainian healthcare system and maternal healthcare sector at the time of the Swiss\u2013Ukrainian program and subsequent reforms. In addition, they provide information on CME in Ukraine, i.e., the system of postgraduate education that was transformed into a continuous professional development (CPD) system in 2018. The case of the two Swiss\u2013Ukrainian collaboration initiatives is described. This is followed by the presentation of the research methods applied in the study, as well as the results of the Swiss\u2013Ukrainian program in mother-and-child health and the subsequent Ukrainian\u2013Swiss project \u201cMedical education development\u201d. Discussion and conclusions focused on complex CME approaches in Ukraine close the article.Ever since 1991, when Ukraine became an independent state, socio-political and institutional changes have been taking place, with gradual and sometimes contradictory tendencies to move away from the Soviet Union structures, practices, and attitudes while trying to approach Western European standards. Many aspects of society have been impacted, including the healthcare and medical education systems. Initially, vertical institutional self-nourishing hierarchies maintained coercive and paralyzing attitudes and rules , which sIndeed, informal patient payments penetrated the healthcare system of Ukraine at most levels. On the side of the provider, informal payments led to higher income for underpaid medical staff while coercing incentives in service provision and restricting knowledge sharing within teams, which led to an outflow of patients to more competent providers (as subjectively perceived by the patients) . By natuApart from the health financing transformation, in 2018 the once-in-five-years postgraduate development system was changed into a more modern one linked to the CPD system of collecting points based on the training and conference activities of medical doctors . PreviouAs reported in the recent WHO country profile , the numFurthermore, human resource practices are not always transparent and provide limited educational opportunities. As noted in the literature, process quality, i.e., how care is provided, is moving ahead of structural quality ,19. The After the collapse of Soviet Union, maternal healthcare services were largely underfunded . Maternal care was provided at healthcare facilities  that were state-owned and had line-item budgets ,20. AfteThe composition of the childbirth teams has not changed over the 30 years of Ukraine\u2019s independence: the obstetrician\u2013gynecologist has remained the leader of the team, with a midwife as a member (who has never been an independent service provider), along with other medical specialists when relevant, such as an anesthesiologist, neonatologist, surgeon, and others . Most poIn the Ukrainian maternal healthcare sector, the delayed reforms in the healthcare system and the shortage of healthcare professionals have frequently meant inadequate care and cure, for instance due to insufficient capacity to treat severe maternal and neonatal cases. Over the years, there have been various reports on over-diagnosis and over-medicalization of pregnant women and infants with no or minor diseases, as well as iatrogenic complications through aggressive long-lasting treatments ,21,22. PAlthough the health outcomes in Ukraine continue to lag far behind the European Union average, recent data on mother-and-child health reveal positive tendencies, namely, exclusive breastfeeding until six months after the puerperium has become more spread, which increased from 6% in 2005 to 20% in 2015 ,23. In tMajor national institutional achievements pairing with international initiatives have resulted, among others, in: (1) the creation of third-level perinatal centers with the aim of regionalization of perinatology services ; (2) the introduction of new registration criteria for newborns in 2007 , and (3) the establishment of updated clinical protocols within national health programs and recommendations ,26,27.The idea of regionalizing of perinatology services was adopted from an international approach to providing perinatal services for mothers and newborns at different levels of healthcare depending on a risk assessment of the mothers\u2019 health conditions, thereby decreasing neonatal mortality . This waEach of the above improvements has been assisted by international partners. A total of six international initiatives and one national project have worked for the improvement of mother-and-child health in Ukraine over the past two decades. These initiatives are presented in In Ukraine, as a former Soviet Union republic, CME has been a matter of great importance in the maternal healthcare sector. In the 1990s, many health professionals lacked information on perinatal regionalization, modern obstetrics and neonatology, and critical care approaches . The conTraditional post-graduate medical education included obligatory category improvements or recertification; every five years, medical doctors were expected to apply for a higher category delivered after points-based assessments  and training attended, as well as exams submitted at state-recognized post-graduate institutions. According to Belli and colleagues , 60% of Hence, until 2020 the CME and organization of healthcare service provision were characterized by top-down hierarchical decision-making and rather outdated (and most probably unsafe) medical practices. Despite the sporadic innovative approaches in healthcare services made possible by individual international collaborations, the overall environment did not facilitate local leadership for an efficient response to the multiple needs of healthcare users and medical doctors. The CPD system is now being modernized; however, because this revision is ongoing, certain aspects lack clarity, e.g., in terms the responsibility for accrediting CPD providers.The Swiss\u2013Ukrainian program in mother-and-child health was initiated in Ukraine in 1997. The goal of the initiative was to improve availability, quality, effectiveness, and access to promotional, preventive, and curative maternal and child health services in selected Ukrainian oblasts (regions), rayons (districts), and communities linked in a system of regionalization . During More specifically, activities on CME in obstetrics and neonatology were implemented with the collaboration and support of the Ministry of Health of Ukraine (MoH), the National Medical Academy of Post-graduate Education (NMAPE), medical universities, regional authorities, and international agencies . Based oEducational materials were developed in collaboration with Ukrainian professionals and were accredited by NMAPE, and were later introduced into teaching curricula. This aided in promoting and enforcing dissemination at a national level, eventually reaching different regions of the country that were not initially involved in the project. NMAPE supported the implementation of innovative educational approaches and institutionalized them within its curriculum of certain courses. Capacity-building regarding key mediators, such as representatives of local and regional medical and managerial authorities, was targeted through the program activities as well. Details on the Swiss\u2013Ukrainian program in mother-and-child health are presented in The Swiss Tropical and Public Health Institute was selected as the implementing agency for the \u201cMedical education development\u201d project. Hence, at the level of international partnership, there has been continuity in terms of lessons learned and partnership relations. Moreover, the \u2018Swiss\u2019 cross sign on medical and educational activities was already recognized in 2018 when this project started. The current project is focused on medical education and primary healthcare. The project collaborates with medical and nursing schools, building jointly with partners their capacities in eLearning, teaching excellence, curricula revision, strengthening skill labs (which are key infrastructure investments of the project), and supporting research capacity-building. On the primary healthcare level, the key areas are the advanced nurse practitioner pilot program, CPD activities for family doctors, and development of eRepository and peer groups. In addition, the project is contributing to policy-making , experience exchange , and communication on medical education (via podcast). Details on the MED are presented in We used a case study approach as a methodology for our study. The case study approach allows focus on a particular setting or area . The casDifferent steps of data collection and data analysis were applied. First, data were collected through a review of documents within the Swiss\u2013Ukrainian program in mother-and-child health (2000\u20132015). This document review employed reports of monitoring visits, observation notes, interim reports, and digests. From these documents, we extracted information relevant to the design and implementation of complex educational activities and adherence to evidence-based medicine (EBM).Second, in-depth interviews were conducted during the closing phase of the program at the end of 2015 to explore the opinions of key stakeholders. The sample included 23 professionals, who were representatives of MoH, NMAPE, international organizations, regional decision-makers, chief medical doctors, healthcare staff, and non-governmental organizations who worked within the area of mother-and-child care. The stakeholder representatives were selected based on the method of convenient non-random sampling. The core idea of the program was presented to them by the program developers and managers through personal communication and further correspondence with the stakeholders. The participants had various job positions and levels of embeddedness in the program. We included both medical doctors who were involved in project activities and those who were not . Hence, triangulation of the sources of information was achieved by the representation of various stakeholders within the sample based on the notion that participants dependent on the program  may not be truly open about the program. Participants were asked to share their opinion on the program\u2019s impact and role in mother-and-child care in Ukraine . Based on Bardin\u2019 theoretical approach , contentFourth, we supplemented our analysis of the CME experience within the mother-and-child program with an analysis of the experience in the more recent Ukrainian\u2013Swiss \u201cMedical education development\u201d project (2018\u20132023). We reviewed internal project reports  and publications . This additional review allowed for updates on the perception and practice of primary healthcare providers and medical educators in terms of professional development.It is important to note that a number of the authors of this paper are or have been managers in both the Swiss\u2013Ukrainian program in mother-and-child health (2000\u20132015) and the Ukrainian\u2013Swiss project \u201cMedical education development\u201d (2018\u20132023). This allowed for access to all relevant documents.Our data analysis of the Swiss\u2013Ukrainian program in mother-and-child health showed that educational initiatives in the frame of the program aimed at setting and raising the standards in mother-and-child clinical practices both adapted to Ukrainian reality and were supported by EBM within neonatologists, obstetricians, anesthesiologist, midwives, and other professional groups less represented in the program  were reasons for dynamic evolutions in educational methodologies resulting in multimodal teaching designs. However, in obstetrics and neonatology the teaching content was consistent and could be resumed for repeatedly tackling educational goals and key messages  were important steps in consolidating partnership and adherence of neonatologists and obstetricians in the program.What has changed between the projects regarding the external stimuli for professional development? With a new MoH decree in 2018, physicians started regularly checking opportunities for CPD points collection and became more responsible for their CPD. However, during the first years of the new policies, the system has not been able to flourish due to the COVID-19 pandemic and Russian\u2013Ukrainian war. The MoH has loosened the requirements in light of the challenging environment. Nonetheless, through joint efforts the large number of motivated medical doctors who take care for their professional development has created demand on the one hand, while on the other the small number of providers of high-quality training programs for medical doctors have received the green light for additional capacity-building with respect to their training initiatives.Moreover, the transformation of primary healthcare through new financing principles, greater autonomy for healthcare providers, and access to public finances on the part of private organizations and individual family doctors, has stimulated the development of healthcare providers. As they have obtained more funds, they are able to pay for the services and training they need, and more motivation has been brought about by a competitive environment in which patients select their GPs on their own.Additionally, the Ukrainian\u2013Swiss project \u201cMedical education development\u201d began its educational activities on already-prepared ground, as the medical community had previously heard about the developments facilitated by the Swiss\u2013Ukrainian program in mother-and-child health. Hence, the project had no need to explain the overall logic of the interventions despite the different target audiences .The educational activities launched by the Swiss\u2013Ukrainian program in mother-and-child health are presented in This educational approach impacted the increase of practical knowledge in only a limited number of professionals, and as such had a limited influence on improving the quality of work of clinical teams. Hence, in-country training for obstetricians, neonatologist, midwives, and child nurses in selected (pilot) regions was implemented during the further stages. To ensure efficiency and sustainability, teaching was later expanded to training of trainers; one team per pilot region was trained, and this team then trained other professionals. In this way, it was estimated that more than 1500 participants could be directly or indirectly trained from 2008 to 2015 in the frame of the program.It has to be acknowledged that the development of regional trainers\u2019 capacities was a long-lasting process that demanded regular and documented supervision. However, due to the rigid healthcare system described above, the sustainability of these teams\u2019 work in a given environment was always at risk. For example, when national or regional power shifted after elections or for other context-specific reasons, this could affect the availability of the medical doctors involved in the teams .The content of the training was developed by a team of international and Ukrainian experts, mainly obstetricians and neonatologists familiar with international medical practice and with knowledge of the English language. Professionals from medical universities, referral maternities, and regional hospitals were involved. Obstetrical modules focused on specific topics not covered by standard Ukrainian clinical protocols or manuals, i.e., multiple pregnancies or vaginal childbirth after a previous Caesarean section (C-section). Neonatology modules covered a broader spectrum of topics related to intensive care of newborns , taking into account the major gaps in neonatal care provision described in further detail below.Training was provided in the form of lecturing and hands-on training or articulated within the model of Ongoing Professional Practice Evaluation with concrete case descriptions, review of morbidity and mortality, discussions, and definition of lessons learned. Handouts resuming the main topics and teaching objectives were delivered. During the implementation of the training of trainers, pedagogic materials containing teaching modules, participants\u2019 guidance, and lecturers\u2019 manuals were developed and published see .Manuals for parents and nurses were developed and disseminated among medical facilities in all regions of Ukraine. For example, pediatric nurses received handbooks containing practical issues on emergency procedures with illustrations, photos of medical equipment, advice regarding the choice and correct use of consumables, modern achievements in neonatology and intensive care, current MoH legislative documents, and principles of communication with parents of healthy and sick newborns.\u201cOur training materials have several advantages over those state materials\u2013they are evidence-based, which is new to healthcare personnel here. The training format is different from typical Ukrainian medical conferences and certificate courses because of interactive communication and the ability to actively participate in the discussion of the topics and issues. And this is very important and effective\u201d.Particular attention was devoted to the active participation of attendants in training activities, which was regarded as crucial for the learning processes. This concept represented a rupture with previous social and educational contexts and model success situations with regard to fears of expressing own thoughts. Hence, participants were encouraged to freely express their opinions and to treat such expression respectfully.\u201cThe participants are often afraid to express their opinion openly, and this can be explained by the fact that they are asked their own opinion rarely, they are not sure whether their opinion is valued, they worry whether their opinion is not different from other staff or the trainer\u201d.In order to adapt to the working schedules of medical practitioners, short-term courses were organized lasting 1 to 2 days. Frequently, trainers visited peripheral maternity wards and assisted in clinical activities. In general, this form of training was very appreciated by the participants and perceived as concrete, useful, and effective.\u201cIn most areas, we employed a neonatologist and opted for short-term courses: one or two full days. Our trainers visited the area, and during the day, they worked with doctors who requested to continue this design of the training because of its perceived usefulness and effectiveness\u201d.In addition to in-class training, distance education through e-learning was introduced in order to support medical personnel at partner hospitals in their practice and to transfer basic knowledge and approaches.\u201cWhen a person comes without adapted basic training, you have to spend a lot of time to explain the obvious principles\u201d.(Trainer)E-learning was recognized as a very innovative educational approach. Between 2003 and 2008, computers and internet connections were installed and program participants with computer skills had to be found and trained. While the implementation of distance learning and compliance by program partners were somewhat complex and time-consuming, they stimulated and facilitated the shift from the post-Soviet type of post-graduate medical education to a modern CME approach.\u201cNow, the lexicon of the lecturers and medical doctors contains a new concept\u2013\u201ccontinuous professional development\u201d. This is a new approach to medical education, including distance learning, telemedicine technology, new knowledge in the workplace, the emphasis on practical skills, professional competence, communication and teamwork as well as the modular approach to designing training programs and electronic textbook\u201d.CME in this program was designed for partner regions; however, educational activities covered the whole country through national conferences, guidelines and educational manuals, and courses  were accredited by NMAPE. Consequently, even those regions which were not directly involved in the program received program outcomes through other disseminative channels, including NMAPE.\u201cFirst, training modules were developed by a working group of the Swiss-Ukrainian program. Obstetricians from rayon hospitals in four program partner regions received training on multiple pregnancies, and management of pregnancy, and childbirth in women after C-section. The program was consequently accredited and became the first certified postgraduate training course at the department of obstetrics at the NMAPE, which used the materials developed by Swiss-Ukrainian program in 2012. It was officially integrated in the academy\u2019s training curricula\u201d.In order to foster the introduction of developed materials into medical practice and promote administrative changes related to clinics according to priorities, regional healthcare administrators\u2019 adherence and advocacy were stimulated by the program with the provision of parallel managerial training.\u201cSo as the clinical protocol does not stay in the drawers of chief doctor but worked efficiently\u2013it is an understanding of the chief \u2018what we do\u2019, \u2018why we do it\u2019 and \u2018who wants to get the result. We implement our initiatives more efficiently when \u201cperinatal thinking\u201d is present among heads of healthcare facilities, districts and regions\u201d.The case of vaginal childbirth after C-section illustrates the impact of evidence-based medical practices introduced in obstetrics in 2011\u20132012. Courses on theory and practice implemented through the program were an eye-opener for many medical doctors, as well as for parents-to-be. This newly introduced clinical approach stipulating that natural childbirths were possible after C-section under clearly defined clinical conditions not only raised patient safety and was cost-effective, it helped to reinforce the status of mothers, as their social role might be impaired in case of non-physiological births.\u201cRural women regard a C-section as an action that indicates the inferiority of their body, as a drawback. Therefore, learning from their friends or acquaintances that they can give birth naturally after a previous C-section increases relevant requests to doctors\u201d.(Head of the maternity department)\u201cToday we are using state-of-the-art protocols for the management of pregnancy and childbirth after the previous C-section. It has been proven that assessment of risk factors, quality selection of patients and quality of follow-up, patients\u2019 awareness and striving for success are the prerequisites of a desired outcome. And we see these outcomes ourselves. If previously we had only single cases of vaginal childbirths among women with scars on the uterus from previous C-section, then in the seven months of the current year, we have already had 30 such childbirths\u201d.(Deputy chief doctor)More recent experiences of the Ukrainian\u2013Swiss project \u201cMedical education development\u201d demonstrate that: (a) e-learning has become widely used since 2020 due to COVID-19 related lockdowns, with some return to face-to-face offline activities after summer 2021, while the war put a pause CPD during spring 2022; since summer 2022 CPD has returned to online, though less to offline modes; (b) EBM as a concept is recognized by healthcare providers and medical educators; however, due to the lack of EBM methodology being integrated in medical school curricula and lack of English language, we observe that EBM is not yet a core principle of care provision; (c) \u2018fear of expressing one\u2019s own thoughts\u2019 is especially noticeable among nurses during training with medical doctors, and critical comments from participants and discussions are not yet typical practice during educational activities; (d) the Swiss project\u2019s designed and supported training and conferences are highly appreciated by participants because of the relevance of the topics, interactive approaches, practical instruments, skills which can be applied in professionally, and competence gap-driven rather than available trainer-driven approach; (e) administrative decisions on process improvement are seen as being the result of high-quality training.In the Swiss\u2013Ukrainian program in mother-and-child health, the first training of trainers took place in 2008; since 2014, regular training and CME in the field of Obstetrics and Neonatology has been implemented for medical students and professionals. This teaching activity has enhanced interregional and international exchanges of experience through meetings, workshops, and conferences. Apart from clinical skills, the training is aimed at the development of additional professional skills such as communication and personal management.\u201cCommunication skills when providing care is a new and interesting topic to study and further develop health professionals of the former Soviet Union. Getting basic medical knowledge at universities and colleges, we have not received the knowledge and skills for good communication, e.g., what you say, especially if you are leading a team. The ability to hear the other person, to communicate with the patient and relatives are greatly needed\u201d.(Neonatologist)Establishing Simulation Centers in Ukraine went beyond the usual frames of training in mother-and-child health. This innovative concept needed special assistance in terms of practical, institutional, and economic aspects. Considerations such as infrastructure requirements, defining and procuring required equipment and technology, capacity-building, writing educational curricula, and defining its role in the Ukrainian educational context all had to be taken into account. Except in Odesa, simulation centers had not been established by state universities or hospitals because of a lack of funds. However, the manikins were not the only element of enhancing practical skills via simulation-based learning approaches. An essential role was given to higher learning and development objectives such as evidence-based methodology, scenarios adapted to the Ukrainian clinical reality, and the selection of relevant cases. Hence, after establishing four simulation centers in Vinnytsia, Volyn, Ivano-Frankivsk, and Sevastopol, particular emphasis was placed on improving capacity by employing interpersonal and group coaching as a major attribute in effective learning. A program handbook covering all these aspects was developed and made available nationwide.Institutionalization of medical training through simulation, specifically its introduction as a mandatory step in CME and certification of medical practitioners, is ongoing. However, a number of lessons learned can be defined. For instance, the low-fidelity (simple and cheap) manikins are preferred to higher-fidelity ones. The program experience has clearly shown that the maintenance of expensive manikins places a high financial burden on the teams and their administration. In addition, institutionalization of simulation should include the development of regulatory norms that create a motivating environment for the trainers to devote their time to training courses. At the same time, clear requirements for the trainers should be agreed upon.During the preceding decade, and with the support of the Swiss\u2013Ukrainian program in mother-and-child health, simulation approaches have become popular in medical education. Nonetheless, those who launch simulation centers and skill labs are more focused on infrastructure and manikins than on trainers\u2019 competencies (including an understanding of debriefing), revision of medical students\u2019 competencies, the skills to be a part of skill labs training, and their integration into the curriculum. Hence, the Ukrainian\u2013Swiss project \u201cMedical education development\u201d is not only supporting the development of existing skill labs or new ones, it is concentrating on the professional growth of skill lab trainers and their skills in scenario development and assessment. Presently, policymakers are discussing certification of skill labs and simulation centers to ensure their adequate functioning and the maintenance of national standards.Our review of documents from the Swiss\u2013Ukrainian program in mother-and-child health showed that monitoring and evaluation were important supplementary activities to the training delivered. Follow-up visits were conducted in order to consolidate knowledge and skills gained during theoretical and practical training and to facilitate their implementation. The monitoring scheme included several follow-up visits that were initiated about ten weeks after the training. The follow-up visits usually happened at 6-month intervals. A focus was placed on the evaluation of general factors affecting hospital performances and bringing to light the problems faced by health professionals in the provision of perinatal care, as well as identifying solutions with clear guidelines for action.\u201cSome doctors are already responding constructively. They say that after monitoring visits, they want to change something\u2013do differently and better. I would not say that the attitude towards monitoring visits is 100% positive in hospitals, and probably absolutely positive perception will never be reached. But the perception that \u201cthe chief will come and punish\u201d is not already there. In contrast, most teams say that these visits are an opportunity to have \u201ca fresh view\u201d in terms of where and how you work\u2013as it is difficult to objectively assess the reality when you do the same from the morning till the night\u201d.The program monitoring visits used to be agreed upon two weeks in advance with the administration of the healthcare facilities. Monitoring teams consisted of neonatologists, obstetricians, and/or pediatricians, usually leading regional professionals. They assessed the situation through observation of work and interviews with health professionals and child-bearing women. The visits corresponded to a new horizontal reciprocal exchange of information and expertise, increasing the chances for further dissemination and implementation of the issues addressed during training seminars. Moreover, the program introduced the practice of inter-regional visits, which was accepted positively and implemented by the program teams.\u201cMonitoring visits\u2013when the teams from other regions came to our region, and vice versa\u2013were a unique opportunity to see how other doctors work and what methods and achievements they have. You notice many positive aspects and then you bring these positive insights into your medical institution. Also, after the seminar on the principles of the monitoring visit, I observe that the attitude of medical doctors towards monitoring has changed. Each next visit is conducted much easier\u201d.Monitoring visits on the part of training providers remain a rare practice. From the perspective of the Ukrainian\u2013Swiss project \u201cMedical education development\u201d, such visits seem to be more relevant in the case of practical skill development of healthcare service providers and less feasible for medical educators , in addition to being less functional in revealing changes in primary healthcare facilities due to the distracting matters of uncertain environment and lack of physical safety. Priorities in service provision seem to be more focused on staff retention and mental health support rather than new developmental challenges. Nonetheless, in this difficult context, reflection on the practices and changes is helpful. Student or patient feedback is helpful to review what has changed after the training, as is feedback from providers and trainees (educators or physicians/nurses). For example, in the Ukrainian\u2013Swiss project \u201cMedical education development\u201d, nurses who extended their roles and competencies in service provision could recognize the pre-heart failure condition after recent ECG training, and shared this achievement with the training organizers.The Swiss\u2013Ukrainian program in mother-and-child health placed special attention on integrating training activities in the context of poor healthcare infrastructure, and as such it concentrated on upgrading facilities and procuring essential equipment. Maternity and neonatology wards and neonatal intensive care units (NICUs) of third-level referral centers and second-level regional health structures were involved in facilitating the creation of a functional network with basic minimal infrastructure for perinatal regionalization. Medical communities and local authorities were involved in defining needs, participating financially, and organizing the reconditioning of the premises. Special training on fundraising strengthened the capacities of head doctors and administrators in negotiating state and private funding. Partnerships between government bodies, non-governmental agencies, and private organizations in financing the development of health services were created.\u201cThe recommendations of the program experts who came to our institution helped very much for proper zoning premises, so, we did everything correctly from the first attempt. The local budget funds were allocated to buy a part of the necessary medical equipment, and then our institution was officially included in the program, and the program provided the rest of the equipment. Nowadays, our maternity ward and intensive care unit are well-equipped with everything we need\u201d.(Chief doctor)Renovations of buildings and facilities aimed to create patient-friendly premises. New spacious, comfortable, and bright obstetric wards and patient individual or two-bed rooms were set up with the aim of closeness to medical facilities such as operating theatres and NICUs. These fitting-outs created space for a partner to be present during childbirth, rooming-in, skin-to-skin policies, and the introduction of \u201cresponsible parenting schools\u201d, as well as for \u201copen days\u201d at the hospital.\u201cThe real innovation is the Open Day in the maternity hospital\u2013each Saturday, from 10.00 to 12.00 the couple can visit the maternity ward, its atmosphere and conditions where they will give birth to their baby. Doctors believe that this promotes better psychological preparation for childbirth\u201d.(Obstetrician)\u201cWhen school started, we did not expect high attendance, but now we have to work in several shifts because the demand is very high. Most pregnant women come with partners and we are preparing them for the process of birth. You will not believe it, but in 2007 we had no partner births in our hospital, and already in 2013, the rate increased to 82%\u201d.Value-focused locations have been a focus of the Ukrainian\u2013Swiss project \u201cMedical education development\u201d from 2020\u20132021, e.g., student-centeredness in medical education should be supported by student spaces (hubs) in which students have the opportunity to discuss their ideas, develop projects, and organize informal or additional non-formal educational activities. Such infrastructure developments support the overall strategic values of being client-oriented, and are one of the distinctive features setting the present approach apart from the Soviet approach to education (teacher-focused) and healthcare (doctor-focused). Unfortunately, the reduction in the financial capacity of partner and the damage to infrastructure due to the war have negatively impacted such infrastructure projects designed earlier; however, new construction and reconstruction of hospitals and schools are taking place even during the war.According to the documents of the Swiss\u2013Ukrainian program in mother-and-child health, the progressive advance of practitioners in terms of knowledge and awareness toward international standards could be clearly observed. Among other examples, we note routine continuous monitoring policies in NICUs . Another example is the improved capacity provided for ventilatory assistance of sick newborns, whether invasive or non-invasive . Protocols for the management of multiple pregnancies, primary resuscitation of newborns, prevention and cure of Hyaline Membrane Disease, in-utero transfer, etc., have begun to be routinely implemented. Out of all educational and connected initiatives that contributed to the changes in clinical practices, the main reason for these improvements can be attributed to CME. It is certain that this educational activity has met the needs of clinicians and has helped substantially in improving practical skills as described. Practitioners were unanimous in acknowledging that the new training experiences were helpful in their practical work and that they would certainly recommend the training course to their colleagues. Mothers saw positive changes in the attitude and in the clinical recommendations they received in peripartum.\u201cThere are the same people working here, but their attitude towards the patients, as well as their practices, has changed a lot\u201d.(Mother)\u201cThanks to our joint efforts in collaboration, we achieved a lot, for example, the loss of children up to 1 year of life  has significantly decreased. And I can say that this figure is 90% possible because of the training and improved knowledge and skills of medical staff\u201d.We found during the document review that the obsolete system of healthcare service provision in Ukraine represented an important reason for the slow pace of healthcare improvements. Obstacles included poor financing that led to the lack of medicines, equipment, and consumables; inadequate human resource development and the outdated official system of CME; and rigid management practices, including in the area of quality improvement. Other barriers that the program had to take into account during training and coaching activities were poor knowledge of the English language among the medical and paramedical professionals, which sometimes, especially initially, led to a reluctance to open up to new advice and international standards.A general atmosphere of fear generated by a vertical long-distance hierarchy  contributed to these attitudes. Superiors in Ukraine were often disconnected from clinical reality, and this might have increased over-diagnosis and over-prescription as a mechanism of self-protection on the part of practitioners.\u201cThe approaches offered by the program have not resonated immediately with our doctors because all medical personnel used old approaches, and it was important first to prepare the personnel psychologically for the introduction of new perinatal technologies\u2013to change the usual vision and to learn a lot. Later, during the training and visiting activities, doctors realized that these methodologies and practical skills were really effective, which in turn gave them more motivation to learn and to change\u201d.(Chief doctor)\u201cNot all medical professionals want to change their old stereotypes of pregnancy and childbirth\u201d.It was recognized that the implementation of perinatal regionalization and its network needed further improvement. Scarce institutional commitment and the problematic financial situation hindered the adequate allocation of means for this purpose . More quotations that illustrate these statements are presented in On the clinical level, less encouraging results compared to the achievements already described concerned the understanding of vertical infectious diseases  and especially the misuse of antibiotics, which are prescribed excessively and not in line with international recommendations. This can be imputed to the insufficient capacities of microbiological laboratories to identify pathogens  and the myth of antibiotics being a prophylactic guarantee against  infections. Further training efforts are advisable. In addition, nutritional problems continued to subsist due to little availability of adapted consumables  and enteral feeding formulas.With the recent launch of the NHSU and new healthcare financing principles, healthcare facilities have more available funds and materials. Chronic underfunding has been responded to by these reforms; however, the war has led to lower fiscal capacity on the part of the state. What remains among the achievements are new and more efficient mechanisms of state funding distribution and its relevance to service provision as opposed to simply maintaining beds. New service provision policies require new and more extended competencies on the part of healthcare managers of autonomous facilities. A lack of studies limits the understanding of both areas. Because directors of healthcare facilities must deal with NHSU service packages, funding distribution, and related policies, this may be a sign indicating that managerial knowledge and skills are developing as well.The results presented above show how complex CME approaches , specifically those of the Swiss\u2013Ukrainian program in mother-and-child health, have been applied in the Ukrainian context. The fresher lens of the Ukrainian\u2013Swiss project \u201cMedical education development\u201d can supplement and update the findings of this program. While the implementation of these two international collaboration initiatives has had multiple benefits for the Ukrainian healthcare sector, it has faced various challenges and barriers as well. Below, we discuss these findings in light of the specific healthcare and education systems in Ukraine and the healthcare labor market shortages outlined at the outset of the article.Innovative education programs are required to ensure the necessary competencies of the healthcare workforce for tackling the current health risks . In partAs shown by our results, during the 15-year Swiss\u2013Ukrainian program in mother-and-child health, clinical changes could be witnessed at many levels. Prior to program implementation, assessments clearly found a lack of awareness and expertise on the part of clinicians, as well as a lack of modern equipment ,39. ImpoAs described in the results section, the Swiss\u2013Ukrainian program in mother-and-child health facilitated the rapid implementation of highly innovative simulation medical education that allowed Ukrainian medical doctors to avoid the thorny approach of trial and error, and provided an impetus to the further development of education scenario development. In addition, the program positively impacted the clinical safety of childbirth, and specifically the core concept of service quality. Simulation in medical education, which is a gold standard in many modern teaching institutions worldwide ,42,43, bHowever, chronic shortages in funding and a lack of experienced EBM staff make it difficult to install new simulation centers in Ukrainian educational and medical institutions. As mentioned earlier, the lack of necessary investment undermines the quality of healthcare provision and increases the healthcare labor gap in Ukraine as well as in the Eastern European region more generally ,6. This Healthcare professionals in the Eastern European region often lack motivation due to unsatisfactory working conditions and low reimbursement rates . UnderfuOur results describe numerous interventions in healthcare provision, including reorganization of the maternal department space, monitoring visits, and more patient-oriented approaches. Indeed, the attention paid to professionals , considering and responding to their professional needs, and development of interpersonal skills  and networking have become crucial in establishing a trustful and safe environment where changes in clinical practice have been possible .Before the Swiss\u2013Ukrainian program in mother-and-child health, over-medicalization within care for women and newborns was widely spread. No individual birthing rooms were available; several women were sometimes giving birth in one room at the same time. Partner presence during childbirth and rooming-in was not allowed; newborns were kept all together in separate rooms. Frequently, nutritional needs essential for the healing of sick newborns were not met. Overall, the care provided to sick neonates was unsatisfactory ,38.Most of these practices have become remnants nowadays due to this program, which has improved mothers\u2019 childbearing experience, as illustrated by our case study. More trustful relations between healthcare providers and families can have a large contribution to service providers\u2019 job motivation as well. Moreover, the program has helped to develop communication skills, which contribute to establishing a trustful and safe clinical environment . Thus, tOne of the major obstacles to service improvement in Ukraine, as in other Eastern European countries, is the lack of financial incentives for individual healthcare providers . Recent The Swiss\u2013Ukrainian collaborations, while bringing forward innovative CME and EBM practices, also promoted the values of openness, transparency, and integrity. This is illustrated by the findings of this case study. In the Ukrainian healthcare sector, strong formal and informal parallel structures exist in virtually all areas, including education . This seThe Swiss\u2013Ukrainian collaborations presented in this article are examples of how international projects can contribute to improving the managerial capacities of facility leaders through training and plans for facility development. In fact, chief medical doctors are advised to supplement such collaborative input with funds raised from other sources, e.g., public\u2013private partnerships. Thus, the Swiss\u2013Ukrainian collaborations have indirectly contributed to awareness of the importance of transparent policies in service provision on the part of healthcare staff, as well as the development of fundraising skills and project-based management of healthcare facilities. This example could be helpful for other Eastern European countries where openness, transparency, and integrity need to be promoted among the healthcare workforce.Ukrainian mother-and-child services previously had no tradition of CME. The CME philosophy was hardly even accepted at the beginning of the Swiss\u2013Ukrainian program; however, in spite of the need for complex and integrated activities, this has become possible, as suggested by our findings. A complex approach in CME as a vital step towards better quality and access shows its sustainable nature . First, Importantly for Ukraine and other Eastern European countries as well, clinical and educational solutions cannot be directly transferred from one international project in one country to another. The priorities have to be (and were) defined according to the population\u2019s health needs, feasibility, and local capacities . To provAs mentioned in the case description and explained in the results, the length of the program was almost two decades, which played a key role in achieving a real shift in the clinical practices and engagement levels of all stakeholders. One of the main lessons learned was the importance of the time factor; in order to allow efficient clinical implementation of complex CME in post-Soviet countries, a minimum period of 10 to 15 years has to be allocated . In thisThis study has disseminated the experience with CME in Ukraine, and specifically highlighted the benefits and challenges of the Swiss\u2013Ukrainian collaboration. The main strength of this study lies in its analysis of long-term collaboration focusing on the healthcare environment and the complexity of the activities that bring about change in both the de jure and de facto dimensions. However, this study is not without limitations. As mentioned in the methods section, some of the authors are or have been managers in the two initiatives studied. While this ensured access to all relevant documents, it could have caused researcher bias. However, not all authors were involved in the programs, which means that the possibility of such bias has been reduced.Notably, the lack of a proper nationwide monitoring and evaluation system makes precise quantitative estimations involving the Swiss\u2013Ukrainian collaboration impossible. Nevertheless, the study has generated evidence on the experience of this collaboration and the perceptions of stakeholders. In addition, the experience analyzed in this case study tackles several regions which were the focus of the program. Although the Ukrainian healthcare system is essentially centralized de jure, de facto there are substantial differences across the regions because of different historical, economic, and socio-cultural aspects. Prior to the healthcare reforms, obstetrics and gynecology were very specific areas of healthcare service provision having, e.g., higher status of healthcare service providers and more resources compared with primary healthcare ; therefoThus, the results must be transferred to other sectors and locales with caution and while ensuring careful understanding of differences in the sector features. For example, due to the latest health financing reforms the status of primary healthcare physicians has been substantially increased by financing, communication campaigns, and the organizational performance of primary healthcare centers.Based on the results presented in this article and their discussion, and considering our study aim, we are able to formulate several key conclusions regarding the experience with the implementation of complex CME approaches in Ukraine. We conclude that complex approaches to implementing CME, namely those in the Swiss\u2013Ukrainian mother-and-child health program, have had positive effects according to the stakeholder representatives involved. Therefore, the experience of this program should be considered in further initiatives in the area of mother-and-child health. An example of such initiatives relates to the implementation of similar activities through the new Ukrainian\u2013Swiss medical education development project.The effect of the numerous multi-level educational activities designed to change medical practice and patients\u2019 health outcomes is especially visible in a long-term perspective . Times wAs shown in this study, the program activities in the Swiss\u2013Ukrainian program in mother-and-child health were designed in view of the relatively weak institutional and technical capacities of the county, i.e., post-Soviet mentalities and an unstable political climate in a societal transition period. Despite the number of problematic areas in the Ukrainian healthcare sector, including the lack of funds, their inefficient allocation, constant changes of authority, and an unstable political situation, the program led to the introduction and adaptation of international practices, assuring its sustainable presence in the service provision.The experience of implementing this program clearly shows that the contextualization of educational activities has to be taken into account. The recent experience of the Ukrainian\u2013Swiss project \u201cMedical education development\u201d implemented under the COVID-19 pandemic and war\u2013related restrictions demonstrates that difficult contexts may bring about new insights and foster new practices .This study concludes that a shift from the old and often unsafe clinical practices to evidence-based and efficient mother-and-child care can be realized in the post-Soviet settings notwithstanding poor governance. A complex CME approach based on the values of openness, respect, dialogue, and professionalism can be successfully implemented by an international assistance program despite the resistance of the system towards change.In Ukraine and other Eastern European countries, such initiatives are of crucial importance for ensuring necessary competencies on the part of healthcare professionals, enhancing their job motivation, and improving the quality of healthcare provision. Investments in infrastructure and life-long learning programs can help to train and retain healthcare professionals in national healthcare systems by providing better work conditions and opportunities for professional development."}