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+{"text": "Radio frequency ablation (RFA) has been accepted clinically as a useful local treatment for hepatocellular carcinoma (HCC). However, intrahepatic recurrence after RFA has been reported which might be attributable to increase in intra-tumor pressure during RFA. To reduce the pressure and ablation time, we developed a novel method of RFA, a multi-step method in which a LeVeen needle, an expansion-type electrode, is incrementally and stepwise expanded. We compared the maximal pressure during ablation and the total ablation time among the multi-step method, single-step method (a standard single-step full expansion with a LeVeen needle), and the method with a cool-tip electrode. Finally, we performed a preliminary comparison of the ablation times for these methods in HCC cases.A block of pig liver sealed in a rigid plastic case was used as a model of an HCC tumor with a capsule. The multi-step method with the LeVeen electrode resulted in the lowest pressure as compared with the single-step or cool-tip methods. There was no significant difference in the ablation time between the multi-step and cool-tip ablation methods, although the single-step methods had longer ablation times than the other ablation procedures. In HCC cases, the multi-step method had a significantly shorter ablation time than the single-step or cool-tip methods.We demonstrated that the multi-step method was useful to reduce the ablation time and to suppress the increase in pressure. The multi-step method using a LeVeen needle may be a clinically applicable procedure for RFA. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of HCC patients suffer from virus-induced liver injury and most have underlying liver cirrhosis . Percutain vitro porcine liver model [It has been reported that radio frequency ablation (RFA) is an effective procedure for hepatocellular carcinoma (HCC) as well as for metastatic liver tumors ,8. However model , we demoAs a model of an HCC tumor with a capsule, we prepared blocks of pig liver tissue packed into a rigid plastic case and the blocks were used in this study (see Methods). Figure In contrast, there was no significant difference in the ablation time between the multi-step (118.3 \u00b1 16.4 s) and cool-tip ablation methods (123.7 \u00b1 67.0 s), although the single-step method had longer ablation time (172.0 \u00b1 26.9 s) than the other ablation procedures Figure . AlthougIn a preliminary clinical trial, we compared ablation time among the multi-step n = 14), single-step (n = 13), and cool-tip (n = 13) methods. The multi-step method showed a significantly shorter ablation time than the single-step or cool-tip method  as previously reported . Two bloThe LeVeen needle was used with either a standard protocol (single-step method) or a modified protocol (multi-step method). For the single-step method, the tines were fully expanded after the needle was inserted to the target position. RF energy was then applied to the tissue at an initial power setting of 40 W and was subsequently increased at increments of 10 W per minute to a maximum power of 75 W. The power setting was left at this point until power 'roll-off' occurred; tissue impedance  rose to over 200, at which time the power passively decreased to less than 10 W. If no roll-off occurred, a total of 15 min elapsed. Using the same device, the multi-step method involved expanding one-tenth of the length of the electrode tines at first step, and the current was delivered until power roll-off occurred. At the second step, immediately following roll-off, two-tenths of the length of the tines was expanded and a current was supplied. With stepwise expansion of the tines, the ablation was repeated until the tines were fully expanded. RF energy was applied with an initial power setting of 30 W in first step. When power roll-off occurred within 30 s at a given step, the ablation at the next step was started at the same electrical power. If roll-off took more than 30 s, the next step was started with the power set 10 W up to 75 W. If the stepwise increase in power reached the maximum level before the final step, the ablation at the subsequent step was performed at maximum power. For the cool-tip electrode method, RF energy delivery was started at 40 W. The electric power was then increased by 10 W every minute. The maximum electrical power was 120 W, and the RF energy delivery was continued until the impedance increased beyond the limit of the generator.We performed a preliminary comparison of ablation times for the single-step, multi-step, and cool-tip methods in 36 HCC cases with liver cirrhosis Table . For the2-test for categorical data and the non-paired t-test for numeric data. Regarding the in vitro model, all measurements were performed four times and the results are shown as mean \u00b1 SD. Statistical comparisons for the cumulative ablation time and the pressure at the programmed endpoint in vitro were made using ANOVA and the Scheffe's test, via the Statview software .Baseline characteristics of the patients prior to RFA treatment are shown as mean \u00b1 SD, and the statistical comparisons were performed using the \u03c7The author(s) declare that they have no competing interests.MN, ME, and HS participated in the experimental design, and performed most of the analyses and writing of the manuscript. MK, SM, TY, YM, and KK measured the ablation time and pressure. All authors read and approved the final manuscript."}
+{"text": "Forty-one patients receiving remission induction chemotherapy with vincristine, adriamycin and prednisolone (VAP) for high grade lymphoma or acute lymphoblastic leukaemia were entered into a double blind, placebo controlled trial of oral acyclovir prophylaxis against herpes simplex virus (HSV) infection. The dose of acyclovir was 200 mg four times daily for the duration of chemotherapy (six weeks). Of the 40 evaluable patients, 20 were randomised to each arm. Prophylactic oral acyclovir significantly reduced the incidence of clinical HSV infection from 60% on placebo to 5% acyclovir (P less than 0.001), and the incidence of viral isolates from 70% on placebo to 5% on acyclovir (P less than 0.001)."}
+{"text": "Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In  RETINOBLASTOMA RELATED (RBR) of Arabidopsis for tetraploid genetic analysis to study the function of RBR during the plant life cycle. In diploids, while RBR\u2013deficient female gametophytes with features of aberrant cell fate and differentiation were analogous to what was previously reported for male gametophytes, we provide evidence that RBR controls gametic genome duplication, thus genome integrity in the gametophyte-derived progeny. Quantitative reduction of RBR in tetraploids led to identification of rbr heterozygous plants that displayed novel RBR dosage-dependent phenotypes in differentiation and development of the sporophyte albeit the absence of cell cycle defects. These phenotypes coincided with deregulation of conserved epigenetic factors such as Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the sporophyte, as shown for the gametophytes as well. However, unlike the repression by the PRC2 in gametophytes, RBR is activated by the sporophytic PRC2 subunits, suggesting that distinct modules of the conserved RBR-PRC2-MET1 loop control gametophyte and sporophyte generations in plants.Understanding the convergent developmental mechanisms of core cell cycle genes is highly instructive in biology. When these genes are essential in development, lethality precludes mutation analysis throughout the life cycle of an organism. We subjected a homozygous lethal mutation in  Independent evolution of multicellularity and thus the cell types has implications for adaptation of distinct developmental strategies in plants and animals ArabidopsisThe tumour suppressor Retinoblastoma (pRB) and closely related proteins are primarily known as negative regulators of the cell cycle and for their antiproliferative activity in multicellular organisms Arabidopsis, RETINOBLASTOMA RELATED (RBR) is the single homologue of pRB, and the pRB-E2F pathway is largely conserved Arabidopsis knock-out alleles of RBR are defective in both female and male gametogenesis RBR in development. Studies that down-regulated RBR in distinct tissues using RBR RNA interference, virus induced gene silencing or by mis-expression of a RBR-binding viral protein to compete with the native RBR, have not elucidated the genetic behaviour of a rbr null mutation during gametophyte or sporophyte development RBR mRNA and protein levels were stably reduced throughout development, or aberrantly elevated due to the auto-regulatory function of the pRB-E2F pathway RBR pathway functions distinctly in different cell types to prevent cell division, endoreduplication and stem cell maintenance. Recent work demonstrated that RBR genetically interacts with the conserved epigenetic regulators of the Polycomb Repressive Complex 2 (PRC2) to control development of both male and female gametophytes RBR control of cell fate in the male gametophyte is at least partly coupled to its genetic interaction with the cell cycle associated pollen-specific CYCLIN-DEPENDENT KINASE A1 (CDK A1) RBR is repressed by a maternal and paternal PRC2 complexes during plant reproduction RBR regulatory network can function differently depending on the developmental context. Together, the developmental role of RBR during sporophytic development remains poorly understood, primarily due to the lack of genetic tools.In Arabidopsis RBR knock-out allele rbr female gametophytes supported the role of RBR in gametophytic cell fate control. Further, we performed a tetraploid genetic analysis that provided direct evidence that at reduced levels of RBR sporophyte development is perturbed. When only one out of four functional RBR alleles was present in tetraploids triplex for rbr (rbr/rbr/rbr/RBR), specific stages of sporophytic differentiation and development were affected. The function of RBR is therefore partially haplo-insufficient during sporophytic plant development, as revealed by RBR dosage analysis in tetraploid plants. Furthermore, we provide genetic evidence that RBR functions in concert with the sporophytic PRC2 subunits to control developmental processes in the sporophyte. In short, our work not only illustrates the coordinated function of the RBR pathway in both gametophytes and the sporophyte, it also demonstrates how tetraploid genetics can be exploited to uncover a novel developmental role of an essential regulator during the entire plant life cycle.In this study, we investigated the effects of an Arabidopsis, the fully differentiated female gametophyte (embryo sac) consists of only four cell types of clonal origin RBR knock-out alleles, rbr-3RBR function did not affect the mitotic divisions and cellularization in the female gametophyte rbr-3 cell types was previously assigned based on their positional information within the embryo sac; however, their molecular identity remained questionable. Therefore, we examined the fate of specific cell types in the absence of RBR using cell type-specific molecular markers that are characteristic for the three cell types of the mature female gametophyte. The marker lines ET1119, ET2634, and ET956 express \u03b2-glucuronidase (GUS) in the egg cell, synergid cells, and the central cell, respectively rbr embryo sacs we could not detect GUS expression in the egg, synergid, and central cell  RBR is required for the identity establishment of these gametophytic cell types. In the rare cases where these markers were still present, possibly due to some RBR activity carried over from the rbr/RBR heterozygous megaspore mother cell, they were mis-expressed in the spatial domains of other cell types  and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) proteins, which are members of distinct PRC2 complexes in plants rbr, msi1 and fie mutant female gametophytes aberrantly proliferate and they are defective either in acquiring cellular identity and/or in heterochromatin status rbr mutant gametophytes can be partly attributed to the derepression of MET1rbr and msi1 could be rescued by suppressing MET1 and associated global methylation, suggesting a complex epigenetic control of development RBR-PRC2-MET1 network controls cell fate determination either independently, by co-regulating cell cycle activity, and/or by forming a repressive chromatin modifying complex both in male and female gametophytes.In a previous work we have shown that the lack of cell differentiation in Arabidopsis, impairment of the RBR-E2F pathway by ectopic expression of the viral RepA protein RBR function in a genetically tractable rbr knock-out allele would change developmentally controlled ploidy. Analysis of cellular ploidy in RBR-deficient female gametophytic cell types is difficult due to the problems in isolating these miniature cells from plants that are heterozygous for rbr. During the morphological analysis of diploid rbr/RBR plants using Nomarski optics rbr gametophytic nuclei and, in particular, proliferating nuclei in the central cell region were of unusual size. Therefore, we analysed the ploidy of these nuclei by confocal microscopy and subsequent 3D reconstruction of acquired image stacks. We noticed that several rbr supernumerary nuclei derived from the unfused polar nuclei had a diploid rather than haploid chromosome number , this led us to investigate potential changes in plant genome ploidy in rbr gamete(s)-derived progeny. We had previously shown that a selfed diploid rbr/RBR plant  produced viable progeny segregating for wild-type RBR/RBR and mutant rbr/RBR genotypes, while the female gametophytically lethal rbr allele was not transmitted to the next generation rbr mutant produced viable male gametes with altered ploidy, we would expect that the ploidy of a subset of rbr progeny would be different from the parent plant. Indeed, we found that selfed diploid rbr/RBR plants produced 6% triploids among rbr mutant offspring (n\u200a=\u200a56), which produced an array of aneuploid, diploid, and tetraploid plants in the next generation  rbr knock-out male gametophytes rarely formed sperm cells RBR controls ploidy maintenance in the gametophytic cells and that it is involved in maintaining genome integrity because in its absence or down-regulation polyploid offspring are produced.Since neration . This phArabidopsis meiotic mutants, dyadmitosis instead of meiosis (mime) jasonINDETERMINATE GAMETOPHYTE 1 (IG1) which encodes for a gene with high similarity to ASYMMETRIC LEAVES2 (AS2) in Arabidopsisrbr mutation we report here is the first case in Arabidopsis in which aberrations in gametogenesis could result in triploid offspring due to doubling of haploid gametic genome. A \u201ctriploid bridge\u201d leading to production of diploids, aneuploids and tetraploids may act as a transition between diploids and autotetraploids and therefore could play a significant role in polyploidization rbr knock-out mutants may also occur in the wild-type, should RBR activity be altered by unknown environmental factors. Thus, RBR might have played a crucial role in plant evolution by controlling genome duplication events.Genome-wide polyploidization has played essential role in speciation and thus evolution of plants RBR in female gametophytic cell specification and differentiation as well as in maintaining genome integrity are only valid if the rbr-3 allele is gametophytically recessive and genetic reduction of RBR function had caused the observed effects. Since rbr-3 carries a T-DNA insertion in the middle of the RBR gene rbr in the gametophyte, we subjected the gametophytically lethal rbr mutation to tetraploid genetic analysis. We asked if the rbr-3 mutation behaves recessive or dominant in diploid gametophytes produced by tetraploid plants by analysing seed set phenotypes and segregation of rbr genotypes in the progeny (rbr mutation (rbr genotypes. Seed set and progeny segregation phenotypes of rbr simplex (rbr/RBR/RBR/RBR), duplex (rbr/rbr/RBR/RBR) and triplex (rbr/rbr/rbr/RBR) mutant plants significantly fit the recessive model of rbr-3 inheritance , implicating abortion of homozygous rbr/rbr gametophytes and rbr/RBR gametophytes giving viable seeds  as reported previously rbr/rbr/rbr/RBR plant, three types of FG genotypes are expected: rbr/rbr, rbr/RBR and rare RBR/RBR. Given that the rbr mutation fits a recessive model of inheritance based on the tetraploid seed set phenotype and progeny test (RBR function (rbr/rbr) would be expected to show ectopic nuclear divisions, accounting for 54% proliferating FGs . These data suggest that the viable rbr/RBR female gametophytes are likely phenotypically and functionally identical to RBR/RBR FGs of wild-type tetraploids, and RBR FGs of wild-type diploids.As an additional step to confirm this genetic model, we examined cytological phenotypes of female gametophytes (FG) in these plants. The majority of FGs in both diploid and tetraploid wild-type plants were at stage FG7 upon emasculation, which is typical for wild-type eny test , only thting FGs . The obsrbr mutation behaves recessive in the female  gametophyte(s). Therefore, we can rule out a dominant (negative or positive) effect of a possible truncated version of RBR mRNA or of RBR protein. This situation is perhaps similar to previous reports that premature termination in mouse Rb exons resulted in truncated non-functional proteins rbr-3 allele is a clear loss-of-function mutation of RBR.Together, two independent genetic experiments of seed set and transmission analysis  and quanrbr mutation constrains analysis of RBR function during sporophytic development. Tetraploid analysis is therefore an excellent approach to investigate a dosage dependent function of RBR in the sporophyte. We recovered and analysed rbr tetraploid plants with different numbers of rbr-3 alleles by large-scale genotyping and segregation analysis of the tetraploid mutant progeny (see rbr quadruplex (rbr/rbr/rbr/rbr) genotypes could be identified, confirming the genetic data (see previous section) that they do not survive post-gametophytically. Specifically, we identified second generation rbr triplex plants (rbr/rbr/rbr/RBR) that showed significant quantitative reduction of RBR expression levels in leaves when compared to the wild-type tetraploids (nulliplex RBR/RBR/RBR/RBR)   or hetert manner .rbr triplex plants, which displayed nearly 75% reduction in gene expression compared to the wild type  at the diploid level. Although rbr triplex plants appeared to grow normally during early sporophyte development (rbr nulliplex (rbr plants (not shown) were normal and indistinguishable at similar stages. Thus, the mutant sporophytic phenotypes only occur in rbr triplex plants, indicating that a single RBR copy cannot sustain normal growth and development and, therefore, RBR function is haplo-insufficient in the tetraploid context.We expected that the ild type , could relopment , they shelopment . In compulliplex , simplexRBR would alter cell division and/or cell size as was reported from other systems rbr gametophytes showed ploidy aberrations (as discussed before), we anticipated that ploidy deregulation could also be observed in the leaf sporophyte with decreased RBR dosage. However, neither diploid (rbr/RBR versus RBR/RBR) (data not shown) nor tetraploid (rbr/rbr/rbr/RBR versus RBR/RBR/RBR/RBR) plants  plants  had signRBR dosage change. We examined trichome differentiation patterns in young rosette leaves around 15 days after germination on plates, in diploid and tetraploid plants. As expected, development of trichomes in the 3rd and 4th rosette leaves of diploid rbr/RBR plants did not differ from the corresponding wild-type, confirming haplo-sufficiency of RBR in diploids  and 5\u20136 branched trichomes (1%) along with an increase of less-differentiated 3-branched trichomes (84%)  . In addiperiment . Therefome cells  but not RBR in diploid leaves provided inconclusive results for ploidy-dependent leaf and trichome differentiation. For example, suppression of RBR in Brassica napus led to elevated ploidy levels in leaves and retarded leaf and trichome development Arabidopsis in order to interfere with RBR function revealed only marginal elevation of ploidy levels in mature leaves and supernumerary trichome branching patterns RBR levels in these plants, or if the transcription of RBR was aberrantly elevated due to the autoregulatory function of RBR-E2F pathway rbr triplex trichome cells was comparable to corresponding tetraploid wild type, and that there was no significant difference within ploidy groups across different genotypes (RBR dosage-sensitive manner (RBR in the triplex (rbr/rbr/rbr/RBR) plants does not alter the general leaf, trichome and plant ploidy, cell proliferation and trichome specification, but it appears to be insufficient to complete a full differentiation program. This could be particularly true for trichome differentiation, as is also suggested by a recent report that RBR is a target of the trichome cell specification and differentiation factors GLABRA1 and GLABRA3 rbr trichomes by inducible methods will be required to analyze how RBR controls early specification and/or differentiation. Taken together, the observed sporophytic developmental anomalies including retarded trichome differentiation are a consequence of partial haplo-insufficiency of RBR in tetraploids, but not due to RBR-mediated cell cycle deregulation.It has been proposed that key cell cycle genes that control ploidy restrict trichome branching enotypes . Thus, we manner . RetainiRBR is an essential cell cycle regulatory gene that is expressed in the sporophyte  and the ovule including the embryo sac RBR throughout the plant life cycle, we analysed RBR RNA and protein accumulation by in situ hybridization and a transgenic RBR protein reporter line RBR::RFP, respectively RBR::RFP expression throughout sporophyte development in leaves and seedlings (not shown), also during trichome development  have essential roles in controlling cell differentiation and development both in plants and animals RBR via its inherent H3K27me3 activity and possibly through its continuous association throughout the cell cycle Arabidopsis, there are three distinct orthologues of Ezh2, namely CURLY LEAF (CLF), which positively regulates cell size and elongation in the leaf sporophyte; MEDEA (MEA), which negatively regulates cell proliferation and cell size during seed development; and SWINGER (SWN), which enhances the function of both CLF and MEAArabidopsis genome. FERTILIZATION INDEPENDENT SEED 2 (FIS2) functions similar to MEA during seed development; VERNALIZATION 2 (VRN2) and EMBRYONIC FLOWER 2 (EMF2) are associated with distinct sporophytic pathways MET1 is the Arabidopsis orthologue of Dnmt1, which is a key target of RBR and a modifier of several PRC2 genes, and it is critical for coordinated cell division, specification and differentiation of the embryos, and also throughout the sporophytic development RBR, several PRC2 genes and MET1 are co-regulated by a negative feedback mechanism during gametophyte differentiation and development The dynamic expression of MET1 in rbr triplex and heterozygous diploid rbr mutant leaf tissues in relation to their corresponding tetraploid or diploid wild-type tissues. Our initial expression analysis in tetraploids suggested that plant to plant variation in expression was quite high. Therefore, we analyzed leaves from individual plants as independent replicates. Prior to gene expression in tetraploids, we examined the expression of MET1 and PRC2 genes in the diploid wild type and rbr/RBR leaf tissues, but we did not detect significant differences in expression levels (not shown). In contrast, we observed that CLF and MET1 were upregulated and VRN2 was downregulated in rbr triplex leaves when compared to the tetraploid wild type  while only 15% differentiated to the 3-branch stage. The vrn2;emf2 double mutant showed similar phenotypes  ET956, ET1119 and ET2634 (Ler background) RBR::RFP reporter line clf-50 (Ws), swn-3 (Col), emf2-10 (Ws) and vrn2-1 (Ler) rd and 4th leaves, when the seedlings were at 5\u20136 leaf stage.The Confocal analysis of ovules and spatial analysis of GUS activity in ovules and seed tissues were performed as described earlier 2+ ions Cauline leaves of mutant and wild-type tissues were fixed in a formaldehyde/glutaraldehyde fixative. Intact trichome cells were isolated from leaf epidermis by an established protocol based on removal of CaRBR cDNA expression clone as a template. In situ hybridization was performed as before Semi-thin paraffin sections of inflorescences, emasculated pistils, and siliques rbr-3 allele and (b) maximal double reduction. Transmission efficiency (TE) is an estimate of inheritance of a mutant allele versus the wild-type allele by female or male gametes rbr allele is not transmitted through female gametes (TErbr)\u2640(\u200a=\u200a0) rbr through pollen was estimated as 0.1 based on both TErbr)\u2640. Double reduction describes the situation in polyploids, in which a heterozygous individual produces homozygous gametes RBR locus is \u223c45 cM away from the centromere, these loci can be considered unlinked. Therefore, we used the maximal double reduction frequency of 1/6 for our calculations ; simplex, dominant (D/RBR/RBR/RBRrbr); duplex, recessive (rbr/rbr/RBR/RBR); duplex, dominant (D/rbrD/RBR/RBRrbr); triplex, recessive (rbr/rbr/rbr/RBR); triplex, dominant (D/rbrD/rbrD/RBRrbr)]. These plants were identified as duplex-recessive for rbr (As we did not know how many mutation . Out of  for rbr . Subsequ for rbr .PP2A (At1g13320) RNA extraction and reverse transcription were performed as described Figure S1rbr female gametophyte. (A\u2013D) the enhancer detector ET1119 (egg cell marker) GUS expression in wild-type and rbr female gametophytes. (A) A wild-type embryo sac at maturity showing a typical egg-specific GUS expression pattern of ET1119 at 2 days after emasculation (red arrow). Green arrow marks synergids. (B\u2013D) Mis-expression of the egg-specific GUS marker in rbr mutant embryo sacs. In some small number of cases, the ectopic GUS staining was restricted to the egg (red arrow) and central cell region (black arrow) (B) (2 observations) or the whole FG  (1 observation). (D) In this particular rbr embryo sac, two big cells in the place of an egg were stained (red arrows) (1 observation). Note that the synergids appeared morphologically normal, but they also showed GUS expression (green arrows). (E) Synergid-specific expression of ET2634 in the wild-type (green arrow). (F) Rare mis-expression phenotype of ET2634 in rbr embryo sac. Black arrow points to egg apparatus (mainly synergid-derived proliferation) with a weaker GUS. Scale bars: 30 \u00b5m.Deregulation of cell-specific markers in (2.47 MB TIF)Click here for additional data file.Figure S2RBR function. (A) RBR mediated triploid bridge led to autonomous tetraploidization of diploid plants heterozygous for rbr. Shown are representative flow cytometry histograms depicting the cellular ploidy of young cauline leaves. (B) Progeny testing (n\u200a=\u200a103) of a tetraploid rbr heterozygote identified rbr triplex plants (shaded in yellow) (see rbr triplex plant (n\u200a=\u200a93) (see Schemes of tetraploid genetics dissecting (2.17 MB DOC)Click here for additional data file.Figure S3rbr triplex versus the corresponding tetraploid wild-type confirms gametophytic recessiveness of the rbr-3 allele. (A) Histogram of female gametophyte (FG) phenotypes in diploid rbr plants (rbr/RBR) in comparison to the corresponding wild-type (RBR/RBR). In the expected genetic model we considered that FGs homozygous for rbr hyper-proliferate and they are lethal, typical of the rbr gametophytic lethal mutation rbr/rbr/rbr/RBR) in comparison to the tetraploid wild type (RBR/RBR/RBR/RBR). The expected ratio of FG phenotypes in the triplex plant was calculated based on a genetic model for recessiveness (in our case full loss of function) considering double reduction Click here for additional data file.Figure S4RBR expression and concomitant reduction of the characteristic 4-branched trichomes in tetraploid leaves confirms RBR dosage-dependent trichome differentiation. Note that reduction in RBR levels ultimately correlated with reduction in seed set. Analyzed are four distinct genotypes, RBR/RBR/RBR/RBR ; rbr/RBR/RBR/RBR (n\u200a=\u200a259 and 387); rbr/rbr/RBR/RBR (n\u200a=\u200a214 and 395); and rbr/rbr/rbr/RBR (n\u200a=\u200a217 and 432). *significantly different in the mutant when compared to the corresponding nulliplex (p\u200a=\u200a0.05).Quantitative reduction of (0.03 MB DOC)Click here for additional data file.Figure S5RBR/RBR), (B) diploid rbr mutant (RBR/rbr), (C) tetraploid Col wild-type (RBR/RBR/RBR/RBR) and (D) rbr triplex (RBR/rbr/rbr/rbr). Scale\u200a=\u200a90 \u00b5m.Loss of three functional copies of RBR in tetraploids does not lead to aberrant cell division and cell size in leaves. Shown are scanning electron micrographs of abaxial region of mature cauline leaves in (A) diploid Col wild-type ((6.86 MB TIF)Click here for additional data file.Figure S6RBR in tetraploids does not lead to changes in ploidy of leaf and trichome cells. (A) Leaf ploidy in tetraploid wild type (RBR/RBR/RBR/RBR) and rbr triplex (rbr/rbr/rbr/RBR) recorded by flow cytometry (B) Ploidy classes of the trichomes in reference to the tetraploid (4C) guard cells, upon nuclear DNA quantification by confocal microscopy.Quantitative reduction of (0.51 MB PPT)Click here for additional data file.Table S1Primers used in quantitative real time PCR assays.(0.03 MB DOC)Click here for additional data file.Text S1Tetraploid genetics and double reduction.(0.09 MB DOC)Click here for additional data file."}
+{"text": "The increasing prevalence of hypertension, owing to modern lifestyles and the increasing elderly population, is contributing to the global burden of cardiovascular (CV) disease. Although effective antihypertensive therapies are available, blood pressure (BP) is generally poorly controlled. In addition, the full benefits of antihypertensive therapy can only be realised when target BP is achieved. International guidelines and clinical trial evidence support the use of combination therapy to manage hypertension. In high-risk patients, such as those with coronary artery disease, diabetes and renal dysfunction, BP targets are lower and there is a need for intensive management with combination therapy to control BP and provide additional CV risk reduction benefits.Combinations of antihypertensive agents with different but complementary modes of action improve BP control and may also provide vascular-protective effects. Calcium channel blockers (CCBs) have been shown to be effective in combination with a range of antihypertensive drugs and in different patient populations. As part of a first-line combination strategy, CCBs can provide CV benefits beyond BP control, even in patients at increased CV risk. Benefits include protection against end-organ damage and serious CV events. Indeed, in major intervention trials, these benefits have already been clearly demonstrated. Ongoing studies will provide further data to support the clinical benefits of combination therapy as a first-line treatment approach. Implementation of this approach in clinical practice, together with adherence to global hypertension management guidelines will help ensure patients achieve and sustain BP targets, and reduce the risk of CV events. A literature search was conducted to identify recent randomised studies assessing CCB-based combination therapy strategies.Despite guideline recommendations, combination therapy is an underused strategy for the treatment of hypertension. Calcium channel blocker (CCB)-based combination strategies are effective and well tolerated when used with other classes of antihypertensive drugs, and should be considered a first-line option in hypertensive patients, particularly in those at high cardiovascular risk.Hypertension is one of the most important modifiable causes of premature death worldwide, and is estimated to cause 7.1 million premature deaths. Approximately one billion people worldwide have hypertension and the prevalence is predicted to increase dramatically in the next few years .Hypertension is a major risk factor for both cardiovascular (CV) and cerebrovascular morbidity and mortality , contribPatients with hypertension are also more likely to have associated CV risk factors ; for exaThe ultimate goal of hypertension management is to reduce CV morbidity and mortality by preventing end-organ damage \u20138. NumerAlthough effective therapies exist, current BP control is still below the \u2018Healthy People 2010\u2019 goal of 50%: only 34% of patients with hypertension have adequately controlled BP, 59% have treated but uncontrolled BP and 30% are unaware of their condition . Poorly Hypertension guidelines have traditionally recommended stepwise regimens to lower BP in patients with hypertension, beginning with lifestyle modification , and adding pharmacological intervention when lifestyle changes are insufficient ,6,7,12. Data from outcome studies show that several classes of drugs, including angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARBs), beta-blockers, calcium channel blockers (CCBs) and thiazide-type diuretics, effectively lower BP and reduce the complications of hypertension ,13\u201316. HIn clinical practice, hypertension management varies greatly and many factors contribute to inadequate BP control, the most important include: patient non-compliance; acceptance of inadequate BP control by clinicians and reluctance to titrate the dose, switch to another drug or add another drug; and the fact that it is difficult to achieve adequate BP control with monotherapy in most patients, even when the dose is optimised . ResponsCompared with high-dose monotherapy, combination therapy is associated with fewer adverse effects. Guidelines advocate combination therapy with once daily treatment regimens that provide 24-h efficacy. The advantages of combination therapy include improved adherence to therapy  and miniCombination therapy was traditionally reserved as a third- or fourth-line approach in hypertension management ; howeverGuidelines recommend various two-drug combinations of different classes of antihypertensive agents based on data derived from controlled interventional trials, but advise that three or four drugs may be required depending on the patient's risk profile. Although older therapies such as diuretics and beta-blockers can effectively lower BP and are included as possible first-line combinations, they are associated with some disadvantages . For exaBy contrast, many studies have shown that newer antihypertensive agents, such as CCBs, ARBs and ACE inhibitors, provide additional benefits by reducing the incidence of CV events in patients with hypertension ,16,25,30Calcium channel blockers are used extensively in clinical practice and data from several clinical studies show that CCBs effectively and safely lower BP and reduce long-term CV risk in a wide range of patient populations ,35,38,39As CCBs have a different mode of action to commonly used inhibitors of the renin\u2013angiotensin\u2013aldosterone (RAAS) pathway (such as ACE inhibitors and ARBs), combination with these agents should provide synergistic or complementary effects, compared with using two agents that inhibit the same pathway. Indeed, in patients with newly diagnosed stage 1 or 2 hypertension or in patients with inadequate BP control after conventional low-dose monotherapy, low-dose combination therapy with CCBs and ARBs was found to provide better BP control than either high-dose monotherapy (p < 0.05 vs. either monotherapy) . FurtherIn the Systolic Evaluation of Lotrel Efficacy and Comparative Therapies study, CCB and ACE inhibitor combination therapy with amlodipine and benazepril was significantly more effective in reducing SBP and pulse pressure in patients with severe systolic hypertension than either monotherapy (p < 0.0001) . SignifiData from outcome trials show that CCB therapy plus additional add-on treatment not only lowers BP but also improves patient outcomes. The Hypertension Optimal Treatment trial showed that intensive lowering of BP with CCB-based therapy (felodipine as baseline therapy with the addition of other antihypertensive agents according to a five-step regimen) was associated with a low rate of CV events . In the Data from ASCOT showed that CCB-based treatment is more effective than a beta-blocker-based regimen for reducing mortality and CV events . ASCOT cIn addition, there are several ongoing studies to determine the optimal antihypertensive combination therapy with the most favourable safety profile for lowering BP and protecting against CV events. The Combination Therapy of Hypertension to Prevent Cardiovascular Events study is a multicentre trial assessing CV outcomes in hypertensive patients treated with various drug combinations including ARBs, beta-blockers or diuretics in combination with a CCB (benidipine) . The AvoSeveral other large, randomised clinical studies have investigated the use of antihypertensive combination therapies in high-risk patient populations, including those with impaired renal function, diabetes or CAD, in whom BP is more difficult to control to target levels. These will be discussed in more detail in the following sections.There is strong evidence to show that hypertension is probably the most important risk factor for stroke  \u2013 the riThe safety and efficacy of CCBs have also been demonstrated in several high-risk patient populations, such as those with impaired renal function, diabetes or CAD.In hypertensive patients at high CV risk, renal dysfunction has been shown to be an important predictor of CV risk and to act as a prognostic marker of progression to CVD . FurtherACTION examined the benefits of additional nifedipine GITS intervention in patients with stable angina and CAD who were receiving best practice therapy. Almost 40% of the patients in ACTION had evidence of renal dysfunction ,55, and In the International Nifedipine GITS Study: Intervention as a Goal in Hypertension Treatment (INSIGHT), patients with mild-to-moderate hypertension received nifedipine GITS or the diuretic combination co-amilozide, with the addition of atenolol  followed by addition of any other antihypertensive drug (other than CCBs or diuretics) if BP targets were not achieved. The study showed that, in hypertensive patients at high CV risk, renal function was better preserved with nifedipine GITS than with diuretics (p < 0.0001). The improved renal function with nifedipine GITS was indicated by better preserved creatinine clearance, which is a marker of renal function, and fewer patients treated with nifedipine GITS had progressive renal deterioration compared with those treated with co-amilozide ,53.post hoc analysis of the changes in estimated glomerular filtration rate with a CCB (amlodipine), a diuretic  and an ACE inhibitor (lisinopril). In this trial, the incidence of end-stage renal disease was similar for all three treatment arms, but estimated creatinine clearance was significantly better preserved with amlodipine than with chlorthalidone or lisinopril  than in patients with primary hypertension ,6. HowevSeveral studies have shown that CCB-based combination therapy can improve clinical outcomes in individuals with diabetes. In INSIGHT, nifedipine GITS reduced the risk of all-cause mortality in patients with diabetes compared with those without diabetes and reduced the frequency of new cases of diabetes compared with diuretic therapy . FurtherIn ACTION, more than 50% of patients had inadequately controlled BP at baseline, despite receiving best practice therapy at study entry . At baseFurther analysis of the ACTION results revealed even greater benefits in the subgroup of patients with inadequate BP control at baseline . There wHypertension is a multifactorial disease, so disruption of a single physiological pathway is often insufficient to control BP. Therefore, a combination of two drugs with different but complementary modes of action is often needed to achieve effective BP control. This is supported by hypertension guidelines, which highlight the need for drugs to be combined effectively and emphasise the benefits of drugs with different mechanisms of action on a multi-regulated variable such as BP ,6,7.Dihydropyridine CCBs and inhibitors of the RAAS pathway  are widely used in patients with CVD. They have complementary mechanisms of action so, when used in combination, have synergistic effects on pathological changes in the vasculature and end organs, providing benefits in addition to BP control.Calcium channel blockers primarily affect the cellular interactions of endothelial cells, smooth muscle cells, monocytes and thrombocytes, which have key roles in the early phases of atherosclerosis development. There is also evidence to show that CCBs affect the nitric oxide system in endothelial cells. Several studies have shown that endothelium-dependent relaxation, which is impaired in individuals with hypertension, can be restored by treatment with dihydropyridine CCBs \u201367. CCBsBoth guideline recommendations and clinical trial evidence support the use of combination therapy in managing hypertension, particularly in patients at increased risk of CV events and those for whom BP targets are lower because of the need for intensive management of additional risk factors, such as those with CAD, metabolic syndrome, diabetes or renal dysfunction. In a large proportion of patients, it is difficult to lower BP to target levels using antihypertensive monotherapy, so more intensive intervention, specifically combination antihypertensive therapy, is often required. Clinical studies have proved the efficacy of CCB-based combination strategies in a wide range of high-risk patient groups.Combination therapy with antihypertensive agents that have different but complementary mechanisms of action not only avoids unnecessary drug interactions and adverse events, but also maximises the benefits of agents that have additional effects beyond BP lowering. For example, there is evidence to show that the combination of CCBs and ARBs provides end-organ protection through synergistic mechanisms. CCBs are effective with all other antihypertensive agents, and this flexibility makes them ideal as part of a first-line combination strategy to achieve target BP and provide additional CV benefits, without compromising safety in patients at increased CV risk.In clinical practice, there is still much inconsistency with regards to stepwise treatment of patients and the decision to increase drug dose, switch therapy or add another drug. Combination therapy may still be considered a last resort in the treatment of hypertension and be rarely used as initial or first-line therapy. The treatment paradigm is now changing and more patients are being treated in line with guideline recommendations, which focus on overall CV risk and therefore recommend multiple-drug strategies early in the course of treatment.Ongoing studies will provide further data to support the benefits of antihypertensive combination therapy on clinical outcomes when used as a first-line strategy. Increased efforts to use combination therapy much earlier in the course of treatment and increased adherence to global guidelines will ensure patients achieve and sustain BP control in addition to reducing the risk of CV events."}
+{"text": "Meticulous control of blood pressure is required in patients with hypertension to produce the maximum reduction in clinical cardiovascular end points, especially in patients with comorbidities like diabetes mellitus where more aggressive blood pressure lowering might be beneficial. Recent clinical trials suggest that the approach of using monotherapy for the control of hypertension is not likely to be successful in most patients. Combination therapy may be theoretically favored by the fact that multiple factors contribute to hypertension, and achieving control of blood pressure with single agent acting through one particular mechanism may not be possible. Regimens can either be fixed dose combinations or drugs added sequentially one after other. Combining the drugs makes them available in a convenient dosing format, lower the dose of individual component, thus, reducing the side effects and improving compliance. Classes of antihypertensive agents which have been commonly used are angiotensin receptor blockers, thiazide diuretics, beta and alpha blockers, calcium antagonists and angiotensin-converting enzyme inhibitors. Thiazide diuretics and calcium channel blockers are effective, as well as combinations that include renin-angiotensin-aldosterone system blockers, in reducing BP. The majority of currently available fixed-dose combinations are diuretic-based. Combinations may be individualized according to the presence of comorbidities like diabetes mellitus, chronic renal failure, heart failure, thyroid disorders and for special population groups like elderly and pregnant females. Achieving recommended goal of blood pressure (BP) {< 140/90 mmHg in all hypertensives, < 130/80 mm Hg in hypertensives with diabetes mellitus (DM) . VariousThe Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure JNC 7) and European Society of Hypertension (ESH) guidelines recommend that therapy with more than one antihypertensive agent be considered in patients with systolic blood pressure (SBP) more than 20 mm Hg or DBP more than 10 mm Hg above goal and among patients at high cardiovascular risk, as determined by elevated BP level and the presence of other risk factors  and Euro. The appThis review focuses the need and basis of combination therapy, different classes of combination agents available at present, rationale for their combination, comparisons of these combinations and their effect on the outcome.National Harris interactive survey for hypertension, in the United States revealed that out of 90% patients taking medication only 50% to 60% were involved in some form of lifestyle change to control BP -14. For Another double blind, parallel group randomized study for 12 weeks comparing the combination therapy of felodipine and metoprolol (5/50mg) with either monotherapy exhibited significantly greater antihypertensive response (98%) with combination compared to monotherapy (felodipine- 79% and metoprolol- 82%). A significant greater reduction in mean systolic/diastolic BP (28/18 mmHg) with combination therapy was evinced compared to either felodipine (18/12 mm hg) or metoprolol (19/12 mm hg) .The long-term (I year) efficacy and safety between lisinopril and trichlormethazide combination therapy and lisinopril monotherapy was investigated in a multi-centre open label trial on 466 patients. It showed effective BP reduction to < 150/90 mmHg in both the groups throughout the study period. Additionally the combination of trichlormethazide reversed the increase in serum potassium observed in the monotherapy group .The results of these studies emphasize that multiple-drug therapy was both safe and effective compared to monotherapy and will be required in most patients to attain BP goals.Many panels including Hypertension in African Americans Working Group (HAAWG), JNC 7, The Task Force for the Management of arterial Hypertension of the ESH and of the European Society of Cardiology have strongly supported that treatment initiation with 2 or if needed 3 drugs is justified in many cases of hypertension management ,19. TherMulti-drug therapy regimens can either be fixed dose combinations (FDCs) or drugs added sequentially one after other. However, choice of combination antihypertensive therapy will depend upon the tolerability and a convenience of dosing/titrating drug regimen. FDCs can enhance adherence to medication regimens compared with treatment given as 2 separate agents. Also they facilitate more prompt reduction in BP. The use of antihypertensive combinations started in the 1960s with hydrochlorothiazide (HCTZ) combined with triamterene, a potassium-sparing diuretic, and has been added with newer and different combinations in due course of time .Available trials have studied different classes of drugs in combination for treatment of hypertension, taking advantage of their complimentary action. Angiotensin receptor blockers (ARBs), thiazide diuretics, alpha and beta blockers, calcium antagonists(CCBs) and angiotensin-converting enzyme inhibitors (ACEIs) have been the commonly used classes of antihypertensive agents. Thiazide diuretics and CCBs are effective, as well as combinations that include rennin-angiotensin-aldosterone system (RAAS) blockers, in reducing BP. Several combinations of an ACEI or ARB with a diuretic or an ACEI with a CCB are available. The majority of currently available FDCs are diuretic-based. Though diuretics have an unparalleled track record of safety and efficacy, recent data documenting low-grade carcinogenicity must be evaluated further .Some of the commonly available combinations are listed in Table 1Some of the combinations which have been studied in detail are discussed below. Refer Table \u03b2-blockers and diuretics have been used for the treatment of hypertension for more than three decades. Although \u03b2-blockers did have a beneficial effect on the BP, \u03b2-blocker therapy failed to favourably affect the cardiovascular events and mortality either alone or in combination with a diuretic . WarmackEarlier it was a widely held belief that beta-blockers should be prescribed for management of hypertension in patients with higher heart rates, an established risk factor for cardiovascular events. But recent Anglo-Scandinavian Cardiac Outcomes Trial-Blood Pressure Lowering Arm (ASCOT-BPLA) trial concluded that, in similar hypertensive populations without previous or current coronary artery disease, higher baseline heart rate is not an indication for preferential use of \u03b2-blocker-based therapy over amlodipine based therapy. ASCOT BP-lowering arm showed outcome inferiority of therapy initiated with atenolol versus that initiated with CCB, amlodipine  . Also thSo based on above evidence beta-blockers alone or in combination should now have more restricted place in cardiovascular therapy and can be possibly indicated in hypertensives with anxiety and fast heart rate.RAAS inhibitor and a diuretic combination will offset the diuretic-induced increase in plasma renin activity. The salt loss will add to the antihypertensive effect of RAAS blocker. Besides, an ARB will also attenuate the metabolic effects of thiazide diuretics like hypokalemia and hyperglycemia. Several studies have demonstrated the antihypertensive effectiveness of this combination in low doses, showing substantially greater reductions in BP and higher response rates than either of the treatments alone ,27.The Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE), trial compared the effects of BP lowering with a perindopril/indapamide combination or placebo, in high-risk type 2 diabetic subjects. The risk of combined primary outcome, a major macrovascular or microvascular event was reduced by 9% (p = 0.041) with a 14% (p = 0.025) reduction in all-cause mortality and an 18% (p = 0.027) reduction in cardiovascular mortality. The study extrapolated saving one death over 5 years for every 79 patients with this ACEI/Diuretic combination . SimilarA trial with patients having uncontrolled BP despite antihypertensive agents including an ARB  randomly assigned to combination therapy with telmisartan 40 mg/day and HCTZ 12.5 mg/day  or to no change in their current drug regimen (n = 32). Both office and home BP was significantly reduced in T+H arm in 12 weeks. Also early morning BP was decreased inferring the long duration activity of combination . ONEAST RAAS blocker buffer CCB-induced activation of the sympathetic nervous system and the RAAS. Also the negative sodium balance caused by CCBs adds to the antihypertensive effect of RAAS blocker. Dose-dependent CCB induced peripheral edema may be minimized in the presence of an RAAS blocker .In patients with both diabetes and hypertension, ACEIs provide clinical benefits that appear to be independent of BP reduction . In the P = .02). The reduction came, however, mostly as a result of a 25% reduction in the relative risk of stroke (P = .001), compared with atenolol-based therapy, yet the between-group difference in SBP was only 1 mm Hg [The rationale for combination therapy with agents that block the RAAS and a CCB or diuretic is well founded . However 1 mm Hg . Moreove 1 mm Hg ,15.In Fogari et al.  study, 4An ACEI/ARB regimen theoretically may provide the advantage of a more complete blockade of the RAAS. ARB will reduce the ACEI escape phenomenon, a mechanism whereby angiotensin II returns to pretreatment levels despite continuous ACEI treatment. Furthermore, angiotensin II generated by ACEI-independent pathways will get blocked by ARBs. Additionally the ACEI itself inhibits bradykinin degradation .Clinical studies of properly dosed ACEI and ARB combinations have demonstrated significant improvement with regard to target organ damage, specifically heart failure and proteinuria. The first major study in this area was the CALM  trial, which was designed to compare the effect of candesartan 16 mg or lisinopril 20 mg or both on BP and urinary albumin-creatinine ratio in 197 type 2 hypertensive microalbuminuric diabetic patients. All three treatments resulted in a significant decrease in both BP and albuminuria. The combination treatment was significantly more effective than monotherapy in reducing BP and resulted in a greater decrease in albuminuria, although this was statistically significant only when the combination was compared with candesartan monotherapy .In the Combination Treatment of angiotensin II receptor blocker and Angiotensin-Converting Enzyme inhibitor in nondiabetic renal Disease (COOPERATE) trial, ,46 the iHowever, the potential hazards of ARB plus ACEI combinations must also be taken into consideration: such combinations often produce worsening of hyperkalemia,  and may Various randomized studies have been conducted to compare fixed combinations of one class with fixed combinations of another class -58. CombACEI/CCBs, ACEI/diuretics and \u03b2-adrenoceptor antagonist/diuretics all are significantly effective than placebo and helpful in achieving DBP < 90 mm Hg . ACEI/CCP < .014) and 17% (P < .018), respectively, in risks for the primary end point and stroke [The BP component of ASCOT-BPLA was stopped prematurely after 5.5 years of median follow-up because there was significantly less risk of secondary end points, including nonfatal myocardiac infarction(MI), total cardiovascular end points, all-cause mortality, stroke, and heart failure in patients treated with amlodipine/perindopril compared with those treated with atenolol/bendroflumethiazide. There was also a nonsignificant trend toward reduced risk for the primary end point  favoring amlodipine/perindopril treatment . A subsed stroke .In patients with metabolic syndrome, ACEI/CCBs are preferred over \u03b2-adrenoceptor antagonist/diuretic and ARB/diuretic combination. ARB/diuretic are associated with marked changes in glucose parameters and a higher incidence of new onset diabetes (26%) as compared to ACEI/CCBs (11%)  In patieThe ACCOMPLISH trial compared the ACEI benazepril plus the diuretic hydrochlorothiazide  and benazepril plus amlodipine  on a composite cardiovascular mortality and morbidity end point . This coARB/diuretic is similarly effective to ACEI/CCBs in controlling 24 hours BP on ambulatory blood pressure monitoring but less effective in achieving SBP < 140 mm Hg and also associated with poorer metabolic control and new onset diabetes as discussed above .Hypertension may act synergistically with diabetes in increasing the risk of both macrovascular and microvascular comlplications of diabetes . VariousThe results have consistently shown a beneficial renoprotective effect of ACEIs and ARBs in diabetic nephropathy. The combined therapy with an ARB and a CCB has a potentially useful antiproteinuric effect in patients with type 2 diabetic nephropathy, even when their renal function is reduced. This was also shown in Fogari et al study also . AlthougHypertension and dyslipidemia are conditions that can coexist frequently. National Health and Nutrition Examination Survey (NHANES III) has shown that 64% of patients with hypertension also have dyslipidemia and conversely, approximately 47% of patients with dyslipidemia have hypertension. Hypertension and hypercholesterolemia are the two leading risk factors for heart disease, These two together cause an increase in coronary heart disease related events .In addition to its anti-hypertensive effect through antagonizing AT1 receptors, telmisartan has a unique property that activates peroxisome proliferator-activated receptor-\u03b3 (PPAR-\u03b3) and is suggested to improve insulin sensitivity and reduce triglyceride levels, leading to a reduction of the risk for atherosclerosis. Miura et al.  demonstrSaga Telmisartan Aggressive Research (STAR) study evaluated 197 patients being prescribed 20 to 80 mg of telmisartan for 6 months. Total cholesterol (TC) levels decreased from 200 to 188 mg/dl (p < 0.05). Triglyceride levels were decreased 270 to 175 mg/dl (p < 0.005) in patients with TG levels \u2265 150 mg/dl . TelmisaTreatment of hypertension in patients with heart failure must take into account the type of heart failure, systolic dysfunction or diastolic dysfunction, in which there is a limitation to diastolic filling and therefore in forward output due to increased ventricular stiffness. Diuretics, beta blockers, ACEIs, ARBs, and aldosterone antagonists are indicated in the management of heart failure and have been shown to reduce morbidity and mortality in appropriately selected patients with heart failure. Hyperkalemia could be the side effect of some of these drugs so the drugs like ACEIs, ARBs, and aldosterone antagonist in combination should not be used. The choice of agents is based on severity of heart failure, left ventricular ejection fraction, history of myocardial infarction and any other associated comorbidities.In these patients, treatment with ACEIs  and \u03b2-blAngiotensin II type 1 receptor blockers have favourable effects on haemodynamic measurements, neurohumoral activity, and left-ventricular remodelling when added to in patients with ACEIs CHF. The primary outcome of the CHARM- Added study was the composite of cardiovascular death or hospital admission for CHF. Candesartan reduced each of the components of the primary outcome significantly, as well as the total number of hospital admissions for CHF. The benefits of candesartan were similar in all predefined subgroups, including patients receiving baseline beta blocker treatment. The addition of candesartan to ACEI and other treatment leads to a further clinically important reduction in relevant cardiovascular events in patients with CHF and reduced left-ventricular ejection fraction .ONTARGET showed that the ARB telmisartan and the ACEI ramipril are equally effective in preventing cardiovascular events in high-risk patients and that the combination provides no added benefit and causes more adverse effects than either monotherapy in this patient population. However, ONTARGET does not rule out use of the ACEI plus ARB combination in severe heart failure, where angiotensin escape mechanisms are expressed and dual blockade may be needed .2 (body surface area), and loop diuretics are used in GFR less than or equal to 40 to 50 mL per minute per 1.73 m2[Hypertension can be caused by chronic kidney disease(CKD) but it itself can worsen the renal failure. The guidelines state that management of hypertension in CKD should focus on reducing BP, with some also emphasizing reducing protein excretion.Choice of agent will primarily depend on the presence of proteinuria as there is a direct relationship between the degree of proteinuria and progression to end stage renal disease. In proteinuric kidney first line agents include an ACEIor ARB, and often requires the addition of a diuretic or a calcium channel blocker. Diuretics are a useful alternative for non-proteinuric patients or as an add-on to renin-angiotensin system blockade. Multiple drug therapy is often needed to maintain BP below the 90th percentile target, but adequate BP control is essential for better renal and cardiovascular long-term outcomes. Thiazide diuretics can be used if glomerular filtration rate (GFR) is greater than or equal to 40 mL per minute per 1.73mr 1.73 m2 Various Recently, combined therapy with an ACEI and an ARB has been shown to provide a greater reduction in urinary albumin excretion (UAE) than monotherapy with either of these agents in patients with diabetic nephropathy. However, such combinations may be associated with potential hazards, including increased serum potassium concentrations and worsening of renal anemia, especially in patients whose kidney function is mildly to moderately impaired -47.Fogari et al.  evaluateThe prevalence of hypertension among patients with hypothyroidism is approximately 3%. Hypertension is much more frequently associated with hyperthyroidism, the prevalence is estimated at 20% to 30%. Hypothyroid state has been shown to accelerate the age-related increases inBP. Studies have shown a significant correlations between DBP and either T4 or T3 suggesting that thyroid hormone deficiency contributes to increase in BP when it is slight to moderate. The mechanism of increased BP in hypothyroidism is not known, but suggested mechanism could be acceleration of structural change of vascular tissue by thyroid hormone deficiency and alteration of autonomic nervous function by thyroid hormone deficiency leading to hemodynamic changes. In patients of thyrotoxicosis, systolic pressures are typically elevated and diastolic pressures are often low, which results in a widened pulse pressure. These findings are attributable to increased cardiac output, stroke volume, heart rate, and cardiac contractility. Although many symptoms of thyrotoxicosis can be controlled with beta-adrenergic blockers, catecholamine levels are usually normal or even decreased. Despite the fact that the activity of the RAAS is increased in patients with thyrotoxicosis, ACEIs and angiotensin II receptor blockers do not always reduceBP. Thus, the role of the RAAS in hypertension associated with thyrotoxicosis remains to be defined.69 Treating hypertension in older patients requires attention to their altered physiology and to concomitant cardiovascular and renal disease, which may indicate use of particular antihypertensive drugs. No specific guidelines exist for hypertension management for this particular population. However studies have shown requirement of two or more drugs in most of them. Combination therapy is often necessary to treat isolated systolic hypertension, but control is only reached in 70% of patients in clinical trials following an algorithm [The estimated prevalence of hypertension in the United States is 66% in men and women aged 60 years and older, which is the highest among all age groups . A metaalgorithm ,75.Till date, the most encouraging data supporting aggressive management of hypertension in the elderly population comes from the Hypertension In the Very Elderly Trial (HYVET), a randomized, double-blind placebo trial that enrolled 3,845 patients from 195 centers in Europe, China, Australia, and North Africa. Patients were started with indapamide/placebo and were added with perindopril if BP of 150/80 mmHg was not achieved. Fatal stroke, cardiovascular death, heart failure got reduced by 39%, 23% and 64% respectively in a median follow up of 1.8 years. Thus HYVET and other trials favor mono-therapy or combination therapy with thiazide diuretics, ACEIs and CCBs for hypertension in the elderly .Hypertension complicates 5% to 7% of all pregnancies. A subset of preeclampsia, characterized by new-onset hypertension, proteinuria, and multisystem involvement, is responsible for substantial maternal and fetal morbidity and is a marker for future cardiac and metabolic disease .Drugs preferred during the pregnancy areIst line - Methyl dopa, Beta blocker (propranolol) and LabetalolIInd line - Metoprolol, atenolol and Calcium channel blocker (nifedipine)IIIrd line agents-clonidine, diureticsThree short acting antihypertensive agents-hydralazine, labetalol, and short acting  nifedipine-are commonly used to control acute, very high blood pressure in women with severe hypertension in pregnancy.Maternal antihypertensive drugs usually compatible with breastfeeding are Captopril, diltiazem, Enalapril, Hydralazine, Hydrochlorothiazide, Labetalol, Methyldopa, Minoxidil, beta blockers like Propranolol and timolol, spironolactone and verapamil. Individual side effects of drugs have to be looked for, while prescribing these drugs in lactation.Few patients may require a third or fourth drug to adequately manage BP. Preference should be given to the selection of an agent from a different class than the initial 2 drugs in the combination therapy. Addition of the third drug may be in the form of spironolactone , minoxidil, hydralazine, carvedilol and rest of the drugs depending on the specific conditions being treated. Centrally acting drugs should be the last option due to potential side effects.ACEIs- Pregnancy, angioneurotic edema, hyperkalemia, renal artery stenosisDiuretics- Gout, Hypokalemia, Pregnancy, Impaired glucose tolerance,Beta blockers- Asthma, marked bradycardia, abnormal glucose tolerance, obstructive pulmonary disease, peripheral artery diseaseARB- Pregnancy, hyperkalemia, renal artery stenosisCa channel blockers- Heart failure, bradyarrythmiasIt is generally accepted that reducing the pill burden improves adherence and/or compliance to therapy, though very few data is available to support this theory. Wald and Law introduced the term \"polypill\" in 2003. Polypill has been thought as a single daily pill to prevent CVD by simultaneously reducing four risk factors . It usually is composed of a statin, three pressure-lowering drugs, each at half of its standard dose, aspirin, 75 mg, and folic acid. The polypill was suggested to reduce ischemic heart disease by 88% and stroke by 80% if taken by everyone over 55 years of age .However, our patients present with a puzzle of clinical features, for which variable doses of the specific medication is required. The polypill provides fix combination of substances, possibly resulting in undertreatment of the main condition(s) and overtreatment of secondary conditions. It also neglects differences in metabolism due to age, race and sex. Even after some studies showing its effectiveness the idea is still under investigation and needs to be studied further .Hypertension is now considered as a part of a complex syndrome of changes in cardiac and vascular structure and function. All of the current guidelines suggest that \u2265 1 antihypertensive agent is required in most patients with hypertension to reach BP goals that will effectively reduce the cardiovascular risk. Therapy with 2 drugs separately or with fixed combinations that include agents with complementary actions. Many combinations have been shown to improve cardiovascular outcome and include a diuretic with the RAAS blocker. Choice of combination therapy depends upon the risk factors, presence of comorbidities like diabetes, renal dysfunction and the adverse effects and tailored according to individual patient.BP: Blood Pressure; DM: Diabetes Mellitus; DBP: Diastolic Blood Pressure; JNC: Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure; ESH: European Society of Hypertension; SBP: Systolic Blood Pressure; FDC: Fixed Dose Combinations; HCTZ: Hydrochlorothiazide; ARB: Angiotensin Receptor Blockers; CCB: Calcium Channel Blockers; ACEI: Angiotensin-Converting Enzyme Inhibitor; RAAS: Renin-Angiotensin-Aldosterone System; ASCOT: Anglo-Scandinavian Cardiac Outcomes Trial-Blood Pressure Lowering Arm; CRF: Chronic Renal Failure; MI: Myocardial Infarction; CVD: Cardio Vascular Disease; AT1: Angiotensin II-type 1; TC: Total Cholesterol; CHF: Congestive Heart Failure; CKD: Chronic Kidney Disease; GFR: Glomerular Filteration Rate; UAE: Urinary Albumin Excretion; and UAER: Urinary Albumin Excretion Rate.The authors declare that they have no competing interests.All authors have contributed equally to literature search and paper writing. All authors read and approved the final manuscript"}
+{"text": "To explore the various perceived reasons for farmers\u2019 suicides in the Vidarbha region of Maharashtra, their common factors, and to suggest solutions.The present formative research was undertaken in the 23 villages surrounding the Anji Primary Health Centre, located in the Vidarbha region of Maharashtra, India. A triangulation of free list and pile sort exercises was used. The data was analyzed by Anthropac 4.98.1/X software. This was followed by a semi-structured focus group discussion. To increase the validity of the results, these findings were presented to the participants and later they were circulated to the 26 farmers\u2019 clubs in the villages for comment and discussion during their monthly, village based meetings.Farmers perceived debt, addiction, environmental problems, poor prices for farm produce, stress and family responsibilities, government apathy, poor irrigation, increased cost of cultivation, private money lenders, use of chemical fertilizers and crop failure as the reasons for farmers\u2019 suicides. Participants suggested solutions such as self-reliance and capacity building among farmers, a monitoring and support system for vulnerable farmers, support and counseling services, a village-level, transparent system for the disbursement of relief packages.Farmers\u2019 suicides in Vidarbha are caused by the complex interplay of social, political and environmental constraints. Hence, a comprehensive intervention to ensure self reliance and capacity building among farmers in modern farming techniques , monitoring and support system for vulnerable farmers, a village-level, transparent system for disbursement of relief packages is required to prevent farmer suicides in the near future. Apart from this, there is a need to strengthen the National Mental Health Program at primary health care level to offer support and counseling to vulnerable farmers in rural area. Studies in India, Sri Lanka, Canada, England and Australia have identified farming as one of the most high-risk industries when it comes to having a suicide rate higher than in the general population.2In India, national data show that the suicide rate was 9.7/100000 population per year in 1995. The population of the Vidarbha region is 1 200 000, so the expected number of suicides was 116 per year, but it was found to be 572 in 2005, 1065 in 2006 and 600 in year 2007.3A report by the Tata Institute of Social Sciences, Mumbai identified the reasons for farmer\u2019s suicides: repeated crop failures, inability to meet the rising cost of cultivation, and debt.4Even when the government announced relief packages for the affected families and remedial measures, this did not lead to any immediate positive effect on suicide behavior. It was reported that farmers\u2019 concerns were not taken into account while designing these relief packages.5In India, farmer suicides had been reported from various states, viz. Andhra Pradesh, Punjab Karnataka and Orissa.6As a step towards self reliance and sustainability, KVMs under KRHTC were up-graded from informal groups to self-help groups. KVMs guaranteed savings and ensured loans on flexible conditions to its poor members and subsequently could prevent attempted suicides by two indebted farmers. In September 2008, we invited KVM members from the 23 surrounding villages to participate in a discussion to explore the various perceived reasons for farmers\u2019 suicides in the Vidarbha region of Maharashtra, their mutual relationships and to suggest solutions.The Kasturba Rural Health Training Centre (KRHTC) in Anji successfully implemented the \u2018Community Led Initiatives for Child Survival\u2019 (CLICS) program in the 23 villages surrounding the Anji Primary Health Centre in the Wardha district during the period 2003 to 2008. As a part of community mobilization, Kisan Vikas Manch  were formed in each village to ensure the involvement of men in the program. This community-based platform was used for Participatory Research and Action, health message dissemination and village-based agricultural guidance.7Wardha is a sister city for Sevagram, and both were used as major centers for the Indian Independence Movement, especially as headquarters for an annual meeting of the Indian National Congress in 1934, and Mahatma Gandhi\u2019s Ashram. In recent time, repeated crop failures, the rising cost of cultivation and debt have created a situation that is leading to farmers committing suicide in the Vidarbha region.The present formative research was undertaken in the 23 surrounding villages of KRHTC, Anji, which is located in the Wardha district of the Vidarbha region, about 758 km east from the state capital, Mumbai. This economically backward region is located in the north eastern part of Maharashatra state and its economy depends on agriculture.8After obtaining written consent, a triangulation of free list and pile sorting exercises was used to identify various perceived reasons for farmers\u2019 suicides, their perceived relationships to these factors and possible solutions to address these problems.9In the free list exercise, the participants were asked to make an individual free list of the various reasons for farmers\u2019 suicides. Eleven reasons with relatively high Smith\u2019s S value were then pile sorted. Smith\u2019s S  refers to the importance, representativeness or prominence of items to individuals or to the group, and is measured in three ways: word frequency across lists, word rank within lists and a combination of these two.10In the pile sorting exercise, the individual participants were asked to group those selected reasons which they felt went together and suggest the solutions to prevent them. It was followed by a semi-structured focus group discussion (FGD) with these ten members. A note taker recorded the discussion. The data was analyzed by Anthropac 4.98.1/X software.11To get the collective picture, multi-dimensional scaling and hierarchical cluster analysis of pile sort data was undertaken. To increase the validity of the results, the findings of the free list, pile sort exercise and FGD were presented to the participants.There were 26 KVMs in 23 villages. Each KVM had 15-20 small scale male farmers as members. Out of this, 17 KVMs had been active in organizing village-level technical agricultural guidance for all other farmers. We invited in one member from each of these 17 KVMs, who was willing to participate and talk freely on this issue. A one day meeting was organized at KRHTC, Anji on a day and at a time convenient to the participants. Representatives from ten KVMs attended the meeting. This sample size was adequate for pile sorting as after fifth participants, results were likely to get repeated with more than 0.75 correlations of results.Later, the findings were translated and typed out in the local language Marathi and then circulated to 26 KVMs (with 15-20 members each) in surrounding villages for sharing and discussion during their monthly village-based meetings. A social worker facilitated this meeting in all KVMs and obtained feedback from the other group members. These monthly meetings were of one to two hours duration and were held in the evening when most of the members are back from their agricultural work. This activity was carried out over the period of one month.Based on the pile sorting exercise, a focus group discussion with ten representative KVM members and feedback from all 26 KVM members were summarized. The third column presents the action proposed or being undertaken by the state and central government. Italic text signifies direct quoting from the participants.In the free list exercise, the various eleven reasons identified for farmers\u2019 suicide in our area in descending order of Smith\u2019s S value were as follows. 1) debt, 2) addiction, 3) environmental problems, 4) poor prices for farm produce, 5) stress and family responsibilities, 6) government apathy, 7) poor irrigation, 8) increased cost of cultivation, 9) private money lenders, 10) use of chemical fertilizers 11) crop failure. These items were then subjected to pile sorting exercise .As found in the analysis of the pile sort data, eleven perceived reasons for farmers\u2019 suicides were clustered into five groups, which they thought of as mutually related to each other and suggested solutions for each group of problems. The solutions suggested for each group of problems was compiled in the second column of Subsequently, in a focus group discussion, a participant said, \u201cNowadays there is no respect and dignity for farm workers. The government announced the relief package for the farmers but they are mere passive recipients of it and no efforts are being made in the direction of farmers\u2019 self-reliance for the future\u201d. In order to ensure self-reliance, farmers wanted capacity building and training on newer techniques of farming. We should be taken on study visits to other states or neighboring countries where farmers are working successfully against the adverse environmental conditions.Even when the government has announced the relief package for farmers, suicides are still going on. In response to this one of the participating farmers suggested the need to develop a \u2018support system\u2019 for the farmers and said, \u201cThis has become a disease now. Similar to disease conditions such as malaria, filarial etc, there should be a monitoring system to identify vulnerable, poor, small-scale farmers and solve their problems\u201d.The government encourages farmers to develop alternative sources of income. In response to this, farmers remarked, \u201cthe produce of such alternative sources should have market demand. Relief packages in the form of farm equipment are being distributed through the district or taluka level bureaucratic government system. Poor and needy farmers avoid going to district or taluka level as that requires frequent visits. Hence, to make the service accessible and bring transparency, participants said that such distribution should be done at village level \u2018Gram-sabha\u2019 [local self-help government] where all the villagers assemble and watch the process. This process will prevent manipulation by the distributing officers and the siphoning off of the poor farmers\u2019 benefit by rich farmers\u201d. Another farmer said, \u201cThe benefit from the government package goes to middle level or large scale farmers. Arranging a loan from the bank is a lengthy procedure and banks avoid giving loans to small farmers who have poor capacity to repay the loan. Hence, these poor small farmers go to private money lenders who verbally negotiate the business. He will be repaying such loans until his death.\u201dIn the present study, farmers perceived debt, addiction, environmental problems, poor prices for farm produce, stress and family responsibilities, government apathy, poor irrigation, increased cost of cultivation, private money lenders, use of chemical fertilizers and crop failure as the most significant reasons for farmers\u2019 suicides. Participants suggested solutions to these groups of problems. The major themes that emerged from the FGD were self-reliance and capacity building of farmers, a monitoring and support system for vulnerable farmers and a village-level, transparent system for the disbursement of relief packages.12In our study, participating farmers perceived farmer suicides as a complex interplay of eleven reasons which cover social, political and environmental constraints. Participants perceived that the human activities such as excess use of chemical fertilizers and use of genetically modified seeds cause a loss of land biodiversity and repeated crop failure, which subsequently lead to high costs of cultivation and debt. It was aggravated by government policies related to market prices, exploitation by private money lenders and its ultimate health consequence was frustration leading to suicide.According to a study conducted by the Indira Gandhi Institute of Development Research, Mumbai, the major reasons for farmers\u2019 suicides are debt, crop failure and low return, illness of family members, failure to arrange marriage of daughters and a lack of alternative sources of income.13In the present study, farmers have suggested the development of a monitoring system to identify vulnerable farmers and offer them timely help.As found in our study, farmers are losing faith in the government due to its failure to design and implement pro-poor policies for the majority of small farmers who survive on agriculture. Many states have offered financial relief packages only to the families of deceased farmers who were unable to manage payments on their bank loans. Provision of relief facilities alone is not sufficient as it has been observed in the case of Andhra Pradesh where farmers committed suicide to enable their families to partake of the benefits of relief packages.14 As suggested by study participants, to ensure transparency, its disbursement should be done through village-level Gram-sabha which is crucial to regain the faith of poor small scale farmers and ensure their survival. In the United States, there was a rise in farmers\u2019 suicides after the Great Depression. To counter this, the government started a farmers\u2019 insurance program, which is the only major federally-managed insurance program in existence.1In the absence of institutionalized finance, the farmers normally resort to borrowing from private money lenders. Significantly, the loans taken from the private moneylenders are difficult to repay due to high interest rates. Hence, the government should ensure institutional finance and crop insurance to small farmers. Maharashtra state government also plan to alleviate debt, ensure fresh crop loans for both small and big farmers, disburse loans through the farmers\u2019 Self Help Groups, provide subsidy of the crop insurance premium, encourage the promotion of agro-processing industry, provide financial assistance for community marriage and encourage organic farming.15In the present study, participants suggested the promotion of organic farming and reducing dependency on commodities such as chemical fertilizers, pesticides, and genetically modified seeds. In rural China, chronic pesticide exposure was found to be associated with suicidal tendencies, which supports findings from previous studies.16Given the high level of pesticide exposure and high suicide risk, a clarification of the causal mechanisms underlying this association and development of appropriate interventions are priorities for public health and health policy.Farmers expressed their concerns for chemical fertilizers and environmental degradation. The nutrients of the soil are being destroyed by the over-use of pesticides and chemical fertilizers needed to successfully grow the genetically modified seeds. This repeated degradation will result in the loss of land productivity thus putting future generations of farmers at even greater risk of poverty and famine.17In Australia, a strong correlation between droughts and suicide rates among farmers was found. Hence, if a drought was predicted, there was rapid mobilization of social workers, psychologists and psychiatrists to the drought-hit region along with other supportive measures while in India action is predominantly limited to political announcement of exgratia benefits and not towards prevention strategies.1In India, there is a need to strengthen the National Mental Health Program at primary health care level so as to offer support and counseling to vulnerable farmers in rural areas.The need for stress relief camps and counseling services for farmers was expressed. Walker et al reported that even in the absence of psychiatric morbidity, farmers were more likely to report that life is not worth living compared with the general population.5 Apart from this, there is a need to strengthen the National Mental Health Program at primary health care level to offer support and counseling to vulnerable farmers in rural areas.The present field-based formative study explored the farmers\u2019 perceived causes of suicides and their solutions. These findings may be useful for policy formulation at local level. The limitations of the present study should be kept in mind. It was a small scale study conducted in a limited geographical area. Hence, further research at a wider level is required to confirm our findings. In conclusion, the farmers\u2019 suicides in Vidarbha are due to the complex interplay of social, political and environmental constraints. Hence, a comprehensive intervention to ensure self-reliance and capacity building of farmers in modern farming techniques, a monitoring and support system for vulnerable farmers and a transparent, village-level system for disbursement of relief packages is required to prevent farmers\u2019 suicides in the near future. These suggested interventions are consistent with the recent recommendations by an autonomous administrative training institute by the government of Maharashtra."}
+{"text": "Both groups underwent haematological and biochemical examinations and were administered two neurobehavioral tests, namely the Mini-Mental State Examination (MMSE) and the Montgomery-\u00c5sberg Depression Rating Scale (MADRS). Sociodemographic, personal medical, nutritional and lifestyle data were recorded. According to personal statements, farmers suffered from hypertension, cardiovascular, orthopaedic and ENT problems in higher frequency. Haematocrit, haemoglobin and serum cholinesterase\u2019s activity were found to be lower among farmers. Lower prevalence of hypertension and better performances on MMSE and MADRS tests were recorded in young farmers in relation to young non-farmers, while these findings were reversed in older ages. Odds Ratios were calculated through multivariate logistic regression models. Factors affecting these impairments remain to be clarified.Investigating the health status of agricultural workers is a challenging goal. Contradictory outcomes concerning farmers\u2019 health are reported in the literature. In this cross-sectional study, certain clinical and neurobehavioral health outcomes were compared between farmers and non-farmers living in the same rural area. Farmers (328) and non-farmers (347), matched  Farmers and agricultural workers are believed to be healthier and have lower morbidity and mortality rates than non-farming rural and urban populations ,2,3,4,5.On the other hand, farming itself and farming-related tasks entail significant hazards to the health and well-being of farmers. Although not well appreciated, farming is among the most hazardous of occupations . FarmersMoreover, stress in farmworkers has been recently recognized as an important public health concern. Stressors inherent in farm work and lifestyle, such as uncertain and fluctuating economic prospects are associated with poor physical and mental health outcomes and result in deleterious effects on cognitive function, depression and high rates of suicide ,13,15. In Greece, about 20% of the labour force population is engaged in agriculture. While agriculture is one of the most promising sectors of the Greek economy, little interest has been demonstrated in studying health and safety of the farming population. Farmers in Greece are involved in all kind of agricultural tasks .The aim of the present study was to assess the health status, as this is reflected by the haematological and biochemical parameters and neurobehavioral and cognitive function, among farming population in relation to non-farmers living under similar environmental conditions. per sex and age category. Non-farmers were selected randomly from the local telephone directories. A first phone contact took place, in order to explain the aims of the study to the recruits. The response rate was 85%. Finally 328 farmers  and 347 non-farmers  were recruited.A two-year (2008\u20132009) cross sectional study was conducted in the prefecture of Aitoloakarnania, an agricultural area in West Greece, where about 30% of the total population deals with various cultivations. It is worth noting that the majority of the farming population in this area is indigenous and non-immigrants. The study population was acquired from 36 rural communities of the prefecture. The number of farmers selected in each community was proportional to its total agricultural population. Farmers were selected randomly from the community\u2019s farmer registry. To participate in the study the farmers had to be at least thirty-five years old, so as to satisfy the criterion of long-term farming (at least 15\u201320 years of farming), given that farmers start dealing with intensive agricultural work at the age of 15 to 20 years old. Non-farmers had to live in the same communities, not to be occupationally involved in farming and to match the recruited farmers i.e., physical activity (type and duration of exercise), smoking habits (packs per day), alcohol consumption (frequency and kind of drinks per day) and coffee consumption (cups per day) were recorded. The smokers were classified as moderate (up to one pack or 20 cigarettes per day) and as heavy (more than one pack per day). Based on daily intake of ethyl alcohol the subjects were classified as abstainer, light, moderate and heavy drinkers [i.e., meat, fish, vegetables, legumes, dairy products, potatoes, candies and fruits were assessed by a 7-day food frequency questionnaire. Furthermore, lifestyle characteristics drinkers . Weekly Following the home visit, all subjects were invited to the local health centre or rural dispensary to conduct clinical and laboratory examinations. Measurements of body weight and height, systolic (SBP) and diastolic blood pressure (DBP) and blood examination to determine various biochemical parameters were performed. The participants were classified as hypertensive if SBP >140 or DBP >90.Finally, the participants were administered two validated questionnaires, the Mini-Mental State Examination (MMSE) and the Montgomery-\u00c5sberg Depression Rating Scale (MADRS). The MMSE is a measure of cognitive components, namely orientation, linguistic and visual functions, with possible scores ranging from 0 to 30 . Lower pThe MADRS is not a diagnostic tool, but can be used to evaluate mood, anxiety, sexual function, appetite, sleep, functional status, ability to think and concentrate, existing physical symptoms or physical complaints, as well as general psychiatric and potential distress . The oveThe study was approved by the plenary meeting of the Medical School of Patras and the Ethics Committee of the University Hospital of Patras.Statistical analysis was performed by SPSS v.17 statistical software . Continuous variables are presented by range and median and categorical variables as frequencies. Mann and Whitney U test was employed to compare groups. Differences in proportions were tested using either the Pearson chi square test or Fisher\u2019s exact test depending on cell frequencies. The statistical significance level was set at a = 0.05. In order to investigate the effects of the various characteristics of the study population as well as the nutritional and other habits on MMSE, MADRS and hypertension, multivariate logistic regression models were applied. The independent variables were dichotomized at the respective cut-off values. The coefficients of retained parameters and/or interactions of logit functions were calculated. Coefficients with a p < 0.100 were kept in the model. Because of the hierarchy principal, parameters in statistical significant interaction terms were retained in the model independently of their significance. The characteristics of the two examined groups, farmers and non-farmers, are shown in i.e., meat, fish, vegetables, legumes, dairy products, potatoes, candies and fruits did not differ between farmers and non-farmers (data not shown). Differences in the educational level were observed, with farmers showing extremely low percentages, especially regarding secondary or higher degrees. Farmers were more likely to have three or more children in comparison to the non-farmers. Furthermore, higher income levels were observed in male farmers than in male non-farmers, whereas the opposite was observed for females. The prevalence of obesity was very high for both farmers and non-farmers. The percentage of heavy smokers was higher in male farmers while the percentage of female smokers was higher among non-farmers for both categories. Similarly, consumers of two or more cups of coffee per day were higher among female non-farmers. The consumption frequencies of major food groups Comorbidities, according to participants\u2019 statement, are presented in The haematological blood examinations for farmers and non-farmers are presented by sex, as some basic haematological parameters like haematocrit and haemoglobin differ considerably between men and women . HaematoThe biochemical blood examinations are presented in Since the prevalence of hypertension was increasing and the participants\u2019 performances in neuropsychological tests were getting worse while aging, the corresponding classifications of subjects are depicted per age group in As it is shown in The retained factors from multivariate logistic regression models of logit function for MMSE, MADRS and Hypertension are presented in versus (vs.) non-farmers after controlling for other characteristics. For instance, the OR for male farmers in the first age category (\u226439 years) with no children vs. non-farmers with all the other characteristics being the same is the exponential of the coefficient of occupation i.e., OR = e\u22120.755 = 0.47. The corresponding OR for farmers in the age category 50\u201359 years old is the exponential of the sum of the occupation coefficient (\u22120.755) and the interaction coefficient of occupation x age category (1.500). The resulting OR is e(\u22120.755+1.500) = 2.11, reflecting the worse scores of farmers in older ages. Furthermore, the coefficient of the interaction term occupation x gender indicates that the OR was increased for female farmers. The OR was increased with the number of children, but this factor acted protectively for farmers as it is indicated by the coefficients of the interaction term number of children x occupation. Additionally, moderate smoking (one pack per day) reduced the OR. The results of As for MMSE, similar results were found, except that instead of smoking the income was retained into the model. The higher the income, the lower was the OR for cognitive impairments.vs. non-farmers were lower than one in younger ages and got higher than one in the older ones. The ORs were lower in females compared to males. Similarly, the OR was reduced by income. Finally, consumption of two or more cups of coffee daily increased the OR. Hypertension was influenced by occupation, gender, income and coffee consumption. The ORs for farmers The present study gives evidence of several clinical and neurobehavioral alterations among farmers who have been involved in cultivation for a long time-period, indicating their susceptibility to certain impairments of their health status in comparison with non-farmers living in the same area.Based on the participants\u2019 statements regarding their health problems, farmers mention more health issues than non-farmers at a statistically significant level (p = 0.044). Hypertension and other cardiovascular disorders occur more frequently among farmers in comparison to non-farmers. The results of blood pressure records confirm partially the personal statements given that the percentage of hypertension in younger ages is lower in farmers than in non-farmers of the same age, but this situation is reversed in older farmers  have been reported previously in a farming population in United Arab Emirates and were attributed to exposure to pesticides . MoreoveThe number of reported miscarriages among women farmers is slightly higher (on the borders of significance) in comparison with non-farmers. Previous studies with similar results have attributed that to pesticide exposure ,27,28. As far as the haematological blood examinations are concerned, a slight, still statistically significant, decrease in haematocrit and haemoglobin levels between both males and females farmers is observed, indicating possible suppression of heme biosynthesis. This finding is consistent with previous studies ,30,31,32Biochemical blood examinations revealed an increase in certain serum enzymes  as well as in some serum components  accompanied by a simultaneous slight decrease in pseudocholinesterase levels in farmers compared to non-farmers. The raise of serum biochemical markers, which suggest liver dysfunction, or at least impairment of normal liver function, in those occupationally engaged to farming, has been previously reported ,30,35, eAs for the addressed neuropsychological tests, it is obvious that farmers in younger ages \u201cstart\u201d with better scores than non-farmers. While farmers do achieve higher scores in MMSE when younger, they start to perform poorer than non-farmers during their middle-age and the differences become more obvious while aging. Respectively, young farmers gain lower scores and better performance in the MADRS test compared to young non-farmers, while this fact is reversed in aged subjects. i.e., education, income, number of children and coffee consumption. Taking into account the analyses results, the main outcome is that farming is related to hypertension, emotional burden and cognitive impairments. The multivariate analysis  confirmsMost of the literature dealing with the health status of farmers worldwide presents contradictory findings. There are studies that measure better health in farming populations ,3,4,6,11The overall conclusion that can be drawn from the literature review is that even if farmers are \u201chealthier\u201d than the general population ,3,11, thThe main limitation of the study arises from its nature. The measures of association that can be estimated by a cross-sectional study are Odds Ratios and no causal inferences can be drawn. Nevertheless, the findings of the cross-sectional studies are a good starting point for further investigation. Another issue of concern is the \u201chealthy worker effect\u201d. However, that seems not to be the case for the present study since young farmers have better health indices than non-farmers, at least for the three main outcomes. Additionally, male farmers are wealthier than non-farmers. Another possible limitation is the potential misclassification of self-reported morbidities, though these were concurrent with the literature. Despite these limitations, this study still managed to discern the discrepancies between farmers and non-farmers in various age groups. What remains to be clarified is which are the factors that contribute to this unique health pattern that is presented into the farming population. Farming is a valuable resource worth preserving. Policies for supporting the agriculture workers are necessary globally but especially in countries such as Greece, in which economy is closely related to agriculture."}
+{"text": "This paper addresses the general concern in plant pathology that the introduction of quantitative resistance in the landscape can lead to increased pathogenicity. Hereto, we study the hypothetical case of a quantitative trait loci (QTL) acting on pathogen spore production per unit lesion area. To regain its original fitness, the pathogen can break the QTL, restoring its spore production capacity leading to an increased spore production per lesion. Or alternatively, it can increase its lesion size, also leading to an increased spore production per lesion. A data analysis shows that spore production per lesion (affected by the resistance QTL) and lesion size (not targeted by the QTL) are positively correlated traits, suggesting that a change in magnitude of a trait not targeted by the QTL (lesion size) might indirectly affect the targeted trait (spore production per lesion). Secondly, we model the effect of pathogen adaptation towards increased lesion size and analyse its consequences for spore production per lesion. The model calculations show that when the pathogen is unable to overcome the resistance associated QTL, it may compensate for its reduced fitness by indirect selection for increased pathogenicity on both the resistant and susceptible cultivar, but whereby the QTLs remain effective. Cultivar resistance is an efficient, environmentally benign, method of disease control that could allow for a reduction in the use of fungicides in agriculture. Conventional resistance breeding has however mainly focussed on qualitative resistance Johnson , a form The most intuitive effect of using a cultivar with quantitative resistance is that the pathogen will in time overcome the resistance mechanisms. Quantitative resistance is usually described by quantitative trait loci (QTLs) having an effect on the pathogen development rate and it is assumed that, like for qualitative resistance, the resistance determined by QTLs can be overcome by the pathogen. Some experimental studies have now been published showing how a pathogen population can adapt to cultivars with quantitative resistance whereby selection leads to the pathogen overcoming the resistance QTLs (see e.g. Lehman and Shaner 2), and spore production capacity, defined as the amount of spores produced per unit lesion area (micrograms of spores/mm2), have independent genetic support and can evolve separately in Puccinia triticina , but the pathogen would compensate for its effect by indirect selection. More specifically, we consider the case of a QTL acting on spore production capacity (spore production per unit lesion area), resulting in a reduction of the number of spores produced by a lesion, which is an important component of pathogen fitness accounting for the pathogen's transmission capacity. Considering that the spore production per lesion is a composite trait that depends both on the spore production capacity of the infected tissue and on the lesion size  is highly specialised to common wheat and durum wheat and has a worldwide distribution  depends both on the lesion size and spore production capacity of the pathogen. It is assumed that the spore production capacity is limited by the presence of quantitative resistance in the host, specifically affecting this trait. We then focus on the capacity of the pathogen to restore its fitness by adapting towards an increased lesion size. Therefore, we will first consider the relationship between spore production per lesion and lesion size (for a fixed spore production capacity), whereby the first part of the paper describes the analysis of a large data set to establish the existence of cultivar-specific relationships between lesion size and spore production per lesion for a wheat pathogen, 2) was calculated as the sporulating area divided by the total number of lesions. Spore production per lesion (\u03bcg of spores) was calculated as the amount of spores produced between the collection dates divided by the number of lesions and the spore production capacity per lesion (\u03bcg of spores per mm2 of lesion) was calculated as the amount of spores produced between the collection dates divided by the sporulating areas.Here, we only give a brief description of the experimental procedures, which are described in full in Pariaud  and PariSoi) and Morocco (Mor) are susceptible to all isolates tested, whereas the other cultivars, Festival (Fes), Scipion (Scp) and Th\u00e9see (The) are only susceptible to a subset of the isolates (see Table S1 in ESM). There are therefore three classes of isolate\u2013cultivar interactions: (i) Mor,Soi,Fes and Scp have a total of six isolates in common; (ii) Mor,Soi and The also have six isolates in common and (iii) Soi and Mor have all twelve isolates in common.The above-described data set is used to characterise the relationship between spore production per lesion and lesion size and investigate the effect that host resistance has on this relationship. Cultivars Soissons , A, and that this relationship is cultivar specific. The full model is thus given byWe used a linear model to test our first hypothesis that there is a significant positive relationship between the spore production per lesion, vi represents the intercept of the regression line for cultivar i,xi represents the slope of the regression line for cultivar i and eij represents the residual for the jth replicate of the ith cultivar.whereby, Our second hypothesis that the isolates lie in a fixed order across the regression lines, which would suggest that adaptation results in a shift along the relationship between lesion size and spore production per lesion, was tested by a Spearman's ranking coefficient test Sprent  of the r2), comprises a susceptible, HS, and a quantitatively resistant, HR, cultivar. A fraction, \u03b8, of the fields is planted with the quantitatively resistant cultivar, whilst the remaining fields are planted with the susceptible cultivar. In an agricultural system, this fraction is controlled by farmers. The leaf area densities of the cultivars are assumed to be directly related to their proportions in the landscape. The plants grow with a cultivar-specific intrinsic growth rate gi, with i\u00a0=\u00a0{S(usceptible), R(esistant)}, and has a total carrying capacity of K. It is convenient here to assume continuous harvest, that is, host plants have a cultivar-specific constant mortality rate \u03c9i. Such a simplification is frequently applied in epidemic models and is assumed acceptable for studying long-term dynamics. The plants in the landscape are affected by a pathogen that can infect both the susceptible and the resistant cultivar, whereby infection by a pathogen spore results in latent tissues ES and ER on the susceptible and the resistant cultivar, respectively. The latent tissue becomes infectious after a cultivar-specific latent period of 1/\u03b3i, resulting in infectious tissues IS and IR on the susceptible and the resistant cultivar, respectively. Once infectious, lesions produce a host-cultivar-specific number of spores per unit time, \u03b1i. The total number of spores produced per unit time on cultivar i thus depends on the spore production capacity (i.e. spores per mm2 lesion surface area) of the pathogen on cultivar i, i,Ii. Lesions have a cultivar-specific infectious period, 1/\u03bci and the infection efficiency of a spore produced by a lesion on cultivar n with n\u00a0=\u00a0{S,R} that lands on cultivar m with m\u00a0=\u00a0{S,R} is denoted by \u03b2mn. The full model is given by,The model represents a crop\u2013pathogen system whereby the total host population, expressed in leaf area densities (leaf surface area per mThe model variables and parameters are summarised in Table\u00a0\u03b1i in the model) and lesion size (LSi in the model). In practice, this relationship is, however, likely to be asymptotically bounded, because firstly the size of a leaf rust lesion is limited by mechanical or physiological constraints  or the slope of the relationship (resistance scenario 2). The relationship between spores produced per lesion as a function of lesion size on the resistant cultivar can thus be given by\u03c3 and \u03c1 denote the relative strength of resistance with 0\u00a0<\u00a0\u03c3\u00a0<\u00a01 and 0\u00a0<\u00a0\u03c1\u00a0<\u00a01. Note that low \u03c3 or \u03c1 values both denote a high level of resistance.where LSS, and eqns\u00a0We assume that lesion size adaptation occurs in such a manner that a change in lesion size on one cultivar results in an identical lesion size increase or decrease on the other cultivar. Because the presence of quantitative resistance in the host may affect the pathogen pleiotropically . Moreover, the regression lines for the individual cultivars have significantly different intercepts (P\u00a0<\u00a00.001) and slopes (P\u00a0=\u00a00.044), indicating that on some cultivars the pathogen strains generally have a reduced spore production per lesion . This host effect can be interpreted as a consequence of the resistance factors affecting the spore production capacity. The relationship thus reveals that spore production per lesion and lesion size are dependent quantitative traits linked through a positive relationship.The regression analysis of the raw data on spore production per lesion (mg) and lesion size relationship as presented in Fig.\u00a0cf. Table S1 in ESM), a Spearman's ranking correlation coefficient test revealed that, for all cultivars except The, the order of the isolate-means is significantly correlated (\u03c1\u00a0>\u00a00.829) for all significant correlations) across the different cultivars. Ranking according to isolate medians rather than means resulted in a slight change in the order of the isolates across the cultivars, but these differences were not significant .The cultivar-specific regression lines are shown graphically in Fig.\u00a02 lesion area). In a homogeneous landscape containing only one cultivar, the pathogen fitness is directly related to the spore production capacity, that is, the trait targeted by the resistance QTL (note that this is not necessarily the case when both cultivars are present in the landscape). Therefore, as in our evolutionary analysis we attempt to maximise pathogen fitness, the maximum spore production capacity then determines the evolutionary output of the PIP analysis (and the optimum lesion size).The model is used to study the adaptation of lesion size in an agricultural landscape with both a susceptible and a quantitatively resistant host cultivar. However, let us first consider the case of a homogeneous landscape containing only plants of the susceptible cultivar. In this case, we find that the lesion size adapts towards an optimum value and will not continually increase. This is because the relationship between spore production per lesion and lesion size is bounded for large lesion sizes. As illustrated in Fig.\u00a0Figures\u00a0\u03b8\u00a0\u2248\u00a01 (i.e. mainly plants of the resistant cultivar), the CSS lesion size on the resistant plants is close to the CSS lesion size in a homogeneously resistant landscape and the CSS lesion size on the susceptible plants has increased accordingly , the CSS lesion sizes are equal for all values of \u03c3 , the CSS lesion sizes differ for different values of \u03c1  and spore production capacity, defined as the amount of spores produced per unit lesion area (micrograms of spores/mm2). They compared three P. triticina pathotypes  for these traits on a wheat variety and found that they presented different pathogenicity profiles: P2 produced large lesions but had a low spore production capacity, which suggests a good ability for growth within host tissues but a poor ability for host resource exploitation for spore production. P3 presented the opposite profile, with small lesions but a high spore production capacity, and P1 presented high values for both traits. The authors concluded that lesions size and spore production capacity are under independent genetic control and are likely to evolve independently. In a study on Phytophthora infestans, Carlisle et\u00a0al. . Further data analysis revealed that isolates lie in a fixed order across the cultivar-specific regression lines in the sense that infections by an isolate that result in small lesions on a susceptible cultivar generally also result in relatively small lesions upon infection of a more resistant cultivar , resulting in an isolate\u2013cultivar interaction for the measured quantitative trait. Such isolate-specific QTLs have been found in several host\u2013pathogen systems . Despite this limitation, the pathogen is able to increase the number of spores it produces in a lesion and therewith to restore its transmission capacity, through the selection of strains with larger lesion sizes. Figure\u00a0In this study, we assumed that the spore production capacity of the pathogen, expressed as the amount of spores it can produce per unit of sporulating tissue, is limited by the action of a resistance QTL in the host. In the classical studies into the evolutionary consequences of the introduction of quantitative resistance into the landscape, it is considered that the pathogen might overcome the resistance QTLs. Our simulations differ from this classical approach in that the resistance QTL remains effective throughout the simulations , but in this paper, we have shown that this is not necessarily the case and that the observed increase in disease severity could instead be due to the selection of increased pathogenicity through selection on a pathogen trait that is not affected by the resistance QTLs. In such a case, both the resistant and susceptible cultivar are affected Fig.\u00a0. When moThis study is based on the relationship between two quantitative traits of the host\u2013pathogen interaction. Although published data on this question are still limited, such relationships, positive or negative, can be found amongst other traits. For example, Pariaud et\u00a0al.  have estA general concern in plant pathology has been that the presence of quantitative resistance selects for an increased pathogenicity as accounted for by quantitative traits (Garrett and Mundt"}
+{"text": "The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single-molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a \u2018one stop shop\u2019 explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties. E) and magnetic (H) components perpendicular to each other and to the direction of propagation, defined by a wave vector k , separated from its source by a distance much greater than its wavelength, is well approximated by a plane wave, described by infinite parallel planes of constant peak-to-peak amplitude normal to k. When an incident monochromatic plane wave described by an electric vector Ei strikes the boundary between two optically transparent, isotropic media with refractive indices ni and nt, there are reflected and transmitted monochromatic plane wave disturbances described by the electric vectors Er and Et. These can be described as:oE is the maximum amplitude of the disturbance, k.r = kxx + kyy + kzz = kr cos \u03b8 is the dot product (or cosine projection) of k on r and r\u03b5 and t\u03b5 are phase constants relative to Ei (as the position of the origin is not unique).Light is energy emitted by charged particles and manifests itself, as it travels through space, in the form of particles (photons) and electromagnetic waves. Electromagnetic waves have their oscillating electric , light turns towards the boundary (\u03b8c = sin\u22121 (nt/ni), at which \u03b8t has the largest possible value, which is \u03c0/2. At the critical angle, the refracted disturbance does not propagate through the medium with the lower reractive index, being \u2018totally internally reflected\u2019 at the interface. In these conditions, the expression describing the electromagnetic disturbance at the lower refraction index medium can be derived as follows: Let us make the incidence plane y = 0 and the interface z = 0. As kt is in the incidence plane, it does not have a y component:2\u03b8t + cos2\u03b8t) we have:\u03b8t > sin \u03b8c = nt/ni. This makes the radicand in (5) negative. Given that Eotez\u03be\u2212. Equation \u03b8i > \u03b8c the electromagnetic field decays exponentially as it penetrates deeper into the media with the smaller refraction index. A near-field \u2018evanescent\u2019 wave therefore forms at the boundary, which propagates along the surface in the x-direction.At an interface with a lower refractive index material . Figure The intensity t = Ee) we need to know the form of the electric vector Eoe =  at the boundary. This can be written as a function of its components parallel and perpendicular to the incidence plane , they allow us to write:\u03b4\u22a5 and \u03b4|| are phase factors given by:To fully describe the evanescent wave . By contrast, the evanescent field amplitude Eoe = Eox + Eoz is elliptically polarized in the incidence plane when Eoi is \u2225 to the incidence plane, also known as p-polarization, and \u2018cartwheels\u2019 along the boundary, as the wave propagates in the x-direction, with a spatial period x0 .Note that, the direction of the evanescent field is perpendicular to the \u03b4\u22a5 and \u03b4||, which give rise to a small but measurable longitudinal shift known as the Goos\u2013H\u00e4nchen shift  E. In dielectric isotropic media , the magnetic permeability \u03bc = 1. Since \u03b8i = \u03b8r and n = c/v, we can write:From electromagnetic theory, H =  and a film of aqueous (buffer) solution between the cover slip and cells adherent to it (nt = 1.33). There are two main approaches to creating an evanescent field: one is prism-based and the other objective-based.A TIRF microscope is designed to induce an evanescent field at the interface of two transparent isotropic media with different refractive indices close to or within a microscope sample. Most systems use laser beams as incident light. In cell biology applications, the interface at which the evanescent field is created is usually between the glass of a cover slip  = 61\u00b0. Total internal reflection also requires that the light emerges as a parallel beam (i.e. a train of electromagnetic plane waves); otherwise, one would only achieve partial internal reflection for those rays with \u03b8i > \u03b8c. To achieve this, the laser light must be focussed at the objective's back focal plane ; this is comparable to standard epi-illumination. By translating the focus beam away from the optical axis along the back focal plane one can increase the value of \u03b8i. The maximum value of \u03b8i that can be achieved is determined by the numerical aperture (NA) of the objective. This is a number that characterizes the range of angles over, which the lens can emit or accept light, and is defined as:nm is the refractive index of the medium the objective is immersed in, and \u03b1 is the field aperture, i.e. the maximum half-angle of the light cone accepted by the objective lens from the sample  and \u223c1.38, which is the refractive index of the primary cellular component (cytosol) in contact with the glass. Finally, the larger the NA of the objective, the larger the percentage of the peripheral area of the lens that can be utilized for total internal reflection, making coupling of the laser into the rear aperture less challenging.Given that sinNA \u2265 1.45, the objective must be immersed in a medium with nm > 1.45. Oil immersion is used because its refractive index (noil \u2243 1.51) matches that of glass, reducing unwanted reflections. Note that, the higher the NA, the lower the possible penetration depth of the evanescent field. The optimal NA may therefore depend on the separation expected between the molecules to be excited by the evanescent field and the interface, which may vary as in molecules at the plasma membrane of cells that show ruffles and invaginations  to characterize short range (<8 nm) intermolecular interactions and intramolecular conformational changes  is unique to TIRF microscopy \u2013 with epi-illumination, the field propagates along this axis and only has components in the x\u2013y plane. When the incident light is p-polarised (in the x\u2013z plane), the x-component is much smaller (about 5%) than the z-component and so Eoe \u2243 Eoz. The corresponding s-polarization is parallel with the y-axis.  Given that the probability of photon absorption depends on the dot product of the fluorophore's excitation dipole and the polarization of the excitation field, the fluorescence intensity from the molecule must also be dependent on its orientation and the evanescent field polarization. By switching the polarization of the incident light between the s- and p-directions to alternate the evanescent field polarization between the y- and z-directions and recording for each the fluorescence intensity from the molecule, one can determine the orientation of its excitation dipole (. The exix and y components of its emission. When performed at the single-molecule level, this enables the three-dimensional orientation of individual macromolecules immobilized on glass to be measured quantitatively to derive structure-function relationships. Suitable fluorophores include (rhodamine, fluorescein, Cy3, etc., In conjunction with dyes that align orthogonally to the membrane, this has been used to measure the local orientation of cell membranes to show ruffles and invaginations. Provided that the fluorophore's excitation and emission dipoles are aligned, i.e. the fluorescence anisotropy in solution is close to 0.4, then the orientation of the molecule may be quantified by resolving the"}
+{"text": "The identification of developmental curve patterns generated by a simulation-based educational method and the variables that can accelerate the learning process will result in cost-effective training. This study describes the learning curves of a simulation-based instructional design (ID) that uses ex vivo animal models to teach laparoscopic latero-lateral small bowel anastomosis.Twenty general surgery residents were evaluated on their performance of laparoscopic latero-lateral jejuno-jejunal anastomoses (JJA) and gastro-jejunal anastomoses (GJA), using swine small bowel and stomach on an endotrainer. The ID included the following steps: (1) provision of references and videos demonstrating the surgical technique, (2) creation of an engaging context for learning, (3) critical review of the literature and video on the procedures, (4) demonstration of the critical steps, (5) hands-on practice, (6) in-action instructor\u2019s feedback, (7) quality assessment, (8) debriefing at the end of the session, and (9) deliberate and repetitive practice. Time was recorded from the beginning to the completion of the procedure, along with the presence or absence of anastomotic leaks.The participants needed to perform 23.8\u2009\u00b1\u20096.96 GJA (12\u201335) and 24.2\u2009\u00b1\u20096.96 JJA (9\u201343) to attain proficiency. The starting point of the learning curve was higher for the GJA than for the JJA, although the slope and plateau were parallel. Further, four types of learning curves were identified: (1) exponential, (2) rapid, (3) slow, and (4) no tendency. The type of pattern could be predicted after procedure number 8.These findings may help to identify the learning curve of a trainee early in the developmental process, estimate the number of sessions required to reach a performance goal, determine a trainee\u2019s readiness to practice the procedure on patients, and identify the subjects who lack the innate technical abilities. It may help motivated individuals to become reflective and self-regulated learners. Moreover, the standardization of the ID may help to measure the effectiveness of learning strategies and make comparisons with other educational strategies. During the past decade, many studies have strengthened the evidence supporting the use of simulation-based training in surgery , 2. ReseIn response to these results, the American College of Surgeons (ACS) and the Association of Program Directors in Surgery (APDS) in the United States of America (USA) have made efforts to enhance resident training by implementing the National Surgical Skills Curriculum in the USA. This curriculum has been carefully structured and designed by content experts to enhance resident training through a web-based open platform and reproducible simulations . As a stDespite the benefits of the National Surgical Skills Curriculum, the overall rate of its adoption in surgical residency programs is still lagging behind. Many institutes have incorporated simulation labs, but training opportunities are still limited due to the lack of faculty protected time, significant costs, and resident work-hour restrictions . ConsequUntil now, studies have not focused on implementation strategies or ways to improve the efficiency of simulation-based training. Nevertheless, evidence-based instructional design (ID) has become increasingly important when aiming for specific learning outcomes over a specific period of time . In surgThis study describes the types of developmental curves generated by a defined simulation-based ID using ex vivo animal models to teach laparoscopic latero-lateral small bowel anastomosis. Small bowel anastomoses were selected because they are one of the most common procedures performed in gastric and intestinal, elective, and emergency general surgery. Laparoscopy has gained wide acceptance because it allows a faster return to normal activity and diet, reduced hospital stay, and reduced rate of complications  when compared to open access . NeverthThe research was conducted at an ACS/AEI at a university hospital in Spain. The hospital offers resident physician training as well as two fellowship programs in surgery, 900 beds and more than 50 clinical specialties and is affiliated with medical and nursing schools . After 6Twenty general surgery residents  participated in the study. The resident program at our institution includes 11 sequential modules that follow the National Surgery Commission Curriculum and the ACS/APDS Surgery Resident Skills Curriculum. Each module is conducted during a specific period of the residency program and integrates knowledge acquisition and simulation lab practice with clinical practice. The study took place during the \u201cBasic Laparoscopic Intestinal Anastomoses Module,\u201d which includes small bowel and gastric laparoscopic anastomosis. This module is conducted during the second and third years of residency and comprises six sessions (4\u00a0h each) of individual practice in the simulation lab each year (48\u00a0h). The module is scheduled in two consecutive sessions during the first, second, and fourth trimesters. Practice is mandatory and takes place in the afternoon, once clinical work is completed. The rationale behind this program is that daily practice for several consecutive days results in the completion of a standardized exercise while minimizing fatigue for novices , and subResidents were evaluated on their performance of laparoscopic latero-lateral jejuno-jejunal anastomosis (JJA) and gastro-jejunal anastomosis (GJA). All anastomoses were 5\u00a0cm long. They were performed using ex vivo swine small bowel and stomach on an endotrainer. All samples had a standard length of 12\u00a0cm. Continuous absorbable (3/0) suture was performed.In a previous prospective study, the time required to perform an anastomosis with no loose sutures, edge eversion, or leakage in relation to training time was almost parallel for both procedures .Provision of a bibliography and prerecorded videos demonstrating the surgical technique to facilitate knowledge acquisition of the targeted objective . The materials were sent 15\u00a0days in advance for the residents to study and watch on their own. The journal articles and book chapters described and demonstrated actual clinical standards in small bowel anastomoses .Introduction to the simulation session  in order to establish and maintain an engaging context for learning (CMS approach). The specific practices that were identified as useful included clarifying objectives, the simulation environment , roles, confidentiality, and expectations. This involved establishing a \u201cfiction contract\u201d , attending to logistic details, expressing respect for the learners, and showing interest in their perspective.Discussion of the articles and a review of the procedure using the video to facilitate the comprehension of the information previously provided .Live demonstration of the critical steps of the surgical technique by the instructor to facilitate procedural knowledge .Hands-on practice to advance the ability to physically manipulate the laparoscopic instruments and skillfully perform a quick, accurate, and highly coordinated surgical technique .In-action structured feedback (within practice encounters) to promote the learning of a complex skill in the early stages including trial and error (Bloom\u2019s psychomotor domain).Time measurement and quality assessment of the anastomosis following the clinical standards described below.Debriefing at the end of each training session to reflect on the experience and conceptualize what went well and what should be done differently next time (second and third stages of Kolb\u2019s theory). The debriefing followed a \u201cgood judgment\u201d approach, which combined feedback with genuine inquiry. It specified a rigorous reflection process that would help trainees identify and resolve pressing clinical and behavioral dilemmas raised by the simulation (CMS concepts and theories).Deliberate and repetitive practice with the specific objectives detected during the previous session to facilitate expert performance (Ericsson\u2019s theory).The ID was based on Kolb\u2019s model of experiential learning , a revisTimes were recorded from the beginning to the completion of the procedure, and the presence or absence of anastomotic leaks was noted. Samples were filled with colored water to test the tightness of the anastomoses. A previous study established the benchmark for performance standards in the lab following extensive training with abdominal simulators to master the technique. It was defined by the mean anastomotic time for end-to-end anastomoses (50\u00a0min) in animals that survived for 2\u00a0weeks. At the end of that period, a reoperation was undertaken to verify whether there were any leaks, obstructions, or adhesions , 31. TheThe most important technical factor that can influence anastomotic healing and the prevention of leakage is the accurate union of the two bowel ends. This is best achieved by meticulous technique that includes accurate seromuscular apposition, size and spacing between each suture, and tension between the ends . These fU test was utilized to compare medians. The learning curve of each trainee was determined by using the time of the procedures along with polynomial regression and was adjusted to the fifth degree [x values moved farther from the x value of the local point [p\u2009<\u20090.05 was considered statistically significant.Means, median, standard deviation (SD), and interquartile range were employed for numerical variables. In addition, the Wilson Score was used to determine CIs and percentages for qualitative variables, while the Mann-Whitney h degree . To obtaal point . R progral point ; we applal point . p\u2009<\u20090.0The study protocol (code: 2014.216) was approved by the Ethics and Research Committee of the Autonomous Region of Cantabria (Spain), and informed consent was obtained from all participants.p\u2009=\u20090.176 with the Mann-Whitney U test). The average training time was 73.7\u00a0min  for GJA and 57.4\u00a0min  for JJA.A total number of 476 GJA procedures and 483 JJA procedures were performed. Each participant completed an average of 48\u2009\u00b1\u200915.3 anastomoses, including the cases performed after reaching the standards. The number of procedures was slightly lower among male residents (42\u2009\u00b1\u200914.6) compared to female residents (52\u2009\u00b1\u200915.0), although the difference was not significant (The participants needed to perform 23.8\u2009\u00b1\u20096.96 procedures (range\u2009=\u200912\u201335) to attain proficiency. Eighty-five percent of participants attained the standards after 30 anastomoses. Eight residents required between 12 and 20 repetitions, nine residents between 21 and 30 repetitions, and three residents between 31 and 35 repetitions.An individual analysis of the data resulted in the description of four types of learning curves Table\u00a0: type 1:The participants needed to perform 24.2\u2009\u00b1\u20096.96 procedures (range\u2009=\u20099\u201343) to achieve proficiency. Eighty-five percent of participants attained standards after 34 anastomoses. Nine residents required between 9 and 20 repetitions, four residents required between 21 and 30 repetitions, and seven residents required between 31 and 43 repetitions.The individual analysis also enabled identification of the same four types of learning curves Fig.\u00a0: type 1:Figure\u00a0The characteristics of the ID of a simulation-based training course may influence the performance level that the participants can achieve as well as the time needed to attain that level . Our resIn another study that used a standardized technique to teach laparoscopic bowel anastomoses, the learning process required 40 procedures with a simulator , insteadIn our study, time was the principal criterion for determining the learning curve. In a systematic review of minimally invasive abdominal surgery, the procedural time was also the most commonly used variable (86\u00a0%). Other outcomes frequently measured included intraoperative outcomes (56\u00a0%), postoperative outcomes (54\u00a0%), intraoperative technical skills (17\u00a0%), and patient-oriented outcomes . In our study, leakage, an intra- and postoperative outcome, was the main variable assessed, as observed in the majority of the articles revised . The oveDespite the fact that different surgeons are likely to learn at different rates, most studies compare mean duration of the operation between groups . In our Figures\u00a0These findings have potential implications for designing training programs for residents or for experienced surgeons aiming to learn new procedures.This study has implications for how simulation training is implemented in educational curricula. To date, many simulation-based educational programs have been designed to include as many competencies as possible within the time frame available for training, without taking into consideration learning outcomes . This apAnother implication of this study is that it suggests a more effective use of resources available for training. The deep understanding of the different learning patterns generated by a specific ID enables early identification of individual learning needs. This can be detected early in the developmental process (after eight procedures in our results). This helps to plan the probable number of procedures required by a particular individual. Trainees who reach the desired level early can move on to another module, and trainees who need more practice may seek specific advice and feedback early on .Another finding of this performance-oriented individual approach is the need to identify reliable, objective, long-term outcomes, and also develop valid and reliable tools to assess performance . This isproficiency in terms of learners\u2019 achievement will determine a trainee\u2019s readiness to proceed with patients. This also may impact patient safety, as it sets a definable milestone for transitioning to clinical practice.The precise definition of Finally, creating opportunities for individualized instructor-guided training and reflection may help motivated individuals to become reflective and self-regulated learners. They can potentially have the tools to improve their own and other team members\u2019 performance throughout their career .There are several limitations of this study. The learning curve patterns resulted from an individual analysis of the participants and not from integration of all results into groups. This was due to a limited number of trainees, and additional cases are needed to validate the patterns we identified. The 2-year duration of the study was based on the timing of the actual resident training program. Future research should be planned during a shorter time period to prevent the influence of confounding effects while practicing other minimally invasive techniques. Participant characteristics such as previous experience with laparoscopic techniques, video game use, concurrent surgical activity during the study, or innate psychomotor ability testing were not evaluated and might have correlated to the different learning curves. Finally, this study evaluated one ID\u2014there was no comparison group\u2014and the results were compared to other ID studies described in the literature.These findings suggest several paths for future research. Once learning curve patterns have been identified and correlated with a specific ID, studies can search for evidence of the most effective strategies within a design. Findings can be related to the sequence and elements of the ID used to build the training activity or to the educational methods within each element so that they can better support the learning needs of trainees while accelerating learning.We propose several strategies to analyze the impact on the rate of learning. One is \u201cpart-task\u201d training, wherein tasks are deconstructed into parts to be learned separately before practicing the procedure as a whole . AnotherIn our series, an average of 24 procedures were needed to attain proficiency in latero-lateral JJA and GJA according to measurable objectives, clinical standards of care, and a standardized ID. However, individually, the number of cases completed did not correlate well with time and errors. In fact, trainees were able to learn laparoscopic skills at different rates. In this regard, four types of learning curves were identified that could be predicted after eight procedures. These findings suggest that determining progression from a novice level to master level for laparoscopic small bowel anastomosis does not mainly depend on the number of cases, but rather the training needs have to be individualized. While our results indicate several ID principles for developing effective simulation-based experiences, there is still a promising area for research to better understand what drives consistent learning outcomes."}
+{"text": "Material properties depend critically on the packing and order of constituent units throughout length scales. Beyond classically explored molecular self-assembly, structure formation in the nanoparticle and colloidal length scales have recently been actively explored for new functions. Structure of colloidal assemblies depends strongly on the assembly process, and higher structural control can be reliably achieved only if the process is deterministic. Here we show that self-assembly of cationic spherical metal nanoparticles and anionic rod-like viruses yields well-defined binary superlattice wires. The superlattice structures are explained by a cooperative assembly pathway that proceeds in a zipper-like manner after nucleation. Curiously, the formed superstructure shows right-handed helical twisting due to the right-handed structure of the virus. This leads to structure-dependent chiral plasmonic function of the material. The work highlights the importance of well-defined colloidal units when pursuing unforeseen and complex assemblies. Colloidal self-assembly is a unique method to produce three-dimensional materials with well-defined hierarchical structures and functionalities. Liljestr\u00f6m et al. show controlled preparation of macroscopic chiral wires with helical plasmonic superlattice structure composed of metal nanoparticles and viruses. It is well established that for example shape8, charge9, specific nearest neighbor interactions11, and patchiness12 can be used to control the arrangement of colloidal nanoparticles into higher order structures5. Controlling the assembly conditions and interaction strengths is particularly important. The assembly kinetics play a significant role in achieving well-defined structures, as rapid self-assembly can yield kinetically trapped particle configurations even for spherical particles4. To extend the concept of supracolloidal self-assembly to include an increasing number of practical applications, the methods need to be robust and yield desired structures reproducibly, that is, to be deterministic13.Supramolecular self-assembly provides a method to control structural features from the sub-nanometer to the nanometer level. At longer length scales, supracolloidal self-assembly14. Another example shows that cooperative complexation of polyelectrolyte-surfactant systems leads to well-defined self-assembled periodicities, independent of the exact polyelectrolyte-surfactant composition15. Cooperative self-assembly dominated by nucleation and growth can be also utilized on the supracolloidal level. The assembly could result in hierarchical periodic structures, even though the colloidal constituents have a considerably lower mobility than in the polymer-surfactant system. In addition, the supracolloidal cooperative self-assembly can lead to superstructures even at nanoparticle stoichiometries, which could assumedly lead to a complete lack of order.A self-assembly pathway can be deterministic and yield a higher degree of order if the subunits assemble in a cooperative manner. In the context of supramolecular polymerization, cooperative assembly allows non-covalently connected subunits to form ordered structures instead of randomly ordered aggregates18. Furthermore, there are a number of sophisticated examples that show how chiral colloidal particles can be used to guide the formation of finite nanoparticle assemblies with structure dependent optical properties21. In this study, the combination of building units forms processable macroscopic wires with structure dependent optical properties.Here the cooperative self-assembly is demonstrated in a colloidal system consisting of anionic tobacco mosaic virus (TMV) nanorods, which direct the assembly of cationic gold nanoparticles (AuNPs) Fig.\u00a0. NanoparDcore\u2009=\u200912.4\u2009\u00b1\u20090.9\u2009nm), which were functionalized with a covalently linked (11-Mercaptoundecyl)-N,N,N-trimethylammonium bromide (MUTAB) ligand giving the AuNPs a hydrodynamic radius of ~\u200915\u2009nm, a highly cationic surface, and a high colloidal stability at a range of ionic strengths  structural order of multiple components.In this work we used AuNPs with a narrow size distribution  that the electrostatic attraction between them is efficiently screened and the like-charged AuNPs are significantly aggregated. We then sequentially dialyzed the mixture against stepwise decreasing ionic strength, which reinstalls efficient electrostatic attraction and causes the AuNPs to form assemblies with TMV  and cryogenic electron microscopy (cryo-TEM) images. In all the studied samples, the superlattices had a fibrillary morphology. Samples with a relative excess of TMV (nAuNP/nTMV ~\u20091) contained unbound TMVs, whereas all the AuNPs were bound into highly ordered AuNP-TMV superstructures or between two or more TMVs forming intermediate zipper-like configurations  still showed similar superlattice structures  equaled that of a 2D square lattice  hexagonal close packing is typical, as it is the most efficient form of close packing, and it has been reported that at high concentrations TMV forms a 2D hexagonal lattice in inclusion bodies found in infected cells\u03c9  was estimated to be ~\u20090.13\u00b0/nm at its steepest. A similar right-handed helical structure of the superlattice wires is also observed in scanning electron microscopy (SEM) images  and 17\u2009nm (\u2248 a/\u221a2) respectively (23.2\u2009nm and 16.4\u2009nm according to SAXS data). The distance between the observed (10) and (11) directions is roughly 1.5\u2009\u00b5m, corresponding to \u03c9 ~\u20090.03\u00b0/nm as the angle between (10) and (11) planes is 45\u00b0. From fast Fourier transform (FFT) analysis, the periods corresponding to the (21) and (31) lattice planes are also identified, and in total the FFT analysis yields a lattice constant of 24.9\u2009nm. Observing smaller \u03c9 for larger superlattices suggests that the initial nanoparticle level interaction leads to helicity of the superlattice, but \u03c9 is reduced upon superlattice growth.In TEM sample preparation, the superlattice wires settle on the TEM grid perpendicular to the viewing direction, thus inhibiting the direct observation of the cross section of superlattice wires, and hence the 2D square lattice. Therefore, we conducted cryogenic electron tomography (cryo-ET) reconstruction of the samples to verify and amplify our understanding of the superlattice structure. The 2D square lattice was evident from the cryo-ET 3D reconstruction Fig.\u00a0 and 2. Fges Fig.\u00a0, from whThe location of AuNPs relative to TMV in the superlattice axis direction is not particularly constrained, but the distance between adjacent AuNPs, in the same interstitial space, is relatively constant Figs.\u00a0i and 4a.The growth of helical nanoparticle wires with a 2D cross-sectional lattice is explained by an assembly mechanism, which assumes that AuNPs are dynamically moving in the interstitial space. In this model, the right-handed helical twist can be explained in terms of electrostatic interaction between a positively charged 0D object and a negative helical charge distribution; the helical superstructure is a net effect of the strong electrostatic attraction between AuNPs and TMV, and the helical symmetry of TMV  measurements of the water-borne superlattice wires at different stages of the assembly. From the SAXS studies Fig.\u00a0 we confi35. The simulation was based on 400 AuNPs arranged into a helical superlattice structure with a lattice constant of 23.15\u2009nm and an interparticle distance of 16\u2009\u00b1\u20091.6\u2009nm. A right-handed helical structure (\u03c9\u2009=\u20090.13\u00b0/nm) was assumed  were further functionalized with cationic magnetic Fe3O4 nanoparticles  mapping  was directly proportional to (sin\u03b8 \u2219 cos\u03b8)2\u2009+\u2009constant. For a perfect linear polarizer I(\u03b8) would be directly proportional to (sin\u03b8 \u2219 cos\u03b8)2. Functionalization by Fe3O4 nanoparticles induces partial aggregation of superlattice wires, which explains the deviation from an ideal polarizer  yielding collective properties that exceeds the properties of the individual nanoparticles. This requires, however, that both the nanoscale structural features and the colloidal interactions are sufficiently well controlled. It can be expected that the advances in the field of designed protein cagesHere, the geometry of the nanoparticles and the ionic-strength-controlled interparticle interaction leads to a distinct wire-like habit of the superlattices. In addition, we show that functionalizing the superlattice wires with magnetic nanoparticles enables a global control of the anisotropic optical properties exhibited by the superlattice wires. In principle the wire-like habit of the structures also enables industrial scale roll-to-roll fiber processing of hierarchically structured materials. The described self-assembly approach utilizes exactly defined colloidal units and addresses the need for screening new self-assemblies that are required for future materials.Nicotiana tabacum (var. Samson) plants grown in greenhouse were inoculated by rubbing the leaves with wild type TMV stock. After 22 days from inoculation, the upper leaves showing the most severe TMV symptoms were collected from six plants. TMV purification was made starting from 52\u2009g of leaf biomass according to Chapman39. Briefly, the leaves were homogenized in 0.5\u2009M phosphate buffer (pH 7.2) with 1% (v/v) 2-mercaptoethanol. Filtered leaf juice was extracted with butan-1-ol, and following centrifugation, the aqueous phase was collected. PEG 8000 was used in a concentration of 4% to precipitate the TMV. The virus pellet was washed with ice cold 10\u2009mM phosphate buffer (pH 7.2) containing 0.7\u2009M NaCl and 4% PEG and finally resuspended.A month-old \u22121)\u22121 i.e. 118200\u2009cm\u22121\u2009mM\u22121\u200940. On the basis of the UV\u2013vis results the TMV concentration in the starting solution was 34.5\u2009mg\u2009ml\u22121, i.e., 0.876\u2009\u00b5M. The A260/A280 ratio R was measured as 1.03, which indicates a slight excess of protein . In TEM intact TMV particles with a length of 300\u2009nm or longer were observed. The coat protein is known to form single helices at acidic (pH\u2009<\u20096) conditions, which may extend the TMV particles43.The TMV was further purified by sucrose gradient centrifugation. The TMV band was collected and finally dialyzed three times against 10\u2009mM sodium acetate buffer . The TMV concentration and the particle composition were characterized using UV\u2013visible spectrophotometry (UV\u2013vis) and TEM. The absorbance at 260\u2009nm and 280\u2009nm was measured from a dilution series  using PerkinElmer LAMBDA 950 UV/Vis spectrophotometer and a quartz cuvette. At 260\u2009nm TMV has an extinction coefficient of 3.0 was then poured into the boiling solution under vigorous magnetic stirring. The color of the initially yellow solution turned immediately clear. 1\u2009min later the solution turned black and after 7\u2009min the solution was dark ruby red. Heating was stopped after 14\u2009min and the AuNP solution was cooled down at room temperature.The synthesis of cationic AuNPs followed mainly the AuNP synthesis described by Hassinen et al.3) as follows. 275\u2009ml of the citrate stabilized AuNP solution was poured into a separatory funnel. First the pH was adjusted by adding 2.75\u2009ml 0.1\u2009M NaOH. Then 13.75\u2009ml of CHCl3 and 688\u2009\u00b5l OLAM (70% purity) was added and the mixture was shaken vigorously after which the CHCl3 phase was let to separate. The CHCl3 phase was then collected and the residual AuNPs were washed from the remaining aqueous phase by adding 3\u2009ml of CHCl3. The mixture was again shaken vigorously. The red CHCl3 phase was again collected. This was repeated three times after which the aqueous phase was transparent. The collected volume was centrifuged to further separate any residual water from the solution.In the first phase transfer AuNPs were conjugated with oleylamine (OLAM) and transferred into an organic solvent (CHCl2O and 550\u2009\u00b5l of 100\u2009mM MUTAB (dissolved in ethanol) were added to the collected CHCl3 phase. The mixture was shaken resulting in a foamy emulsion, which was dissolved by adding 110\u2009\u00b5l of 1\u2009M HCl and shaking. The sample mixture was then centrifuged to separate the aqueous phase. After centrifugation the mixture consisted of a clear colorless CHCl3 phase (bottom), a foamy emulsion (middle), and a dark ruby red aqueous phase (uppermost). The uppermost phase was collected and washed twice with 5\u2009ml of CHCl3  to remove residual OLAM. Finally the MUTAB conjugated AuNPs were dialyzed three times against milliQ water to remove excess MUTAB and to neutralize the pH.In the second phase transfer AuNPs were conjugated with (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide (MUTAB) and transferred back to aqueous phase as follows. 4.4\u2009ml HDH was measured using DLS (see below). The AuNP number density nAuNP was calculated from the known mass concentration, by assuming a spherical shape for the AuNP, and an average MUTAB surface density of 5.6 MUTAB/nm2. Because of the errors in AuNP size, shape, and MUTAB surface density, the calculated nAuNP are considered to have an accuracy of\u2009\u00b1\u200910%.The AuNP mass concentration in the stock solution was measured by lyophilizing a known volume of AuNP stock solution and weighing the mass, whereas the AuNP core-size distribution was measured using TEM (see below), and the hydrodynamic diameter 3O4) nanoparticles were prepared according to a modified procedure reported by An et al46. In short, 0.86\u2009g of Fe(II)Cl2 \u2219 4H2O and 2.35\u2009g of Fe(III)Cl3 \u2219 6H2O (in 1: 2 molar ratio) were dissolved in 40\u2009ml of degassed MQ H2O. 3.4\u2009ml of 28\u201330% NH4OH was added dropwise into the solution under vigorous stirring (400\u2009rpm) and N2 degassing. Black precipitate formed instantly. The particles were washed with three cycles of magnetic decantation followed by redispersion in degassed MQ H2O. The particles were cationized by coating with -trimethylammonium chloride (EPTMAC). For the EPTMAC functionalization, the magnetic decantation was repeated once more and the particles were redispersed in 60\u2009ml of degassed 1.75\u2009M NaOH. 20\u2009ml of the nanoparticle solution was used to prepare the cationic Fe3O4 particles used in this study. The cationization was carried out according to a modified procedure reported by Hasani et al.47 EPTMAC was added to obtain roughly 1:1 molar ratio of iron and EPTMAC. The vials were filled with N2, sealed and placed in an oil bath at 65\u2009\u00b0C for 4\u2009h under stirring. The vials were removed from the oil and washed with three cycles of magnetic decantation followed by redispersion in degassed MQ H2O to remove unreacted EPTMAC. The pH of the mixtures was lowered to 3.0 with HCl and the vials sonicated for 2\u2009min. Centrifugation (5000 RCF) for 3\u2009min was used to separate and discard large aggregates. The supernatant was collected and characterized by DLS . The mode size (volume fraction) was 58.9\u2009nm and the zeta potential was at +43.6\u2009mV.The iron oxide (FeA800\u2009nm/A522\u2009nm) as measured using UV-vis spectrophotometry (PerkinElmer LAMBDA 950 UV/Vis spectrophotometer). 1\u2009M NaCl solution was sequentially added to 500\u2009\u00b5l of dilute (maximum absorbance <2) AuNP solution to obtain the different NaCl concentrations. After each titration step the sample was mixed properly after which the UV-vis spectrum was measured. After reaching 250\u2009mM NaCl concentration the ionic strength was increased by adding 5\u2009M NaCl solution instead of 1\u2009M NaCl solution to avoid excessive dilution of the sample.The AuNP colloidal stability upon increasing ionic strength was characterized by the aggregation index  with a 4\u2009mW He-Ne ion laser at a wavelength of 633\u2009nm and an avalanche photodiode detector at an angle of 173\u00b0. Experiments were carried out at 25 \u2009\u00b0C. Plastibrand semi-micro PMMA cuvettes were used in the experiment and Zetasizer software  was used to obtain the particle size distributions.The hydrodynamic diameter Conventional transmission electron microscopy (TEM) imaging of TMV particles, AuNPs and AuNP\u2013TMV superlattices was carried out with JEM-2800 High Throughput transmission electron microscope (JEOL) using an acceleration voltage of 200\u2009kV. 3\u2009\u00b5l of the sample was added on 200 mesh copper grid with carbon support film (CF-Quantifoil) and the excess sample was blotted away with filter paper. AuNP samples and AuNP\u2013TMV superlattices were imaged without staining, whereas ammonium molybdate negative staining was used in imaging of the TMV particles.The Cryogenic transmission electron microscopy (Cryo-TEM) images were collected using JEM 3200FSC field emission microscope (JEOL) operated at 300\u2009kV in bright field mode with Omega-type Zero-loss energy filter. The images were acquired with Gatan Digital Micrograph software while the specimen temperature was maintained at \u2212187\u2009\u00b0C. The Cryo-TEM samples were prepared by placing 3\u2009\u00b5l aqueous dispersion of the sample on a 200 mesh copper grid with holey carbon support film (CF-Quantifoil) and plunge freezed using vitrobot with 3\u2009s blotting time under 100% humidity.48. Samples were tilted between\u2009\u00b1\u200969\u00b0 angles with 2\u20133\u00b0 increment steps. Prealignment of tilt image series, fine alignment and cropping was executed with IMOD49. The images were binned 2\u20134 times to reduce noise and computation time. Maximum entropy method (MEM)50 reconstruction scheme was carried out with custom made program on Mac or Linux cluster with regularization parameter value of \u03bb\u2009=\u20090.001.Electron tomographic tilt series were acquired with the SerialEM-software packagecNaCl. The AuNP and TMV (cNaCl\u2009=\u2009500\u2009mM for both) were mixed and set into the dialysis cup. The cNaCl for the dialysate was 500\u2009mM at start and the cNaCl was decreased by 50\u2009mM every 30\u2009min until the desired cNaCl was reached. After reaching the final cNaCl the dialysate was changed one more time and the dialysis was continued for 2\u2009h.The superlattices were formed during dialysis in floating dialysis cups  that floated on the dialysate, which consisted of 10\u2009mM sodium acetate buffer (pH 5.5) and a specific NaCl concentration nAuNP/nTMV\u2009=\u200950) was prepared by mixing AuNP and TMV at 100\u2009mM NaCl concentration without further dialysis.The sample for conventional TEM (\u03bb\u2009=\u20091.54\u2009\u00c5). The beam was monochromated and focused by a Montel multilayer focusing monochromator (Incoatec). The X-ray beam was further collimated by four collimation slits (JJ X-ray) resulting in a final spot size of less than 1 mm at the sample position. A Hi-Star 2D area detector (Bruker) was used to collect the scattered intensity. Sample-to-detector distance was 1.59\u2009m, and a silver behenate standard sample was used for the calibration of the length of the scattering vector q. One-dimensional SAXS data was obtained by azimuthally averaging the 2D scattering data and the magnitude of the scattering vector q is given by q\u2009=\u20094\u03c0 sin\u03b8/\u03bb, where 2\u03b8 is the scattering angle. The measured structure factor S(q) was obtained by dividing the scattering intensity I(q) by the I(q) measured from dissolved AuNPs according to the local monodisperse approximation51. The theoretical S(q) was calculated with PowderCell52.The aqueous samples were sealed between two Kapton foils. The sample environment was evacuated to reduce background scattering from air. The small-angle X-ray scattering (SAXS) was measured using a Bruker Microstar microfocus rotating anode X-ray source  and energy dispersive X-ray spectroscopy (EDX) included liquid propane plunge freezing and subsequent freeze drying of suspensions deposited on TEM grids. SEM imaging was carried out using Zeiss Sigma VP electron microscope and EDX mapping was carried out using JEOL JSM-7500FA analytical field emission scanning electron microscope.\u22121 of AuNP and 3.9\u2009\u00b5g\u2009ml\u22121 of TMV except for the reference AuNP sample which contained 31\u2009\u00b5g\u2009ml\u22121 of AuNP. The samples were measured multiple times using a short measurement time and the sample was gently stirred between measurements to avoid the effect of sedimentation. The final data is an average of at least five short measurements.The circular dichroism spectrometry (CD) measurements were carried out using a Chirascan (AppliedPhotophysics) spectrometer and a high quality quartz cuvette with a 10 mm light path (Hellma QS). The UV-visible spectrophotometry (UV-vis) data was measured simultaneously. The samples were prepared by dialysis according to the description above. All the samples contained in total 62\u2009\u00b5g\u2009ml35. Each nanoparticle is modeled as a 12.5\u2009nm diameter gold sphere, which interacts with an external, circularly polarized electromagnetic field and the fields produced by the other nanoparticles in the system. Quasistatic approximation is used to obtain the polarizability of an individual nanoparticle\u03b1m is the radius of m:th nanoparticle, and \u03b5s=1.8 is the relative permittivity of the surrounding media. For the relative permittivity of the nanoparticle \u03b5mNP, we use tabulated data53. The dipole moment induced in a single nanoparticle is given bym:th particle and Ed \u2212 d is the electric field due to all other dipoles in the systemSimulations of the circular dichroism (CD) signal are done with a coupled-dipole approachrmn = rm \u2212 rn, and nmn = rmn/|rmn|. Eqs.  \u2212 1\u03b1Eext. We solve the matrix inversion numerically. Once the inverted matrix (1 \u2212 \u03b2) \u2212 1 is known, the response of the structure to circularly polarized (\u2009\u00b1\u2009) external fields can be evaluated. The absorption cross-section for the nanoparticle assembly isIn equation  rmn = rn|. Eqs.  and 3)  rmn = rThe CD signal is then obtained by calculating the difference in the absorption cross-sections for different circular polarizations, averaged over solid angle \u03a9 as the structures are free to move in the solution:The authors declare that all data supporting the findings of this study are available from the corresponding authors on request.Supplementary InformationPeer Review FileSupplementary FilesSupplementary Movie 1Supplementary Movie 2"}
+{"text": "HeadCoach trial is to evaluate the effectiveness of a newly developed online training intervention to determine whether it is able to build managers\u2019 confidence to better support individuals within their teams who are experiencing mental ill health, and the confidence to promote manager behaviour likely to result in a more mentally healthy workplace.Within high income countries, mental health is now the leading cause of long term sickness absence in the workplace. Managers are in a position to make changes and decisions that have a positive effect on the wellbeing of staff, the recovery of employees with mental ill health, and potentially prevent future mental health problems. However, managers report addressing workplace mental health issues as challenging. The aim of the HeadCoach, an online training intervention for managers with a focus on the mental health of their employees, compared to a waitlist control. The target sample is 168 managers, and their direct employees. Managers and employees will be assessed at baseline and at 4-month follow up. Managers will have an additional, intermediate assessment 6-weeks post-baseline. The primary outcome is change from baseline in managers\u2019 self-reported confidence when dealing with mental health issues within their team and promoting a mentally healthy workplace. The difference between the intervention and waitlist control groups will be assessed using linear mixed effects repeated measures (MMRM) analysis of variance (ANOVA). Secondary managerial outcomes include mental health literacy, attitudes towards mental health issues in the workplace and managerial behaviour in dealing with mental health matters with their staff. Employee outcomes will be perceived level of manager support, engagement, psychological distress, and rates of sickness absence and presenteeism.We will conduct a cluster randomised control trial (RCT) to evaluate the effect of To our knowledge this will be the first RCT of a purely online training intervention developed specifically for managers that promotes confidence to both support staff experiencing mental ill health and create a mentally healthy work environment. If successful, this intervention has the potential to provide an effective and efficient method of training managers in workplace mental health and to enhance employee wellbeing.ACTRN12617000279325Australian and New Zealand Clinical Trials Registry  Mental ill health has become the leading cause of long-term sickness absence and work incapacity in many high income countries \u20133. The mManagers are in a unique position to manage work-based mental health risk factors and improve the mental health of their workers. Their knowledge of workplace issues, their ability to implement adjustments to working conditions, and their position to lead by example with regards to acceptance and understanding of mental health issues can influence changes and decisions to benefit their workers. As a result, managers may be able to implement reactive and preventative managerial strategies through primary, secondary and tertiary interventions as a means to enhance their employees\u2019 mental health and to aid the recovery process of those workers who have become unwell , 11.Best practice guidelines are available outlining the role managers should play in sickness absence, regardless of the underlying cause of the illness , 13. TheA small body of research, comprising a number of pilot studies and a few small scale controlled trials, has evaluated specialised training programs for managers to promote understanding of mental health problems among workers, with evidence suggesting that managers value and benefit from such initiatives , 15\u201317. There is increasing recognition that best practice in workplace mental health requires an integrated approach that prevents harm, promotes positive mental health, and addresses mental health in the workplace regardless of the cause of the illness . HoweverHeadCoach. The use of online technology through a mobile and desktop compatible website will provide a number of benefits compared with traditional modes of workplace training [The current trial will test an integrated and comprehensive online training intervention for managers called training . AlthougHeadCoach will be delivered to managers with a focus on how to best support the mental health needs of employees reporting directly to them. The primary aim of this trial is to evaluate the effectiveness of the HeadCoach intervention to improve managers\u2019 confidence to effectively respond to the needs of staff experiencing mental health issues and to implement evidence-based managerial techniques that promote a more mentally healthy workplace.HeadCoach will be evaluated via a cluster randomised controlled trial (RCT) conducted within three industry partner organisations. Clusters will comprise of groups of managers based on organisational-specific pre-existing geographical work zones to reduce the risk of contamination between the intervention and control groups. Stratification will occur based on the number of managers within each zone for each industry. A researcher independent from the trial will use a computer-generated program to conduct the stratified randomisation of workplace zones prior to recruitment. Outcome data will be collected from the managers and from employees who report directly to them.HeadCoach online training intervention aims to build managers\u2019 confidence in dealing with mental health issues in the workplace. This includes managers\u2019 confidence when responding to mental illness and/or distress amongst their staff, and the implementation of behaviours supporting more mentally healthy workplaces to help prevent new mental health problems. Building confidence across these reactive and preventative managerial strategies to better manage mental health issues in the workplace is a key step in altering managers\u2019 behaviours across these domains [The  domains . Improvi domains .Common mental illnesses, provides an introduction to mental health issues commonly found in the workplace. The next topic, How to help an employee you are concerned about, discusses ways managers can identify people within their team who may be at risk of mental health issues, how managers can support their workers, and direction around how to best have a conversation with employees who may be experiencing mental ill health. It also discusses how to help employees stay at work or return to work faster following a period of sickness absence due to mental illness. The third and final topic, Minimising mental health risks in the workplace, examines managerial skills that are useful in reducing mental health risk factors in the workplace in order to create a mentally healthy workplace for all employees.The intervention is self-guided and divided into three broad topics to be completed sequentially. The first topic, Each topic comprises between three and seven 10-min modules featuring text, activities, short videos, and practical exercises for individuals to complete. The course outline listing the twelve modules, three topic consolidation exercises and three topic toolboxes providing quick access to useful resources presented in that topic, is outlined in Table\u00a0We aim to recruit both managers and their direct reports from three large industry partner organisations collaborating with this trial. These industry partners come from the emergency services and construction sectors. Together they have 690 managers and 9100 employees reporting directly to these managers who could potentially participate in the trial.To be included in the study, participants must be 18\u00a0years or older; currently residing in Australia; have good English comprehension; and be a current employee of one of the three collaborating industry partners. In addition, participants in the manager group must supervise a team of three or more employees.The process of recruitment will commence with each industry partner distributing an introductory email about the trial to managers at a certain level within each organisation and their direct employees describing the purpose of the research, outlining what is involved in the trial participation, and emphasising the voluntary nature of participation. It will discuss the proposed benefits of the trial to the individual and the industry, outline the independence of the research team from their employer, and advise that all components of the trial may be completed during work hours. Confidentiality of participation and data from their employer is assured.Within the following week, all direct report employees of the managers within each cluster will receive an email from the research team inviting them to participate in the research study. This email will contain a link to the online research platform which employees can click to opt into the study and commence participation. At the completion of the recruitment period for direct report employees, participants at the managerial level will receive a manager-specific email invitation. This staggering of recruitment is designed to ensure that baseline employee data is obtained prior to the managers receiving any intervention. This email will invite managers to take part in the evaluation of an online educational training program about mental health in the workplace. As with the employee recruitment email, this email will include a link for managers to click if they choose to opt into the study.Figure\u00a0Direct report employees who opt into the trial and provide consent via an online consent form will complete a 10-min baseline questionnaire. Demographic details, as well as information relating to own mental health, perceived level of manager support, self-reported absenteeism and presenteeism, psychosocial safety climate and work engagement will be collected. The completion of this baseline questionnaire will be the only requirement for employee level participants until the 4-month follow-up questionnaire. This follow-up questionnaire will be distributed by email and will contain the same assessment items as the baseline questionnaire. A unique code assigned to employee email addresses will be used to maintain participants\u2019 privacy while allowing pre-post data to be linked. Employees who complete the 4-month employee questionnaire will indicate at the completion of the questionnaire whether they wish to be entered into a prize draw for one of three $250 vouchers by providing their name and contact details, with reassurances that this information will be kept separately from their responses and not disclosed to their employer.HeadCoach registration account and complete a baseline assessment questionnaire. Demographic details and self-reported information on mental health literacy, attitudes towards mental health issues, confidence in dealing with mental health issues within the workplace, managerial practices used to support the mental health needs the team, and demographic details will be collected. Managers in the intervention group will then be directed into the online HeadCoach manager training program while those managers in the control group will be advised they will receive access to the training after a waiting period.At the completion of the baseline data collection period for employees, managers will be invited by email to participate in the study irrespective of whether their employees responded to the baseline questionnaire. Managers who agree to participate and provide online consent will create their individual HeadCoach. If a manager finishes all components of the online program earlier in the 6-week training period, they will be invited to complete the questionnaire at that earlier point of completion. This approach is expected to prevent non-response and ensure an accurate measure of the post intervention effect.Post intervention questionnaires will be disseminated by email to the intervention and control groups at 6-weeks after completion of the baseline questionnaire. This 6-week period comprises the training period allocated to managers to complete HeadCoach program.At the 4-month post-baseline assessment, a final data collection for the primary outcomes and other measures will be conducted for all managers and employees participating in the trial. An email will be sent to all manager and employee level participants inviting them to complete the final questionnaire, and to advise of a prize draw offered for its completion. Managers who complete the 4-month post questionnaire will be entered into a separate prize draw from the employees, for one of three $250 vouchers. Following the completion of all data collection, managers in the waitlist control group will be provided with online access to the Managers and employees will complete separate questionnaires at each time point. Data will be collected via a secure, encrypted online research platform and de-identified to maintain the privacy of participants.not at all to extremely confident. This results in an overall confidence score ranging from 6 to 30. The types of scenarios asked about include \u201cInitiating contact with staff on sickness absence leave that you believe might be due to mental illness\u201d and \u201cCreating a work environment that prevents and reduces stress within my team\u201d. The wording of these vignettes aims to focus on managers\u2019 self-efficacy and perceived confidence. As noted above, self-efficacy and confidence are thought to be key determinants of behaviour modification [The primary outcome is managers\u2019 self-reported confidence when responding to staff members experiencing mental health problems and when considering how to alter the workplace environment in order to enhance employee mental health and well-being. Managers\u2019 level of confidence will be assessed at 6-week and 4-month follow-ups, with the primary outcome being considered at 4-months post baseline. Confidence in managing mental health issues and promoting a mentally healthy workplace will be measured using a modified version of a\u00a0previously published supervisor scale . This scfication , with evfication .) with options on a five-point Likert scale ranging from strongly disagree to strongly agree. Managers will also be asked how often they apply a range of managerial techniques that promote a mentally healthy workplace using an adapted version of the HSE Management Standards Indicator Tool [At each of the data collection points, information on a range of secondary outcomes will be assessed at the manager level. Measures that will\u00a0be used\u00a0to help understand the reasons for any shift in confidence include: knowledge about common mental health problems ) ; stigmattor Tool . Finallytor Tool .Secondary outcomes will also be assessed at the employee level, although we acknowledge the possibility that any beneficial impact on employees may not be seen within the timeline of this type of study. Employees\u2019 self-reported levels of well-being and psychological distress will be assessed by the 7-item Short Warwick-Edinburgh Mental Well-Being Scale  and the Demographic information including age, gender, job role and length of service in this role will also be collected for managers and employees.Primary analyses will be undertaken within an intent-to-treat framework, retaining all participants as randomised, regardless of extent of engagement with training or withdrawal from the study. Likelihood based methods (mixed-model repeated measures (MMRM)) will be used to assess significance of change in the two primary manager outcome measures. Clustering will be accommodated by a random cluster membership (work site) factor. A priori planned comparisons of change from baseline to the 4-month endpoint will test the primary hypothesis. An unstructured variance-covariance matrix will be used to accommodate relationships between observations at different occasions of measurement. Stratification variables and any variables found to be substantially imbalanced between intervention arms post randomisation will be tentatively included in these models and retained if statistically significant and influential on outcomes. Length of time from baseline to complete training and associated post-intervention assessment will also be introduced tentatively into models to evaluate the effects of adjustment for possible effects associated with some managers undertaking these assessments earlier than others and earlier than control group participants.Similar analyses of scaled secondary manager outcomes will assess differential change due to intervention arm. Mathematical transformation or categorisation of raw scores may be undertaken to meet distributional assumptions and address any violation of assumptions attributable to outliers. Comparable analyses of employee measures will additionally accommodate the nesting of employees within managers by including a random effect for managers where this is possible.HeadCoach has a benefit comparable to the complete program.Additional per-protocol analyses to assess effectiveness of the program will focus on managers who completed at least one of the three topics. This subgroup will be compared to all responders in the waitlist control group and, secondarily, with managers who completed less of the training material. The latter comparison will inform the dose-response profile of the intervention and, in particular, whether completing only a subset of the earlier components of All tests of treatment effects will be conducted using a two-sided alpha level of 0.05 and 95% confidence intervals. A member of the research team who is blinded to the intervention and waitlist control allocations will perform this analysis. The nature of the intervention will make blinding managers to their group allocation impossible.Our industry partners comprise a total of 42 geographical clusters with an average of 20 managers in each cluster. Participation in this study will be voluntary. Based on pilot data, we assume a conservative participation rate of 20% of all managers, with a drop-out rate of 25%. Based on these assumptions, we anticipate being able to recruit a minimum of 168 managers and to successfully follow up 126. Assuming an intraclass correlation of 0.05 yields a design effect of 1.15, the forecast achievable sample size would have 80% power to detect a moderate effect size of 0.54 using an alpha value of 0.05.This protocol describes a cluster RCT of an online workplace mental health training intervention developed specifically for managers. To the best of our knowledge, this will be the first evaluation of an online intervention that simultaneously addresses how managers can best support and respond to employees experiencing mental health issues, while also promoting primary prevention strategies that may be valuable in creating a mentally healthy workplace for the whole team. Workplace mental health interventions have been found to be effective for particular manager level outcomes such as mental health literacy and mental health promotion behaviours within the workplace , as wellHeadCoach by designing the program to be easily integrated into a managers\u2019 workload and own learning style. The twelve modules and three topic consolidation exercises are approximately 10\u00a0min each in duration. Managers\u2019 may choose to complete one at a time, or to work through as many as they can depending on the time available to them, thus potentially increasing the opportunity for program adherence. Data on program adherence and engagement will be available through the research platform.The dissemination of workplace training via the internet can offer many potential benefits as it offers flexible, yet standardised delivery of educational material, the opportunity for revision of modules, reduced organisational expenses associated with staff attending face-to-face training, and the opportunity to monitor progress through the research platform . HoweverA cluster randomisation design has been selected for this RCT. Although this reduces the power of the sample, which has been estimated as capable of detecting a moderate effect size, the method of randomising clusters of managers based on their geographical location reduces the risk of intervention contamination within worksites. However, in the event of substantial movement of managers between work sites, such contamination may be unavoidable. Within the time frame of this study, such movement is unlikely, but demographic data obtained at baseline and follow up will allow the exploration of any organisational movement.It is also recognised that assigning worksites to the waitlist control condition may also produce some problems. Level of interest and motivation to participate in the trial may wane over time for those in the waitlist control as a result of the delay in being accorded access to the program, and dropout rates have the potential to be higher in the waitlist control group, as incentive to respond to follow up questionnaires may be reduced.A further potential limitation is the selection of a primary outcome based on self-report, particularly as confidence levels may be prone to a social desirability bias. Although confidentiality of data from participants\u2019 employer is assured, some participants may still hold concerns that could influence their choice to respond, or bias their responses towards more favourable reporting of their level of confidence in the performance of certain managerial behaviours.HeadCoach online program may provide a practical and efficient method of training a large number of managers in best workplace mental health practices.Given managers can play a key role in promoting the mental health and well-being of their staff, but that they often report uncertainty about how to best address these matters, it is critical that managers are upskilled and educated, through the latest evidence-based information, in how they can best identify and support the mental health needs of their staff. If effective, the Recruitment commenced in 2017 from all three industry partners. Data collection is ongoing. It is anticipated that data collection will be finalised in early 2018, with data analysis due to be completed by mid-2018."}
+{"text": "CCDC168 has no mutations in the stomach adenocarcinoma (STAD) cohort despite its common presence in other tumor types. Surprisingly, we found that the lack of called mutations was due to a systematic insufficiency in the number of sequencing reads in the STAD and other cohorts, as opposed to differential driver biology. Using strict filtering criteria, we found similar behavior in four other genes across TCGA cohorts, with each gene exhibiting systematic sequencing depth issues affecting the ability to call mutations. We identified the culprit as the choice of exome capture kit, as kit choice was highly associated with the set of genes that have insufficient reads to call a mutation. Overall, we found that thousands of samples across all cohorts are subject to some capture kit problems. For example, for the 6353 samples using the Broad Institute\u2019s Custom capture kit there are undercalling biases for at least 4833 genes. False negative mutation calls at these genes may obscure biological similarities between tumor types and other important cancer driver effects in TCGA datasets.The Cancer Genome Atlas (TCGA) provides a genetic characterization of more than ten thousand tumors, enabling the discovery of novel driver mutations, molecular subtypes, and enticing drug targets across many histologies. Here we investigated why some mutations are common in particular cancer types but absent in others. As an example, we observed that the gene  The Cancer Genome Atlas (TCGA) has been a valuable resource for shining light on tumor genetic and molecular biology, allowing for the move towards targeted therapy oncology clinical trials like NCI\u2019s MATCH . One of CCDC168 is a protein-coding gene with poorly understood function known to be mutated in several cancer types. Studies based on TCGA data have reported that this locus is susceptible to microsatellite-instability events resulting in frameshift mutations in colorectal cancer but not in gastric cancer, and this has been interpreted as a functional distinction between the tumor types [CCDC168 frameshift mutations to occur in stomach adenocarcinomas as well. We therefore hypothesized that the lack of CCDC168 mutations in stomach adenocarcinoma might be due to measurement bias.To understand which processes are most important in cancer development, it is critical to have accurate assessments of which mutations recur in different cancer types. However, there may be other spurious mutation annotations in TCGA due to systematic biases. For example, or types , which wCCDC168 mutations and other TCGA mutations are impacted by measurement bias by considering features in each cancer sample associated with a failure to call mutations. We show that measurement bias associated with the exome capture platform explains the CCDC168 effect. Moreover, we demonstrate how these platform biases affect mutation calling throughout TCGA data. Our results indicate that potentially false negative somatic mutation calls due to insufficient coverage recurrently impact at least 701 genes. Over 8000 samples across a wide variety of TCGA tumor cohorts used the implicated capture kits. Due to these false negatives, different tumor types may be more mutationally similar than previously reported, and the impact of these genes on cancer may have been underestimated.In this work, we have investigated whether CCDC168 mutations, we first manually inspected read depth in individual STAD samples. This revealed low numbers of reads aligning to the CCDC168 locus  lack 68 locus . We then68 locus . Given tCCDC168 lacked mutation calls in 17 cohorts, i.e. over half of all cohorts in TCGA. Other genes with similar behavior included SETD1B and SOX11, which lacked called mutations in 20 and 14 cohorts, respectively.We then searched for other genes with a systematic lack of mutation calls and analyzed whether they had cohort-specific biases. To do this we analyzed non-silent mutations using all TCGA MAF files. 22017 of 22022 genes had at least one cohort where no mutation was called in any of the cohort samples. Here we use the term cohort to refer to TCGA samples from different tissues, e.g. stomach adenocarcinoma, colon adenocarcinoma, etc. To distinguish potential genes subject to cohort-specific measurement biases from those with true low mutation rates, we considered only genes that met minimum criteria for mutational prevalence in the overall TCGA set (Methods). This yielded 136 high-confidence genes having strong cohort-specific biases for mutational absence . We set a minimum average depth threshold of 25x across all bases in a gene\u2019s canonical exons to assess if a gene had sufficient coverage to call a mutation. At this threshold, Mutect has a sensitivity approaching 0.99 for an allele fraction of 0.3 [ cohorts  at the 2CCDC168, i.e. where large numbers of samples within multiple cohorts had insufficient coverage to call mutations. We defined undercovered cohorts as those in which over 75% of samples had insufficient coverage to call mutations for a given gene. The four genes which had multiple undercovered cohorts were the long non-coding RNAs (lncRNAs) MALAT1 and XIST and the protein-coding genes SETD1B and SOX11. The undercovered cohorts of the three protein coding genes had strong similarities, with PRAD tumors in particular uniformly showing insufficient coverage at all three gene loci  Search and Retrieval API (Methods). We found that cohorts annotated as assayed with the Custom V2 Exome Bait capture kit exclusively were undercovered for at least one of SETD1B and SOX11 loci (SOX11 when studied with Roche NimbleGen\u2019s SeqCap EZ Human Exome Library v2.0 kit. In ovarian serous cystadenocarcinoma (OV), where 512 samples lacked sufficient coverage in one of the three protein-coding genes, the 10 samples with sufficient coverage were all measured with Roche NimbleGen\u2019s SeqCap EZ Human Exome Library v3.0 kit  cohort, the usage of the custom kit explained all 120 cases with insufficient coverage at the X11 loci . For theX11 loci , notablyv3.0 kit . These fCCDC168, MALAT1, and XIST each had at least one mutation called in the KIRP cohort.We then expanded our scope to include TCGA cohorts where mutations had been observed, focusing on the five genes described above. Again we found that coverage bias was impacted by capture kit choices. The KIRP cohort used the Custom V2 Exome Bait kit, SeqCap EZ HGSC VCRome kit, and derivatives such as the VCRome V2.1-PKv1 kit. As expected from the intercohort analysis, the samples using the Custom V2 Exome Bait kit had insufficient coverage at the three protein coding loci, whereas the samples using the SeqCap EZ HGSC VCRome kits had insufficient coverage at the two lncRNA loci . This efMALAT1 and XIST. Therefore, these genes are particularly susceptible to underestimation of the mutation rate. Similar effects were observed for the exon capture kits Gapfiller_7m, a proprietary capture kit developed by Roche NimbleGen and used by Baylor University, and Agilent\u2019s SureSelect Human All Exon 38 Mb v2 in these two cohorts.As other examples, we also checked the extent of the capture kit effect in the COAD  and rectMALAT1 and XIST which were due to a complete lack of coverage in their loci  cohort (Methods). We adjusted the definition of an undercovered gene such that 80% of bases must have an average sequencing depth greater than 20 across the 57 samples. We found 4833 undercovered genes using this modified definition, which included  than 20 . SOX11 hing exon , with 55CRLF2 and U2AF1, were a result of absent probe coverage whereas the rest, such as VHL, were due to incomplete probe coverage  were common events across multiple tumor types, including STAD. In each of these cases, such as with ACVR2A  [eoplasms . XIST haated AKT , and as ated AKT . SETD1B  cancers . Framesh cancers  in ways ia (MLL) . Histoneia (MLL) , althougCCDC168 and SMAP1 are sites of microsatellite instability in colon adenocarcinoma but not stomach adenocarcinoma [The finding of poor coverage at 4833 genes, including 53 known cancer genes, by the Custom V2 Exome Bait kit presents an important problem to be aware of in cancer genomic analysis. 1258 of these genes have absent or poor probe coverage spanning the entire coding region, a nontrivial fraction of the genome with little interpretable information. Kit dependencies can bias comparisons between tumor histologies, and likely explain a prior report that arcinoma . The remSeveral strategies should be considered to allow for comparison between samples and cohorts assayed with separate capture kits. The first would be to restrict analyses to regions and genes where sufficient reads occur in both groups, reducing the occurrence of falsely-identified differences. This compromise is acceptable for experiments using newer exome kits where the union of poorly-covered regions will be smaller but may limit analyses with TCGA cohorts using the Custom V2 Exome Bait kit. Another potential strategy would be to pool reads in poorly-covered regions at the cohort level to rescue mutation calls. This would allow for some comparisons that include Custom V2 Exome Bait kit cohorts without discarding information at the expense of cohort-level resolution as opposed to sample-level resolution. The drawback for this method is the increased rate of false-positive associations in order to increase the true-positive rate.In summary, our findings reveal strong undercalling of TCGA mutations in cancer genes due to problems in capturing their exons for sequencing. The five genes that we focused on are merely the most extreme of more systematic biases, as we found at least 4833 other genes that are undercalled in the samples assayed by the Custom V2 Exome Bait capture kit and 2353 in samples assayed by the SeqCap EZ HGSC VCRome capture kit. Such biases may hide shared driver mechanisms in tumors of different histologies, obscuring key differences and similarities between tumors as well as samples within cohorts. In both cases, this would lead to spurious subtyping based on mutational status. TCGA is an invaluable resource for understanding the genetics of cancer, but it is important to be cognizant of its biases. Otherwise measurement issues such as choice of exome capture kit will confound attempts at broad understanding across cancers.To identify genes with potential false negative mutation calls, we analyzed each TCGA cohort\u2019s MAF file obtained from the Genomic Data Commons (GDC). We searched for genes with at least a minimal mutation rate across all cohorts and then identified cohorts that appeared to have a spurious lack of called mutations for that gene. For each non-silent mutation, we looked for cohorts where zero patients have a called mutation in a gene. As a first filtering step, we eliminated rarely mutated genes by retaining only those with at least a 5% mutation rate across cohorts having non-zero mutation calls. There are also many small cohorts where it would not be unlikely for a gene to have zero called mutations, even at a 5% true mutation rate. Therefore only genes where three or more cohorts had no called mutations were considered further, eliminating cases isolated to smaller cohorts. The remaining 136 genes are shown with their respective sets of cohorts lacking mutation calls in Table B in To identify all genes affected by the Custom V2 Exome Bait capture kit, we chose genes with no called mutations in the 14 cohorts using the kit exclusively. Only those genes which had an associated Gencode v22 name were chosen to probe further as GDC\u2019s BAM Slicing API uses Gencode v22 gene names to retrieve reads.We used GDC\u2019s BAM Slicing API to download gene-specific reads for each sample based on the gene-cohort associations in Table B in We used GDC\u2019s Search and Retrieval API to query TCGA sample information for all cohorts. We restricted our query to the WES BAM files for tumor samples only. Retrieved information consisted of filename hash ids necessary for the BAM Slicing API and metadata regarding the whole-exome capture kit used for sequencing.BED files obtained from the respective manufacturer\u2019s websites were converted from the hg19 genome assembly to hg38 with UCSC Genome Browser\u2019s LiftOver, using the default webtool parameters. TCGA genes, defined as a gene with a called mutation in any TCGA cohort, were identified by aggregating all mutations across all 33 tumor types\u2019 MAF files. Exon and CDS coordinates were drawn from canonical exons used previously and retrieved from UCSC Genome Browser\u2019s Table Browser using assembly GRCh38, track GENCODE v24, group Genes and Gene Predictions, and table knownGenes. Of the 22042 genes with a called mutation, 20974 mapped to UCSC canonical exons.https://github.com/TheJacksonLaboratory/GDCSlicing. Bed files of canonical exon coordinates are available as a supplemental file  loci previously identified in TCGA [CCDC168 in this sample do not overlap well with the MSI loci.IGV plot of reads aligned to the (TIF)Click here for additional data file.S2 FigCCDC168 locus. Other genes share this same behavior, namely SETD1B, and SOX11 in the PRAD cohort (B), and MALAT1 and XIST in the LIHC cohort (C).The STAD cohort (A) had no samples with sufficient coverage of the (TIF)Click here for additional data file.S3 FigMALAT1 (A) and XIST (B), corresponding to the poor depth in samples using the kit. On the contrary, the VCRome kit does contain probes for CCDC168 (C) which does have reads in samples using this kit.The VCRome exome capture kit does not contain probes for the loci containing (TIF)Click here for additional data file.S4 FigACVR2A (A), a previously-identified stomach adenocarcinoma MSI loci, appears to have reasonable coverage of the MSI region (third track) in two TCGA-STAD samples. SMAP1 (B) and SPINK5 (C) are MSI loci associated with colorectal adenocarcinoma but not stomach adenocarcinoma. TCGA-STAD samples appear to have poor coverage of the SMAP1 MSI loci whereas the SPINK5 appears to have much higher coverage of the MSI loci.(TIF)Click here for additional data file.S5 FigThe co-cleaning step in the Genomic Data Commons pipeline that incorporates local realignment around indels handles this issue. For example, we performed BWA alignment of reads to microsatellite instability loci in CCDC168 for TCGA-COAD sample TCGA-AD-6889 without the co-cleaning step (top read track). Reads are grouped in 3 sets based on their nucleotide at a known indel . The red circle indicates a locus with multiple read support for an SNV prior to co-cleaning. After co-cleaning (bottom read track), these reads no longer support SNV status.(TIF)Click here for additional data file.S1 TablesSupplemental Tables A-I Table A. STAD CCDC168 CoverageTable B. Genes of InterestTable C. Capture Kit by CohortTable D. Capture Kit Frequency by CohortTable E. SeqCap EZ HGSC VCRome Undercovered Genes and ExonsTable F. xGen Exome Research Panel Undercovered Genes and ExonsTable G. Custom V2 Exome Bait Undercovered Genes and ExonsTable H. Undercovered Cancer Genes and ExonsTable I. STAD MSI Loci.(XLSX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S2 Data(ZIP)Click here for additional data file.S3 Data(ZIP)Click here for additional data file."}
+{"text": "Retinitis pigmentosa (RP) is an inherited retinal dystrophy that courses with progressive degeneration of retinal tissue and loss of vision. Currently, RP is an unpreventable, incurable condition. We propose glycogen synthase kinase 3 (GSK-3) inhibitors as potential leads for retinal cell neuroprotection, since the retina is also a part of the central nervous system and GSK-3 inhibitors are potent neuroprotectant agents. Using a chemical genetic approach, diverse small molecules with different potency and binding mode to GSK-3 have been used to validate and confirm GSK-3 as a pharmacological target for RP. Moreover, this medicinal chemistry approach has provided new leads for the future disease-modifying treatment of RP. Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa (RP) are disorders that have different etiology and pathogenesis and where the progressive impairment of diverse retinal cell types leads to vision loss. However, at the cellular and molecular level, the response to retinal injury is similar in all of them, and results in morphological and functional impairment of retinal cells. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these diseasesAmong other retinal degenerative disorders, RP and glaucoma course with neuronal death of photoreceptors and retinal ganglion cells, respectively. RP, together with other inherited retinal dystrophies, comprises a genetically heterogeneous, although clinically similar, group of neurodegenerative retinal disorders caused by mutations in more than 250 different genes. These genetic defects disrupt the function of photoreceptor cells which gradually causes night blindness, progressive constriction of the visual field and eventually blindness,The retina is a highly specialized sensory tissue that belongs to the central nervous system. It may be considered as an accessible, external part of the brain, as both derive from the most rostral neural tube,50\u2009=\u20092\u2009mM) has been used to reduce intraocular pressure in a rat model of glaucoma when it was intraperitoneally administered,Glycogen synthase kinase 3 (GSK-3) is a constitutively active Ser/Thr kinase ubiquitously expressed in the human body. Although it was first identified as an enzyme involved in glycogen synthesisex vivo studies that show the potential of GSK-3 inhibitors to protect retinal cells and, thus, their potential to be translated into treatments to delay vision loss, particularly in RP.Based in our previous experience with GSK-3 inhibitors as drug candidates for neurodegenerative disorders1, 2 and tideglusib have been synthesized in our laboratories following their previous described proceduresCompounds rd10 mouse model of RP is a homozygous recessive mutant for phosphodiesterase 6b (rd10/rd10Pde6b) on a C57BL/6J background. It was kindly provided by Bo Chang from The Jackson Laboratory . Wild type (WT) control mice of the same background were obtained from The Jackson Laboratory. All animals were housed and handled in accordance with the ARVO statement for the use of animals in ophthalmic and vision research, European Union guidelines, and those of the local ethics committees of the CSIC and the Comunidad de Madrid. Mice were bred in the CIB core facilities on a 12/12-h light/dark cycle.The rd10 and P42\u201343\u2009WT mice were euthanized, and their eyes were enucleated. Retinas were dissected and cultured on Teflon filters in R16 medium as describedrd10 mouse retinas were cultured for 24\u2009h. Compound 1 was employed at 3.2\u2009\u00b5M, compound 2 at 10\u2009\u00b5M and tideglusib at 10\u2009\u00b5M. Retinas were subsequently fixed in 4% (wt/vol) paraformaldehyde in phosphate buffer 0.1 M, pH 7.4 for 1\u2009h at RT and processed for the detection of cell death.P23 z-axis retinal sections, to ensure that TUNEL-positive nuclei belong to ganglion cells or to photoreceptors, respectively. Direct counting of TUNEL-positive cells was carried out on merged images using ImageJ v1.48.s software. Paired statistical analysis was performed using the Wilcoxon signed-rank test.Ganglion cell and photoreceptor cell death was visualized by DNA fragmentation assay terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling  as describedex vivo studies. The chemical genetic rationale postulates that different small chemical probes assayed in different in vitro, ex vivo and/or in vivo studies contribute to decipher the role of a potential therapeutic target,,1, 2 and Tideglusib have previously been tested in cell cultures and animal models showing no toxicityIn order to confirm the therapeutic relevance of GSK-3 modulation on retinal pathologies, we employed a chemical genetic approach in 1 and 2) in retinal explants obtained from rd10 and cultured over Teflon discs (Millipore), as exemplified in First we assayed the two more novel inhibitors Next, the retinal explants were obtained from wild type (WT) mice and cultured as above in the presence of NMDA treatment induced massive cell death in the ganglion cell layer that was significantly reduced when the retinas were co-incubated with either one of the two GSK-3 inhibitors . These r1 and 2) and shows a non-ATP, non-substrate competitive mechanism of GSK-3 inhibition. The treatment of rd10 mouse retinal explants with tideglusib significantly reduced photoreceptor cell death (Finally, we confirmed the pro-survival effect of GSK-3 inhibition in the RP model with a third GSK-3 inhibitor, tideglusib. This compound has a chemical scaffold different to the other two inhibitors described above (compounds ll death , an obseex vivo models of retinal pathologies, including RP. The use of mechanistically and structurally diverse GSK-3 inhibitors allows us to state that the neuroprotective effect was produced by the target inhibition, highly reducing the possibilities of cross-reactivity based on the structure or inhibition type. The results of these studies suggest that GSK-3 inhibitors are a potential pharmacotherapeutic strategy to rescue photoreceptor cells, as well as retinal ganglion cells, from a progressive degeneration such the one present in RP. Furthermore, results obtained upon tideglusib treatment in the rd10 model leads us to propose this compound as a clinical lead for RP.During the last years a large body of evidence shows that GSK-3 pathway is altered in neurodegenerative conditions of the central nervous system and small molecules targeting GSK-3 selectively have been developed as disease-modifying drugs for several unmet diseases. However, the role of GSK-3 inhibitors in retinal pathologies has not been extensively studied although they may represent potential therapeutic candidates for retinal degeneration therapy. Hereby, we have shown that GSK-3 inhibition results in a protection of retinal ganglion cells and photoreceptor cells, an observation made with three different inhibitors in two different Finally, the transient benefit observed in RP patients after oral intake of indirect GSK-3 inhibitor valproic acid, suggests and supports the clinical therapeutic potential of GSK-3 inhibitors for the treatment of RP. Together with the results here shown, selective GSK-3 modulation emerges as a new therapeutic target for the treatment of RP, opening new lines of research to discover an effective pharmacotherapy for RP and other retinal diseases."}
+{"text": "Age-associated macular degeneration (AMD), which leads to loss of vision at its end stage, is one of the most common neurodegenerative diseases among the elderly. However, to date, no effective drug therapy is available for the prevention of AMD. Here, we report the occurrence of AMD pathology and its prevention by chronic treatment with the neurotrophic peptidergic compound P021, in aged rats and 3xTg-AD mice. We found photoreceptor degeneration, lipofuscin granules, vacuoles, and atrophy in retinal pigment epithelium (RPE) as well as Bruch\u2019s membrane (BM) thickening; in aged rats, we even found rosette-like structure formation. Microgliosis and astrogliosis were observed in different retinal layers. In addition, we also found that total tau, phosphorylated tau, A\u03b2/APP, and VEGF were widely distributed in the sub-retina of aged rats and 3xTg mice. Importantly, chronic treatment with P021 for 3 months in rats and for 18 months in 3xTg mice ameliorated the pathological changes above. These findings indicate the therapeutic potential of P021 for prevention and treatment of AMD and retinal changes associated with aging and Alzheimer\u2019s disease. Age-associated macular degeneration (AMD) involves the degeneration of the small central area of the retina, called macula, that controls visual acuity. AMD is a leading cause of vision loss in the elderly. In AMD, the macula is irreversibly destroyed, which leads to loss of vision required for activities such as driving, recognizing faces, reading, and seeing in color , choriocapillaris, and photoreceptors. The wet AMD is neovascular. It occurs when new blood vessels invade from the choroid and penetrate Bruch\u2019s membrane (BM), causing vascular leakage, hemorrhage, and scarring. Wet AMD is less common than the dry form but choroidal neovascularization (CNV) in this form leads to vision loss. Dry AMD is believed to result from the aging and thinning of macular tissues, deposition of pigment in the macula, or a combination of these two processes  that is orally bioavailable and is blood-brain barrier (BBB)\u2013permeable; it enhances dentate gyrus neurogenesis and neuronal plasticity by competitively inhibiting the leukemia inhibitory factor and by increasing the expression of brain-derived neurotrophic factor . Other chemicals were from Sigma-Aldrich .The primary antibodies used in this study are listed in AG-NH2; mol. wt. of 578.3) corresponds to a biologically active region of human CNTF (amino acid residues 148\u2013151) to which adamantylated glycine was added at the C-terminal to increase its stability and lipophilicity  and in artificial intestinal juice (>95% for 120 min). BBB studies on P021, which were carried out through a commercial service , demonstrated that a sufficient amount of P021 crossed the BBB to exert its effect in the brain  weighing approximately 300 g were used. Fisher rats are commonly used for studies on aging, and the aged animals are commercially available. Unlike males, which continue to add weight, female rats present a relatively stable weight of ~300 g from ~3 months to 24 months, and we successfully used these animals for our previous study, in which we investigated the chronic effect of compound P021 on cognitive aging  in pathogen-free facilities with 12-h light/12-h dark cycles. All animal studies were carried out according to the National Institutes of Health guidelines for the care and use of laboratory animals. Oral treatment of rats with P021 or vehicle was carried out as a paid service at Charles River Labs, Finland, and was approved by the National Animal Experiment Board, Finland.Animals were housed at a standard temperature (22 \u00b1 1\u00b0C) and in a light-controlled environment (lights on from 7 AM to 8 PM), had access to food and water n = 7) were given P021 per os by gavage (10 ml/kg body weight) once a day for 88 days. The dose of P021 was 500 nanomoles (289.15 \u03bcg)/kg body weight daily; the dose was based on our previous study, in which we found that this dose could rescue cognitive impairment in aged Fisher rats  and a group of young adult female rats  were treated identically, but with normal saline (vehicle) only  Fisher rats (le) only . Adminisn = 6\u20137 mice per group; At approximately 3 months of age, the female wild type and 3xTg-AD mice were divided into five groups . Eyeballs were dissected out from the carcasses, immersion-fixed for 24 h in 4% paraformaldehyde at 4\u00b0C, and then transferred to 70% alcohol for storage; in the case of mice, the eyeballs were prefixed by injecting 4% paraformaldehyde, followed by removal from the carcasses and immersion fixation as above. After the cornea and lens of the left eyes were removed, the remaining eye cup was embedded in paraffin, with the orientation parallel to the optic nerve in longitudinal position. In all cases, 5-\u03bcm serial sections were cut by using a rotary microtome and were mounted on Superfrost Plus slides. Processing and analysis of the retinal tissue were conducted blind.n = 5\u20137 rats per group; n = 5\u20136 mice per group) were manually counted in three columns per image by using a 40\u00d7 or, where necessary, a higher magnification objective. The thicknesses of the ONL and INL of the central and peripheral retina in each group  were measured and quantified by using the ImageJ/NIH image analysis system. Processing and analysis of the retinal tissue and the data were conducted blind to the genotypes and the treatment of the animals.Every 25th retinal section was deparaffinized and subjected to hematoxylin and eosin (H & E) staining to evaluate the morphology. The H & E staining images were captured with a light microscope using 2\u00d7, 40\u00d7, and 100\u00d7 objective lenses. Because there is no significant difference between the 1/5th to the 1/3rd of the retina from the optic nerve head and the most peripheral part, we chose the 1/5th position of the retina from the optic nerve to the most peripheral part of the retina as the \u201ccentral,\u201d and the most peripheral part of the retina as \u201cperipheral.\u201d Rows of the outer nuclear layer (ONL) and the inner nuclear layer (INL) of the retina in each group , as described previously .Paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling for 20 min in 10 mM sodium citrate solution (pH 6.0). The sections were then washed three times in 10 mM PBS for 15 min each and incubated in 0.3% Triton X-100 for 30 min. The sections were again washed in 10 mM PBS  and blocked in blocking solution  for 45 min. The sections were then incubated overnight at 4\u00b0C with the corresponding primary antibodies . Based on the experimental design, At as early as 12 months of age, OXYS rats and Wistar rats are known to show AMD-like pathology, such as BM thickening and lipofuscin accumulation  or vehicle feed until they reached 21 months of age. We used WT-3 m and 3xTg-3 m mice without any treatment as young controls . ParaffiDisruption and degeneration of RPE are well known to occur in AMD , but also in the Cho of the ~22- to 24-month-old/Veh rats , but theAltogether, these findings indicate that microglia are distributed more widely in the whole retina of aged mice and rats, especially in the ONL, which is associated with photoreceptor degeneration shown in , 3, and Astrocytes are another key cell type involved in macular degeneration. Previous studies showed activated astrocytes in the retina of Tg2576 mice at 14 months  that exhibit several features of AD , rosette-like formation in the photoreceptor cell layer, RPE disruption, accumulation of lipofuscin and vacuoles in RPE, increase in auto-fluorescence of RPE, BM thickening, and the formation of basal deposits between RPE/BM and Cho in aged rats, mice and 3xTg-AD mice. P021 conferred protection for the retina against this age- and disease-related damage. Even neuroinflammation detected by microgliosis and astrocytosis was ameliorated by P021 treatment. Furthermore, P021 prevented the increase in tau and A\u03b2 pathologies and VEGF deposition in the sub-retina. These findings are the first demonstration of AMD-like pathology in aged rats, and 3xTg-AD mice and its rescue by a neuro-regenerative compound, P021.SWE/PS1\u0394E9 mice at ~9\u201312 months of age , impairments of retinal structure and function  and were carried out according to the guidelines of the National Institutes of Health.YL carried out the study and wrote the article. WW studied the autofluorescence and analyzed the data. NB treated the animals and extracted the eyeballs and fixed them in paraformaldehyde. FL helped in discussing the data and writing the article. KI conceived, designed, supervised the study and wrote the article.The authors have a U.S. patent on neurotrophic peptides, which includes compound P021, and additional patent-related applications are in the process. The animal studies conform to the National Institutes of Health guidelines and were approved by our institutional IACUC.This paper is dedicated to Dr. Inge Grundke-Iqbal, who along with KI conceived this study. Dr. Grundke-Iqbal passed away on September 22, 2012.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": "Indocyanine green videoangiography (ICG-VA) is a near-infrared range fluorescent marker used for intraoperative real-time assessment of flow in cerebrovascular surgery. Given its high spatial and temporal resolution, ICG-VA has been widely established as a useful technique to perform a qualitative analysis of the graft patency during revascularization procedures. In addition, this fluorescent modality can also provide valuable qualitative and quantitative information regarding the cerebral blood flow within the bypass graft and in the territories supplied. Digital subtraction angiography (DSA) is considered to be the gold standard diagnostic modality for postoperative bypass graft patency assessment. However, this technique is time and labor intensive and an expensive interventional procedure. In contrast, ICG-VA can be performed intraoperatively with no significant addition to the total operative time and, when used correctly, can accurately show acute occlusion. Such time-sensitive ischemic injury detection is critical for flow reestablishment through direct surgical management. In addition, ICG has an excellent safety profile, with few adverse events reported in the literature. This review outlines the chemical behavior, technical aspects, and clinical implications of this tool as an intraoperative adjunct in revascularization procedures. Revascularization procedures are considered in the management of various cerebrovascular disorders, including complex intracranial aneurysms, moyamoya disease (MMD), symptomatic cerebral ischemia recalcitrant to standard medical therapy, and cranial base tumors that require a proximal major vessel sacrifice. MMD is a pathological condition that is characterized by a bilateral progressive narrowing at the terminal portion of the intracranial internal carotid arteries. This stenosis may lead to occlusion of the vessel lumen and a consequent abnormal, compensatory collateral neovascularization at the base of the brain. Superficial temporal artery (STA)-middle cerebral artery (MCA) bypass is the main direct revascularization procedure performed, and it is effective in reducing the risk of ischemic and hemorrhagic strokes as well as in improving cerebral ischemia \u20133.Microscope-integrated indocyanine green videoangiography (ICG-VA) is a relatively recent technique used as an intraoperative adjunct for real-time assessment of anastomotic patency. Indocyanine green was approved by the US Food and Drug Administration (FDA) in 1956 for the evaluation of cardio-circulatory and liver functions. In addition, the FDA approved ICG for use in ophthalmic angiography in 1975 . Since t43H47N2NaO6S2) is a tricarbocyanine dye with near-infrared fluorescent properties.ICG  light range. The NIR peaks allow greater transmission of the fluorescence signal through the native tissue compared with the fluorophores, with peaks in the visible light range , 8\u201310. IOn intravenous administration, ICG binds to alpha-1 lipoproteins, belonging to the globulin family, and remains intravascular as long as the vascular wall integrity is preserved. This fluorescent compound does not enter the enterohepatic circulation and is not metabolized in the body. It has a plasma half-life of 3\u20134 min and is excreted exclusively by the liver. Despite its relatively good safety profile, the ICG molecule or the 5% sodium iodine contained in the drug can potentially cause adverse reactions  that blocks both the excitation and the ambient light. An infrared-sensitive camera on the surgical microscope detects the ICG fluorescence and translates the signal into a real-time white image on a black background. Recently, an innovative dual-image videoangiography system was developed to overlay visible light and NIR fluorescence images and allow the simultaneous intraoperative visualization of both ICG fluorescence and surrounding anatomical structures .Before activating the INFRARED 800 mode to perform ICG-VA, the focus distance and the magnification should be set at <300 mm and less than 5\u00d7, respectively. Afterward, the surgeon can adjust these two parameters during the INFRARED 800 recording to optimize the image. However, modern surgical microscopes usually assist the user with an automatic function that fine-tunes the settings to render the highest quality images. The KINEVO 900 (Carl Zeiss) provides an AutoZoom function that automatically adjusts the total magnification and diaphragm opening to the preconfigured values at the start of ICG-VA and an AutoGain function that automatically adjusts the camera gain to fit the NIR signal intensity within the dynamic range of the camera as the ICG-VA progresses. Chromatic aberration due to higher magnification and surgical field depth account for the inability of electromagnetic radiation of different wavelengths to focus on the same positions in the focal plane. Through the use of special lenses, apochromatic optics adjust light arrays of different colors to converge to the same focal point. This method achieves a variable degree of compensation that is more significant in the visible light spectrum (400\u2013700 nm). Chromatic aberration of NIR light, like the ICG emission light (820\u2013900 nm) can be compensated the most at working distances of <300 mm and at low magnification (<5\u00d7). The use of zoom and focal values other than those recommended can increase the focal point discrepancy between the NIR and visible light images and affect the quality of ICG-VA, resulting in a blurry NIR image.The usual dose of ICG used is 0.2\u20130.5 mg/kg. At the time of administration, 25 mg of the dye is dissolved in 5 mL water and flushed with 10 mL of normal saline as a single intravenous bolus. Once the INFRARED 800 mode is activated, the ICG solution is injected through a peripheral line. ICG can cause lower values of peripheral oxygen saturation to be falsely displayed. The surgeon should manipulate and mobilize anatomical structures through the operating oculars of the surgical microscope to ensure that the bypass graft is visible. Although NIR fluorescence has significantly greater penetration depth than visible light, the blood flow can be appreciated only in exposed blood vessels that are not obscured by anatomical structures, brain parenchyma, surgical instruments, or patties. The ICG fluorescence signal cannot be visualized directly through the operating oculars but is seen on the monitor of the surgical microscope following image processing , 14. TheAn ICG-VA image takes ~10\u201330 s to appear on the screen. Once the recording is ended by the user, the automatic playback mode of the operating microscope allows the surgeon to assess the quality of the imaging in detail. ICG-VA can be repeated over time without any significant interference from the residual fluorescence of the previous injection. However, ICG should be reinjected after 20 min to guarantee adequate clearance of the tracer and optimize contrast enhancement , 14. RepICG-VA can also analyze the intraluminal flow rate and flow velocity in selected regions of interest. In addition, a graph is generated to display the results of this quantitative analysis in a map delay time. The time characteristics of the graph allow the evaluation of the time to peak, defined as the interval from uprise of a wave to highest signal intensity, and washout time, represented by the interval from uprise of a wave to the time of rapid fall of signal intensity.During bypass surgery, the assessment of intravascular graft patency is essential to reduce the rate of graft failure. Early intraoperative identification of an occluded graft artery is critical for prompt surgical correction and for early restoration of blood flow to ensure a successful bypass. Several characteristics of the donor and graft vessels have been found to affect bypass function, including the graft diameters and the extent of focal atherosclerotic change . Other cDirect intraoperative visual inspection of the anastomotic site cannot always accurately determine the patency of the graft vessel. The postoperative graft patency rate reported in the existing literature for an extracranial-intracranial (EC-IC) bypass is 87\u201396% \u201322. HoweHowever, these techniques have major drawbacks in terms of spatial resolution and image quality, such that blood flow in small perforating arteries cannot be evaluated.MR and CT angiography are effective non-invasive radiological modalities for evaluation of the patency of the bypass in the early postoperative period . ConventAs mentioned earlier, ICG-VA is a cost-effective and easy-to-use technique that provides real-time visualization of the fluorescent tracer inflow within cerebral vessels. ICG-VA is ideal for the intraoperative assessment of graft patency, and it seems to be superior to standard angiographic techniques . It consThe remarkable spatial resolution of ICG-VA allows assessment of the morphology of graft patency and the hemodynamic of the bypass functioning . This diIn a previous series, Woitzik et al. reported bypass function in 31 of 35 (89%) STA-MCA bypasses, 2 of 2 (100%) STA-PCA bypasses, and 6 of 8 (75%) high-flow bypasses . Four STJanuszewski et al. described three different types of flow through a graft: Type I, robust anterograde flow; Type II, delayed anterograde flow; and Type III, anterograde delayed flow but with no continuity to the bypass site or no convincing flow. This study analyzed 36 EC-IC and intracranial-intracranial (IC-IC) bypasses performed in 33 ischemic patients with hemodynamic compromise despite maximal medical therapy . In theiA quantitative analysis of flow was performed by Awano et al. using a specialized image analysis software . The ICGIn recent years, several studies investigated the role of semi-quantitative and quantitative analysis of intraoperative ICG-VA findings in predicting the occurrence of postoperative neurological events after revascularization surgery in MMD. A variety of transient neurological events (TNEs) have been reported in the immediate postoperative period after STA-MCA bypass, which is most commonly employed in MMD treatment. TNEs occur in 14\u201377% of patients after MMD bypass surgery  and inclSimilarly, Uda et al. analyzed ICG-VA using specialized software  that allows the recording of several parameters in different regions of interest around the anastomotic site, including CBF, mean transit time, mean gradation time (Grad), and time to peak (TTP). Percentage change of these parameters after bypass was calculated using the formula (\u0394X = (Xafter - Xbefore)/Xbefore). They demonstrated that patients with \u0394TTP \u2264 \u221212, \u0394CBF \u2265 8, or \u0394Grad \u2265 30 have a higher risk of developing postoperative TNEs . Larger EC-IC bypass surgery, in particular STA-MCA anastomosis, is the most common revascularization procedure for the treatment of cerebral ischemia. Designed and successfully performed for the first time by Ya\u015fargil in 1967 \u201350, thisPe\u00f1a-Tapia et al. first described a new application of ICG-VA to identify a cortical target point and find an adequate recipient vessel . They usPrevious studies have shown that the application of ICG-VA is not limited to the identification or safe dissection of the STA in bypass procedures. In fact, this intraoperative technique has also been used to selectively target the most suitable cortical M4 recipient artery for flow-preserving bypass to trap complex MCA aneurysms . AlternaThe senior author has previously introduced the \u201cflash fluorescence\u201d technique , 2 to seFluorescence videoangiography has gained significant traction in the recent years in the practice of complex cerebrovascular neurosurgery. Despite the widespread acceptance of ICG-VA in neurosurgical procedures, the recent reemergence of fluorescein sodium as a fluorescent tracer has prompted authors to investigate and compare fluorescein videoangiography (FL-VA) with ICG-VA. An objective analysis was recently conducted by applying quantitative metrics to data derived from video capture images, which were subsequently elaborated by pixel intensity-analyzing software . In thisThe widespread acceptance of ICG-VA is attributable to its ease of use along with its diagnostic accuracy, low cost, and good safety profile . AnotherIn recent years, a few novel techniques in the application of ICG-VA have been described. These techniques include the introduction of a \u201csecond window\u201d ICG technique for glioblastoma resection that capitalizes on the inherent property of permeability of the tumor vasculature. A high dose of ICG is delivered ~24 h before surgery, leading to non-specific accumulation of the tracer within the tumor tissue. This technique may allow real-time intraoperative tumor identification to optimize surgical resection . AdditioThe contribution of ICG-VA has also been investigated for other types of revascularization procedures including coronary artery bypass. The calculated sensitivity and specificity of ICG in detecting 50% stenosis were 83.3 and 100%, respectively . HoweverICG-VA has been widely established as a useful technique to perform qualitative analyses of graft patency and CBF during revascularization procedures. The widespread incorporation of ICG-VA is attributable to its ease of use, diagnostic accuracy, low cost, and excellent safety profile.CC: literature review, manuscript draft, revision, and final draft approval. SG: manuscript draft, revision, and review of final manuscript. XZ and EB: manuscript draft and review of final draft. DV: literature review and final draft review. PN, MP, and ML: study supervision, review, and revision of the final draft.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": "The ideal prophylaxis duration for transrectal ultrasonography-guided prostate biopsy is incompletely defined.To compare the infectious complications of transrectal ultrasonography-guided prostate biopsy with and without extended antibiotic prophylaxis. The secondary aim was to evaluate the risk factors for infectious complications.Prospective observational study.rd and 7th day after the procedure. Medical histories of all patients were collected prior to biopsy. Information on medical history include the following: hospitalization, urethral catheterization, or urinary tract infections within the past 12 months; antibiotic use within the last 3 months, prior urinary tract interventions, and previous transrectal ultrasonography-guided prostate biopsy and Charlson comorbidity indexes. Ultrasound-guided biopsy was carried out using General Electric\u2019s 7 MHz transrectal ultrasound device in the left decubitus position. Patients received one of the two ciprofloxacin-based prophylaxis regimens. Subsequent transrectal ultrasonography-guided prostate biopsy to all patients were followed-up for 30 days. Further follow-up of patients was carried out on the second and fourth weeks after transrectal ultrasonography-guided prostate biopsy, and symptoms, such as dysuria, rectal bleeding, fever, hematospermia, hematuria, and pollakiuria, were recorded.Four hundred patients who underwent transrectal ultrasonography-guided prostate biopsy were recruited. Patients orally received either 750 mg ciprofloxacin 60 min before the procedure or 500 mg ciprofloxacin twice a day for a duration of 7 days with the initial dose administered 24 h prior to the procedure. All patients were followed-up for 4 weeks after the transrectal ultrasonography-guided prostate biopsy procedure for infectious complications. Screening of urine was carried out in all patients on the 3Both groups presented similar baseline characteristics and medical history. Infectious complication rates within the 4-week follow-up were similar in both groups (single dose: 3% vs prolonged: 3%) (p>0.05). In both groups, infectious complications significantly increased than that at previous antibiotic usage . Non-infectious complication ratios showed no significant variation (p>0.05).Pre-operative single dose of 750 mg oral ciprofloxacin compared with 7 days prolonged treatment resulted in similar infectious complication outcomes in patients undergoing transrectal ultrasonography-guided prostate biopsy. The use of antibiotics within the last 3 months increases the risk for post-transrectal ultrasonography-guided prostate biopsy infectious complications. Within the last few decades, the prevalence of transrectal ultrasound-guided prostate biopsies (TRUS-bx) has increased worldwide owing to the use of prostate-specific antigen (PSA) for screening of prostate cancer (PCa) ,2. AlthoThe increase in antimicrobial resistance (AMR) is a threat toward the success of TRUS-bx prophylaxis and to the whole health care system. An important reason for the increase in AMR is the high environmental antibiotic pressure. For this reason, antibiotic stewardship programs and controlled prophylaxis practice are important tools to use. Unfortunately, the misuse of antibiotics for prophylaxis commonly occurs in the field of urology . This siWe conducted a prospective observational study in our hospital between February 2015 and December 2017. All patients who required a TRUS-bx for the suspicion of PCa were eligible for the study (Supplement 1). These patients received either a single-dose prophylaxis or prolonged antibiotic administration (details explained below). The exclusion criteria of the study are as follows:- Previously known hypersensitivity to ciprofloxacin; - Gastrointestinal diseases preventing oral ciprofloxacin treatment;- Diagnosis of UTI prior to TRUS-bx;- History of endoscopic manipulation or catheterization within the last 10 days;- Conditions that require particular antibiotic prophylaxis practices ;- Immunosuppression .Institutional ethical approval was received from the local ethical board. All patients were informed about the procedure, and possible complications and patient consenting for the study were included in the study. Overall, 437 patients agreed to join the study, and 400 completed the follow-ups. Medical histories of all the patients were collected prior to biopsy. The risk factors associated with the increased risk of health care-associated UTI were collected ,20,21,22Ultrasound-guided biopsy was carried out using General Electric\u2019s 7 MHz transrectal ultrasound device. The biopsy was performed with patients in the left decubitus position and with their knees pulled up to their abdomen. Disposable needles were used for the procedure. Patients were asked not to use any anticoagulant or non-steroid anti-inflammatory medicine during the preceding week of the biopsy. Periprostatic local anesthetic was administered to all patients prior to the biopsy procedure . FollowiPatients received one of the two ciprofloxacin-based prophylaxis regimens. The first regimen included oral administration 500 mg ciprofloxacin twice a day for 7 days with the first dose started 24 h prior to the biopsy. The second regimen included a single oral dose of 750 mg ciprofloxacin 60 min before biopsy without any additional antibiotics.rd and 7th days, all patients were examined at the outpatient clinic and assessed for any symptoms of infection or complications related with the TRUS-bx. At these time points, urinalysis and urine culture were performed to capture asymptomatic urine findings. Further follow-up of patients was carried out on the second and fourth weeks after TRUS-bx, and symptoms, such as dysuria, rectal bleeding, fever, hematospermia, hematuria, and pollakiuria, were recorded. Any complications arising until the 30-day follow-up were recorded. These complications included hematuria, hematospermia, rectal bleeding, pain, dysuria, UTI, and retention. Diagnostic criteria of UTI and classification are provided in Supplement 2.Subsequent to TRUS-bx, all patients were followed-up for 30 days. On the 3Mean, standard deviation, median, lowest and highest scores, frequency, and ratio values were used for the descriptive statistics of the data. Distributions of variables were measured using Kolmogorov-Smirnov test. For the analyses of independent quantitative data, Mann-Whitney U test or chi square test was utilized; when these methods were inapplicable, Fischer\u2019s exact test was used. All analyses were conducted with SPSS 22.0. The accepted level of statistical significance was p<0.05. The statistical power of our study was calculated post-hoc by free-for-public-use software: Post-hoc Power Calculator . Using this software, the statistical power of the study was calculated to be 90.6%.Age (63.5\u00b17.2 and 62.2\u00b18.1. p=0.821), PSA levels (9.4\u00b117.3 and 11.1\u00b118.5. p=0.549), prostate volume (52.05\u00b127.1 and 53.08\u00b128.6. p=0.741), risk factors, Charlson score (2.34\u00b11.3 and 2.15\u00b11.1. p=0.477), and cancer detection rates  were similar for both groups . PCa wasrd  and 7th days  post TRUS-bx were similar for both prophylaxis groups (Follow-up urine culture screening with colony formation on the 3s groups . Symptoms groups .The most common non-infectious findings comprised microscopic hematuria [single: 57 (28.5%) vs prolonged: 58 (29%) p=0.001] followed by rectal bleeding [single: 42 (21%) vs prolonged: 38 (19%) p=0.001] and dysuria [single: 20 (10%) vs prolonged: 23 (11.5%) p=0.001]. Non-infectious complication rates were similar in both groups (p<0.05) . One patOur study has identified no additional benefit of extending the antibiotic administration in patients who undergo TRUS-bx. Urology guidelines strongly recommend antimicrobial prophylaxis. However, the choice of regimens and duration of prophylaxis remains debatable . Further"}
+{"text": "The velocity of settling particles is an important determinant of distribution in extinct and extant species with passive dispersal mechanisms, such as plants, corals and phytoplankton. Here, we adapted dynamic scaling, borrowed from engineering, to determine settling velocity. Dynamic scaling leverages physical models with relevant dimensionless numbers matched to achieve similar dynamics to the original object. Previous studies have used flumes, wind tunnels or towed models to examine fluid flow around objects with known velocities. Our novel application uses free-falling models to determine the unknown sinking velocity of planktonic Foraminifera \u2013 organisms important to our understanding of the Earth's current and historic climate. Using enlarged 3D printed models of microscopic Foraminifera tests, sunk in viscous mineral oil to match their Reynolds numbers and drag coefficients, we predicted sinking velocity of real tests in seawater. This method can be applied to study other settling particles such as plankton, spores or seeds. Summary: A novel method to determine the sinking velocity of biologically important micro-scale particles using 3D printed scale models. The transport of organisms and biologically derived particles through fluid environments strongly influences their spatiotemporal distribution and ecology. In up to a third of terrestrial plants , reproduMeasuring the individual settling velocity of small particles directly is challenging, especially when they are too small to be imaged easily without magnification e.g. . Here, wmodel/Lengthreal, is constant \u2013 this is the scale factor (S) of the model. Less obvious is the additional requirement of dynamic similarity, signifying that the ratios of all relevant forces are constant. For completely immersed objects sinking steadily at terminal velocity , dynamic similarity is achieved by matching the Reynolds number (Re).Engineering problems such as aircraft and submarine design often are approached using scaled-down models in wind tunnels or flumes to examine fluid flows around the model and the resulting fluid dynamic forces it is subjected to. To ensure that the behaviour of the model system is an accurate representation of real life, similarity of relevant physical phenomena must be maintained between the two. If certain dimensionless numbers  that describe the system are equal between the life-size original and the scaled-down model, \u2018similitude\u2019 is achieved and all parameters of interest (e.g. velocities and forces) will be proportional between prototype and model . IntuitiRe is a measure of the ratio of inertial to viscous forces in the flow (fluid is the density of the fluid (kg\u00a0m\u22123); L is a characteristic length (m) of the object; U is the object's velocity (m\u00a0s\u22121); and \u03bc is the dynamic viscosity  of the fluid. In cases where LU\u03c1fluid is large compared with \u03bc, e.g. whales, birds and fish , viscous forces dominate. Finally, when LU\u03c1fluid is of comparable magnitude to \u03bc, Re is intermediate and one cannot discount either inertial or viscous forces. If the scaled model and original system exhibit identical Re, the relative importance of inertial versus viscous forces is matched between the two and any qualitative features of the flows (e.g. streamlines) will also be identical.the flow , and is Re as the original systems have been used in a number of biological studies. Re when enlarging small organisms, the fluid flow must be slower and/or the fluid more viscous, and when making smaller models of large organisms, the fluid flow must be faster and/or the fluid less viscous. For instance, E. coli flagella at a scale factor of \u223c61,000, submerged in silicone oil (105 times more viscous than water), and rotated at 0.002\u2005rpm compared with the 600\u2005rpm observed in real bacteria  of the original system was known, designing experiments to achieve similitude was relatively straightforward because the Re was also known a priori \u2013 in these cases, the model size, speed and working fluid properties were simply interrelated through Re (Eqn\u00a01). For instance, once a working fluid and the model size were chosen, the required towing speed was obvious. However, in the case of sedimentation of small particles , the sinking speed (U) is the key unknown. With an unknown sinking speed, the operating Re is also unknown, so it is not straightforward to design experiments that achieve similitude with the original system. Here, we present an iterative methodology leveraging 3D printed dynamically scaled models that allows determination of the sinking speed of small objects of arbitrarily complex shape.In all the above studies, basic kinematics such as speed in the original system were relatively easy to measure, and the experiments aimed to reveal the forces involved (e.g. hydrodynamic drag) or details of the fluid flow such as the pattern of streamlines. As the representative speed (3) .Aparticle projected  area perpendicular to flowArArchimedes numberCDcoefficient of dragCDERe,CD) experimental datainterpolating spline through CDwallsCD with walls present (i.e. measured in the tank)Fbuoyancybuoyant forceFdragdrag forceFweightparticle weightgacceleration due to gravityHV versus Scubic spline interpolant for measured Kwall effects correction factorLmaximum length of particle parallel to the flowMparticle massNiteration numberOvalue for real particle at natural operating pointReReynolds numberSmodel scale factorUsinking speed of particleVparticle volumeZ(t)depth of the sphere\u03bbtank to particle diameter ratio\u03bcfluid viscosityfluid\u03c1fluid densityparticle\u03c1particle densityF\u03a3sum of external forces\u03a83D shapeWe used planktonic organisms of the subphylum Foraminifera (hereafter referred to as the group \u2018foraminifera\u2019) as an example of a small (200\u20131500\u2005\u00b5m) biological particle for which the settling velocity is important and typically unknown. Foraminifera are marine amoeboid protists . By secr3) , an impo3) . Of part3) . While tL \u2013 defined as the maximum length parallel to the settling direction, A \u2013 defined as the projected frontal area, and V \u2013 the particle volume not including any fluid-filled cavities), 3D shape  of the original sinking particle are known, while the sinking speed (U) is unknown. The properties of the fluid surrounding the original particle  are also known, and our goal is to design experiments in which we sink a scaled-up model particle in a working fluid of known \u03c1fluid and \u03bc in order to determine the model particle's sedimentation speed and, via similitude, U of the original particle.We assume that the size  in order to match this Re in the experiments and ensure similitude. Solving for both Re and S simultaneously requires additional mathematical relationships beyond Eqn\u00a01.While previous work  suggestsLO, VO, AO, UO) and ReO, O). While the fluid dynamics of flow around a particle of particular shape \u03c8 can be considered theoretically over a range of Re, only the dynamics at ReO and UO.Throughout, we use a superscript O to refer to the original values of dimensioned variables at life size (e.g. F) acting on the particle is zero  and buoyant force  must balance the weight of the particle :CD, a dimensionless descriptor of how streamlined an object is. Both CD and Re must be matched to achieve similitude. CD depends on the shape of the object \u03c8, including its orientation relative to the freestream flow; for instance, CD of a flat plate oriented parallel to laminar flow is as low as 0.003 while CD of a flat plate oriented perpendicular to the flow is \u223c2.0  in the expression for Fdrag (Eqn\u00a03). For brevity, we will omit \u03c8 hereafter and write the drag coefficient as CD(Re).When a particle is sinking steadily at its terminal velocity, the sum of the external forces  and weight  are both expressed using particle volume (V), gravitational acceleration (g) and density of the fluid (\u03c1fluid) or particle (\u03c1particle), respectively:U via the definition of Re (Eqn\u00a01) yields an expression for the drag coefficient obtained through a force balance (indicated by a superscript Ar=gL3Ar\u03c1fluid (\u03c1particle\u2212\u03c1fluid)/\u03bc2 if the cubed length scale L3Ar=VL2/A, yielding L explicit.The buoyant force  in a viscous fluid is measured directly, allowing us to calculate Re via Eqn\u00a01 and then S and the original test parameters :L=SLO and A=S2AO. While one would also expect V=S3VO for 3D printed models, limitations of our 3D printer led to variation in V that we overcame using a more general empirical relationship between S and V based on mass measurements \u2013 see \u20183D printer limitations\u2019, below. Eqn\u00a07 represents a constraining relationship between CD and Re for the sinking particle, which we use to collect  experimental data points at several S. Once sufficient data are collected, we can construct a new, empirical relationship  between CD and Re for a particular particle shape, which we term ReO, UO by finding the intersection point between the S=1 and https://github.com/matthewwalkerbio/Dynamic-scaling.If minifera  coupled http://webdb2.museum.tohoku.ac.jp/e-foram/), with a micro-computed tomography (\u00b5CT) scan resolution between 2.5 and 3.6 pixels\u00a0\u00b5m\u22121, and were exported as 3D triangular mesh (STL format) files. Specimens of an additional three species were scanned using synchrotron radiation-based micro-computed tomography (SR\u00b5CT). Imaging was performed at the Imaging Beamline P05 (IBL) (https://www.morphosource.org/Detail/ProjectDetail/Show/project_id/1167). Meshes of all foraminifera were manually checked in Meshlab   operated05 (IBL) . For pro05 (IBL) , and aga Meshlab  for inteFor species where more than one scan was available, the scan that contained the best-preserved specimen was chosen. By only including one specimen per species, this approach neglects phenotypic plasticity which is demonstrated in planktonic foraminifera e.g. , but wasS) physical models of each specimen using a FormLabs Form1+  3D printer, using FormLabs Clear Resin Version 2 with a layer thickness of 50\u2005\u00b5m , we measured the projected area of the sinking foraminifera. Referring to high-resolution images of the sinking model , a digital model of the foraminifera was manually aligned to measure the projected area in a plane perpendicular to the sinking direction . We used the same procedure to measure the maximum length parallel to the flow (L) for the calculation of Re . These choices facilitated objective comparisons of CD across morphologically diverse species, to be detailed in a future study.Following convention, when defining the area V=S3VO, because of inherent limitations of the 3D printer as well as difficulty in removing excess resin from small models, we found that this expectation was usually not satisfied, and weighing the models showed that M/\u03c1particle>S3VO where M is particle mass . Therefore, we estimated V of each model by weighing models on an Entris 224-1S mass balance (\u00b10.001\u2005g) and assuming \u03c1particle was 1121.43\u00b113.73\u2005kg\u2005m\u22123, based on the average mass of five 1\u2005cm3 cubes of printed resin. Furthermore, whenever a predicted value for V at a given scale factor S was needed, i.e. in Eqn\u00a07 , we based this on cubic spline interpolation of our V(S) data for existing models when sufficient data were available, with extrapolation based on cubic scaling of V(S) if required :N is the number of existing volume measurements (i.e. the current iteration number) and H represents the cubic spline fit of V versus S. Note that because we always directly measured V by weighing after printing each model, and it is not necessary to achieve the exact Re and CD of the operating point  filled to a depth of 1.18\u2005m (approximately 750\u2005l). The tank was fitted with a custom-designed net and net retrieval system  to allow easy retrieval of the models after their descent, allowing each model to be sunk 5 times. Integrated into the net retrieval system was the release mechanism, which was held centrally over the tank, with the grasping parts submerged below the oil level. This ensured that each model was released in a controlled and repeatable fashion.The models were released in a cylindrical acrylic tank (0.9\u2005m in diameter and 1.2\u2005m in height) of mineral oil  and recording at 960 pixels\u00d7720 pixels and \u223c30\u2005frames\u2005s\u22121, allowing monitoring of the position and orientation of the particle in 3D as it fell. As these consumer-grade webcams use a variable frame-rate system, a custom-written MATLAB script was used to initiate camera recording, recording both frames and frame time stamps. Videos were recorded for 500\u2005frames (\u223c17\u2005s). Sinking velocity was calculated over a central 0.8\u2005m depth range, ensuring the model was at terminal velocity  whilst also avoiding end effects which could slow the model as it reached the bottom of the tank. Based on observations of suspended dust, there was no discernible convection in the tank during any trials that might potentially affect sinking velocity. The curved walls of the tank introduced distortion, which was removed using the MATLAB toolbox \u2018Camera Calibrator\u2019  were accurate to within 0.5\u2005mm (0.06% of the traversed depth).The sinking models were recorded using two Logitech C920 HD webcams , placed at 90\u2005deg to each other (ibrator\u2019 . Pixel shttps://zenodo.org/record/45559#.W6z6c2j0nIU). The per-frame centroid coordinates obtained were then paired with the time stamp values recorded to calculate average settling velocity components in 2D for each camera . A resultant velocity magnitude was then calculated for each camera, and these two values were averaged to yield a single estimate for U per experiment:U was calculated from these replicates. Replicates beyond a threshold of \u00b15% of the median sinking velocity were discarded from this average. Each model was dropped one additional time and photographed using a Canon 1200D DSLR camera  mounted on a tripod close to the tank, to obtain high-resolution (18 megapixels) images which were used to determine model orientation (and thus L and A) during settling .Models were tracked in distortion-corrected frames using a modified version of Trackbac  system can be non-intuitively large and lead to substantial errors if not accounted for  and model diameters of the order of 1\u2005cm. To reduce potential errors further, we applied the method of d/D where d is the diameter of the sinking particle and D is the tank diameter; we take d=L. While Eqn\u00a010 is not exact, it substantially reduces the error otherwise incurred if one were to neglect wall effects entirely. Note that Eqn\u00a010 is only valid up to about Re=50, beyond which different corrections can be used  are needed. These first three S were chosen by using an existing empirical CD(Re) relationship for a sphere, valid for 0<Re<106  relationship for that shape could provide better initial guesses and faster convergence.While morphologically complex particles such as foraminifera tests  are not S=1, V=VO, and fzero function) for our first estimate of the operating ReO. Substituting this Re back into Eqn\u00a07 or Eqn\u00a011 yields an estimate of the operating Re and CD in the first experiment, except that we accounted for wall effects by distinguishing between Re into Eqn\u00a07 but now with \u03c1particle corresponding to the resin model and \u03c1fluid and \u03bc corresponding to mineral oil, and combine this expression with Eqn\u00a010, assuming our estimated CD=SLO/D. The resulting expression can be solved numerically for the first scale factor, termed S1. Two more scale factors (S2 and S3), one smaller and one larger than S1, were chosen to span expected Re values for foraminifera from published literature  to these three initial  data points, constrained to be monotonically decreasing and concave-up within the limits of the data to match expectations for drag on objects at low to moderate \u00adRe. Three optimally spaced spline knots were used as this yielded excellent fits to the data as the number of data points increased. These details of the spline as well as its order (i.e. cubic versus linear) are somewhat arbitrary but we ensured that our results were sufficiently converged as to be insensitive to them .An empirical cubic spline curve ReO, S=1 and ReO numerically using a root finding algorithm (MATLAB's fzero function) on the objective function ReO into Eqn\u00a07. Finally, UO was easily determined from the definition of ReO  the variation between the predicted ReO and the closest experimentally measured Re was less than 15%.The iterative process  relationship is already well known. In order to achieve low density (and thus low sinking velocity and low Re), these spheres were hollow and filled with oil via two small holes (of diameter 0.8% of the sphere diameter). Our empirically generated R2=0.875, Re\u224825. While the error grows larger at lower Re, we expected most foraminifera species to operate at Re\u224818\u201355 based on previous work  for which the theoretical l  curve   settling in seawater. This size range corresponds to Re=12\u201327, the area where our UO based on our empirical UO was 11.5% at Re=16 (corresponding to a sphere 860\u2005\u00b5m in diameter) while the minimum difference was 6.5% at Re=27  from vanishingly small Re (when the particle is at rest) up to the terminal velocity. Hence, we will assume a spherical particle as an approximation to the model foraminifera, so that Morrison's empirical equation (Eqn\u00a011) can then be substituted for CD:During the transient acceleration phase, Eqn\u00a02 does not hold. Instead, we can revert to the more general form of Newton's second law:U/dt on the right-hand side of the equation. If the definition of Re=(LU\u03c1fluid) /\u03bc is inserted into Eqn\u00a014 (not shown for brevity), one obtains an ordinary differential equation (ODE) for the unsteady velocity U(t). The depth of the sphere Z(t) can then be obtained by solving a second much simpler ODE:ode45 function, subject to the initial conditions U(t=0)=0 and Z(t=0)=0.Here, we have isolated dRe approaches zero in the limit of inertia-less Stokes flow, unsteadiness can only occur as a result of time-varying boundary conditions. Thus, a microorganism that stops actively swimming will almost instantly come to a stop, and a heavy micro-particle released from rest will almost instantly begin sinking at its terminal velocity  truncatulinoides to operate at Re=42 . The depth corresponding to where speed equals 99.9% of the terminal velocity is approximately 4.6\u2005cm, which is much smaller than the 19\u2005cm between where the models were released and the edge of the cameras' field of view for data collection. Hence, the transient acceleration of each model foraminifera should have had no effect on our data or results. Most of our models should have reached terminal velocity even sooner as they sank at lower Re, e.g. within 2.2\u2005cm for Catapsydrax dissimilis operating at Re=36 . In ReO, UO of a typical foraminifera species. There was little variation in the number of iterations required to reach convergence . We suspect the higher end of this range was due to these species having forms that were particularly challenging to clean residual resin from, or to the incomplete removal of air bubbles once submerged in oil.Here, we present a novel method of determining settling speed by leveraging dynamically scaled models falling under gravity rather than being towed at a controlled speed. Applying our method to foraminifera-inspired spherical particles A, we preFig.\u00a0S1; Fig.\u00a0S1). This could be due to our ability to observe enlarged models of sinking foraminifera more accurately compared with actual specimens, and the lack of control for wall effects in previous work, which would tend to underestimate sinking speeds. There could also be considerable natural variation, which our single specimen per species (excluding Sphaeroidinella dehiscens) does not capture.Our predicted sinking speeds of foraminifera fall within aggregated existing data for 14 species Fig.\u00a0S1;  and compin situ  curve that describes the flow dynamics (and thus sinking speeds) that would occur if various fluid and/or particle parameters were varied, offering a degree of flexibility not seen in other studies. For example, phytoplankton blooms can increase both the density and viscosity of water due to exudates  to enable sufficiently large models to be produced . The method can also be applied to terrestrial systems such as settling spores , important in the study of wind erosion and particle transport and deposition . Similar"}
+{"text": "The IMPRESSION machine learning system can predict NMR parameters for 3D structures with similar results to DFT but in seconds rather than hours. The IMPRESSION  machine learning system provides an efficient and accurate method for the prediction of NMR parameters from 3-dimensional molecular structures. Here we demonstrate that machine learning predictions of NMR parameters, trained on quantum chemical computed values, can be as accurate as, but computationally much more efficient (tens of milliseconds per molecular structure) than, quantum chemical calculations (hours/days per molecular structure) starting from the same 3-dimensional structure. Training the machine learning system on quantum chemical predictions, rather than experimental data, circumvents the need for the existence of large, structurally diverse, error-free experimental databases and makes IMPRESSION applicable to solving 3-dimensional problems such as molecular conformation and stereoisomerism. The prediction of these parameters, especially in studies of 3-dimensional molecular structure, are increasingly moving towards quantitative comparison between computed values for proposed chemical structures and experiment. In such comparisons, the use of fast and accurate NMR prediction methods is crucial.NMR spectroscopy remains the pre-eminent analytical technique for elucidating molecular structure in solution, with the prediction and interpretation of e.g. flat-but-stereochemically-aware HOSE codes1H\u20131H coupling constants are more directly linked to 3-dimensional structure, however generically applicable Karplus-style empirical relationships, such as the widely used equation reported by Haasnoot et al.,e.g. carbohydrates,1H\u201313C scalar coupling constants, 1JCH, which are sensitive to both chemical connectivity and 3-dimensional structure are rarely used in isotropic studies precisely because there are no general fast predictive methods for 1JCH.Fast empirical predictions of chemical shifts for 2-dimensional chemical structures have been used for decades, with the additivity rules exemplified by Pretsche.g.1JCH predicted with <4 Hz accuracy to experiment1H and 13C chemical shifts respectively. The substantial downside of DFT is the significant computation time required when using methods that can provide sufficient accuracy in NMR predictions. Accurate DFT-based predictions of chemical shift and scalar couplings typically take hours to days of CPU time for a single rigid molecule of even relatively low (\u223c500) molecular mass. The largest proportion of this CPU time is occupied by the NMR computations, especially when computing scalar coupling constants. Naturally, in cases where multiple conformers or isomers must be considered (and thus predictions for multiple structures are required) this becomes days to months of computation for a single study.For all of these reasons, the accurate prediction of NMR parameters in modern 3-dimensional structure determinations relies increasingly on the use of quantum chemical calculations, typically based on Density Functional Theory (DFT).1H and 13C chemical shifts based on 2-dimensional structures are well-established.e.g.1JCH values) and they can be critically dependent on 3-dimensional structure . On the other hand, a machine could be trained using large datasets of DFT-computed NMR parameters, such as chemical shifts and scalar couplings, derived from 3-dimensional structures. Such large DFT-derived datasets can be generated systematically with minimal effort and are not limited to offering accuracy only for structures that are similar to previously experimentally determined molecules. With a large enough training database, such a machine would be expected to approach the accuracy of DFT calculation of NMR parameters for 3-dimensional structure analysis, but with several orders of magnitude reduction in time for the NMR predictions. This approach was recently reported for solid-state chemical shift predictions by Paruzzo et al. , trained on DFT-predicted values rather than relying on scarce or error-prone experimental data. We have chosen to demonstrate the versatility of machine learning of NMR parameters using both 22.1\u03b41H, \u03b413C, 1JCH), computed using DFT in the Gaussian09 software package.et al.1JCH; 18\u2009383 \u03b41H; 17\u2009081 \u03b413C values/environments) were selected by an adaptive sampling (active learning) procedurei.e. how much a given parameter changes when predicted from 5 separate machines each trained on a different 80% subset of the current training set). The 100 structures in the superset which show the highest variance are then added to the training dataset and the cycle is iterated  harvested from the CSD-500 dataset recently reported by Paruzzo et al.22In order to train and test IMPRESSION, we developed a dataset of NMR parameters  improved steadily with increasing training set size, as illustrated in the learning curves (\u03b41H/\u03b413C/1JCH/predictions and root mean squared error (RMSE) of 0.35 ppm/3.88 ppm/1.39 Hz against the independent test set  of 11.22 ppm. Similar outcomes were observed for the other parameters with 187 \u03b413C values (\u223c2.5%) with errors >10 ppm (MaxE = 63.33 ppm) and 14 (\u223c0.2%) of the 7788 predicted 1JCH values having errors of >10 Hz (MaxE = 24.63 Hz). Diagrams of the structures containing the five most significant outliers for each NMR parameter are shown in Fig. S19\u2013S21 in the ESI.Notably however, a very small number of predictions for the test set were much less reliable. For example, 186 (\u223c2.3%) of the a priori identify poorly described environments using the same variance-based approach used to generate the training set. By assessing the variance in the prediction of a given NMR parameter across a 5-fold cross-validation, we can quantify our confidence in each individual prediction since environments which are poorly described by the chemical structures in the training set will have high variance in this cross-validation. There is indeed a clear correlation of variance against prediction error for the independent test set . For \u03b41H the 0.1 ppm filter excludes 5 environments (<0.1%) and improves the fit to MAE = 0.23 ppm, RMSE = 0.32 ppm; MaxE = 2.16 ppm. For \u03b413C a 5 ppm filter provided a good fit  while excluding 538 (\u223c7.2%) of the environments. For 1JCH a 1 Hz filter improved the fit to MAE = 0.81 Hz, RMSE = 1.17 Hz; MaxE = 13.37 Hz while discarding only 207 (<3%) of the environments.We therefore identified variance cut-offs for IMPRESSION predictions that provide a good compromise between accuracy and excluded values for the test set, which were found to be 1 Hz for i.e. 3D structure generation + NMR prediction, could be reduced further by using 3D structures derived from molecular mechanics rather than DFT. While not the key focus here, the use of molecular mechanics structures as inputs to a re-trained IMPRESSION machine was explored. While practical, this resulted in a \u223c30\u201350% increase in the average prediction errors for \u03b41H and 1JCH presumably arising from a mismatch between the detail of molecular mechanics geometries and those used to calculate the DFT NMR parameters . This overall time, 2.31JCH, a validation set of 608 experimental 1JCH values were taken from structures collated by Venkata et al.et al. reported for each compound. While not making the predictions entirely experimentally relevant, it allows direct comparison between DFT and IMPRESSION NMR predictions for this data. Calculating the 608 couplings with \u03c9b97xd/6-311g took 156 CPU hours and initially gave a relatively poor fit to experiment (MAE = 10.92 Hz) but with a systematic offset from the experimental data by an average of \u201310.91 Hz. Adding this systematic offset to the DFT-predicted values provided a good fit between DFT and experiment  and this was used for all subsequent comparisons to experiment based on this DFT method. As IMPRESSION is trained on DFT data computed with this same \u03c9b97xd/6-311g method and both methods use only single conformer predictions for each molecule, then these statistics represent a practical limit for the accuracy that we might expect from IMPRESSION on this experimental data.Naturally, a key test of IMPRESSION is its ability to reproduce DFT predictions of experimental values of relevant compounds. To test this for 1JCH values but with some substantial outliers . Applying the 1 Hz variance filter gave: MAE = 2.01 Hz, RMSE = 2.69 Hz, MaxE = 10.01 Hz  which was essentially identical accuracy to that obtained from the DFT method for these same filtered environments: MAE = 1.83 Hz, RMSD = 2.60 Hz, MaxE = 14.63 Hz. An overlay of the error distributions for DFT and the 1 Hz variance-filtered IMPRESSION vs. the experimental values  described by Buevich et al. as typical for DFT methods, with <25% of the values being tagged as unreliable by the variance filter. Of course, if a slight loss in prediction quality is acceptable for a given study, then more predicted values could be retained by using a slightly looser variance-filter.IMPRESSION took only 60 CPU seconds to predict the full set of 612 l values  demonstr1H chemical shifts for 36 structures reported by Smith and Goodman\u03b413C using the 5 ppm variance filter identified during training and testing of the machine compared slightly less well to the DP4 experimental dataset  than DFT . A tighter 1 ppm variance filter for the \u03b413C predictions was examined, but gave only a slight improvement in prediction quality MAE = 3.20 ppm, RMSE = 4.00 ppm, MaxE = 13.03 ppm while removing 120 out of the 458 carbon environments.Similar accuracy could be obtained for IMPRESSION predictions of 734 \u03b413C predictions have a wider distribution of errors than the other NMR parameters when compared to the quality of the DFT from which they are trained. This is unsurprising given that the structural environments of 13C nuclei in molecules are inherently more complex than 1H given the higher valency and thus more complex bonding environments and geometries, so in future development, larger training datasets focussed on optimising \u03b413C predictions will be beneficial.At every stage in this study we found that the IMPRESSION 2.4i.e. those that differ only in their 3-dimensional structure, but not connectivity. Even though IMPRESSION has not been explicitly trained to deal with multiple conformers/isomers of any one compound, 3-dimensional variation is implicit within the varied chemical structural space of the adaptively sampled training set. Buevich et al. recently demonstrated1JCH values can successfully discriminate the naturally occurring structure 1 of the polycyclic alkaloid strychnine . The error for the correct structure is \u223c30% lower than the diastereomer with the second lowest error 6  and this is very similar to the discrimination offered by \u03c9b97xd/6-311g , and 1b which is the second lowest energy conformer (3% population in solution).1JCH values for strychnine (MAE = 1.87 Hz) is slightly lower than that obtained from the DFT method (MAE = 1.31 Hz), its discriminating power between structural isomers is nearly the same.A demanding test of IMPRESSION is in its ability to predict and discriminate experimental NMR data for stereoisomeric compounds rychnine , centre 1JCH with 1H and 13C chemical shifts also provides correct identification of the naturally occurring structure, but IMPRESSION and DFT now both see structure 2 as the next best candidate (\u03b41H values having better agreement with the predictions for diastereomer 2 than 1a for DFT and also IMPRESSION. While this is obviously problematic for structure elucidation purposes, it clearly arises because of a deficiency in the DFT prediction of 1H chemical shifts, which is then faithfully reproduced by IMPRESSION. For the individual MAE values across all three parameters see ESI Section S5.Combining IMPRESSION predictions for andidate , right. 1JCH for the diastereotopic protons in strychnine were consistently in line with each other (details can be found in Section S4 of the ESI1JCH values both methods correctly assign these protons (Fig. S16Similarly, we found that IMPRESSION predictions can be used to correctly assign the diastereotopic protons in strychnine. IMPRESSION and DFT predictions of  Fig. S16.Finally, we validated IMPRESSION chemical shift predictions for natural product structures. We conducted DFT and IMPRESSION predictions on structures from a recent report which suggested structural reassignments for oxirane-containing natural products on the basis of DU8+ DFT calculations.3In summary, this first generation IMPRESSION machine, trained on DFT-computed NMR parameters derived from a set of 3-dimensional structures is capable of reproducing DFT-predicted NMR parameters for a range of experimentally relevant systems with high accuracy but in a fraction of the time. Accurate and generalised prediction of NMR parameters for 3-dimensional applications has not been addressed by previous machine learning systems but the confidence provided by the variance-filtered IMPRESSION results makes this tool essentially as robust for 3-dimensional applications to experimental systems as DFT. At this stage, the two primary sources of error in IMPRESSION predictions of experimental data are errors in the underlying DFT method on which it is trained (of which there can be severalThere are no conflicts to declare.Supplementary informationClick here for additional data file."}
+{"text": "Cyclodextrin/poloxamer-soluble polypseudorotaxane-based nail lacquers have demonstrated significant capacity for promoting the permeation of drugs into the nail plate. Furthermore, previous studies have shown that the use of hydroalcoholic blends as vehicles promotes drug permeation. The work described herein studies the effect of the type of alcohol used in the lacquer preparation, and the composition of the vehicle is optimized to obtain soluble doses of 8% and to promote the diffusion of ciclopirox base and olamine across the nail. Permeation studies on different types of alcohols show that optimum results are achieved with short-chain alcohols, and that results become less satisfactory the higher the number of alcohol carbons. In addition, solubility and penetration studies on the bovine hoof have enabled the composition of the lacquer to be optimized for both forms of ciclopirox. The results suggest that optimized lacquers have better ciclopirox diffusion and penetration properties than the commercial reference lacquer. Lastly, in vivo studies in which optimized ciclopirox olamine lacquer was applied for 45 days to the nails of healthy volunteers showed that it caused no negative effects or changes to the nail surface. These results demonstrate the significant potential of cyclodextrin/poloxamer-soluble polypseudorotaxane-based nail lacquers for the ungual administration of drugs. Onychomycosis is a fungal infection of the nail caused by dermatophytes, nondermatophyte molds, and yeast that can affect 2\u201318.5%, or higher, of the world\u2019s population .Most of today\u2019s topical treatments for onychomycosis are based on nail lacquer compositions containing ciclopirox, amorolfine, or tioconazole , and rec\u00ae nail lacquer  and Ciclochem 8%, which contain the drug  dissolved in a blend of ethyl acetate and isopropyl alcohol and butyl monoester of poly as the film-forming polymer. The use of organic solvents is associated with problems as regards their safety, toxicity, complicated disposal and the formation of hydrophobic and impermeable films on the nail surface. The formation of a hydrophobic polymer film in which the active ingredient is dispersed in the form of small, solid precipitates does not favor the delivery of the active ingredient to the nail matrix, thus limiting its penetration. Furthermore, the formation of an impermeable hydrophobic film prevents nail transpiration, which encourages fungal growth. In addition, these types of lacquer require removal between applications using organic solvents or nail files, which causes damage and irritation to the nail and surrounding skin area [Independently of the drug used, the composition of most of the lacquers sold is based on the use of hydrophobic polymers dispersed or dissolved in volatile organic solvents, designed to promote the formation of even, impermeable films on the nail surface. Examples are Penlackin area .Some different physical and chemical enhancement techniques have been proposed to improve the efficacy of topical treatments for onychomycosis. These techniques include the abrasion or the ablation of the nail plate , the useNumerous studies suggest that nail hydration plays a highly significant part in the ability of active ingredients to permeate the nail plate ,15,16,17\u00ae, Galderma, Lausane, Switzerland), the composition of which includes a methacrylate acid copolymer, triacetin, butyl acetate, ethyl acetate, and ethyl alcohol. The experimental amorolfin lacquer displayed better diffusion than the commercial water-insoluble polymer option, which suggests better efficacy of the hydroxypropyl chitosan lacquer. Moreover, lacquers prepared with ciclopirox showed better antimycotic efficacy than lacquers prepared with amorolfin. In 2010, Monti et al.  compared\u00ae and \u03b2-cyclodextrin derivatives, with the aim of improving the penetration of antimycotic agents and corticoids into the nail. The presence of the poloxamer facilitates the formation of thermosensitive hydrogels in situ on the nail surface, and their combination with soluble cyclodextrins increases the solubility of the drugs contained in the lacquers. In the study described herein, the authors propose the inclusion of acetylcysteine to enhance nail absorption, since it favors nail hydration and increased porosity, in turn favoring permeation of the drug [In 2013, Nogueiras Nieto et al.  developethe drug . The drathe drug  studied To dissolve better still the lipophilic active ingredients, in 2018 Cutr\u00edn-G\u00f3mez et al.  studied \u00ae, Alfasigma, Alanno, Italy). In addition, an in vivo study was carried out on healthy volunteers to determine any changes in the nail structure as a result of prolonged treatment with optimized lacquers. This study, based on the latter formulations, examines how the type of alcohol used in the formulation affects drug permeability, the optimization of vehicle composition to achieve a concentration of 8% ciclopirox base in the lacquers, and examines its effect on drug diffusion and nail penetration, using as reference formulation a commercially available hydroalcoholic lacquer containing chitosan in its base and 8% ciclopirox  with a molecular weight of 207.27 g/mol, logP 2.59, pKa 6.84, and aqueous solubility of 0.22 mg/mL. Ciclopirox olamine is a salt of ciclopirox with a molecular weight of 268.35, logP 2.15, and aqueous solubility of 14.41 mg/mL .\u00ae F127 , hydroxypropyl-\u03b2-cyclodextrin , sodium lauryl sulfate , ethanol , distilled water , ciclopirox , and ciclopirox olamine  were used. Bovine hooves were obtained from the local abattoir . They were thoroughly cleaned with water and hydrated for 24 h to facilitate obtaining fine slices (0.3\u20130.7 mm thick) . For the study, we selected slices of even thickness free from defects or splits.PluronicThe saline phosphate buffer solution was prepared as per the 8th Edition of the Pharmacopoeia European using potassium phosphate dihydrogen, sodium chloride, and sodium dihydrogen phosphate dodecahydrate, all of analytical grade. The sodium azide added to the buffer solution to prevent microbial growth was supplied by Panreac Quimica SA , and methanol (Prolabo) was used to extract the ciclopirox olamine that penetrated the hooves and nails. Ethanol, butanol, hexanol, and octanol were from Sigma-Aldrich, Germany.\u00ae F127, 10% HPB, 1% sodium lauryl sulfate, and different water and ethanol ratios, and the addition of ethyl acetate was tested in order to dissolve 8% ciclopirox in base or salt form, obtaining stable lacquers without any nucleation or precipitation taking place.Lacquers were prepared with 5% Pluronic\u00ae F127). Under constant agitation, ethyl acetate and sodium lauryl sulfate were added. Lastly, ciclopirox olamine or ciclopirox base 8% was added. Ciclopirox concentrations in the lacquers were determined by UV spectrophotometry .To prepare the lacquers, first the HPB was dissolved in water or a water\u2013ethanol mix under constant agitation. The solution was then cooled to around 4 \u00b0C to promote the correct dissolving and homogenization of the poloxamer  due to it being impossible to fully dissolve the 8% dose. Ethanol, butane, hexanol, and octanol were used in this study, and the amount of ciclopirox base diffusing into in the bovine hoof model was measured over 6 days. In view of the difficulty of obtaining human nail samples of sufficient size for carrying out diffusion studies, in the first stage of evaluating the different lacquer vehicles, bovine hoof models were used. 2. This arrangement was assembled in Franz vertical diffusion cells  with a receptor compartment volume of 7 mL. The dorsal and ventral surfaces of the nails were arranged facing the donor and receptor compartments, respectively. The donor compartment contained 2 mL lacquer. The receptor medium was a pH 7.4 saline phosphate buffer. Sodium azide (30 mg/l) was added to prevent microbial and algae growth. The receptor compartment was kept at a constant temperature (37 \u00b1 0.5 \u00b0C). At 24 h intervals, 1 mL samples were taken from the receptor compartment, this volume being replaced with fresh receptor medium.The samples were hydrated for two hours, then dried and arranged between two cylindrical polytetrafluoroethylene (PTFE) adaptors , giving an effective diffusion area of 0.196 cmThe concentration in the receptor medium was quantitated following filtration through nylon filters. Ciclopirox was determined by UV spectrophotometry (Hewlett Packard 8452A diode array spectrophotometer) at 308 nm.2 and plotted against time.The thickness of each hoof was measured using a micrometer (Mitutoyo) before the experiments began. The cumulative amounts diffused into the receptor compartment were standardized for an area of 0.049 cmAt the end of the diffusion studies, the amount of ciclopirox present in the hooves was measured. To do so, the diffusion cells were dismantled, and the samples carefully cleaned with distilled water and dried with cellulose paper. Then, the sections of hooves or nails that had been exposed to lacquer were separated and cut into small pieces that were weighed in vials. Then, 5 mL of 5% methanol solution was added to each vial, and they were incubated under agitation for 6 days at 25 \u00b0C so that the drug could be extracted. A 1 mL sample was taken from each vial and filtered through 0.45 \u00b5m nylon filters, and the drug was quantitated as described above. The study was carried out on 14 healthy male and female volunteers presenting no nail damage or alteration and without relevant derma conditions. The study was carried out as per the criteria laid out in the Structure and Content of Clinical Study Reports from ICH Harmonized Tripartite Guideline, International Recommendations IH Topic E6, CPMC/ICH/135/95 of May 1st 1996, European Parliament and Council Guideline 2001/20/EC-DOCE of May 1st 2001. Each volunteer was informed beforehand about the type and procedure of the study and signed an informed consent form.The optimized nail lacquer containing ciclopirox olamine was applied once daily to the nails on one hand of the volunteers for a period of 45 days. After this period had elapsed, pieces of the treated nails were cut (2\u20133 mm size), and pieces of the untreated nails on the other hand were taken for control purposes. Samples were fixed to microscope slides with an adhesive. An S neox non-contact 3D Surface Profiler from SENSOFAR Surface Metrology  was used in confocal mode to acquire 3D images using the Nikon EPI 20X v35 lens and with the following parameters: topography, 1360 \u00d7 1024 p; area, 877.20 \u00d7 660.48 \u03bcm; ZSCAN, 100\u2013200 \u03bcm; acquisition time, 00:30\u201301:30 min.; pixel size, 0.64 \u03bcm/pixel.Surface roughness was characterized using the 3D (S-parameters) and functional (volume) parameters according to ISO 25178 : The bovine hoof model has been used in different works in permeation studies as an alternative to human nail. Microstructural characterization of bovine hoof and human nail has shown that the bovine hoof surface is more porous than the nail surface, although the internal porosity is similar for both substrates . Neverth2, and the bovine hoof slices used were 0.8 cm in diameter. Palliyil et al. in 2014 [To conduct diffusion experiments, we used two cylindrical PTFE adapters with an O-shaped ring of 0.5 cm diameter, giving an effective diffusion area of 0.196 cm in 2014  demonstrWalter et al. ,25 study\u00ae F125, and 1% SLS, different vehicles were prepared to change the proportions of water, ethanol, and ethyl acetate in the blends. For the purpose of their evaluation, direct macroscopic observation was performed to check in which blends the 80 \u03bcg/mL dose of ciclopirox olamine or ciclopirox base was fully dissolved, subsequently measuring in same the final concentration by means of UV\u2013vis spectroscopy to confirm that it had fully dissolved. Ternary diagrams were drawn using the results in order to establish the areas of interest  and amount diffused (R2 = 0.9520) can be observed as the proportion of water in the blend increased. A previous study [Nail permeation kinetics showed that a higher proportion of water in the blend led to a higher amount of drug being diffused. To investigate the relationship between the amount of water and trans-hoof diffusion, us study  showed tThe increase in diffusion observed when the proportion of water ions the vehicle increased may be associated with nail hydration capacity and increased chemical activity of the drug, promoting its trans-hoof diffusion. Lack of ethyl acetate on diffusion may be associated with the solvent\u2019s rapid evaporation once the dose was administered in the donor chamber of the diffusion cells. This may be advantageous because it would allow the lacquer to dry more quickly after application to nails and would have no effect on drug penetration.As Trichophyton rubrum DSM 4167 (15.6 \u00b5g/mL) [T. rubrum strains (24.13 \u00b5g/mL) [The amounts of ciclopirox and ciclopirox olamina recovered from the bovine hoof slices after the experiment were 29.7 \u00b1 0.36, 45.8 \u00b1 2.93, and 53.26 \u00b1 0.89 for reference, 34/43.7/0.8 8% ciclopirox olamine, and 49.6/25.9/2.0 8% ciclopirox base, respectively. According to Hui et al. , the fin6 \u00b5g/mL)  or sevenWith a view to studying any changes in nail surface structure because of prolonged treatment with the optimized lacquer, the formulation containing ciclopirox olamine was administered daily to 14 volunteers over a period of 45 days, and their nails were analyzed using non-contact 3D surface metrology based on confocal optical microscopy. Using the 3D images shown in The permeability studies carried out on cyclodextrin/poloxamer-soluble polypseudorotaxane lacquers prepared using different alcohols have demonstrated the superiority of short-chain alcohols in promoting drug permeability in the bovine hoof model. A decreasing linear logarithmic relationship was observed between the amount of ciclopirox diffused per unit of area and the number of carbons in the alcohol carbon chain. In view of the lower topical toxicity and permeability values observed, ethanol appears to be the most appropriate alcohol for preparing these types of vehicles. Taking these results into account, the composition of the lacquer base was optimized to achieve a soluble dose of ciclopirox base and ciclopirox olamine of 8%, and to obtain better nail permeation efficacy. In the case of ciclopirox base, given its lower solubility in the lacquer, higher proportions of ethanol need to be used. However, diffusion results show that a higher proportion of alcohol is detrimental to permeability. Therefore, both solubility and permeability must be taken into consideration when selecting the proportion of alcohol. The results obtained show that by selecting the appropriate composition for the hydroalcoholic base of the nail lacquer, diffusion profiles can be optimized, and significantly higher levels of nail penetration by anti-mycotic agents can be achieved than those obtained with the commercial reference lacquer.Furthermore, the results of in vivo studies obtained after daily administration of the optimized lacquer to healthy volunteers over prolonged periods show that the lacquer is safe and does not alter the nail surface structure.These results demonstrate the huge potential of the cyclodextrin/poloxamer-soluble polypseudorotaxane base for ungual administration of antifungal medicines. Nevertheless, more in vitro and in vivo studies are necessary to validate the results obtained using the bovine hoof model.This work is part of the family patent application EP 15725664 A 20150603 \u201cHydroalcoholic system for nail treatment\u201d."}
+{"text": "At elevated temperature, the SPP\u2010treated TiO2 exhibits a 300 times higher conversion rate for CO2 reduction under solar light irradiation and a 7.5 times higher removal rate of acetaldehyde under UV light irradiation, suggesting the effectiveness of the proposed strategy to enhance the photoactivity of colored wide\u2010bandgap oxides for energy and environmental applications.Colored wide\u2010bandgap semiconductor oxides with abundant mid\u2010gap states have long been regarded as promising visible light responsive photocatalysts. However, their catalytic activities are hampered by charge recombination at deep level defects, which constitutes the critical challenge to practical applications of these oxide photocatalysts. To address the challenge, a strategy is proposed here that includes creating shallow\u2010level defects above the deep\u2010level defects and thermal activating the migration of trapped electrons out of the deep\u2010level defects via these shallow defects. A simple and scalable solution plasma processing (SPP) technique is developed to process the presynthesized yellow TiO 2. The H dopant bridges the gap between the oxygen vacancy (Ov) and conduction band (CB), resulting in photothermal extraction of trapped electrons at the Ov to CB. The solution plasma strategy endows a 300 times higher conversion rate for CO2 reduction under solar light at 393 K than that at 298\u00a0K.Solution plasma brings H doping into oxygen vacancy type TiO Recently developed solar\u2010induced photothermal catalysis demonstrated significantly better results than solar\u2010induced photocatalysis alone, especially when performed using semiconductor oxide catalysts enriched with defects. The defect at surface can facilitate the adsorption and activation of substrate molecules to boost the sluggish reaction thermodynamics and kinetics. Defects may also activate lattice oxygen, which participates in the photothermal coupling catalysis. The electrons can be trapped at defects and thermally released to the surface, which, in turn, improves charge utilization and accelerates reaction rate at the surface. These advantages of defects are the major factors behind the recent renewed interest to semiconductor oxide photocatalysts in solar\u2010induced photothermal processes.Solar\u2010driven photocatalytic processes attract substantial attention because they offer significant energy savings and because of their environmental friendliness.4, 52 materials in past decades for pursuing visible light photocatalysis. Oxygen vacancies (Ov) are the most exploited defects of TiO2, which as the native defects of oxides, can be easily incorporated into the lattice of TiO2 by annealing in oxygen\u2010deficient atmosphere or as a co\u2010product of anion or cation doping. Enriched Ov can redshift the absorption spectra of TiO2 and changes its color from white. However, the Ov levels, \u22480.75 eV below the conduction band bottom of TiO2, are deep levels and work more likely as recombination centers, which impairs the photocatalytic activity of colored TiO2. Hydrogen\u2010doping are another important means to modify TiO2 photocatalysts. Hydrogen is a shallow donor in TiO2, with defect levels closer to the conduction band of TiO2 than Ov. Hydrogen\u2010doping is reported to enhance the photocatalytic activity of TiO2, and even produces black TiO2 under severe conditions by the formation of amorphous TiO2:H or defect complex. It is interesting to note that incorporating shallow hydrogen\u2010donor levels above Ov deep levels might be a promising way to enhance the photocatalysis over Ov\u2010TiO2, by which the trapped electrons can be extracted out of Ov deep levels via the shallow levels bridge, and the enhancement should be more remarkable for the photothermal catalysis due to the thermal activation of electron extraction. However, up to now few reports have investigated the synergistic effect of Ov and hydrogen dopants on enhancing the visible light photocatalysis of TiO2. And to the best knowledge of us, almost no report has dealt with the synergistic effect of Ov and hydrogen dopants on solar photothermal catalysis over TiO2.Defect engineering has been intensively investigated on TiOn doping.19 Enr2 is indeed enhanced by incorporating shallow donor levels of hydrogen. We employ a novel solution plasma processing (SPP) to do hydrogen doping of Ov\u2010rich TiO2. Plasma\u2010in\u2010liquid can produce high flux of hydrogen radicals via H2O splitting at the open end of the discharge zone of SPP. In comparison with conventional plasma\u2010in\u2010gas, SPP can be performed at room temperature and normal pressure, without the direct use of dihydrogen. The Ov\u2010rich TiO2 treated is a home\u2010made yellow\u2010colored TiO2, named as TiO2(AB), which is a mixture of anatase and TiO2(B) phases with anatase\u2010TiO2(B) heterojunction for efficient charge separation. As\u2010synthesized TiO2(AB) phase absorbed visible light up to 700 nm due to the presence of rich Ov. SPP treatment realized hydrogen doping into this Ov\u2010TiO2, which retained its visible light absorption and promoted charge separation. Activity of SPP\u2010treated TiO2(AB) in relationship to acetaldehyde degradation and CO2 reduction was significantly enhanced under both UV and visible light irradiation. Particularly, CO2 conversion rate can be enhanced by two orders of magnitude for photothermal catalytic reduction of CO2. We believe that SPP technique demonstrated in this work can be employed as a general strategy to activate other Ov\u2010rich wide\u2010bandgap semiconductor oxides, which are colored but weak in visible light photocatalysis, for solar photothermal catalysis applications.Herein, we report that the solar photothermal catalysis over colored Ov\u2010rich TiOe of SPP.36 In 2 as possible because TiO2 enriched with oxygen vacancies (Ov) is a prerequisite for achieving simultaneously strong visible light absorption. Thus, we synthesized TiO2 with oxygen vacancies by a solvothermal method and then treated the resulting product with SPP to incorporate hydrogen doping into this TiO2   phases. This TiO2(AB) had high surface area (136 m2 g\u22121) as well as high porosity with the average pore size equal to 3.5 nm . Electron Spin Resonance (ESR) spectrum showed a signal at g = 2.003 , which is related to the interaction of surface Ov with adsorbed atmospheric O2. This confirms the presence of Ov in the as\u2010synthesized yellow TiO2(AB). Thus, prior to SPP treatment, TiO2(AB) synthesized by solvothermal method contained abundant oxygen vacancies. Yang and coworkers also reported that creating Ov activated new catalytic sites and/or promoted the exposure of active sites. Accordingly, the catalytic performance with augmented reaction sites by the introduction of Ov vacancies will exceed that of the pristine catalyst.As\u2010synthesized yellow TiO2 Figure\u00a0, which i2 Figure\u00a0, left sh) Figure\u00a0, X\u2010ray dheric O2.25 Thi2(AB) after SPP treatment, it was characterized using combination of techniques. After SPP, TiO2(AB) became darker  particles , which will be discussed below (Vide infra).To understand structural changes occurring with TiOs Figure\u00a0, right. s Figure\u00a0, XRD, Ra2(AB) before and after SPP treatment , the signal originated from Ov decreases after SPP treatment. This indicates the decrease of Ov content in TiO2(AB). As reported, plasma\u2010in\u2010liquid can instantaneously generate high temperature radiation (up to 104\u2013105 K), which can eliminate some Ov. Herein, it is believed that the instant high temperature in liquid plasma contributes to the decrease of Ov content during the SPP treatment.Compared the ESR spectra of TiOTiO2(AB).41 As 2(AB). After SPP treatment, there is a subtle shift to 530.16 eV of Ti\u2010O. Additionally, the peak assigned to Ti\u2010OH synchronously shifts to higher binding energy. Moreover, the relative content of Ti\u2010OH is increased from 26.68% to 30.64%, indicating that H is effectively doped into the lattice. To further address this point, solid 1H NMR measurements were conducted  after SPP treatment may be caused by the incorporation of H atoms at bridging sites at the disordered phase produced during the SPP treatment process. Additionally, the stronger peak at 6.15 ppm suggests stronger hydrogen\u2010bonded bridging hydroxyl groups in TiO2(AB) after SPP treatment. In contrast to pristine TiO2(AB), TiO2(AB) after SPP treatment shows that two additional sharp resonances of H2 and H3 at chemical shifts of 0.82 ppm and 0.87 ppm are observed on the baseline. The additional signals can be assigned to the terminal and internal hydroxyl groups of TiO2, respectively, which are associated with hydrogen located in TiO2(AB) as a result of hydrogenation. In combination with above analysis, the SPP treatment not only gives rise to a slight decrease of Ov, but also realize hydrogen doping into TiO2(AB). The introduced H\u2010doped into TiO2(AB) lattice can make the color darker, which had same phenomenon in the previous studied about H\u2010doped TiO2.In high resolution of X\u2010ray photoelectron spectroscopy (XPS) spectra of Ti 2p , a slight shift higher binding energy from 458.75 to 458.80 eV has been detected. Compared the high resolution XPS spectra of O 2p before and after SPP treatment , the peak located at 530.14 eV can be assigned to Ti\u2010O for as\u2010synthesized yellow TiO lattice.42 To d Figure\u00a0. Both sa2(AB) catalytic activity and to test applicability of the resulting TiO2(AB), we conducted two reactions: 1) degradation of gaseous acetaldehyde and\u00a02 using irradiation by UV, visible, solar, blue, and green light. Photocatalytic tests were performed at room temperature. Photothermal catalytic tests were performed at different temperatures depending on the reaction enthalpy. Degradation of gaseous acetaldehyde is an exothermic reaction, thus, elevated temperatures are not favorable for the direct reaction to proceed. Therefore, photothermal catalytic reactions involving gaseous acetaldehyde were performed at 343 K. Because CO2 reduction is an endothermic reaction, its temperature during photothermal tests was maintained at 393 K.To evaluate feasibility of SPP treatment as a strategy to enhance TiOat 393 K.182 was tested under solar and visible light irradiation. Reaction rates, at which CO and CH4 were generated over differently prepared catalysts, are shown in FigureTable2(AB) (named T\u20100 h) demonstrated some activity judging by the amount of generated CO and CH4. These rates were 7.9 and 8.2 times higher, respectively, when CO2 reduction reaction was performed over TiO2(AB) treated by SPP for 2 h (named T\u20102 h). Thus, T\u20102 h enhanced its catalytic activity toward photoinduced reduction of CO2.Photo\u2010 and photothermal catalytic reduction of CO4 formation rates over T\u20102 h were 300 and 108 times higher during photothermal reactions, respectively, than those during photocatalytic reaction over T\u20100 h. Compared with other TiO2, the yield of CO and CH4 are at a high level . Besides CO and CH4, other products such as C2H4, C2H6, C3H6, and C3H8 can be detected . However, in comparison with CO and CH4, the yields of other products are very trace. CO2 reduction by H2O vapor mainly produces CO and CH4 and whereas CO2 reduction by H2O liquid mainly produces CH3OH. In our work, the photothermal catalytic reduction of CO2 is performed in H2O vapor. CO and CH4 are main products, which is in consistence with previously reported . More importantly, an m/z of 29 is definitely detected, which can be solely produced via reduction of 13CO2 .Both samples, T\u20100 h and T\u20102 h, demonstrated activity during photothermal catalytic reactions induced by solar light. Activities of T\u20100 h and T\u20102 h were on the different order of magnitude as their activities during corresponding photocatalytic reactions. Apparently, CO and CH2 reduction efficiency and the selectivity of products, rate of the reacted electrons (RREs) and the selectivity of CH4 were calculated taking into account formed products:To further analyze COproducts:44(1)\u22121 h\u22121) was 152 times higher than rate over T\u20100 h photocatalyst (Table\u00a02(AB) is an efficient strategy capable to enhance both photo\u2010 and photothermal catalytic properties. SPP treatment enhanced photothermal catalytic properties of TiO2(AB) more than its photocatalytic properties. The yield of CO and CH4 is too low to accurately evaluate the selectivity in the photocatalytic test. Accordingly, the selectivity is not discussed in the case of photocatalysis. As for the photothermal catalytic test, the solution plasma treatment of TiO2 improves CH4 selectivity from 45.3% to 55.0% under solar light and 52.3% to 56.9% under visible light, respectively. In spite of the improved selectivity over solution plasma activated TiO2, the increased magnitude is slight and inferior to the increase in rate of reaction electrons. Herein, it is difficult to conclude that solution plasma treatment can simultaneously improve the reaction rate and product selectivity of CO2 reduction. It should be noted that this work studies pure TiO2 and turns to no cocatalyst. In our previous work, we also compared the selectivity of Ov\u2010TiO2 in photocatalytic and photothermal catalytic reduction of CO2. The results similarly revealed that the selectivity of CH4 can be hardly improved over pure TiO2. However, the grafting of cocatalyst  can significantly improve the CH4 selectivity in photothermal catalytic reduction of CO2. The selectivity of CH4 is expected to be significantly improved in the future work on cocatalyst loaded TiO2 rather than pure TiO2.Rate of reacted electrons over T\u20102 h during photothermal catalysis . As can be seen, the yield of CO and CH4 is decreased before the 5th cycle. However, the yield is maintained at a stable level after the 5th cycle, suggesting the good stability for practical application. Moreover, the spent photocatalyst of T\u20102 h was characterized by XRD, Raman, XPS and ESR . No obvious structural change can be observed after cycling tests, again confirming the good stability of T\u20102 h. The better stability of T\u20102 h may benefit from hydrogen doping. As is reported, the hydrogen doped oxides can be of good stability due to the formation of stable Ov\u2010H. The Ov\u2010H binding is more stable than either interstitial hydrogen or oxygen vacancy. This tends to avoid the filling of oxygen vacancy by atmospheric oxygen.The stability of T\u20102 h toward CO vacancy.46 Thiv\u2010TiO2 in photocatalytic CO2 reduction, the theory calculations of reaction pathway were performed. We construct two stable structural models: (101) surfaces of anatase TiO2 with oxygen vacancies (Ov\u2010TiO2) and (101) surfaces of H\u2010doped anatase TiO2 with oxygen vacancies (H\u2010doped Ov\u2010TiO2), referring to previous studies . Figure\u00a02 reduction into CH4. With regard to the reaction pathway from CO2 to CO, the Ov\u2010TiO2 and H\u2010doped Ov\u2010TiO2 display different potential determining steps (PDS). That is, the PDS of Ov\u2010TiO2 is from *COOH to *CO with an energy barrier of 0.28 eV, while the PDS of H\u2010doped Ov\u2010TiO2 is from CO2 to *COOH with an energy barrier of 0.12 eV. By comparison, the lower energy barrier of PDS for CO product over H\u2010doped Ov\u2010TiO2 indicates a faster reaction rate than that of Ov\u2010TiO2. Moreover, the initial step over H\u2010doped Ov\u2010TiO2 is of positive energy barrier, suggesting the increased reaction rate at elevated temperature. On the contrary, the initial step over Ov\u2010TiO2 is of negative energy barrier, which is thermodynamically unfavorable for enhanced reaction rate at elevated temperature. On the basis of above lower energy barrier and endothermic reaction characteristics over H\u2010doped Ov\u2010TiO2, it is reasonable to understand the more significant promotion of CO production over H\u2010doped Ov\u2010TiO2 in photothermal catalysis. With regard to the reaction pathway from CO2 to CH4, hydrogenation of *CHO to *CHOH is of the highest energy barrier and becomes the potential determining steps over both Ov\u2010TiO2 and H\u2010doped Ov\u2010TiO2. By further comparison, H\u2010doped Ov\u2010TiO2 exhibits lower energy barrier (0.57 eV) than that of Ov\u2010TiO2 (0.73 eV). Therefore, the lower energy barrier of PDS for CH4 can better explain the enhanced CH4 yield over H\u2010doped Ov\u2010TiO2 than Ov\u2010TiO2 in photothermal catalytic experiment. In order to further unravel the reaction mechanism at the atomic level, we also calculate and discuss the transition state for *CHO by adding *H to *CHOH (PDS\u2010CH4) over both Ov\u2010TiO2 and H\u2010doped Ov\u2010TiO2 . The activation energy (Ea) of H\u2010doped Ov\u2010TiO2 is 0.86 eV, which is much lower than that of Ov\u2010TiO2 (2.29 eV). This decreased energy barrier of H\u2010doped Ov\u2010TiO2 agrees with the result that H\u2010doped Ov\u2010TiO2 is more conducive to CO2 reduction reaction in comparison with Ov\u2010TiO2.To investigate the reactivity fundamental of the H\u2010doped Ormation).48 Fig2 evolution over TiO2(AB) with (for 2 h) and without SPP treatment are performed. Apparent rate constants (k) of acetaldehyde degradation are listed in Table2 evolution than T\u20100 h , which indicates an enhanced oxidation capacity of lattice oxygen in TiO2 treated by SPP. Stability of SPP treated TiO2(AB) was also evaluated via UV light photocatalysis . Photocatalytic activity of T\u20100 h after five cycling tests decreased by 75% while T\u20102 h maintained its initial photocatalytic activity. T\u20102 h demonstrated no activity loss after exposure to air for 80 d and after additional six cycling tests, which demonstrates excellent stability of SPP\u2010treated TiO2(AB).Apparent rate constant for the photothermal catalytic reaction with T\u20102 h was 7.5 times higher than the constant for the photocatalytic reaction with T\u20100 h as a catalyst  resulted in very active photo\u2010 and photothermal catalyst functioning upon exposure to both solar and visible light.We also measured apparent quantum efficiency (AQE) during the photocatalytic acetaldehyde degradation to determine solar energy conversion efficiency at different single wavelengths. AQEs for acetaldehyde degradation at 365 nm were 4.1% and 12.8% when T\u20100 h and T\u20102 h were used, respectively . AQE of acetaldehyde degradation in the visible light region with T\u20102 h as a catalyst was \u22481%. Thus, based on all results mentioned above on CO2(AB) after SPP treatment . To confirm this hypothesis, we used HRTEM to analyze unannealed TiO2(AB) nanoparticles with weak crystallization, which were exposed to SPP for 8 h . Lattice fringes and FFT images of unannealed TiO2(AB) before SPP were not very clear , which indicates weak crystallization. HRTEM of unannealed TiO2(AB) treated by SPP for 8 h demonstrated much clear lattice fringes and sharp FFT images , which confirmed that SPP treatment indeed improved crystallinity of TiO2(AB) and annihilate some Ov. To further confirm, we also collect the Raman spectra of the weak crystallized TiO2 before and after solution plasma treatment . The Raman bands of weak crystallized TiO2 exhibit stronger signal after solution plasma treatment, indicating the improved crystallinity by solution plasma. Thus, SPP treatment is proven effective for slight decrease of Ov content and introduction of shallow energy level via hydrogen doping.Change of color observed for TiOt Figure\u00a0 can be m Typically, PL intensity directly correlates with location of defects in TiO2. Contributions of different defects to PL spectra can be clearly seen at elevated temperatures. Difference in PL intensity at any given temperature depends on competition between charge separation at shallow energy state and charge combination at deep energy states. Thus, as temperature increases, electrons trapped by shallow energy state of hydrogen dopant tend to migrate to the surface, and electrons trapped by deep energy state of Ov recombine with holes. PL spectrum of T\u20100 h showed gradual intensity decrease as temperature increased from 93 to 483 K, which indicates that hydrogen dopant predominated over Ov  was used to study distribution of defects.52 Typv Figure\u00a0. T\u20102 h sv Figure\u00a0. This su2(AB). RDB\u2010PAS can measure energy\u2010resolved distribution of electron traps (ERDT) and provide valuable information on their density and content. Electron trap density during catalytic reaction over T\u20102 h was higher than over T\u20100 h . Positive \u0394electron\u2010trap\u2010density was observed at shallow levels near the conduction band  after SPP treatment is induced by introduction of hydrogen dopant. As shown in Figure\u00a02(AB), T\u20102 h, display broad absorption in the near\u2010infrared region, suggesting SPP can introduce additional intermediate level between conduction band and oxygen vacancy of TiO2. Meanwhile, the reversed double\u2010beam photoacoustic spectroscopy (RDB\u2010PAS) shown in Figure\u00a02(AB) originates from hydrogen doping.Reversed double\u2010beam photoacoustic spectroscopy (RDB\u2010PAS) was conducted to further understand defect distribution in TiO content.57 Eleh Figure\u00a0,e. This d Figure\u00a0. Since h2 materials was analyzed using transient surface photovoltage (TPV) technique. This technique can provide insight on dynamic properties of photoinduced charge carriers in semiconductors, including generation, separation, and recombination of photogenerated charges. TPV spectra showed stronger photovoltaic response from the system containing T\u20102 h than from the T\u20100 h . Time to achieve maximum photovoltage value in the system containing T\u20102 h was longer than for T\u20100 h, which demonstrates better charge separation ability of TiO2(AB) catalyst treated by SPP. Such behavior is very beneficial for catalytic activity enhancement upon exposure of the system to solar light.Behavior of photogenerated charges during catalytic reactions involving different TiO charges.59 TPV2(AB) catalytic activity under solar irradiation. TiO2(AB) treated by SPP demonstrated strong visible light absorption and high charge separation capability, which are also very beneficial for improving TiO2(AB) catalytic ability. These two phenomena should equally enhance TiO2(AB) activity during photo\u2010 and photothermal catalytic reactions. However, higher activity over SPP modified TiO2(AB) was observed for photothermal catalytic reactions than for photocatalytic ones. Better performance in photothermal reactions is very likely because of electrons near shallow energy state released at high temperatures. The higher content of shallow energy state, the easier it is for the electrons to be released from their traps. All these freshly freed electrons accelerate surface reduction of adsorbed species.All the results mentioned above support our hypothesis that SPP treatment caused defect redistribution, which significantly enhanced TiO As a result, such excellent photothermal catalytic activity of T\u20102 h can be attributed to the synergy between strong visible light absorption, high charge separation capability, thermal release of trapped electrons and thermal oxidation of the absorbed species by lattice oxygen.Thermal oxidation of acetaldehyde in the dark  implies very strong oxidation ability of lattice oxygen, which is also induced by Ov presence. To verify this assumption, we recorded oxygen TPD spectra . Thermal escape of lattice oxygen observed for the T\u20102 h was detected at lower temperature than for the T\u20100 h. Stronger signal from T\u20102 h indicates higher amount of escaped lattice oxygen. Thus, T\u20102 h material possessed lattice oxygen with lower activity than T\u20100 h. H doping in T\u20102 h makes it very prone to oxidative half\u2010reactions, which perform better in photothermal catalysis.61 As 2(AB) under solar light is shown in Figure2(AB) contained deep energy states of oxygen vacancies. In this defected phase, photogenerated electrons transition either from the valence band to the conduction band under UV light or to the defect level of oxygen vacancies under visible light. However, oxygen vacancies tend to recombine photogenerated electrons and holes. Despite the strong visible light absorption, serious charge recombination at Ov limits TiO2 photo\u2010 and photothermal catalytic activity. After SPP, TiO2(AB) contained less Ov and newly formed hydrogen dopant. Such redistribution of defects results in formation of continuous energy levels below the conduction band, which trap more electrons, promoting charge separation and supporting visible light absorption. At the same time, activation of lattice oxygen is accelerated, and release of electrons trapped by Ov is enhanced. All these processes significantly enhance photo\u2010 and photothermal catalytic activity under solar light.Schematic of the mechanism of enhanced catalytic activity of SPP\u2010treated TiO2(AB) to understand how solution plasma interacts with TiO2(AB). To study how SPP discharge time affects final TiO2 properties, as\u2010synthesized T\u20100 h was SPP\u2010treated for 4 h (named T\u20104 h). XRD, Raman, HRTEM, and XPS analyses showed no obvious changes in phase composition as SPP\u2010treatment time increased . UV\u2013vis spectra showed weaker adsorption for T\u20104 h in the visible to near\u2010infrared spectrum region in comparison with the T\u20102 h . \u0394electron\u2010trap\u2010density between T\u20104 h and T\u20102 h demonstrated that hydrogen dopant at the shallow level increased and Ov at the deep level decreased  catalysts decreased gradually as SPP\u2010treatment time increased confirming decreased concentration of Ov .We also studied discharge time and Ov content in TiO h Figure5a. Howed Figure\u00a0. \u0394electrd Figure\u00a0 while am2 than T\u20102 h judging by the amount of CO or CH4 formed under solar . The optimal SPP treatment time for hydrogen doping can be understood by a balance between elimination of defects at deep level and introduction of hydrogen dopants at shallow level of Ov\u2010TiO2 in solution plasma process. The solution plasma process is accompanied by instant high temperature and flux of hydrogen radical. The instant high temperature is beneficial for elimination of defects, which is verified by improved crystallinity of SPP treated weaker crystallized TiO2 . In addition, the electron\u2010trap\u2010density determined by RDB\u2010PAS is decreased from 302 \u00b5mol g\u22121 over T\u20102 h to 281 \u00b5mol g\u22121 over T\u20104 h. The combined results confirm the role of elimination of defects by solution plasma. The flux of hydrogen radical is beneficial for introduction of hydrogen dopant. As shown in Figure\u00a02 in solution plasma process.T\u20104 h showed worse performance during photothermal catalytic reduction of COr Figure\u00a0 and visi2, a pure anatase phase was obtained by annealing at 450 \u00b0C, while other conditions kept the same as TiO2(AB). The solution plasma treatment on anatase TiO2 was lasted for 2 h, which is the same as that TiO2(AB). For simplicity, the as\u2010calcined sample at 450 \u00b0C is named as 450\u20130 h. The 450\u20130 h sample subjected to solution plasma treatment for 2 h is named as 450\u20132 h. HRTEM, XRD, and Raman analysis  confirm the single phase of anatase and solution plasma treatment does not brings significant structural change of TiO2, which is similarly revealed by aforementioned study on solution plasma treated TiO2(AB). XPS comparison  on 450\u20130 h and 450\u20132 h exhibits hardly observed shift of binding energy, further confirming the insignificant structural change. ESR analysis  demonstrates a slight decrease of signal intensity related to oxygen vacancy after solution plasma treatment, which is in consistence with the regular on solution plasma treated TiO2(AB). The photothermal catalytic results toward reduction of CO2 over 450\u20130 h and 450\u20132 h under solar light are shown in Figure S35 (Supporting Information). The evolution rate of CO and CH4 over 450\u20132 h is 1.40 and 1.44 times higher than 450\u20130 h, respectively. In other word, the solution plasma treatment effectively promotes CO2 reduction over anatase TiO2. Accordingly, besides TiO2(AB), the solution plasma also works well in activation of TiO2 with single anatase phase for photothermal catalytic reaction of CO2.To prepare single phase Ov\u2010TiO3, and Ta2O5. Their extensive characterization demonstrated no changes in their phase compositions  and no noticeable shifts of the intrinsic band edges  after SPP treatment. Photo\u2010 and photothermal degradation of acetaldehyde using these SPP\u2010treated oxides as catalysts demonstrated enhanced activity for all of them . Thus, SPP can be universally applied to activate and to enhance catalytic properties of variety of semiconductor oxides.To demonstrate applicability of SPP treatment to other semiconductor oxides for photo\u2010 and photothermal catalysis, we treated ZnO, WO2 to be used as a catalyst for solar\u2010induced photo\u2010 and photothermal degradation of acetaldehyde and CO2 reduction. This strategy involved synthesis of TiO2 with rich oxygen vacancies using hydrothermal method followed by hydrogen doping in SPP. UV\u2013vis absorption, temperature\u2010dependent PL, RDB\u2010PAS, and ESR spectra demonstrated that SPP increased hydrogen dopant content and decreased content of oxygen vacancies. Moreover, SPP technology does not cause any structural or chemical changes and allows initial material to maintain its strong visible light absorption. TPV spectra demonstrated enhanced charge separation of the resulting TiO2 after redistribution of defects. Because of this enhanced charge separation and visible light absorption, catalytic activity was significantly improved. It was confirmed by the catalytic acetaldehyde degradation and CO2 reduction under visible and solar light. This was also demonstrated for both photo\u2010 and photothermal\u2010catalytic reactions. For the best SPP effect, initial material needs to be rich with oxygen vacancies. We demonstrated applicability of SPP treatment to variety of semiconductor oxides used as catalysts for solar\u2010induced photo\u2010 and photothermal reactions.In summary, we developed a strategy to activate TiO4 was added into 45 mL of ice water and stirred until a clear colorless solution was obtained. Then, 5 mL of 25% NH3\u00b7H2O, 60 mL of deionized water, and 10 mL of 30% H2O2 were added and mixed until a clear yellow solution formed. A portion of 0.5 g glycolic acid was added to this yellow solution, which was then heated at 80 \u00b0C for 390 min to remove excess H2O2 and NH3. The resulting yellowish gel was mixed with 50 mL of deionized water. pH value of the resulting yellow suspension was adjusted to 2.0 by H2SO4. The suspension was transferred into two 50 mL autoclaves and heated at 160 \u00b0C for 50 min. After the hydrothermal reaction completion, the resulting product was centrifuged, washed several times with ethanol and deionized water, dried at 80 \u00b0C, and finally annealed at 370 \u00b0C for 1 h. The resulting material was named TiO2(AB), and it was composed of anatase and TiO2(B). The two materials were also synthesized using the same procedure but with a 450 \u00b0C final calcination step and without any calcination. The sample of 450 \u00b0C is a monophase of anatase. The sample of without any calcination is a weak crystallized TiO2.A portion of 0.8 mL TiCl A 0.4 g portion of each material was placed into a reactor containing 200 mL of KCl solution with 300 \u00b5S cm\u22121 electrical conductivity. A tungsten rod, 3 mm in diameter, and a tungsten tube  were used as electrodes. Each electrode was inserted into a silicone plug and placed in the middle part of the reactor. The distance between two electrode tips was < 0.5 mm. N2 was introduced into the reactor through the hollow tungsten electrode. Plasma was generated by a bipolar\u2010DC pulsed power supply . The pulse width and frequency were 2.0 \u00b5s and 20 kHz, respectively. The temperature of solution (which was 10 \u00b0C) was controlled by a water chiller. Discharge time was 2 h. Samples underwent SPP treatment were marked as T\u2010x, where \u201cT\u201d stands for TiO2(AB) and \u201cx\u201d represents discharge time. All samples were dried at 60 \u00b0C.SPP was performed as described in detail elsewhere.39 A 0The authors declare no conflict of interest.Supporting InformationClick here for additional data file."}
+{"text": "The AV gel-treated fruits showed reduced increments in total sugar, malondialdehyde, and total carotene contents compared to untreated controls. AV gel-treated fruits exhibited higher contents of ascorbic acid, flavonoids (quercetin and rutin), and total phenolics in comparison to control fruits. Moreover, AV gel-treated fruits displayed greater activities of superoxide dismutase, catalase, and ascorbate peroxidase, along with higher antioxidant capacity and higher levels of total soluble solids, than untreated fruits. These results demonstrate that AV gel coating, especially at high concentrations, can be considered an eco-friendly and non-chemical substitute treatment for maintaining the postharvest quality of guava fruit.Guava is an important climacteric fruits in terms of taste and aroma, which contains various vital nutrients such as minerals, carotenoids, ascorbic acid, and polyphenols. At ambient conditions, it exhibits a short shelf life, which makes it difficult for marketing and subsequent storage. Therefore, it is necessary to develop procedures to extend its shelf life and conserve quality. For this purpose, an aloe vera (AV) gel coating was assessed for its potential to enhance the shelf life of guava fruits. Guava fruits coated with AV gels (0, 20, 40, 60 and 80%,  Psidium guajava L.) fruit is highly palatable and a rich source of vitamin C. Pakistan, Brazil, South Africa, Mexico, India, Venezuela, Egypt, and Columbia are major guava-producing countries. In Pakistan, the total area of guava cultivation is 62,300 ha, producing 512,300 t of fruits per annum. After citrus, mango, and apple, guava ranks fourth in Pakistan in terms of area and production [The guava  family. It commonly grows in arid regions of most continents . The AV plant stem has a high tendency to survive in severe circumstances because it can retain moisture in warm and dry climates [Staphylococcus aureus, Salmonella, Streptococcus, Escherichia coli, Aspergillus niger, Candida, etc. [2 consumption and CO2 production, thereby preventing anaerobic conditions [2, resulting in a slow respiration rate and conserving fruit quality [Citrus sinensis L) fruit [Aloe arborescens) has also been used for postharvest application on various fruits. Recently, it was used as an edible coating for strawberries, reducing total soluble solids (TSS) increasing the concentrations of total phenolics compounds (TPC) and ascorbic acid (AsA), and improving the antioxidant activity in comparison to non-coated fruits [Aloe vera (AV) is a short-stemmed succulent plant species belonging to the Asphodeleceae  fruit . The geld fruits .2 showed greater firmness and better shelf life than untreated fruits [2 also reduces the ripening rate and color loss without changing the qualities of the products and maintaining a high content of TSS [w/v) in fruit has been reported in many studies. AsA has great anti-browning and antimicrobial effects and is used as an additive in the formulation of edible coatings [Calcium salts have been widely used in the food industry, especially in the fruit and vegetable industry, as a preservative and firming agent. It plays a vital role in maintaining the cell wall structure . Papaya d fruits ,19. In rt of TSS . Additiocoatings . Many aucoatings , jackfrucoatings , and papcoatings .The present research was planned to assess the potential of an AV gel to enhance the postharvest quality and shelf life of guava fruits. The antioxidant profiles of AV gel-coated fruits were also analyzed to understand the potential mechanisms of quality and shelf life improvement.Fine-quality guava fruits of the commercial cultivar \u201cGola\u201d at full green color and firm stage were purchased from local farms  located in Faisalabad, Pakistan. Fruits without any sign of injury, free from insects, pests, and diseases, and of uniform size, shape, and color were selected. All fruits were washed before application of the AV gel.Aloe barbadensis Mill) were grown in clay pots (5 kg soil) in a net house at Nuclear institute for agriculture and biology college (NIAB-C), Faisalabad, Pakistan. At maturity (180 days old), the leaves were cut with a sharp knife and brought to the Food Toxicology Laboratory, NIAB. The leaves  were washed with tap water and shade-dried, and the gel was separated and blended to obtain a homogeneous mixture. Glycerol, citric acid, carboxymethyl cellulose (CMC), and calcium chloride  were used for gel preparation. The pH of the gel was standardized to 3.75 with citric acid (5%), pasteurized at 90 \u00b0C for 15 min, cooled, and filtered using a muslin cloth. AV gel coating solutions  were made up to the volume of 1 L by adding distilled water. AsA (4% w/v) as an antimicrobial agent, CaCl2 (3% w/v) as a firming agent, glycerol (1% v/v) as a plasticizer, and CMC (3% w/v) as a thickening agent were added to each solution. All the ingredients were homogenized to smooth mixtures of coating gel solutions. Tween 20, 0.03% (v/v), was added to the gel for adhesive purposes.Plants of aloe vera  were used in this study. Fruits were treated with AV gel solutions in triplicate. All fruits were dipped for 3 min in AV gel solutions, dried at room temperature, packed in sintered polyurethane plastic baskets  and stored up to 12 days at 23 \u00b1 2 \u00b0C and 70\u201375% relative humidity for visual observations and biochemical analyses. Data on quality parameters and shelf life were recorded at 3-day intervals.TSS were determined by using a digital hand refractometer  and expressed as \u00b0Brix . The reaTotal chlorophyll content was measured by a spectrophotometer  at 663 and 644 nm .The total and reducing sugar contents of the fruits were determined by following a previously described method . Non-redLipid peroxidation level in the guava fruits treated with the AV gel formulations and in the control fruits was measured by the reaction of thiobarbituric acid (TBA) and trichloroacetic acid . AbsorbaThe Folin\u2013Ciocalteu (F\u2013C) colorimetric method was used for the determination of TPC in guava juice . The TPCThe TCC of all samples was determined spectrophotometrically on the basis of their chlorophyll content after measuring the absorbance (OD) at 663, 645, and 480 nm. The TCC was calculated by using the following formulas :ChlorophAsA content was measured by adding dichloroindophenol (DCIP) and metaphosphoric acid to the samples; after dilution with distilled water and thorough mixing, absorbance was measured at 520 nm . The TAC3) and potassium acetate (CH3CO2K) [TF were determined by diluting the samples with aluminum chloride (AlClCH3CO2K) . A lineaGuava juice samples were prepared and analyzed to determine the concentrations of rutin and quercetin. Extraction of flavonoids from guava juice samples for HPLC analysis was carried out by taking samples of guava juice (10 mL) in 50 mL Eppendorf tubes; 25 mL of 100% methanol was added to each tube . The samv/v) were prepared. An equal mixture of these solvents (50:50 v/v) was used for the separation of flavonoids in isocratic mode with the following parameters:Qualitative and quantitative determination of quercetin and rutin was conducted using HPLC . Two solvents (A: 3% trifluoroacetic acid (TFA) and B: acetonitrile and methanol ; analytical column: C18 ; delivery pump: LC-10AS; system controller: SCL-10A; acquisition software: CLASS LC-10. The determination of individual flavonoids was made by comparing the retention times and peak areas to those of certified reference standards .HPLC system: LC-10 A, with a UV-Vis detector (SPD 10-A (\u03bb 360 nm); flow rate: 0.8 mL/min; pressure: 67 kgf/cmFlavonoids (rutin and quercetin) were purchased from Sigma-Aldrich, USA. Different concentrations of standard were prepared in 100% methanol. A series of solutions  was prepared to determine the limit of detection (LOD) and linearity of detector (response).Activities of SOD, CAT, and APX were assayed in the outer skin of the fruits. Fruit samples were homogenized in 2 mL potassium phosphate buffer (pH 7.0). Sample homogenization, extraction of enzymes, and centrifugation at 10,000 rpm for 10 min were the steps involved in performing the enzymes assay. After centrifugation, the collected supernatants were used for enzyme activity assays. All the steps were executed at 4 \u00b0C .http://www.graphpad.com) was used.Analysis of variance was used to analyze the data. All the parameters were assessed at the 5% significance level by using Tukey\u2019s multiple comparison test by and the SAS 9.4 software. The complete experiment was conducted by factorial layout. The two factors were coating concentrations and storage days. Results are presented as mean \u00b1 SD. GraphPad Prism 8  than for the non-treated fruits (0 to 3.93%; Guava fruits changed color from green to yellow during the storage period. The change in color was quantified by measuring the chlorophyll content of the fruits\u2019 peel. AV gel treatments and storage time significantly influenced the chlorophyll content of guava fruits. The chlorophyll content decreased in AV gel-treated fruits as well as in control fruits, but the reduction rate was lower in treated fruits compared to the controls C. Fruit 2 treatments during storage [The effect of storage periods and AV gel treatments on reducing, non-reducing, and total sugars was significant. Regardless of the treatments, the total sugar content in guava fruits increased throughout the storage period A but rem storage  of guavaAn increase in MDA content is associated with membrane injury in fruit, resulting in membrane rupture or shriveling of the fruit\u2019s skin. Lipids in the epidermis are oxidized into MDA, which is indicative of damage to the fruit membrane . The MDAThe highest phenolic content was observed at the beginning of the storage period, and no change was found in control fruits up to the third day. However, later, an abrupt decrease in TPC was observed B in the Total carotenoid contents increased significantly in untreated control samples, whereas AV gel-treated samples exhibited smaller increases in total carotenoids during storage A. Oxidat2O2 and O2.TAC is the activity of total antioxidants against oxidative stress. We observed that TAC progressively decreased in AV gel-treated and control samples during the entire period of storage B. AV gel2 availability for oxidative breakdown, thereby reducing fruit degradation and fruit senescence [Ascorbic acid plays a role as an antioxidant as it helps to scavenge free radicals and, in that way, prevents the deterioration of fruit during ripening, owing to oxidation. The AsA content in control samples decreased significantly during storage, and this decline was also observed in AV-treated guavas. No significant difference in AsA concentration was observed in AV gel-treated and control guavas from the third to the nineth day of storage C. Howevenescence . AV gel nescence .Total flavonoid content decreased during storage in the control fruits. This reduction in TFC was retarded by AV gel treatment in a dose-dependent manner A. The AVThe quercetin and rutin contents decreased throughout the storage period regardless of the treatment, but it was observed that in AV gel-treated fruits their decline was comparatively slower than in control fruits B,C. The 2O2 in tissues [APX enzyme activity in AV gel-treated samples decreased from the third day to the nineth day during storage, whereas, in control fruits, the activity declined at a noticeable rate throughout the storage period A. Moreov tissues . In our  tissues .The results of this research demonstrate that chemical-free edible coatings like AV gels have potential to prolong the marketability of guava fruit. AV gel coating was found to be effective in maintaining the postharvest quality of guava fruit. Treatments with the AV gels prevented storage-induced changes such as weight loss, changes in total soluble solids and total chlorophyll content, while maintaining the contents of sugars. In general, the 80% AV gel treatment proved to be the most effective in improving the postharvest life, as evidenced by the values of the parameters assessed in this research. AV gel treatment reduced lipid peroxidation, consistent with its maintenance of antioxidants, i.e., TPC, TAC, TF, AsA, and carotenoids during the storage period. AV gel coating also prevented the decline in enzymatic antioxidants including APX, CAT, and SOD. AV gel coating can be considered an effective and environment friendly, non-chemical treatment for delaying ripening processes and retaining guava fruit quality through redox homeostasis during postharvest storage."}
+{"text": "Staphylococcus aureus in collagen-elastin matrices with CAP for 2 min resulted in a 4 log reduction. CAP treatment was less effective on S. aureus on dermal samples. CAP did not affect cellular activity or DNA integrity of human dermal samples when used for up to 2 min. Repeated daily CAP treatments for 2 min lowered cellular activity of dermal samples to 80% after 2 to 4 days, but this was not significant. Repeated treatment of ex vivo human burn wound models with CAP for 2 min did not affect re-epithelialization. Intact skin of 25 healthy volunteers was treated with CAP for 3\u00d7 20\u201d to determine safety. Although participants reported moderate pain scores , all volunteers considered the procedure to be acceptable. Severe adverse events did not occur. CAP treatment resulted in a temporarily increased local skin temperature (\u22483.4\u00b0C) and increased erythema. Lowering the plasma power resulted in a significantly lower erythema increase. Good log reduction (2.9) of bacterial load was reached in 14/15 volunteers artificially contaminated with Pseudomonas aeruginosa. This study demonstrated the in vitro and in vivo safety and efficacy in bacterial reduction of a flexible cold plasma device. Trial registration number NCT03007264, January 2, 2017Cold atmospheric plasma (CAP) devices generate an ionized gas with highly reactive species and electric fields at ambient air pressure and temperature. A flexible dielectric barrier discharge (DBD) was developed as an alternative antimicrobial treatment for chronic wounds. Treatment of \u2022 CAP strongly reduced bacterial numbers both in vitro and in vivo.\u2022 Re-epithelialization of burn wound models was not affected by repeated CAP.\u2022 CAP treatment of intact skin was well tolerated in volunteers.The online version contains supplementary material available at 10.1007/s00253-021-11166-5. Pseudomonas aeruginosa and Staphylococcus aureus  devices have been tested clinically as an alternative treatment to reduce bacterial load and support wound healing. Various plasma devices have been shown to be highly effective against a multitude of bacterial species including clinically relevant pathogens and multidrug-resistant bacterial strains, while buildup of resistance against CAP has not been observed  devices at the treatment site in ambient air by applying a high voltage. DBD devices use no counter electrode (surface DBD) or use the body as counter electrode (volume DBD). The antibacterial effects of surface DBDs have been shown in several in vitro studies  generates plasma in the wound in a closed system. The creation of plasma is accompanied by an electric current in the skin and by electromagnetic fields that can stimulate blood vessel formation and cell proliferation  Fig. . Plasma Ozone density was determined by absorption spectroscopy at 266 nm, using an Nd:YAG laser (ULTRA CFR Nd:YAG Laser System) coupled through optical fibres and collimated using quartz lenses  were harvested using a dermatome.P. aeruginosa  and S. aureus (Methicillin-resistant S. aureus (MRSA), strain LUH14616, culture collection NCCB 100829, obtained from P. Nibbering, LUMC) were routinely cultured on Luria-Bertani broth  agar at 37\u00b0C. Bacteria from a proliferating or logarithmic culture in 5 ml LB were diluted in 0.85% NaCl to the required colony forming units (CFU)/ml, based on OD600. To determine bacterial numbers, collagen-elastin matrices, dermal samples or the viscose ends of swabs were placed in a vial with 1 ml PBS and a metal bead and were placed in a TissueLyser LT  for 4 min at 45 Hz  were pipetted on dry porous collagen-elastin matrices ; CEM prewetted with 100 \u03bcl 0.85% NaCl or on skin . These samples were placed on agar, metal or chicken meat to which the reference electrode was connected. Chicken meat represented tissue that is present underneath wounds. To mimic the in vivo situation, ex vivo human skin with a hole (\u00d8 15 mm) was placed around the samples  were treated. Three punch biopsies (\u00d8 4 mm) were taken for viability assessment.2. Formazan was dissolved by shaking skin samples in the presence of 1 ml DMSO and a metal bead during 4 min at 50 Hz (TissueLyser). The resulting supernatant was quantified in a spectrophotometer . Results were expressed relative to untreated controls (in %). For repeated treatments, skin samples were incubated in-between treatments in FBM at 37\u00b0C, 5 CO2 for 24 h.To estimate tissue viability after treatment, MTT assays were performed  attached to a soldering iron . This would correspond to a full-thickness burn. The burned wound models (BWMs) were placed epidermis up on stainless steel grids and were cultured air exposed at 37\u00b0C with 5% CO2 using DMEM/ Ham\u2019s F12 (3:1) supplemented with 2% (v/v) P/S, 2% (v/v) FCS (Gibco), 1 \u03bcM hydrocortisone, 1 \u03bcM isoproterenol, 0.1 \u03bcM insulin, 1 \u03bcM L-carnitine, M L-serine, 1 \u03bcM DL-alpha-tocopherol, 130 \u03bcg/ml ascorbic acid, a lipid supplement containing 25 \u03bcM palmitic acid, 15 \u03bcM linoleic acid, 7 \u03bcM arachidonic acid  and 24 \u03bcM BSA (Thermo Fisher Scientific).To determine the effect of plasma on wound healing, an ex vivo human skin model was used , 20 \u03bcM 5-bromo-2-deoxyuridine (BrdU) (Merck KGaA) was added to the culture medium.Skin samples were processed for paraffin embedding. Sections (5 \u03bcm) were deparaffinized and rehydrated for haematoxylin and eosin (H&E) staining, using standard techniques. Sections from BWMs were stained for BrdU  . The study followed the tenets of the Declaration of Helsinki . A total of 25 healthy subjects were included from the general public; informed consent was obtained. Exclusion criteria were skin disease, infected wounds, implanted electrical medical devices, (possible) pregnancy, life-threatening cardiac conductivity abnormality and active malignancy. A research nurse assisted the procedures. Sixty percent of volunteers were male, average age was 58 \u00b1 12 years and Fitzpatrick skin type 3 was most prevalent (56%) , registered at %) Table . SubjectPain scores were determined on a numerical rating scale (NRS) of 0\u201310. A pain score less than 4 is considered acceptable pain . This reliable narrowband spectrometer generates an erythema index based on the differences in light absorption of red haemoglobin . TEWL estimates the flux density of evaporated water from the skin surface ; ECG patches  were placed on the right arm next to the pad to connect the reference electrode. Plasma treatments were applied (high power setting) on the right arm: 3 times for 20 s with plasma on separated by 2 intervals for 10 s with plasma off. The contralateral arm was treated exactly the same except plasma was not applied. One pad was used per arm, and pads were not changed between intervals. Pads and reference electrode were removed directly after treatment. Measurements were done immediately after treatment and after 30 min.7 CFU/ml) was applied on the area of interest on both arms and left to dry for a few minutes. Plasma pads and ECG patches were placed as in group A. Plasma treatments were applied 3 times for 20 s with plasma separated by 2 intervals for 10 s with plasma off, at high (group B) or low power setting (group C). Pads and reference electrode were removed directly after treatment, and thermal images were taken instantly and after 30 min. The scrub wash method  test was used to determine significant differences due to CAP treatment or between groups. Wilcoxon matched-pairs signed-rank test (WMP) was used for paired samples . Results of MTT assays were expressed relative to their respective untreated controls before averaging to circumvent donor variations. Volunteer characteristics sex and skin type were compared using the chi-square test for categorical data.Statistical analysis was performed with SPSS . The Mann-Whitney A flexible DBD plasma pad was developed to enable treatment of curved surfaces. Here we report the bactericidal and safety effects of in vitro tests. An interventional study on intact skin of human volunteers was conducted to study safety and efficacy in vivo.Initial tests were performed with bacteria spread on agar plates. These tests showed that the volume DBD pad inactivated bacteria effectively after short treatment times . To simulate the wound environment and for a better quantification of the bactericidal effect, we tested the plasma pads on bacteria in a 3D-collagen/elastin matrix . Collagen and elastin are major components of the dermis. CAP treatment of bacteria in dry CEM also resulted in high reduction (log 4 reduction) after <2 min treatment (data not shown).To mimic the in vivo wound situation even more, CEMs were placed on chicken meat, while human skin was placed around the CEM see Fig. . Plasma p<0.05).For safety assays, a similar setup was used. Dermal samples were placed on chicken meat, while human skin was placed around the sample to mimic the in vivo situation Fig. . Dermal 2. Viability was decreased after 2 and 4 daily CAP treatments to 80%  and plasma pad directly after CAP treatment. To limit the buildup of heat, the plasma treatment was divided in three periods of 20 s separated by intervals of 20 s with plasma off. Temperature of the plasma pad increased with treatment time Fig. . As a reOzone density measurements were performed using plasma treatment divided in three periods of 20 s separated by 2 intervals of 60 s with plasma off. During successive plasma periods of 20 s, ozone is generated at a concentration up to 600 ppm  were reported by the subjects: warmth sensation  and itching until one day after treatment (1 subject in group A). SAEs did not occur.p<0.05, MWU). Pain scores were 0 after 30 min for all subjects.The primary outcome pain estimated with NRS directly after CAP treatment was on average 3.3 Table . All subBaseline values were recorded on both inner volar arms. The mean absolute skin temperature at baseline was 32.0 \u00b1 3.8\u00b0C. CAP treatment significantly increased local skin temperature by on average 3.4\u00b0C compared to baseline in all groups Fig. . After pp<0.05, WMP). The lower power setting in group C resulted in a significantly lower erythema index (8% increase) compared to the high power setting . Thirty minutes after treatment, erythema index of the treated arm was still significantly increased in all groups . No changes were observed in erythema index on the left arm.Plasma treatment also significantly increased the redness of the skin Fig. . Erythemp<0.05, WMP). Significant differences between groups were not noted. TEWL after 30 min was lower on both arms in groups B and C than in group A, which was most likely the result of the swabbing and cleaning procedure used to determine bacterial survival in groups B and C.TEWL measures the evaporation of water from the skin, which characterizes the barrier function of the stratum corneum. TEWL was significantly increased directly after treatment in all three groups Fig. . BecauseP. aeruginosa. CAP was only applied to the area on the right arms. Surviving bacteria were collected from both arms by scrub wash and counted after plating. Data from one subject were excluded because of low bacterial survival on the untreated left arm. CAP significantly reduced the number of bacteria in both groups  was contaminated with ups Fig. . The meaIn this study, we have shown the bactericidal property and safety of a flexible CAP device, both in vitro in wound models and in healthy volunteers on intact skin. Short plasma treatments were able to greatly reduce bacterial load and did not cause DNA damage or reduce cellular activity or wound healing.The fast bactericidal effect of CAP was reduced by moisture and nature of the treated target such as skin vs. CEM Fig. , which h3 for 8 h per day. The PLASOMA prototype produces \u22483 \u03bcg ozone per 20-s period, which does not pose a problem.One of the reactive species involved in CAP-mediated bacterial killing is ozone. Up to 600 ppm ozone was locally produced by the DBD plasma pads Fig. . Ozone hSingle treatment with CAP for 120 s slightly reduced the cellular activity in dermal samples to 86% Fig. , while 7A systematic review showed that plasma treatment in general was safe . Application of CAP or heat will result in vasodilation and increased TEWL. Because TEWL values were increased in both arms in our study, this was most likely related to the temporary occlusion by the pad and not to the CAP treatment. TEWL values returned to normal levels after 30 min in groups B and C, which was probably related to the swabbing and cleaning procedure used to determine bacterial survival. The reason for the sustained high TEWL levels in group A of our study remains elusive. Literature reports on TEWL related to plasma treatment are inconclusive: TEWL values were reduced by pulsed plasma jet and DBD by about 20%   the temperature effect AE; device errors were generated when the safety circuit of the device intervened. Nevertheless, short CAP applications were found safe and demonstrated good bactericidal properties.ESM 1(PDF 476 kb)"}
+{"text": "Salmonella Typhimurium, and treatment with 1% AA\u00a0+\u00a08\u00a0ppm ozone caused browning of lettuce leaves and stimulated increases in aerobic mesophilic count (AMC), aerobic psychrotrophic count (APC), S. Typhimurium, and Escherichia coli O157:H7. Treatment with 2% LA\u00a0+\u00a08\u00a0ppm ozone resulted in the lowest S. Typhimurium, E. coli O157:H7, Listeria monocytogenes, AMC, APC, and molds and yeasts during storage (0\u20137\u00a0days at 4\u00a0\u00b0C). Quality analysis indicates that LA\u00a0+\u00a08\u00a0ppm ozone and SH\u00a0+\u00a08\u00a0ppm ozone did not negatively affect L*, a*, b*, polyphenolic content, weight loss, and sensory properties; however, the levels of two individual phenolic compounds , responsible for phenylpropanoid synthesis, were significantly increased after treatment with 2% LA\u00a0+\u00a08\u00a0ppm ozone. These findings provided insights into the use of LA combined with gaseous ozone for application in disinfecting fresh produce.Reduction of sanitizer dosage and development of non-immersion disinfection methods have become major focuses of research. Here, we examined the disinfection efficacy of combining gaseous ozone (4 and 8\u00a0ppm) with aerosolized oxidizing sanitizer [sodium hypochlorite ] and aerosolized organic acid [acetic acid  and lactic acid ]. Notably, 1% AA and 4\u00a0ppm gaseous ozone were ineffective for disinfecting  However, few reports have described disinfection methods that can simultaneously minimize sanitizer dosage and meet non-immersion characteristics . In addition, changes in microbial growth and fresh produce quality after washing can only be controlled by packaging, storage environment, coatings, and microbe-microbe interactions. However, during practical application, microbe-microbe interactions and coating film methods are complicated and expensive.Many recent studies have explored the minimum free chlorine (FC) concentration to prevent cross-contamination during fresh produce washing. For example, Luo et al.Ultrasonic-assisted aerosolization has the characteristics of consuming a low amount of sanitizer and the formation of micron-sized particles, which can attach to the leaf surface and continuously control the microbe levels after treatment. Among all aqueous sanitizers, the disinfection efficacy and cost of chemical disinfectants are most suitable for practical application In this study, we aimed to evaluate the disinfection effects of combinations of gaseous ozone  and aerosolized chemical sanitizers  using fresh-cut green leaf lettuce as a model.22.1Lactuca sativa L. var. crispa) was purchased from a local market on the day of the experiment. After rinsing for 30\u00a0s to remove dirt, the two outer leaves, inner baby leaves, and stems were removed, and a circle knife (diameter 5.2\u00a0\u00d7\u00a010\u22122 m) was used to cut the sample Green leaf lettuce (2.2E. coli O157:H7 (NCTC12900), L. monocytogenes (ATCC19115), and Salmonella Typhimurium (ATCC14028) were inoculated into nutrient broth  and shaken overnight at 37\u00a0\u00b0C. The bacterial suspension was adjusted to 109 CFU/mL, and 5\u00a0mL of this culture was then added to a stomacher bag containing 200\u00a0mL sterilized 0.85% NaCl solution. Then, 10\u00a0g of the lettuce sample was placed into the bag and massaged for 20\u00a0min. The sample was then placed on a sterilized plastic tray in a biosafety cabinet and air dried.The inoculation procedure was carried out according to our previous report 2.3The sanitizers used in this study were SH , AA , and LA (Macklin). The concentration of FC was adjusted to 100 or 200\u00a0ppm using a DPD test kit . The concentrations of AA and LA were adjusted to 1% and 2%. The concentrations of GO were 4 and 8\u00a0ppm, respectively.A schematic of the equipment used in this study is shown in 2.4E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium, respectively, and incubated for 24\u00a0h at 37\u00a0\u00b0C. For naturally present microbes, 0.1\u00a0mL of the diluted bacterial suspension was surface-plated on Rose Bengal agar (Hopebio) and incubated at 30\u00a0\u00b0C for 3\u00a0days to quantify molds and yeasts (M&Y). In addition, 1\u00a0mL of the suspension was pour-plated onto plate count agar (Hopebio) and incubated at 7\u00a0\u00b0C for 10\u00a0days to obtain the aerobic psychrotrophic count (APC) and or at 37\u00a0\u00b0C for 2\u00a0days to obtain the aerobic mesophilic count (AMC). All results are expressed as log CFU/g.Samples were analyzed at 0, 3, and 7\u00a0days. A 25-g sample was homogenized with 225\u00a0mL sterile NaCl solution for 1.5\u00a0min in a stomacher bag. Then, the suspension was serially diluted. The suspension (0.1\u00a0mL) was surface-plated on modified sorbitol MacConkey agar (Hopebio), Listeria chromogenic agar , and xylose lysine deoxycholate agar (Hopebio) to analyze 2.52.5.1At the end of the storage period (day 7), 10 leaves were randomly selected from each package for instrument color analysis. L*, a*, and b* values were detected at two locations per piece using a colorimeter . Before use, the colorimeter was calibrated using a white standard plate .2.5.2Fifteen panelists (ages 25\u201340\u00a0years) from Liaoyang, Liaoning, China were invited to evaluate sensory color, flavor, and crispness. A 3-point scale method was used for evaluation, where 0 was very bad (not characteristic of the product), 5 was the acceptability threshold, and 10 represented very good product characteristics 2.5.3Weight loss during the storage period (0\u20137\u00a0days) was analyzed according to the following formula:2.5.4The content of polyphenols was analyzed at the end of storage (7\u00a0days) using the Folin-Ciocalteu method 2.5.5Targeted metabolomics was applied to analyze the individual phenolics at the end of storage (7 d). The group of 2% LA\u00a0+\u00a08\u00a0ppm ozone and control were selected. Standard solutions of individual phenolic compounds were prepared at ten different concentrations  using 50% methanol. The fresh sample was homogenized and 0.1\u00a0g of the resulting homogenate was extracted using 2\u00a0mL of 1\u00a0N NaOH for 2\u00a0h, followed by the addition of 0.5\u00a0mL of 5\u00a0M HCl. Then, 2\u00a0mL ethyl acetate was used to purify the sample in three rounds of purification, followed by blow drying using nitrogen. Subsequently, 0.5\u00a0mL of methanol was added to obtain a sample for liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS) analysis. UHPLC  system equipped with a Waters HSS T3 column  was applied for analysis. Injection volume was 2\u00a0\u03bcL, and the column temperature was 40\u00a0\u00b0C. Mobile phase A was 0.1% formic acid (FA) with acetonitrile, and mobile phase B was 0.1% FA with water. The flow rate was 0.3\u00a0mL/min, and the gradient elution was as follows: 0\u20132\u00a0min, 10% A; 2\u20136\u00a0min, A was linearly increased from 10% to 60%; 6\u20138\u00a0min, 60% A; 8\u20138.1\u00a0min, A was linearly decreased from 60% to 10%; 8.1\u201310\u00a0min, 10% A. The separated sample was then subjected to a Q-exactive mass spectrometer (Thermo). ESI negative and single ion monitoring modes were used for detection. ESI source conditions were as follows: spray voltage, 3\u00a0kV; source temperature, 350\u00a0\u00b0C; full ms resolution, 70000; sheath gas flow rate, 40 Arb; aux gas flow rate, 10 Arb.2.6Differences between group means were evaluated using SPSS v.20 , and differences in mean values were analyzed using Duncan's multiple range tests. Results with P values of <0.05 were considered significant. All experiments were independently replicated three times.33.1Salmonella spp. is the pathogen that causes the most foodborne diseases S. Typhimurium. The results indicated that 1% AA and 4\u00a0ppm ozone were ineffective at disinfecting S. Typhimurium ; these values were significantly lower than those for SH\u00a0+\u00a0GO. The antibacterial activities of organic acids are traditionally attributed to cellular anion accumulation, which is associated with the dissociation constant (pKa). Compared with dissociated anions, undissociated acidic molecules have stronger lipophilicity, allowing them to penetrate the microbial cell membrane more easily. After penetration, the higher intracellular pH in the environment promotes dissociation of acid molecules, and the dissociated anions accumulate in the cell and exert toxic effects on DNA, RNA, and ATP synthesis Many studies have shown that hurdle technology cannot provide synergistic disinfection effects, but it can enable additional microbial reduction when compared with single disinfection methods himurium B and C. E. coli O157:H7, L. monocytogenes, and S. Typhimurium in the control and tap water groups were not significantly increased, consistent with a previous study S. Typhimurium and E. coli O157:H7 in the 1% AA\u00a0+\u00a0GO group were significantly increased, indicating that this combination could stimulate the growth of S. Typhimurium and E. coli O157:H7. Similarly, another study found that L. monocytogenes was stimulated to grow on lettuce after washing with 0.5% propionic acid (PA), whereas 1% PA significantly reduced the counts of this bacterium. The authors suggested that this result may be explained by the observation that, compared to the native microflora, L. monocytogenes is more resistant to 0.5% PA and more competitive, whereas 1% PA can create an acidic environment that exceeds the upper limit of resistance of the bacterium Ka value; AA has a pKa value of 4.75, which is similar to that of PA (4.87). With the increase in storage days, the counts of E. coli O157:H7, L. monocytogenes, and S. Typhimurium in the LA\u00a0+\u00a0GO group showed a decreasing trend, and the combination of 2% LA\u00a0+\u00a0GO led to the lowest counts on days 3 and 7; these values were significantly lower than those for SH\u00a0+\u00a0GO. Similarly, researchers in a previous study L. monocytogenes on broccoli showed a decreasing trend during storage after washing with organic acid\u00a0+\u00a0oxidizing sanitizer . In summary, the combination of 2% LA\u00a0+\u00a0GO appeared to be the best choice for controlling foodborne pathogens on fresh-cut lettuce.During storage, the counts of For naturally present microbes, among the five treatment combinations, the 2% LA\u00a0+\u00a0GO group had the lowest counts  on day 0 A\u2013C. Acco3.33.3.1Purchase decisions for minimally processed leafy greens are strongly influenced by their color, which directly affects consumer visual perception Previous studies have shown that GO can cause color deterioration in lettuce leaves. For example, in one study Compared with oxidizing sanitizers, organic acids typically have fewer negative effects on color quality. Berm\u00fadez-Aguirre and Barbosa-C\u00e1novas 3.3.2Ganoderma lucidumPolyphenolics are important secondary metabolites of plants and function as key nutrients to prevent oxidative damage in the human cells. After 7\u00a0days of storage, the polyphenolic content was 312.34\u00a0mg/kg GAE in the control group B, consistrans-ferulic acid  groups were compared. As phenolic acids are the major phenolic compounds in lettuce lic acid , which ilic acid C and E, During storage, the weight loss of fresh produce was mainly due to nutrient loss and water segregation 3.3.3Sensory color, flavor, and crispness are crucial factors affecting consumer acceptance 4S. Typhimurium levels. Additionally, 1% LA\u00a0+\u00a08\u00a0ppm GO, 2% LA\u00a0+\u00a08\u00a0ppm GO, 100\u00a0ppm FC\u00a0+\u00a08\u00a0ppm GO, and 200\u00a0ppm FC\u00a0+\u00a08\u00a0ppm GO did not negatively affect the quality and sensory properties of fresh-cut lettuce. Overall, LA\u00a0+\u00a0GO and SH\u00a0+\u00a0GO could significantly disinfect foodborne pathogens  and naturally present microbes , with 2% LA\u00a0+\u00a08\u00a0ppm GO resulting in the lowest counts. This additional microbial reduction caused by LA\u00a0+\u00a0GO may be due to the different antibacterial mechanisms of action of LA and GO . After LA penetrates the cell membrane, the higher intracellular pH environment promotes its dissociation, which leads to inhibition of DNA, RNA, and protein synthesis; additionally, ozone oxidizes the outer cell membrane. Similarly, SH destroys the cell membrane; thus, the additional microbial reduction caused by SH\u00a0+\u00a0ozone may be attributed to the accelerated destruction of cell membrane. In future studies, omics technology should be applied to analyze the changes in bacterial biological processes. Furthermore, scanning electron microscopy, propidium iodide staining, K+ leakage analysis, and protein leakage analysis should be carried out to reveal the mechanism of action of the additional microbial reduction caused by LA\u00a0+\u00a0GO and SH\u00a0+\u00a0GO. Finally, our study showed that 1% AA\u00a0+\u00a08\u00a0ppm GO stimulated increases in AMC, APC, S. Typhimurium, and E. coli O157:H7. These findings provided important insights into the use of organic acids combined with GO for application in disinfecting fresh produce.In this study, we proposed a non-immersion disinfection method for lowering sanitizer dosage and the risk of cross-contamination. Aerosolized sanitizer and GO were combined to disinfect fresh-cut lettuce. Our results showed that 2% AA and 1% AA\u00a0+\u00a08\u00a0ppm GO could cause browning of lettuce leaves, and 1% AA and 4\u00a0ppm GO did not significantly reduce Jiayi Wang: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing. Yangyang Zhang: Investigation, Methodology, Writing - original draft. Yougui Yu: Writing - review & editing. Zhaoxia Wu: Data curation. Hongbin Wang: Formal analysis.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "Ischemic retinal dystrophies are leading causes of acquired vision loss. Although the dysregulated expression of the hypoxia-responsive VEGF-A is a major driver of ischemic retinopathies, implication of additional VEGF-family members in their pathogenesis has led to the development of multivalent anti-angiogenic tools. Designed as a decoy receptor for all ligands of VEGFR1 and VEGFR2, Aflibercept is a potent anti-angiogenic agent. Notwithstanding, the molecular mechanisms mediating Aflibercept\u2019s efficacy remain only partially understood. Here, we used the oxygen-induced retinopathy (OIR) mouse as a model system of pathological retinal vascularization to investigate the transcriptional response of the murine retina to hypoxia and of the OIR retina to Aflibercept. While OIR severely impaired transcriptional changes normally ensuing during retinal development, analysis of gene expression patterns hinted at alterations in leukocyte recruitment during the recovery phase of the OIR protocol. Moreover, the levels of Angiopoietin-2, a major player in the progression of diabetic retinopathy, were elevated in OIR tissues and consistently downregulated by Aflibercept. Notably, GO term, KEGG pathway enrichment, and expression dynamics analyses revealed that, beyond regulating angiogenic processes, Aflibercept also modulated inflammation and supported synaptic transmission. Altogether, our findings delineate novel mechanisms potentially underlying Aflibercept\u2019s efficacy against ischemic retinopathies. Representing leading causes of vision loss worldwide2, retinal microvasculopathies are characterised by the emergence of new retinal blood vessels in a process known as neovascularization3. Such vessels, however, are immature and leaky. Consequently, nourishment and oxygenation to retinal cells becomes deficient4. Together with the accumulation of waste products, the latter ultimately leads to retinal malfunction and vision impairment.Retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) are prominent microvasculopathies that severely compromise retinal function5 in the retina7 and in tumours8 led to the development of multiple pharmacological compounds for its neutralization. Despite their efficacy in the treatment of cancer10 and neovascular retinopathies12, VEGF-A inhibitors exhibited unforeseen side effects and limitations, including toxicity, short systemic life-times, and limited diffusion across the tissue13. The latter, together with findings suggesting that the placental growth factor (PGF)\u2014another member of the VEGF-family\u2014played a major role in driving pathological neovascularization14, boosted interest in the development of multivalent anti-angiogenic therapies15. To simultaneously inhibit both PGF and VEGF-A, the decoy receptor Aflibercept  was created. AFL is a fusion protein formed by the extracellular domains of VEGF receptor 1 (VEGFR1) and VEGFR2 joined to the crystallizable fragment of human IgG17. Thereby, AFL is capable of neutralizing the ligands of VEGFR1 and VEGFR218 and has received FDA approval for the treatment of age-related macular degeneration (AMD), diabetic macular edema (DME), macular edema following retinal vein occlusion, and background DR2. However, AFL has not yet been authorized for use against PDR (off-label only) or ROP, although phase III clinical trials are ongoing for the latter (NCT04004208).The identification of the vascular endothelial growth factor A (VEGF-A) as a hypoxia-responsive driver of pathological angiogenesis19, which represents the most widely studied in vivo model of retinal proliferative microvascular disease21, to investigate the effects of AFL administration on the ischemic retina22. Our study revealed that AFL potently inhibits retinal neovascularization while permitting the concomitant revascularization of the tissue and partially protecting dopaminergic amacrine cells from hypoxic damage. Additionally, transcriptional profiling during the proliferative phase of the OIR protocol  using two distinct AFL treatment regimens , and at positive effects of AFL on synaptic transmission in OIR retinas, where its administration seems to protect retinal ganglion cells (RGCs) from hypoxic damage.Previously, we used the oxygen-induced retinopathy (OIR) mouse model20. Here, we used the mouse model of OIR to investigate the transcriptional changes undergone by retinal cells in response to hypoxia and to subsequent anti-angiogenic therapy. To this aim, we administered AFL, a recombinant decoy receptor neutralizing the ligands of VEGFR1 and VEGFR2, at a dose of 25\u00a0mg/kg body weight to mice subjected to the OIR protocol. The drug was injected intra-peritoneally (i.p.) following two distinct regimens: either (1) on P13 and P15, or (2) on P13, P15 and P17  behaviour in health and diseaseP17 Fig.\u00a0\u201d. Retina22 , which increased over 2.5 times for the period between P17 and P19 , potentially supported by enhanced ion transport (e.g. potassium ion transport), and neuronal and synaptic maturation . In contrast, GO terms enriched in OIR retinas at P17 relative to P14 were primarily associated to injury repair , inflammation (e.g. response to interferon-gamma), hypoxia (peptidyl-proline hydroxylation to 4-hydroxy-L-proline), and angiogenesis (e.g. positive regulation of cell migration involved in sprouting angiogenesis). In line with these findings, a recent study reported that sterile inflammation and the recruitment of neutrophils are both essential for the clearance of neovascular tufts from the OIR retina24. Meanwhile, GO terms enriched at P19 compared to P17 were mainly related to metabolic changes in both normoxic and OIR retinas 29.Previously, we described the transcriptional state of the OIR retina at P14 and P17 after AFL administrationglutamatergic synapse) as enriched from P14 to P17 in normoxic retinas , cancer (e.g. melanoma), response to infection (e.g. staphylococcus aureus infection) and diabetes . In agreement with our GO term enrichment analysis, KEGG pathway enrichment analysis also revealed that most changes between P17 and P19 were metabolic, both in normoxic and OIR retinas  or proteins relevant for cell-extracellular matrix interactions . Whereas the synthesis and deposition of basement membrane components such as collagen IV (of which a component is encoded by Col4a1) by ECs represent crucial processes in blood vessel formation and maturation31, Anxa2 expression is known to be regulated by VEGF and has been previously reported to be elevated in OIR retinas, with its knockdown supressing retinal neovascularization32. Further, the up-regulated expression of Angpt2 at all examined time-points might be of key relevance as Angiopoietin-2 (Ang2), the protein it encodes, has been detected in high levels in the vitreous of individuals with DR33 and agents for its blockade (e.g. Faricimab) are currently undergoing clinical trials. Additionally, the PDZ and LIM domain protein 1 (encoded by Pdlim1) has been recently reported to play a role in the assembly of stress fibres and to be potentially involved in cell migration34, a crucial process for the formation of neovascular tufts. While the role of mir-7025 in the context of retinopathy requires investigation, the only gene detected as down-regulated in OIR retinas relative to normoxic controls at all three examined time-points was Pcp4. Encoding Purkinje Cell Protein 4, Pcp4 expression has been reported in ganglion, amacrine and horizontal cells35. Thus, our findings hint at a potentially detrimental effect of OIR on these retinal cell populations. Further, the expression of the Insulin-like growth factor-binding proteins 5 and 7 (Igfbp5 and Igfbp7) was higher in OIR than in normoxic retinas , most were related to the binding of cellular and extracellular components . At this time-point, GO terms enriched in the biological process domain . The latter, however, are likely linked to pathological aspects of blood vessel formation as enriched GO terms also included the negative regulation of vasoconstriction and the regulation of blood\u2013brain barrier permeability. In agreement, we previously reported hyperpermeability and a higher number of dilated vessels in the OIR retina than in normoxic age-matched controls22. Notably, the GO term \u201cpositive regulation of endothelial cell chemotaxis by VEGF-activated VEGFR signaling pathway\u201d was also enriched, evidencing the role of VEGF in the pathogenesis of OIR.To determine whether gene sets associated to specific cellular functions were differentially expressed in OIR retinas relative to normoxic controls, we conducted GO term enrichment analysis at each time-point. Whereas the most strongly enriched terms in the molecular function domain at P14 Fig.\u00a0d, top weain Fig.\u00a0e, top wechemoattractant activity) and VEGF signalling . GO terms enriched in the biological function domain at this time-point also partially overlapped with those detected at P14 . Remarkably, enriched terms also included some related to leukocyte recruitment and to increased apoptosis. These findings are in agreement with previous reports describing a decrease in the number of Foxp3+ Regulatory T cells occurring concomitantly with the emergence of microvascular aberrations in the OIR retina40, as well as a central role for neutrophils in repairing such aberrations by promoting the apoptosis-mediated clearance of senescent ECs within neovascular tufts24. In particular, 6 genes  belonging to the \u201cpositive regulation of leukocyte migration\u201d GO term were detected among those differentially up-regulated in P19 OIR retinas relative to age-matched controls. Meanwhile, 8 genes belonging to the \u201cleukocyte migration\u201d term were detected, including 5 not associated to the term \u201cpositive regulation of leukocyte migration\u201d, namely Ccl3, Itgb1, Fcer1g, Cd34 and Itga6. Additionally, 9 genes were associated to the term \u201cpositive regulation of chemotaxis\u201d, including 7 not associated to neither of the two previous GO terms: Pdgfrb, Sema7a, Padi2, Angpt2, Tubb2b and Fn1.Although most of the GO terms identified as enriched at P19 within the molecular function domain had already been detected at P17, previously unidentified terms included \u201cinsulin-like growth factor I binding\u201d and \u201cplatelet-derived growth factor binding\u201d Fig.\u00a0d, bottomPecam1 is known to be required for leukocyte transendothelial migration under inflammatory conditions41, Mstn was recently shown to be expressed in bloodstream-derived leukocytes42, and integrin \u03b21 (encoded by Itgb1) was recently described to mediate the adhesion of monocytes to liver sinusoidal ECs and thereby contribute to hepatic inflammation43. Further, detecting not only Itgb1 but also Sema7a as up-regulated is remarkable given that endothelial Semaphorin 7A is known to promote neutrophil migration during hypoxia44 and that vascular Semaphorin 7A up-regulation has been shown to promote atherosclerosis through endothelial \u03b21 integrin45. Similarly, monocyte/macrophage-derived matrix metallopeptidase 14 (encoded by the Mmp14 gene) has been reported to modulate monocyte infiltration in a context-dependent manner46 while CC chemokine ligand 3 (encoded by Ccl3) is known to induce neutrophil chemotaxis under conditions of acute inflammation47. Meanwhile, knockdown of nckap1l in zebrafish has recently been reported to reduce neutrophil migration48, and its upregulation might thus promote neutrophil recruitment. Hence, an increased presence of leukocytes in the OIR retina at P19 is likely. Further, up-regulation of Cx3cr1 might be linked to the significant increase in the number of microglial cells previously reported to occur in the OIR retina49, although it is surprising that the expression of this gene was not up-regulated neither at P17 nor at P14. Considering that Cx3cr1 depletion has been associated to an accelerated progression of retinopathy in the rat model of streptozotocin-induced diabetes50, its up-regulation during the post-P17 phase of the OIR protocol might also play a relevant role in recovery. Together, these findings suggest that multiple genes encoding proteins with pronounced, and in certain cases complementary, regulatory effects on leukocyte migration are upregulated in the OIR retina at P19.Among these genes, To shed light on the transcriptional processes mediating the capacity of anti-angiogenic therapy to counteract pathological neovascularization and reduce hypoxia-induced damage in the retina, OIR animals were treated with AFL -like) protein, a secreted molecule that modulates macrophage chemotaxis and activation54 whose expression levels were higher in OIR\u2009+\u2009AFL than in OIR retinas at P17 but lower in OIR\u2009+\u2009AFLx3 retinas than in OIR, a transcription factor (Sox2), a microRNA (Mirlet7g), the Glycosylphosphatidylinositol-anchored molecule-like (Gml), and an uncharacterized protein . Kcnj8 mutations have been linked to excessive retinal vessel formation and increased vascular tortuosity in retinas of children and adults affected with Cant\u00fa syndrome, respectively55, and Kir6.1 expression has been detected in human retinal pigment epithelium (RPE)56. Further, expression of the microRNA (miR) let-7\u00a0g is downregulated in the heart of mice after coronary occlusion in vivo and in neonatal rat ventricular myocytes subjected to hypoxia in vitro, with its ectopic expression preventing hypoxia-dependent caspase activation and death in both settings57. Similarly, hypoxia has been shown to increase the expression of Sox2 in mesenchymal stem cells58, with higher oxygen levels leading to its reduced expression in embryos in vitro59. Therefore, the reduced expression levels of Kcnj8, Mirlet7g, and Sox2 in response to AFL might evidence its beneficial effects on retinal oxygenation, with their functional relevance in the OIR retina requiring further investigation. Meanwhile, Gml is speculated to be a p53 target involved in the apoptotic pathway and in cell-cycle regulation60. Yet, its relevance to retinal disease, as that of the uncharacterized Pnmal2, remains unknown.Unexpectedly, only 11 genes were differentially regulated at P19 in response to both two and three AFL doses , Mir103-1, and Rps19-ps11. The functions of 2310008H04Rik, encoding a scaffolding protein involved in DNA repair, in the context of retinal disease are presently unknown. Meanwhile, Rps19-ps11 encodes a pseudogene of the ribosomal protein S19 (Rps19). Although pseudogenes are presumed to be non-functional as a consequence of mutations and truncation due to premature stop codons, some are protein-coding61. In this sense, knockout of Rps19 is pre-implantation lethal in mice62 and its knockdown has been shown to result in stagnant blood cells as a consequence of circulatory defects in zebrafish (Danio rerio)63. Notably, by binding the macrophage migration inhibitory factor (MIF), Rps19 reduces the adhesion of monocytes to ECs64, which is a key step in the inflammatory response. In turn, miR103-1, whose expression is downregulated in RPE cells in response to hypoxia65, binds the nuclear factor-\u03baB (NF\u03baB) Interacting LncRNA (NKILA), whose high levels drive the up-regulation of NF\u03baB, lead to neuronal cell death and protect RPE cells from hypoxic damage66. Hence, the upregulation of these genes at P19 in response to AFL might be relevant for the regeneration of the retinal microvascular network, to modulate inflammation, and to protect retinal cells from hypoxia.Three genes were up-regulated in both P19 OIR\u2009+\u2009AFLx2 and P19 OIR\u2009+\u2009AFLx3 retinas compared to OIR controls . HMGCS2 is highly expressed by primary human foetal RPE cells in vitro67 and mHS is one of three mitochondrial enzymes often inactivated in cancer68. Moreover, the product of mHS, \u03b2-hydroxybutyrate, has a role not only in cellular energetics but also as an agonist of the cell surface receptor GPR109A, a tumour suppressor relevant to apoptosis68. As the metabolic transcriptome of retinal ECs undergoes profound changes in pathological conditions69, the relevance of Hmgcs2 as a possible regulator of EC apoptosis in the OIR retina should be addressed in future studies.Interestingly, only one DRG was up-regulated in response to AFL both at P14 and P19 (\u00d7\u20092)  and P19(\u00d7\u20093), three genes were differentially regulated in response to AFL  has been previously detected in mouse kidneys and brain70, with its expression recently reported to be confined to the endothelial compartment in the central nervous system71. Notwithstanding, MCT14 remains an orphan transporter with unknown functions or substrate specificities72. Similarly, the role of 2610528A11Rik in the retina is currently unknown. However, its human ortholog, chromosome 10 open reading frame 99 (C10ORF99), is structurally similar to chemokines of the CC family and modulates the infiltration of T cells to skin transplants in mice73. On this basis, we hypothesize that the upregulation of 2610528A11Rik in response to AFL might be relevant to regulate leukocyte recruitment to the OIR retina. Meanwhile, Abcc9, encoding the ATP-binding cassette subfamily C member 9 protein, has been reported as down-regulated in murine OIR retinas74 and, similar to KCNJ8, gain-of-function mutations in ABCC9 have also been reported in Cant\u00fa syndrome76. As both Kcnj8 and Abcc9 encode subunits of ATP-sensitive potassium channels, which have been linked to enhanced retinal resistance against severe ischemic insults77, their AFL-induced down-regulation at P17 and P19 might be a consequence of improved retinal oxygenation.Five genes were identified as differentially regulated in response to AFL in three treatment regimens Fig.\u00a0d. At P14pt2 Fig.\u00a0c. The laPecam1 , AFL administration led to the reduced expression of Gabra4, Tax2, P2rx2, Gabrb1, Glra1, Shc3, Cplx1, Plat, Drd4, and Gabrg3. From these genes, only 2 (Drd4 and Plat) had higher expression in OIR than in normoxic retinas, with 7 being lower and one (Tac2) non-significant  as differentially down-regulated in OIR retinas, with two of them fully and one of them partially restoring their expression levels relative to normoxia in response to AFL. GABAA receptors are expressed in RGCs, where their blockade has been shown to reduce oxidative stress-induced death83. Whereas this finding by itself could suggest that RGCs engage in a protective behaviour to reduce hypoxia-associated damage and death, the reduced expression levels of Glra1, which encodes the subunit alpha-1 of the Glycine receptor , may instead indicate that RGC functionality and survival is compromised as a consequence of OIR and partially rescued by AFL administration. Finally, among the 10 genes associated to the chemical synaptic transmission term, P2rx2, which encodes a subunit of a ionotropic receptor expressed in starburst amacrine cells86, was the most significantly down-regulated gene in OIR relative to normoxic retinas and subsequently up-regulated in response to AFL at P17. No GO terms in the biological process domain were detected as enriched in P19(AFLx2) or P19(AFLx3) retinas. Overall, our findings suggest that, beyond modulating angiogenic processes in the OIR retina while exerting regulatory effects on apoptosis and ion transport, AFL also aids in preventing hypoxic damage to retinal neurons, with changes in gene expression suggesting the protection of RGCs and amacrine cells.Further, it was striking to detect three distinct subunits of the Gamma-aminobutyric acid (GABA) receptor Type A , has been shown to protect cells from oxidative stress by scavenging oxidative molecules and harmful products of oxidation87, while Lymphocyte antigen 6A-2/6E-1 , encoded by Ly6a, marks a population of multipotent cells in the neonatal murine retina88 and of bone marrow stem cells which differentiate into glial and microglial cells that help preserve visual function in a mouse model of retinal ischemia/reperfusion89. Importantly, as Ly6G inhibition was recently reported to impair the endogenous capacity of the murine OIR retina to clear neovascular tufts after P17 by depleting it from neutrophils24, the up-regulation of Ly6a we observed in P14 OIR\u2009+\u2009AFL retinas relative to P14 OIR ones might reflect an increased presence of neutrophils with the capacity to promptly destroy such tufts. Thus, these findings suggest that AFL may protect the retina from hypoxia-induced damage by increasing the cell\u2019s capacity to tolerate oxidative stress while supporting the presence of microglial cells and tuft-clearing neutrophils.Of the 5 remaining transcripts downregulated both by AFL at P14 and between P17 and P19 in OIR, AFL only restored the expression level of one of them, namely BC046401; for the other 4 transcripts, AFL administration led to an expression level significantly higher than in OIR or normoxia  or partially  restored to normoxic levels by AFL, with only one transcript (Mir467c) maintaining expression levels similar to baseline at all timepoints  and, on the other, allowing rapidly proliferating cells to migrate more effectively via cell\u2013cell and cell-ECM interactions90. Importantly, CD146 (encoded by Mcam) is a co-receptor required for the phosphorylation of VEGFR-2 upon binding with VEGF-A and the subsequent promotion of EC migration and microvascularization91. Thus, its down-regulation by AFL might limit the VEGF-A-induced activation of VEGFR-2 and prevent EC invasion of the vitreous body. Meanwhile, Itgb1, which had been briefly discussed earlier, is essential for the formation of stable and mature blood vessels but can also impair sprouting angiogenesis in certain contexts92. Hence, its down-regulation by AFL might support a more efficient retinal revascularization by promoting sprouting angiogenesis. Additionally, the RNA binding motif protein 24 (encoded by Rbm24) is known to post-transcriptionally regulate the stability of the transcription factor Sox293. As its depletion has been reported to result in anophtalmia or microphtalmia in mice and zebrafish93, which are defects similar to those linked to SOX2 deficiency in humans, reduced expression of this gene in OIR retinas might severely impair correct ocular development. In this sense, Complexin (encoded by the Cplx1 gene) is relevant for the stabilization and timely release of synaptic vesicles94 and its modulation by AFL might be related to improvements in synaptic maturation.From the 15 genes differentially regulated both in P19 OIR retinas relative to P17 OIR\u00a0ones and in P17 OIR\u2009+\u2009AFL retinas relative to OIR ones at P17 but not at P14, the expression levels of 14 of them were either fully 96 expressed also on newly forming vessels in developing tissues97. Therefore, its down-regulation in response to AFL at P17 might be the reflection of reduced levels of hypoxia in the tissue. Interestingly, the expression trends of Fam19a4 and Kcna4, both of which are known to be expressed in RGCs99, change similarly in normoxic and OIR retinas throughout time. The latter suggests that the normalizing effect of AFL on the expression of these genes at P17 might be linked to a protective effect on such cell population. Meanwhile, the expression dynamics of Mir1896, which is upregulated in activated astrocytes and induces the production of inflammatory cytokines and the migration of CD4+ T cells in a mouse model of experimental autoimmune encephalitis100, suggest that a likely consequence of the hypoxic damage induced to the retina by the OIR protocol might be a period of retinal astrocyte activation.Further, Vsnl1 and Nabp1, were also lower in OIR\u2009+\u2009AFL(\u00d7\u20092) retinas than in normoxic controls and only a third dose of AFL restored their expression. While Nabp1 has been primarily linked to the DNA damage response and to transcription termination as part of the Integrator complex101, enriched expression of Vsnl1 has been previously detected in rat RGCs using microarrays102, which suggests that a third dose of AFL prolongs its protective effects on this cell population. From the clinical perspective, this finding might be of the utmost relevance in light of the very recently reported continuous loss of RGCs in individuals affected by diabetic retinopathy (DR)103.To assess the effect of administering\u00a0an additional dose of AFL at P17, we compared the\u00a0transcriptional profiles of P19 animals subjected to the OIR protocol that received two and three AFL injections. This comparison revealed the differential regulation of 92 genes (Supplementary Tables Olfr genes). Indeed, relative to OIR\u2009+\u2009AFL(\u00d7\u20092) retinas, 17 Olfr genes were down-regulated in OIR\u2009+\u2009AFL(\u00d7\u20093) tissues, which is in agreement with the identification of \u201csensory perception of smell\u201d and \u201colfactory receptor activity\u201d as the most and second most enriched GO terms in the biological process and molecular function domains, respectively  retinas, respectively. Considering the prominent role that microRNAs play during retinal development, as well as in pathological conditions107, and the recent report of certain miRNAs gradually changing their expression levels throughout the progression from non-proliferative to PDR in humans103, it is not beyond reason that AFL might partially exert its beneficial effects by modulating the expression level of these transcripts. However, this hypothesis requires future experimental validation.In contrast, from the 73 genes differentially down-regulated in OIR\u2009+\u2009AFL(\u00d7\u20093) retinas relative to OIR\u2009+\u2009AFL(\u00d7\u20092) ones, 19 were dysregulated after a third dose of AFL compared to normoxic controls while only 13 were differentially expressed in retinas of animals that received only 2 injections. Nonetheless, a third dose of AFL effectively normalized the expression levels of 11 of these 13 dysregulated genes, including 3 olfactory receptors  and three  upregulated genes in P19 OIR\u2009+\u2009AFL(\u00d7\u20093) retinas found to also increase their expression in normoxia from P14 to P17 and from P17 to P19, respectively  retinas relative to P19 OIR\u2009+\u2009AFL(\u00d7\u20092) samples with those up and down-regulated either from P14 to P17 or from P17 to P19 in the retinas of mice kept in ambient air conditions. Our comparison revealed no overlap in down-regulated genes, with one , the use of distinct animal species, and/or modifications to the OIR protocol19 (we started hyperoxia one day earlier). For instance, of 662 DRGs we identified at P17 in OIR retinas, only 80 overlapped with those found by Guarischi-Sousa and colleagues at the same time-point112 . In a different study, 43 and 1622 DRGs were identified in OIR retinas at P13 and P18, respectively74; in contrast, we detected 272 and 398 DRGs at P14 and P19. Notwithstanding, the processes regulated by the genes we identified are consistent with those reported in other studies on the OIR retina117, including sprouting angiogenesis, metabolism and inflammation.Considering this limitation, we attempted to maximize the sensitivity of our approach by using a low threshold for identifying DRGs (fold-change\u2009>\u20091.2). The latter is in line with a recent study on the human diabetic retina reporting the magnitude of changes in gene expression levels to be below 50%24. In agreement, a number of GO terms detected as enriched in our study were related to leukocyte migration and chemotaxis. Moreover, the proteins encoded by the genes associated to these terms included important regulators of leukocyte adhesion and transendothelial migration, as well as modulators of monocyte infiltration and neutrophil migration. Additionally, as high VEGF-A levels have been suggested to lead to leukostasis in the eye118, its neutralization by AFL might expedite leukocyte recruitment to neovascular tufts for their subsequent clearance. In this context, our findings are in line with previous reports and hint at the potential regulatory role of AFL on this process.Presently, the mechanisms mediating neovascular tuft clearance remain understudied. In a recent report, leukocytes were described as central players in this processAngpt2, as its expression was consistently elevated at all examined time points in OIR retinas, in agreement with a previous report119, and downregulated by AFL at P14, P17 and P19 after 2 doses; in P19(\u00d7\u20093) retinas its fold-change was -1.16 . Ang2 is an antagonist of the tyrosine kinase receptor Tie2 whose importance in vascular development is difficult to overstate. While the expression of Ang2 is responsive to hypoxia120 and its dysregulation is characteristic of disease121, its blockade has been shown to reduce tumour vascularization and to inhibit metastasis in mammary gland carcinomas and pancreatic insulinomas122. Additionally, Ang2 has been implicated in OIR123 and described to be up-regulated in the serum of type 2 diabetic patients with either non-proliferative DR or PDR compared to patients without retinopathy124. These findings have attracted interest in the possibility of pharmacologically regulating the activity of this agent, with clinical trials assessing the efficacy of neutralizing this molecule currently ongoing125. In this sense, the bispecific anti-VEGF/anti-Ang2 antibody Faricimab has been shown to be more effective than sole anti-VEGF therapy in an animal model of laser-induced choroidal neovascularization126 and studies evaluating the therapeutic efficacy of simultaneously blocking VEGF-A and Ang2 are ongoing127. As Angpt2 expression is increased in response to stabilization of the hypoxia-inducible factor2, our results suggest that AFL is effective in reducing retinal hypoxia.The number of DRGs detected in response to two doses of AFL at P17 (149) was similar to that observed at P19 after three doses (151). Interestingly, however, no single gene was differentially regulated in all treatment regimens. Yet, the most relevant target of AFL identified in our study might be Esm1 and Cd34. Esm1 has been previously reported as a top overexpressed gene in OIR retinas at P17119 and plays a role in both pathological and physiological angiogenesis, modulating VEGF bioavailability and regulating leukocyte extravasation128. Moreover, increased expression of Esm1 enhances the proliferative capacity, colony forming ability, migration and invasiveness of radiotherapy-resistant breast cancer cells all by itself, with its down-regulation reversing these effects129. Meanwhile, Cd34 has been previously shown to promote pathological neovascularization in the OIR retina130. Therefore, the downregulation of these genes might constitute a relevant mechanism by which AFL inhibits the formation of neovascular tufts while promoting their clearance and permitting the regeneration of the retinal microvascular network. Together, our observations suggest that the capacity of AFL to concomitantly modulate the synthesis and deposition of ECM components, which regulate adhesion, together with its effects on Angpt2 levels, might represent key mechanisms by which it orchestrates a more efficient recovery of the retinal microvasculature. Specifically, we hypothesize that lower levels of Ang2 are central to supress the exuberant vasoproliferation characteristic of the OIR retina while the normalization in the composition of the basement membrane might allow ECs to migrate and assemble functional vessels that contribute to reducing tissue hypoxia more effectively. Moreover, besides genes, we reveal that AFL exerts regulatory effects over certain miRNAs. Given the broad spectrum of binding partners of each individual miRNA species, their ease of synthesis and their limited toxicity, these small RNAs might be valuable not just as disease biomarkers but also as powerful therapeutic agents and targets.Two more genes prominently involved in angiogenesis and modulated by AFL were 3, the enrichment of GO terms associated to potassium, calcium, and chloride ion transport in response to AFL administration provides further evidence of its beneficial effects on visual perception, in line with our previous findings22. The latter is of key relevance as damage to retinal neurons can be detected before vascular abnormalities in human patients with diabetes127. Modulating not only angiogenesis but also a broader spectrum of processes relevant to retinal function and apparently protecting not only dopaminergic amacrine cells from hypoxic damage, as we have previously reported22, but potentially also RGCs, AFL might be a more powerful agent than previously recognized. In this sense, our exploratory study might serve as a valuable resource for informing future research aimed at the identification of additional molecular targets whose pharmacological regulation might be beneficial for the treatment of multiple retinopathies, including diabetic retinopathy and retinopathy of prematurity.Beyond its marked regulatory effects on angiogenesis, AFL also modulated inflammatory processes and exhibited neuroprotective features, as revealed by the enrichment of GO terms associated to wound healing and synaptic transmission. Moreover, considering that ion flux into photoreceptors is a critical process for vision often disrupted in diabetesAnimal experiments were performed in strict accordance with German Animal Welfare legislation and adhering to the ARRIVE guidelines. All experimental protocols (TVV26/2017) were reviewed and approved by the Landesdirektion Dresden with the approval number DD24.1\u20105131/394/29. All efforts were made to minimize animal suffering. Anesthesia overdosing prior to eye collection was verified by the absence of interdigital withdrawal reflexes. Pharmacological euthanasia was followed by decapitation. All mice were acquired from Charles River Laboratories , either to breed in\u2010house or as pregnant females. Newborns were allocated to experimental groups arbitrarily and irrespective of their sex.19. The protocol consisted in exposing C57BL/6J  mouse pups and their mothers to 75% oxygen from P6 to P11 (P0\u2009=\u2009birthdate); before and afterwards, all mice were housed in ambient air conditions in cages with wood shavings beddings and using a 12\u00a0h light/dark cycle. Temperature control and water quality was continuously monitored and followed the regulations of the TU Dresden Medical Faculty Animal House. Aflibercept  diluted in sterile PBS  was administered intra\u2010peritoneally to half the pups in each litter at 25\u00a0mg/kg BW on one of two regimens: either on P13 and P15 or on P13, P15 and P17. Non-injected OIR littermates were kept as controls. Half the animals in each litter were arbitrarily allocated by the experimenter to the Aflibercept\u2010treated group and the remaining half to the non\u2010injected OIR control group. No exclusion criteria were enforced and all animals were considered. In total, 12 mice were used in the present study. Non-OIR animals (normoxic controls) were reared in ambient air conditions. Normoxic controls were not injected with any substance before being sacrificed. All mice were sacrificed between 13:00 and 17:00\u00a0h (CET\u2009+\u20091) by an overdose of ketamine  and xylazine . The effectiveness of the OIR protocol was ascertained by confirming the presence of neovascularization and vaso-obliteration by immunofluorescence in flat-mounts of retinas from animals used for a different study which were inside the hyperoxia chamber at the same time-points as the animals used for this study. The oxygen concentration in the hyperoxia chamber was monitored daily to confirm deviations did not exceed\u2009\u00b1\u20092%, which from previous experience is considered an acceptable range.Retinal neovascularization was induced by the OIR protocolg and 4\u00a0\u00b0C. The aqueous phase was subsequently collected and mixed with 1.5 \u00b5l RNA\u2010grade glycogen (Thermo Fisher Scientific) and 375\u00a0\u00b5l isopropanol (Merck). Samples were subsequently vortexed and centrifuged 20\u00a0min at 12,000g and 4\u00a0\u00b0C. Pellets were washed twice in 1\u00a0ml 75% ethanol, air\u2010dried 10\u201320\u00a0min, and re\u2010suspended in 20 \u00b5L RNAse\u2010free water. Aliquots of 1 \u00b5L RNA per sample were kept separate for quality control measurements avoiding unnecessary freeze\u2013thaw cycles.Three P19 mice per condition  were used for RNA isolation. The retinas of each mouse were dissected in PBS at 37\u00a0\u00b0C and subsequently pooled together. Warm PBS was replaced by 750 \u00b5L TRIzol (Thermo Fisher Scientific), and retinal tissues were disrupted by 10 rounds of aspiration/ejection through 18G needles\u00a0and 10 more rounds through 23G needles. After 5\u00a0min incubation at room temperature, 150 \u00b5L chloroform  were added and samples were vortexed, incubated an additional 15\u00a0min at RT, and centrifuged 20\u00a0min at 12,000t test) across time-points or conditions. False discovery rate (FDR) was not used. Transcripts expressed from the X or Y chromosome were not considered for analysis. Data were exported as .csv files for further analysis. The microarray data produced in this study has been deposited in the NCBI Gene Expression Omnibus database (accession number GSE137705). Venn diagrams were created using InteractiVenn131 and refined in Adobe Illustrator. Lists of differentially regulated genes were used as input in PANTHER27 and ShinyGO132 for identifying enriched GO terms and KEGG Pathways, respectively. PANTHER settings were used as default, i.e. using a Fisher\u2019s Exact test and a False Discovery Rate correction. When a high number of terms or pathways were detected, a subset of the most specific terms was arbitrarily selected and used for depiction in figures.Isolated RNA was sent to the Gene Expression Facility of the Max Planck Institute for Cell Biology and Genetics . There, biotinylated cDNA samples were prepared according to the GeneChip WT PLUS Reagent Kit (Cat. Nr. 902280) protocol using 250\u00a0ng total RNA as starting material. 5.5 ug of biotinylated and fragmented single-stranded cDNA were hybridized for 16\u00a0h at 45\u00a0\u00b0C on Affymetrix GeneChip Mouse Whole Transcriptome Arrays according to the GeneChip\u00ae WT PLUS Reagent Kit User Manual. GeneChips were washed and stained in the Affymetrix Fluidics Station 400. GeneChips were subsequently scanned using the Hewlett-Packard GeneArray Scanner G2500A. Scanned images were processed with the Transcriptome Analysis Console (TAC) Software (Affymetrix) using default parameters to obtain background subtracted and summarized probe sets. Data (.CEL files) generated in these microarray chips was imported into GeneSpring v14.9 (Agilent Technologies) together with the .CEL files generated in our previous study (GEO accession number: GSE124956) describing the response of the OIR retina to AFL administration at earlier time-points, namely P14 and P17. By normalizing all datasets together using the Robust Multi-array Average (RMA) approach, we aimed at ensuring comparability across all datasets. No baseline transformation was conducted. Normalized expression levels were used to identify differentially regulated genes in GeneSpring. A gene was considered to be differentially expressed if it exhibited a fold-change above or below 1.2 and a p-value below 0.05 as a result of pairwise comparison (unpaired Supplementary Figures.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.Supplementary Table 8.Supplementary Table 9.Supplementary Table 10.Supplementary Table 11.Supplementary Table 12.Supplementary Table 13.Supplementary Table 14.Supplementary Table 15.Supplementary Table 16.Supplementary Table 17.Supplementary Table 18.Supplementary Table 19.Supplementary Table 20.Supplementary Table 21.Supplementary Table 22.Supplementary Table 23.Supplementary Table 24."}
+{"text": "Manufacturing has been the key factor limiting rollout of vaccination during the COVID\u201019 pandemic, requiring rapid development and large\u2010scale implementation of novel manufacturing technologies. ChAdOx1 nCoV\u201019  is an efficacious vaccine against SARS\u2010CoV\u20102, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large\u2010scale production. We discuss the process economics and the \u201cdistributed manufacturing\u201d approach we have taken to provide the vaccine at globally\u2010relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID\u201019 vaccine supply at low cost. Well over a billion doses of the ChAdOx1 nCoV\u201019 vaccine developed by The University of Oxford and AstraZeneca have been produced and distributed at low cost. This represents a substantial proportion of global COVID\u201019 vaccine supply to date, especially in low\u2010 and middle\u2010income countries. The authors report on the development of the manufacturing process for the vaccine, and the process economics and distributed manufacturing approach involving bulk vaccine (drug substance) production on five continents. To our knowledge the greatest previously\u2010disclosed scale of manufacture of adenovirus vectors for human use was the production of c. 2\u2009\u00d7\u20091017 VP (for 2.7 million doses) of Janssen Vaccines' adenovirus\u2010vectored Ebola vaccine  , followed by a multistep downstream process  . In the second quarter of 2020 we introduced process modifications, removing bottlenecks to enable commercial manufacturing at 1000\u20134000\u2009L scale. The first 1000\u2009L drug substance batch was completed in September 2020, and the billionth dose was released in July 2021.22.1The ChAdOx1 nCoV\u201019, ChAdOx1 Lassa\u2010GP, ChAdOx1 luciferase, and ChAdOx2 GFP vectors used here have previously been described , with a working volume of 25\u201335\u2009ml in a 125\u2009ml flask unless otherwise stated. BioBlu 3c and 14c (Eppendorf) single\u2010use bioreactor vessels were used in accordance with the manufacturers' instructions. A GX bioreactor controller unit and C\u2010BIO software  were used to control both vessel types. Dissolved oxygen (DO) was regulated at a setpoint of 55% air saturation by addition of medical air via macrosparger. pH was regulated in the range 7.2\u20137.3 as previously described  was used in 50 and 200\u2009L STRs. 0.05 culture volumes of BalanCD feed was added when the density reached 1.0\u2009\u00d7\u2009106 culture cells/ml. At a cell density of 4.0\u2009\u00d7\u2009106/ml (range 3.0\u20136.0\u2009\u00d7\u2009106), cells were diluted with 1 volume of medium and infected, using an MOI of 10 unless otherwise stated. 0.05 volumes of BalanCD feed were added 30\u2009min after infection, and again after 22\u2009h.50 and 200\u2009L upstream processes were performed using Pall Allegro\u2122 stirred tank reactors (STRs). For work using the high MOI fed batch process  were added to cell culture at 48\u2009\u00b1\u20094 and 96\u2009\u00b1\u20094\u2009h post bioreactor inoculation. Bioreactor temperature was reduced to a lower set point within 4\u2009h of the second feed. About 140\u2009h postinoculation, the cell culture was harvested and processed for analysis.For the low MOI upstream process Figure\u00a0, bioreac2.3Lysis was performed as previously described . Column equilibration was in accordance with the manufacturer's instructions. After loading, capsule was washed with 10 membrane volumes (MV) of equilibration buffer before the elution step (both at 5 MV/min).For \u201cdirect\u2010load\u201d AEX (loading clarified lysate), the small\u2010scale studies shown in Figure\u00a0For the \u201cdirect\u2010load\u201d AEX purifications from a 10\u2009L bioreactor Figure\u00a0 a perist2, 5% w/v sucrose. After loading, the capsule was washed with 10 MV of equilibration buffer followed by at least 20 MV of wash buffer . Product was eluted with 5 MV of elution buffer . Loading, equilibration, washing, and elution were all performed at 2.5MV/min.For the \u201cdirect\u2010load\u201d AEX purification from a 1000\u2009L bioreactor Figure\u00a0 a PK50 lSmall\u2010scale studies shown in Figure\u00a02.6Product quantification was as previously reported, using quantitative polymerase chain reaction (qPCR) and UV spectrophotometry assays for viral particles in impure and pure samples, respectively, and an immunostaining\u2010based infectivity assay  was quantified using the HEK293 HCP ELISA kit (Cygnus Technologies), according to the manufacturer's instructions. Residual host cell DNA was quantified using a previously reported quantitative PCR method targeting a 94 base pair amplicon within the Alu repeats . A detailed execution protocol for drug substance production  was combined with mass balance data as described in this manuscript to build and parametrize a model. Assumptions made were designed to be conservative: productivity of 2\u2009\u00d7\u20091033.17 cells/ml with high viability  per ml, around fivefold greater than typically obtained in our previous USP , cell density and time of harvest Figures\u00a0. Althougs Figure\u00a0.We have previously demonstrated and discussed advantages of the use of a cell line/promoter combination which represses antigen expression during production  remained in the range 2\u20134\u2009\u00d7\u20091011 VP/ml, with acceptable cell growth and metabolite profiles  of a high MOI species E adenovirus production process is relatively low. Considering the high particle:infectivity ratio of these vectors , after estimating likely losses in processing and aliquoting and adding a margin for safety, we felt we could only rely on production of c. 300 IU of useable seed per cell infected during seed production: this would be sufficient to infect only 30 cells at an MOI of 10. This 30\u2010fold amplification factor implied the need for large numbers of batches of working virus seed (creating a testing burden and consistency concerns), and a possible requirement for multi\u2010step seed expansion (from master virus seed through two working seed production passages). Reduction of the MOI to c. 3 was considered, but we felt this would be unlikely to fully address these concerns.To avoid the complexity arising from a high seed requirement, we investigated use of MOI\u2009<\u20091. By infecting cells at low density within 24\u2009h of bioreactor inoculation with cells, and then continuing culture for a further 120\u2009h, we allowed the initially\u2010uninfected cells to proliferate before becoming secondarily infected by the released virus particles from first round of infection Figure\u00a0. InformeThe reduced MOI, and resulting increased virus amplification factor, has enabled seed from a single 200\u2009L bioreactor batch to supply global manufacturing needs.3.32 per 40\u2009L of lysate) and although we anticipated this could be reduced with optimization, we were concerned that TFF membrane supply could become problematic due to high demand during the COVID\u201019 pandemic.The initial TFF step was identified as the key bottleneck for DSP scale\u2010up. Time did not permit extensive process characterization before scale\u2010up, and we were concerned that excessive concentration of the lysate during TFF would increase risk of formation of aggregates of product with host\u2010cell protein and DNA. Using only modest concentration to favor process robustness meant that multiple process volumes of diafiltration buffer and waste needed to be handled, which we anticipated would be challenging in some facilities. The original process also required large TFF membrane areas , was likely to prove limiting for the speed of global COVID\u201019 vaccination rollout. We were also concerned that so\u2010called vaccine nationalism was likely to impede equitable access to effective vaccines, as had been seen during the H1N1 swine flu pandemic Fidler,\u00a0.We therefore set out to design a global manufacturing strategy to achieve three goals: low cost; speed to large volume supply, including prompt wide geographical availability; and, critically, consistent high quality.Low cost was achieved due to the high productivity of the process and the use of off\u2010the\u2010shelf equipment and materials. Modeling of DS manufacturing using a commercial cost\u2010modeling package suggests a cost of goods of <EUR 1 per dose Figure\u00a0. This foDespite the productivity of the process, it was apparent multiple manufacturing facilities were required to achieve speed to large volume supply: global supply required >1\u2009m\u2009litres of upstream process output and hence a large number of batches. Moreover, location of DS manufacturing appeared likely to be a key determinant of vaccine availability, especially for low and middle income countries. From the outset (Q1 2020), our strategy therefore included simultaneous technology transfer to multiple sites, including in India and China. Through Q2\u20103 2020, this strategy was further developed, resulting in distribution of drug substance manufacturing across a network of existing contract manufacturing and licensee sites capable of single\u2010use bioreactor (SUB) viral manufacturing at 1000\u20134000\u2009L scale Figure\u00a0.To achieve the earliest possible release of commercial product through this distributed manufacturing strategy, we \u201cparallel\u2010tracked\u201d a number of process development, tech transfer, validation and regulatory activities which would more typically run sequentially Figure\u00a0. For sev4Vaccine supply is proving to be the key determinant of timing of COVID\u201019 vaccination, especially in low and middle\u2010income countries.Some aspects of adenovirus\u2010vectored vaccine manufacturing are inherently well\u2010suited to pandemic response. Antigen\u2010repressing promoter/cell combinations, and the absence of the encoded antigen from the adenovirus virion, enable similar upstream, downstream and drug product manufacturing regardless of the encoded antigen, and we had previously described a small\u2010scale process designed for rapid response to emerging pathogens  facilitates the implementation of the process using standard equipment available at most sites, reduced material requirements, and achieved chromatography cycling times as short as 2\u2009h at 2000\u2009L scale. Efficient recovery and high quality of adenovirus from such a \u201cdirect load\u201d AEX has not to our knowledge previously been reported.10 VP) per litre of upstream culture. We believe this productivity is around double that of previously disclosed batch or fed batch processes for adenovirus manufacture . Combined with the biosafety of the vector (BSL1\u20102 dependent upon jurisdiction), the design of the process enabled a distributed manufacturing strategy, with several independent national or regional supply chains. Such strategies have previously been proposed as means of ensuring wide geographical availability of vaccines, but never to our knowledge implemented with this speed and scale. In the context of current calls for waivers of intellectual property relating to COVID\u201019 vaccines, it is important to note the degree of central co\u2010ordination which has been necessary to ensure efficient manufacture and consistent quality across the multiple production sites Figure\u00a0.The process described here has thus provided a substantial proportion of global COVID\u201019 vaccine supply to date (>1 billion doses released for supply as of August 2021). Uniquely among current COVID\u201019 vaccines from high\u2010income country developers, the programme has aimed to achieve \u201cequity by design\u201d: as a result of the process development and technology transfer strategy, the majority of output has been manufactured and/or used in low/middle income countries.C. C. D. J., Y. L., S. F., G. G., A. B., R. R. S., Te. L., S. C. G., A. J. R., and A. D. D. are named inventors or contributors to intellectual property assigned to Oxford University Innovation relating to the ChAdOx1 nCoV\u201019 vaccine and/or manufacturing process, and may receive a proportion of proceeds from out\u2010licensing of the intellectual property. J. J., Th. L., N. B., D. P., R. V., and R. T. are employees of AstraZeneca.Conceptualization: Alexander D. Douglas and Richard Turner. Experimental design, planning, execution, and data acquisition: Carina C. D. Joe, Jinlin Jiang, Teresa Lambe, Yuanyuan Li, Sofiya Fedosyuk, Gaurav Gupta, Adam Berg, Rameswara R. Segireddy, David Mainwaring, Amar Joshi, Paul Cashen, Byron Rees, Jonathan Humphreys, Sarah Davies, Nick Smith, Scott Bruce, Dennis Verbart, Daan Bormans, Carol Knevelman, Mark Woodyer, Lee Davies, Lisa Cooper, Maria Kapanidou, Nicole Bleckwenn, Daniel Pappas, Daniel C. Smith, Catherine M. Green, Raghavan Venkat, Adam J. Ritchie, Richard Turner, and Alexander D. Douglas. Construction of process model: Nitin Chopra and Piergiuseppe Nestola. Provision of critical materials: Teresa Lambe and Sarah C. Gilbert. Funding acquisition: Sarah C. Gilbert, Richard Turner, and Alexander D. Douglas. Writing and review of manuscript: all authors.Supporting information.Click here for additional data file."}
+{"text": "Lactiplantibacillus plantarum N-1 from traditional cheese in China. This study aimed to investigate the effects of N-1 on renal CaOx crystal deposition. Thirty rats were randomly allocated to three groups: control group (ddH2O by gavage), model group [ddH2O by gavage and 1% ethylene glycol (EG) in drinking water], and Lactiplantibacillus group (N-1 by gavage and 1% EG in drinking water). After 4 weeks, compared with the model group, the group treated with N-1 exhibited significantly reduced renal crystals (P < 0.05). In the ileum and caecum, the relative abundances of Lactobacillus and Eubacterium ventriosum were higher in the control group, and those of Ruminococcaceae UCG 007 and Rikenellaceae RC9 were higher in the N-1-supplemented group. In contrast, the relative abundances of Staphylococcus, Corynebacterium 1, Jeotgalicoccus, Psychrobacter, and Aerococcus were higher in the model group. We also predicted that the arginase level would be higher in the ileal microbiota of the model group than in the N-1-supplemented group with PICRUSt2. The arginase activity was higher, while the level of arginine was lower in the ileal contents of the model group than in the N-1-supplemented group. The arginine level in the blood was also higher in the N-1-supplemented group than in the model group. In vitro studies showed that exposure to arginine could reduce CaOx crystal adhesion to renal epithelial HK-2 cells. Our findings highlighted the important role of N-1 in reducing renal CaOx crystals by regulating arginine metabolism in the gut microbiota. Probiotics containing L. plantarum N-1 may be potential therapies for preventing renal CaOx stones.Renal calcium oxalate (CaOx) stones are a common kidney disease. There are few methods for reducing the formation of these stones. However, the potential of probiotics for reducing renal stones has received increasing interest. We previously isolated a strain of  Renal stones are a common kidney disease worldwide that can cause pain, urinary tract infection, chronic renal disease, or even loss of renal function. The prevalence of these stones has been increasing, being approximately 6.4% in China , 6.3% inThe main component of renal stones is calcium oxalate (CaOx), which accounts for approximately 80% of renal stones . Only a Oxalobacter formigenes could degrade oxalate into formate through formyl-CoA transferase and oxalyl-CoA decarboxylase , containing Streptococcus thermophilus, Bifidobacterium, and Lactobacillus, could reduce gastrointestinal oxalate absorption  at an inoculation of 1% (v/v) for 18 h at 37\u00b0C. Then, the suspension of strains was centrifuged for 5 min at 3,000 \u00d7 g. After two washes, the strains were resuspended in 10 mL saline solution. The bacterial suspension was adjusted to 108 CFU/mL with a spectrophotometer and preserved in a centrifuge tube at 4\u00b0C for further use.The We purchased 6-week-old male Sprague\u2013Dawley rats from Dossy Experimental Animals Co., Ltd. . The mean body weight of rats was 198 g. The rats were first acclimatised for 1 week before the experiment in the specific-pathogen-free facility at the Animal Experiment Center of West China Hospital, Sichuan University. The rats were given standard food and sterile water. We obtained ethical approval from the West China Hospital of Sichuan University Medical Research Ethics Committee (protocol numbers: 2017063A).Lactiplantibacillus group. The control group rats were provided with 2 mL/kg ddH2O by gavage. Apart from ddH2O gavage, the rats in the model group had free access to drinking water with 1% (v/v) ethylene glycol (EG). In the Lactiplantibacillus group, the rats had drinking water with 1% EG and a suspension of N-1  by gavage once a day. After 4 weeks, 24 h urine of all the rats was collected using metabolic cages. Each urine sample was divided into three aliquots, which were supplemented with 0.5 mL hydrochloric acid (6 mol/L)/10 mL urine to prevent the degradation of oxalate  to obtain plasma. Renal tissues were fixed in 10% formaldehyde and embedded in paraffin. The remaining kidneys, plasma and intestinal contents in the ileum, caecum, and colon were stored at \u221280\u00b0C.Thirty rats were randomly allocated to three groups: the control group, model group, and  oxalate . Then, wHaematoxylin\u2013eosin (HE) and von Kossa (VK) staining methods were used to observe the renal CaOx crystals. First, kidney sections (3\u20134 \u03bcm) were prepared and stained with HE following the manufacturer\u2019s protocols for the staining kit . Second, VK staining was also performed to detect calcium crystals using a staining kit . We analysed the staining images using a microscope . The HE staining scoring system reported by g). Then, 100 \u03bcL supernatant was added with 300 \u03bcL acetonitrile and centrifuged for 10 min . The oxalate was turned into 2,3-dihydroxyquinoxaline. The samples were examined using an LCMS-8040 system .Urinary oxalate was measured using liquid chromatography\u2013mass spectrometry. Briefly, 10 \u03bcL hydrochloric acid (12 mol/L) and 200 \u03bcL 1,2-diaminobenzene (5 mg/mL) were added to the 100 \u03bcL urine, which was then heated at 140\u00b0C for 40 min. When the sample was cooled to room temperature, it was added with 10 \u03bcL sodium hydroxide (10 mol/L) and centrifuged for 10 min . The sequences were classified into different operational taxonomic units (OTUs) when their similarity was 97% or higher. The Silva 16S rRNA database (version 138) was used to identify the taxonomy of the OTUs.g, 4\u00b0C). Fifty microlitres of plasma and 200 \u03bcL of methanol acetonitrile/methanol  were mixed. We left the mixture to stand for 60 min before centrifugation for 20 min . Then, we applied the supernatant to UHPLC  and a QTRAP 5500 system . If the relative standard deviations of quality control (containing the same amount of contents from each sample) for all amino acids were less than 30%, the measurements were reproducible and stable. The information of amino acids standards used for liquid chromatography\u2013mass spectrometry analysis was shown in Amino acids were measured by liquid chromatography\u2013mass spectrometry. We mixed 100 mg of ileal contents with 1 mL acetonitrile/methanol/water  and sonicated them for 30 min (4\u00b0C), followed by centrifugation for 20 min  according to the manufacturer\u2019s protocol. Briefly, each sample well was added with 20 \u03bcL plasma, 20 \u03bcL assay buffer and 10 \u03bcL substrate mix. Each standard well was added with 50 \u03bcL prepared standards with different concentrations. The 96 well plate was incubated for 20 min at 37\u00b0C. Then each well was added with 50 \u03bcL reaction mix. Finally, the absorbance was measured on a microplate reader at OD = 570 nm in kinetic mode at 37\u00b0C. The arginase activity was calculated based on absorbance values and standard curve.HK\u22122 cells were cultured in DMEM/F\u221212 supplemented with 10% foetal bovine serum at 37\u00b0C, 5% CO2 and saturated humidity. The cells were cultured in six-well culture plates and exposed to sodium oxalate (0.75 mM) and/or arginine  for 24 h . After tt-test. P-values < 0.05 were regarded as significantly different. GraphPad Prism (version 8) and R (version 3.6.1) were used for statistical analyses and to draw graphs.Data are shown as the mean and standard deviation and were analysed by ANOVA and Student\u2019s P < 0.05) (P < 0.05). However, the administration of N-1 did not affect urinary oxalate excretion (P > 0.05) (P > 0.05) (P < 0.05), and they did not change after the administration of N-1 (P > 0.05)  . The lev > 0.05) . The vol > 0.05) . The wei > 0.05) .To explore whether N-1 reduced renal crystals by regulating the gut microbiota, we next examined the gut microbiota. We found that the coverage indices were >0.99 in all groups at the OTU level, indicating that the sequencing depth was sufficient in all samples. The alpha diversity of the gut microbiota in the ileum, caecum, and colon was not different between the control, model, and N-1-supplemented groups .P = 0.019, ADONIS). The beta diversity of the gut microbiota was different between the model group and the Lactiplantibacillus group . The difference in the beta diversity between the control and N-1-supplemented groups decreased    using the Bray\u2013Curtis index. In the ileum, PCoA showed significant separation between the control and model rats ( ADONIS) . In the  ADONIS) . This evLactobacillus was the most common bacterium. The relative abundance of Lactobacillus decreased from 65\u201383% in the ileum to 16\u201336% in the caecum and to 18\u201346% in the colon. In the ileum, the percentage of Lactobacillus decreased from 83% in the control group to 65% in the model group and then increased to 75% in the N-1-supplemented group . At the genus level, ed group . In the ed group . This evStaphylococcus, Corynebacterium 1, Jeotgalicoccus, Psychrobacter, and Aerococcus were more abundant in the model group  to explore the different bacteria between the control, model, and N-1-supplemented groups. In the ileum, el group . In conte caecum  and coloe caecum .L-serine ammonia-lyase). However, there were fewer significantly different enzymes between the model and N-1-supplemented groups in the caecum and colon than in the ileum  to predict the metabolic enzymes of the gut microbiota and analysed their differences between the three groups. Using LEfSe, we found that the levels of 19 enzymes were higher in the ileum model group (such as arginase). In addition, the levels of 12 enzymes were increased in the ileum after the administration of N-1 (such as he ileum .P = 0.040) and N-1-supplemented group (P = 0.029) than in the model group (P = 0.010) than in the control and N-1 groups (P = 0.021)  . The orn= 0.021) . In addi= 0.021)  or blood= 0.021)  was posiin vitro, we added CaOx crystals and arginine to the cell culture medium. The results showed that there was no adhesion of CaOx crystals to renal epithelial HK-2 cells when cells were treated with was arginine alone. The crystals adhesion increased after the exposure to 0.75 mM oxalate, and reduced when cells were exposed to 0.75 mM oxalate + 1.72 mM or higher arginine (P = 0.023)  .L. plantarum N-1 was resistant to acid and bile salt and, compared to other strains, had the highest survival rates of 98.0 and 99.1% under the conditions of pH 3.5 and 0.1% bile salt, respectively. This strain has the ability to adhere to intestines and antibacterial activity. In addition, the culture supernatants of N-1 could inhibit the growth of Escherichia coli  are also metabolised to oxalate as a terminal metabolite production in the liver . The oxa in rats . Howevery levels . Howeverpatients . InteresLactobacillus was the predominant bacterium in the gut, especially in the ileum. We also found that the beta diversity of the gut microbiota and the percentage of Lactobacillus in the N-1-supplemented group were more similar to those in the control group, which indicated that N-1 could reverse the gut microbiota of model rats to relatively normal conditions. However, this phenomenon was not observed in the caecum or colon. The most likely reason may be that the environment in the ileum was more suitable for Lactobacillus.Several studies have reported that the gut microbiota is different between renal CaOx stone patients and healthy controls, which suggests that the whole gut microbiota, instead of a single kind of bacterium, is important in the formation of renal stones. Thus, we further examined the composition of the gut microbiota in the ileum, caecum, and colon between the three groups. We found that Lactobacillus was the main genus of the gut microbiota. Its relative abundance was much higher in the colon of the control group, which suggested that the decrease in Lactobacillus was associated with renal stones. Our study also verified that supplementation with L. plantarum N-1 could reduce renal crystal formation. In addition, short-chain fatty acid (SCFA)-producing bacteria such as Eubacterium and Ruminococcaceae have been found in lower abundances in the gut microbiota of model groups (Staphylococcus, Corynebacterium 1, Jeotgalicoccus, Psychrobacter, and Aerococcus were higher in the gut microbiota of the model group of this study, and these genera are associated with acute or chronic inflammatory diseases, including inflammatory bowel diseases, necrotising lymphadenitis, and diabetes mellitus (We also found that l groups . It was l groups . Howevermellitus . This evmellitus .Lactobacillus was higher in the ileal contents than in the caecal and colonic contents. Thus, we hypothesised that the supplemented Lactobacillus mainly regulated the arginase activity and arginine level in the ileal contents. Further examinations verified that the arginase activity was lower in the ileal contents of N-1-supplemented rats than in the model rats. Arginine levels were higher in the ileal contents and blood of N-1-supplemented rats than in the model rats. Ornithine and citrulline are the key arginine metabolites, where arginine is metabolised to ornithine through arginase. Thus, we also compared the ornithine and citrulline levels in the ileal contents and blood. Though they were not significantly different between three groups, there was a decrease trend of ornithine level in the ileal contents after the supplementation of N-1.In this study, we predicted that a higher level of arginase would be found in the ileal microbiota of the model group than in the N-1-supplemented group. The predictive results also showed that the arginase level in caecal or colonic contents was not significantly different between model group and N-1-supplemented group. Meanwhile, the relative abundance of in vitro study also showed that arginine could reduce CaOx crystal adhesion to renal cells. L-arginine could increase urinary citrate and reduce free radicals and oxalate in the kidney in rats with renal crystals. L-arginine could reduce oxidative stress in kidney epithelial cells exposed to oxalate in vitro. Arginine could also suppress the inflammatory response and apoptosis of intestinal epithelial cells (L. plantarum N-1 may reduce renal crystals by regulating the metabolism of gut microbiota and increasing the level of arginine in the gut.Additionally, an al cells ,b. All tL. plantarum and to discuss its effect on preventing renal CaOx stones by decreasing arginase levels in the gut microbiota. These results will promote further study of the mechanisms underlying renal CaOx stone formation.The present study was performed only on Sprague\u2013Dawley rats. The effect of N-1 on preventing renal CaOx stones in humans needs further research. However, our study also had several advantages. First, N-1, isolated from traditional cheese in China, is not pathogenic to humans and is easily obtained. Second, compared with the study performed by L. plantarum N-1 in reducing renal CaOx crystals by regulating arginine metabolism in the gut microbiota. Probiotics containing L. plantarum N-1 may be potential therapies for preventing renal CaOx stones.Our findings highlighted the important role of PRJNA759335.The data presented in the study are deposited in the NCBI repository, accession number: The animal study was reviewed and approved by West China Hospital of Sichuan University Medical Research Ethics Committee.YL, XJ, QS, and KW conceived and designed the experiments. YL, LT, YM, LC, and YC performed the experiments, analysed the data, and wrote the manuscript. ZJ, XJ, HL, QS, and KW reviewed and edited the manuscript. QS and KW provided guidance for the experiments. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Up to nine treatments with a minimum interval of 8 weeks between each session were performed. A six-month follow-up visit assessed patient satisfaction . Overall clinical result was assessed with a clinical evaluation by two blinded dermatologists using a 5-point scale, comparing pictures before treatment and at follow up. A total of 52 patients were included and analyzed: 30 females (57.7%) and 22 males (42.3%). Mean age was 43.7 \u00b1 12.7 years. According to Fitzpatrick\u2019s skin classification, 16 individuals (30.8%) were type II, 15 (28.8%) were type III, and 21 (40.4%) were type IV. Most of the treated tattoos were carried out by professionals. The mean number of sessions required to obtain a result was 4.6 \u00b1 2.5, and the final tattoo removal rate was 60% or higher, with 51.9% of the patients reporting highest satisfaction scores Q-Switched 1064/532 nm laser may be considered today as the gold-standard treatment for tattoo removal. Our results confirm literature findings of the safety and effectiveness of these devices.Tattoo removal is a well-established procedure in dermatology. Lasers represent the gold standard in the management of this condition nowadays. In this study, we report our experience on the use of a Q-switched nanosecond source. A total of 52 patients were consecutively enrolled in performing tattoo removal at Magna Graecia University of Catanzaro and Tor Vergata University of Rome. Black and blue tattoos were treated with a 1064 nm laser, with a pulse duration of 6 ns and a fluence up to 10 J/cm Individare used .However, this kind of laser induces superficial tissue destruction, therefore risk of scarring and hypopigmentation in tattoo treatment is very high ,6. For tThe medical literature indicates that Nd YAG lasers acting at a 1064/532 wavelength are very effective in managing this condition ,8.Furthermore, different studies show that Q-Switched lasers release high energies in a minimal amount of time (nano or picoseconds). They also act selectively on tattoo pigmentation, sparing surrounding tissues ,10,11. TAll patients presenting for tattoo removal from the 1 January 2018 to the 30 December 2019 at the dermatological clinics of Magna Graecia University  and University of Tor Vergata  were retrospectively enrolled in this study.Exclusion criteria were the following: hypersensitivity to light (visible and near-infrared); medication known to increase sensitivity to light; therapies with anticoagulants and/or immunosuppressants; pregnancy or nursing; personal or family history of skin cancer; sun exposure in the three weeks before treatment (for any skin type); previous tattoo removal treatment; gold-containing medication; and recent exfoliation treatments, surgical treatments, and past skin disorders (including keloids). All patients signed informed consent on the risk of the procedure.\u00a9, Deka M.E.L.A., Calenzano, Italy), which provides ultrashort pulses to achieve selective photothermolysis of the target (tattoo pigments) with minimum thermal damage to surrounding biological structures. The physician chose treatment parameters according to skin phototype, tattoo class, and location. According to their characteristics  treated tattoos were stratified according to the classes listed in Patients included in the study were treated with a Q-Switched 1064/532 nm laser system  and parameters, the same shooting settings, a twin flash, and the same ambient light.Two independent dermatologists evaluated the pictures, providing the final result in tattoo removal with a score in a 5-point scale .A Visual Analogue Scale (VAS) from 1 to 10 was administered to the patients at the six-month follow-up to measure patient satisfaction .Data analysis  was performed using Statistica 14.0 .n = 25\u201348.1%) and were characterized by intense pigments in dark, red, or mixed colors; other represented classes were amateur tattoos (n = 9\u201317.3%), followed by medical, traumatic (n = 7\u201313.5% each), and cosmetic ones (n = 4\u20137.7%). Tattoos were mainly placed on upper trunk/shoulder (n = 18\u201334.6%), lower arm/leg (n = 12\u201323.1%), and face/head (n = 11\u201321.2%), with fewer cases on wrist/hand/ankle/foot (n = 8\u201315.4%) or lower trunk/upper leg (n = 3\u20135.8%). Patients characteristics are reported in A total of 52 patients were included in this study and analyzed: 30 females (57.7%) and 22 males (42.3%). Mean age was 43.7 \u00b1 12.7 years. According to Fitzpatrick skin classification, 16 individuals (30.8%) were type II; 15 (28.8%) were type III; and 21 (40.4%) were type IV. Most tattoos had been performed by professionals , while in phototype III only one-third of cases (33.3%) reached 80\u2013100% tattoo removal . All cosNo serious adverse event occurred, and treatment was well tolerated by all patients. In two cases, a final \u201cghost effect\u201d was observed; seven patients developed petechiae after session treatments and were medicated with an occlusive dressing, antibiotics, and hydrolytic enzymes, resolving the condition in 10\u201315 days.Laser interacts with tissues mainly thanks to three effects: the photothermal effect, characterized by conversion of the energy conveyed by a laser in heat, the photochemical effect, which breaks chemical bonds inside the pigment, and the photoacoustic effect, characterized by the mechanical destruction of the pigment. If wavelength, soot size, and fluence are correct, this will result in ink fragmentation and sparing of surrounding tissue. Whitening of the tissue right after laser treatment represents dermal and epidermal vacuolization due to localized steam formation, with signs of epidermal damage. ,14This interaction creates newly generated unknown chemical compounds that may be disposed from the skin via the lymphatic system and generate a reaction from the immune system. Molecules remaining in the dermis may have different physical and biological characteristics. Metal-containing pigment could theoretically transform into toxic chemicals after light exposure, and cleavage of azo dyes may result in carcinogenic amines ,15.Colorants are the most important part of tattoo inks, consisting of almost insoluble pigments dispersed in water plus additives. A significant problem for the toxicological assessment of tattoo inks is the absence of appropriate data for ink composition and toxicology. Most tattoos are black, made by soot-related compounds  with shading additives such as titanium dioxide or iron oxides. Potentially genotoxic polycyclic aromatic hydrocarbons can be found in skin specimens and regional lymph nodes even years after tattooing . The samThe QS Nd: YAG laser is to date considered a valuable tool for tattoo removal because its technology enables the obtainment of good results with minimal damage to the epidermis, dermis, and skin appendages, with a low risk of scarring and hypopigmentation ,17,18. AOur study confirms the results previously obtained in the medical literature, confirming Q-Switched laser treatments as the gold-standard treatment for tattoo removal. In our cohort of patients, we found that professional tattoos with mixed colors and tattoos on the lower limbs were more challenging to treat, while amateur tattoos and tattoos on the upper part of the body were more easily removed.Of course, a prospective study with a more significant number of patients would be helpful to better analyze all the possible side effects and confirm our paper\u2019s results."}
+{"text": "Most of the protein\u2013protein docking methods treat proteins as almost rigid objects. Only the side-chains flexibility is usually taken into account. The few approaches enabling docking with a flexible backbone typically work in two steps, in which the search for protein\u2013protein orientations and structure flexibility are simulated separately. In this work, we propose a new straightforward approach for docking sampling. It consists of a single simulation step during which a protein undergoes large-scale backbone rearrangements, rotations, and translations. Simultaneously, the other protein exhibits small backbone fluctuations. Such extensive sampling was possible using the CABS coarse-grained protein model and Replica Exchange Monte Carlo dynamics at a reasonable computational cost. In our proof-of-concept simulations of 62 protein\u2013protein complexes, we obtained acceptable quality models for a significant number of cases. Protein\u2013protein interactions are fundamental in many biological processes. Their structural characterization is one of the biggest challenges of computational biology. A variety of docking methods are currently available for structure prediction of protein\u2013protein complexes ,2. They Presently, most of the free docking methods treat the backbone of input protein structures as rigid. This approximation reduces the protein\u2013protein docking problem to a 6D  search space. Rigid-body search for the binding site most often rely on the Fast Fourier Transform ,8. OtherIn this work, we use a well-established CABS coarse-grained protein model  for protThe most accurate models  are characterized in The results analysis below focuses on the sampling performance for the selected low-flexibility barnase/barstar case. An essential and unique feature of the presented docking simulations is the level of backbone flexibility during docking. In the example above, the ligand backbone fluctuations (LoRMSD) were in the range of 2\u20137 \u00c5 b, with tFinally, using structural clustering of contact maps (see Methods), we attempted to select the set of 10 top-scored models for each case. This work demonstrates a significant improvement in the sampling of large-scale conformational transitions during global protein\u2013protein docking compared to other state-of-the-art approaches. We show that modeling the large conformational changes is possible at a relatively low computational cost. The presented simulations took between 10 and 80 h (depending on the system size) using a single standard CPU. The proposed modeling protocol can be used as the docking engine in template-based and integrative docking protocols using experimental structural data and additional information from various sources ,40. We fPresently, the most common approach to account for conformational changes in protein docking is using ENM ,36,37,38In this work, we present the protein\u2013protein docking simulation protocol that relies on the CABS coarse-grained model. The CABS design and applications have been recently described in the reviews on protein coarse-grained  and protPreparing input structures of a protein-ligand and a protein-receptor. The protocol requires the input of two protein structures (single- or multi-chain) in the PDB format. One of them has to be indicated as a ligand and the second as a receptor. The ligand undergoes large conformational fluctuations, translations, and rotations around the receptor within the proposed protocol. The \u201cligand\u201d should be a smaller protein because the computational cost of searching its conformational space rapidly grows with the chain length. That is because the motion of the entire structure  is simulated by a random sequence of local moves. The accuracy of such sampling is acceptable for not too-large proteins. On the other hand, treating the \u201cligand\u201d as a fully flexible object allows approximate studies of entire docking trajectories. In some cases, it would be perhaps worth treating a larger protein (but not too large) as a flexible \u201cligand\u201d, although this was out of range of the present studies.Generating starting structures. Starting conformations are built using C-alpha coordinates only . The alDocking simulations using CABS coarse-grained model and REMC dynamics. During simulations, the protein receptor structure is kept close to the starting structure using distance restraints. Distance restraints are generated using the input coordinates of the C-alpha atoms. Two residues are automatically restrained if two conditions are met. First, their separation along the sequence has to be at least five residues. Second, the distance between their C-alpha atoms must be within the range of 5\u201315 \u00c5. During simulations, the receptor restraints imply small-scale fluctuations of the protein receptor backbone in the range of 1 \u00c5 and, accordingly, more significant fluctuations of the side-chain atoms. A similar restraints scheme is applied to the protein-ligand but with tenfold weaker weights. During simulations, the ligand moves freely within the vicinity of the receptor and internal restraint allows for large-scale fluctuations of its structure. Usually, the ligand fluctuations are within the range of 2 and 12 \u00c5 to the input structure although folding-unfolding events are possible at highest temperatures. The docking simulation is conducted using CABS REMC pseudo-dynamics with simulated annealing. In this work, 20 replicas and 20 annealing steps have been used. All the REMC scheme parameters have been adjusted to allow for large-scale conformational transitions, rotations, and translations of the protein-ligand in a reasonable computational time. The modeling protocol collects trajectories from all 20 replicas. The protocol saves only a small fraction (2%) of the generated models for further analysis i.e., 500 models from each replica, thus 10,000 models in total.Reconstructing to CABS coarse-grained representation. The set of 10,000 models in C-alpha traces are reconstructed to complete CABS model representation using CABS algorithm [lgorithm . In CABSClustering of contact maps. First, for all of the 10,000 models the contact maps between the receptor and the ligand proteins are calculated. Two residues are considered to form a contact if their Side Chain pseudoatoms are at most 6 \u00c5 apart . Next, the algorithm sorts the models according to the number of the receptor-ligand contacts, and the set of top 1000 is kept for further processing. This way the transient and weakly bound complexes are removed from the solutions pool. In the next step, the 1000 contact maps are clustered together to identify the most frequently occurring contact patterns. The complete link hierarchical clustering was used with the Jaccard index as the distance metric between contact maps. Finally, the identified clusters are ranked according to their density, defined as the number of the cluster members divided by the average metric between them.Reconstructing to all-atom representation. Representative models from the ten most dense clusters are reconstructed to all-atom representation using Modeller-based rebuilding procedure [rocedure  (or can rocedure ).The modeling protocol consists of the following steps:In recent years, the CABS model has been used for modeling the flexibility of globular proteins ,45,46,47The presented protocol for protein\u2013protein docking utilizes the CABS-dock standalone package  developeThe docking simulation analysis was performed using Python and NumPy (Python library). Structural differences between experimentally determined structures and generated models were evaluated using Root Mean Square Deviations (RMSDs). Interface RMSD (iRMSD) is an RMSD calculated for interface residues of the receptor and the ligand separated by no more than 6 Angstroms. Ligand RMSD (LRMSD) is an RMSD computed for the ligands after the superimposition of the receptors. Ligand only RMSD (LoRMSD) is an RMSD computed for the ligand structure only. Root Mean Square Fluctuation (RMSF) is a measure of the amino acid\u2019s flexibility. It is calculated for every residue as the square root of this residue\u2019s variance around the reference residue position. The fraction of native contacts (fNAT) was calculated as a number of experimental structure contacts found in the generated structure divided by the total number of contacts found in the experimental structure. Rather restrictive contact criterion, distance up to 6 \u00c5 between side-chain centers, was used. All figures presented in this work were generated using PyMOL, UCSF Chimera, and Matplotlib (Python library).In this docking study, we used protein\u2013protein cases from the ZDOCK benchmark set   and force-field improvements. The proposed approach is also very promising in the refinement applications when searching for the binding site is not needed, and only the protein\u2013protein interface needs to be optimized."}
+{"text": "Correction: Arthritis Res Ther 24, 251 (2022)https://doi.org/10.1186/s13075-022-02943-2Following publication of the original article , an erroThe updated funding is given below and the changes have been highlighted in bold.is funded by NIH/NHLBI R01HL145985, and J.K.L is funded by NIH/NHLBI1F32HL156614-01.A.M.B. was supported by Tobe and Stephen E. Malawista, MD, Endowment in Academic Rheumatology, Rheumatology Research Foundation and by NIH/NHLBI HL155955-01A1. F.E. is supported by NIH 5T32HL007224-45, M.T. is funded by NIAMS P50 AR060780, F.S. The original article  has been"}
+{"text": "Reference and multivariate models did not show significant differences in error rates (p = 0.124), regardless of the nature of predictors  or the pooling method used. In addition, models built over a larger population did not provide significantly more accurate predictions. In conclusion, GNSS features seemed to be of limited relevance for predicting individual A-V profiles. However, new signal processing features open up new perspectives in athletic performance or injury occurrence modeling, mainly if higher sampling rate tracking systems are considered.This study aims to predict individual Acceleration-Velocity profiles (A-V) from Global Navigation Satellite System (GNSS) measurements in real-world situations. Data were collected from professional players in the Superleague division during a 1.5 season period (2019\u20132021). A baseline modeling performance was provided by time-series forecasting methods and compared with two multivariate modeling approaches using ridge regularisation and long short term memory neural networks. The multivariate models considered commercial features and new features extracted from GNSS raw data as predictor variables. A control condition in which profiles were predicted from predictors of the same session outlined the predictability of A-V profiles. Multivariate models were fitted either per player or over the group of players. Predictor variables were pooled according to the mean or an exponential weighting function. As expected, the control condition provided lower error rates than other models on average ( Data collected from these wearable devices provide useful insights for understanding a player\u2019s activity and its relationship with performance outcomes or injury occurrences during practice16.Global Navigation Satellite System (GNSS) is one of the gold standard systems in position measurements in field sports. Widely used for athlete monitoring purposese.g. distance covered at different speed intervals, averaged pace for a given interval, acceleration, and deceleration counts). Beyond a standard set of simple and easily comprehensible features, extra information of mechanical and energetic nature that is derived from the players\u2019 position may be available for customers under the manufacturer\u2019s policy19. However, the validity of GNSS sport receivers should be considered regarding their technical properties, such as sampling frequency. Despite a lack of accuracy for quantifying exercise demand over short distances covered at high speed, including sharp turns, GNSS suffers from error rates according to a relatively low GNSS sampling frequency21. The signal quality of receivers also depends on the spatial configuration of satellites locked for recording 22. Nonetheless, GNSS with embedded IMU stands of interest and remains prone to further technological improvements. Beyond technological aspects, practitioners mostly use summarised GNSS statistics or metrics, whereas raw data are seldom considered for player analysis. The usual data fed back from the GNSS units might be elementary, while new features extracted from raw data could be more insightful for monitoring the physical demand of exercise and the related athlete\u2019s response.In most cases, the information provided by wearable GNSS devices are summarised features over a session or a period  from continuous GNSS measurements for in-game and post hoc analysis. Such profiles come with practical meanings, notably for monitoring changes in athletic properties . They could be used for optimising training plans or proceeding to in-game tactical adjustments in case of significant profile impairments. However, determining in situ A-V profiles for monitoring athletic performances remains at a proof-of-concept stage23. It should be further validated for athletic performance modeling and injury explanation purposes.Using GNSS data for predicting athletic performance in team sports remains challenging. First, it implies defining an athletic performance in which interactions with opponents and the environment are sufficiently lowered. Usually, assessing athletic performances requires specific testing sessions performed in controlled conditions. It comes with challenging issues due to time and investigation costs, injury exposure, psychological state disruptions, and adjustments to training plans. Nevertheless, Morin et al.The predictability of A-V profiles using only related GNSS featuresThe value of common metrics (summarised statistics) and aggregated features that are delivered by GNSS sensors manufacturers for predictive applicationsThe use of raw GNSS data for extracting new features for prediction purposesIn order to investigate these issues, we attempted to predict A-V profiles using data from an elite football team through different modeling approaches. A baseline approach that only considers A-V profiles and dismisses any potential predictors other than historical profiles was carried out. Then, we compared it with two distinct tasks that used commercial GNSS features and features extracted from raw GNSS data.On this basis and according to the literature, the present study considers three research issues: The rest of the manuscript is organized as follows. We introduce the data set that highlights the predictor and outcome variables. Next, we introduce the proposed models and their variants. Accordingly, we present the obtained results followed by exhaustive discussions of these results before concluding our study in the last section.This section introduced a descriptive analysis of the data set used in our experiments. We defined the predictors and outcome variables besides the considered problem formulations before elaborating the proposed models. For clarification, we provided a pseudo-code of the modeling methodology through Algorithms 1, 2 and 3.FC Lucerne football club were collected over a 1.5 season period (2019\u20132021). The team evolves in the Superleague division, the highest division in Swiss professional football. A total of 196 training sessions and 74 games were stored in a cloud-hosted multi-model database . For each session, raw GNSS data  and summarised features (see Appendix A for details) of each player were stored in a database as json files. A total of 42 players were initially recorded, including regular professional players and young hopes. Participants were fully informed about data collection, and their written consent were obtained. The study was performed in agreement with the standards set by the declaration of Helsinki (2013) involving human subjects. The protocol was reviewed and approved by the local research Ethics Committee . The present retrospective study relied on the collected data without causing any changes in the training plans of football players.Data from the X is thus defined as a vector of d dimensions, composed of aggregations of the summarised features given by the GNSS software . Aggregated features can take two forms: The average of summarised features softmax function  preceding the game of interest. Since the frequencies of sessions are heterogeneous, the number of days preceding the game to be predicted may differ over the weeks.For both aggregation methods, we arbitrarily set a window 23 but in a slightly different way. Individual A-V profiles were modeled for each games. From the raw velocity A such thatx(t).In order to investigate the effect of training on athletic performances, we relied on A-V profiles such as provided bye.g. young players) who only played a few games through the season.Then, a first-order Butterworth filter was applied to the acceleration signal with a cut-off frequency of 1 Hz. Velocity observations were binned into j and the day of realisation t. A sample of fitted coefficients is presented in Figure f, the built data set is a sample The performance definition is given by entclass1pt{minimaA descriptive analysis of A-V coefficients reported mean values and standard deviation as a noise estimate. Accordingly, we have 27 generally intended to tipsters or bookmakers, sports popularity28, or performance monitoring29. In our study, we first consider that the auto-regressive component of the target variable may be influential in the prediction of individual athletic performances. Therefore, we started the modeling by defining baseline prediction performances from time-series forecasting, only using games observations (excluding training sessions).Time-series forecasting problems are dominant in sports, applied to game outcomesIn time-series forecasting, models without covariates use a restricted data set in which predictors are merely time information. The forecasting is deduced from the information in trend and seasonality components. In order to find the most performing models for time-series forecasting, we proceeded with a model selection using a simple holdout procedure, according to a split ratio of 0.8 between the training and testing data set. We can righteously expect a linear relationship between changes in theoretical maximal acceleration and maximal running velocity. Consequently, the ensemble Afterwards, we benefited from the selected forecasting models by combining them into a weighted average ensemble for better performances than a randomly selected single model on average.Then, we addressed the problem of predicting the acceleration-velocity profile from GNSS summarised features. Changes in A-V fitted parameters were investigated using a predictive regression approach. For this, a linear model (a ridge regularisation) used features pooled according to the two aforementioned aggregation methods see Eqs.  and 2 an32. It thus prevents biased estimates through penalising estimates of correlated features34. According to the two aggregation methods, the multivariate linear models mean function , and velocity were available. Since commercial features were computed from the raw velocity vector In the previous formulation, the player position was recorded by GNSS at a sampling frequency of 10 Hz. Timestamp, player position .Several features were extracted from the time series in both time and frequency domains through Discrete Fourier Transform. For this purpose, we used the In summary, pseudo-code of the algorithms used in the methods is provided in Appendix 1, Section A.3.p value adjustment was used. The marginal mean difference p = 0.05 and consistently reported within the analysis.In prediction, model performances were characterised by the mean absolute percentage error  computed on test data sets. Repeated measures analysis of variance (ANOVA) and post-hoc analysis highlighted the significance of differences in MAPE distribution between models. Depending on a reference model for comparison, Tukey\u2019s or Dunnett\u2019s control task. Using a set of predictors to predict A-V coefficients of the same session using a ridge regularisation returned an average MAPE of 0.066% and 0.102% for intercept and slope, respectively.The first baseline prediction was described by error rates observed in the As shown in Fig. 47, Theta, FourTheta48, and Fast Fourier Transform based. As expected, the combination of these models into an averaged ensemble provided the best performances , averaged ensembles forecasting models tended to be less accurate . On average, individually fitted models did not provide lower prediction errors than those fitted on the group (p = 0.381). Except for univariate time-series forecasting models which only considered data from games and multivariate LSTM, there was no advantage of using the exponentially weighted aggregation . That discarded any advantage of building models per player for predictions.No significant difference was reported between averaged intercept and slope predictions in models that used features extracted from raw data. Only a slight trend for a lower MAPE was imputed to intercept predictions  provided a lower error rate than any modeling task using past data .An overview of model performances showed that in average, the reference models that account for past events for predicting the A-V profiles of the next game. Beforehand, predictions made using predictors of the game of A-V profile realisation (i.e. control models) informed their predictability in ecological conditions.This study compared two multivariate modeling approaches that use commercial features and features extracted from GNSS raw data to a time-series forecasting approach. We considered the last as Concerning the reference models, performing multi-modal forecasting might provide better results, but it also requires a larger sample size than uni-modal forecasting methods to estimate model parameters correctly. Accordingly, we filtered out players who performed less than 40 games for computing multi-modal forecasting models. Only nine players (out of 42) were retained for prediction, whereas the uni-modal task included data from a larger population (19 players). Therefore, the sample size heterogeneity should be considered when interpreting the forecasting results since a larger sample size might reasonably provide different, if not better, forecasting performances.51.A practical limitation of univariate forecasting models is that we only consider game data for prediction. Hence, interpretations drawn from each forecast are restricted to the effect of preceding games on the next game, and the contribution of training sessions preceding a performance remains unclear. Accordingly, technical and medical staff around players should exploit multivariate models for detecting key performance indicators (KPIs) of A-V changes, or any other outcomei.e. returned by the manufacturer, see Appendix 1, Table A1) or features extracted from the velocity vector, it was likely easier for the model to predict A-V profiles\u2019 intercepts than the slopes. A greater variance allocated to this parameter may reasonably explain that finding, easing the estimation of the coefficient regarding a random error. In practice, a small change in the A-V profile may result in a substantial modification of the theoretical normalised force output (i.e. the acceleration) at the onset of maximal locomotor activities.Being based on commercial summarised statistics , no significant differences in error rates suggest that features describing past sessions were not informative enough to improve predictions. Accordingly, time stands as a significant predictor variable of subsequent events.When comparing multivariate to control models allow us to support a reasonable A-V profile predictability.In addition, ridge regularisation used pooled features according to a simple aggregation by the mean or exponential smoothing. However, changing one pooling method to another did not lower prediction error rates. At first glance, that indicates either a limited relevance of the explanatory features used in the model or a lack of A-V profile predictability. However, the low error rates of predictions provided by 32. Our results did not confirm such benefits since there is no benefit to using player-specific models for predicting A-V profiles with the current data.In a small sample context, using a larger population may lead to more robust estimates of parameter coefficients. One possible way could be to build models over a group of players instead of a model per player52 or their value for explaining outcomes under a substantial opponent influence. It is essential to point out the lack of information for the GNSS signal quality. As mentioned in the introduction, GNSS signal accuracy relies on time/frequency and spatial parameters. The receiver manufacturer used in our study  did not store any spatial accuracy factor such as horizontal dilution of precision. Therefore, we recommend the manufacturer to report signal quality details for practical use and research purposes54. Nevertheless, using features not based on expert hypotheses but fully extracted from signal processing methods appeared to be as valuable as the commercial ones. It leverages information that could be drawn from GNSS data and opens the way to future works on data mining and knowledge discovery in the sports field. However, this perspective comes with feature interpretability issues, particularly those related to the frequency domain.An overall analysis and model comparison highlight that despite slight differences between top and bottom model ranking (see Table reference models (i.e. univariate forecasting models), interpretation of the selected features for practical application should be made with caution.Features retained for regression after the feature selection procedure reveal KPIs of A-V profile changes. Based on a top ten representation , we could state that the distance covered at high intensity is not necessarily the highest value when regarding other variables, such as the number of accelerations and decelerations for specific intensity bands. Such KPIs should help guide field and resistance training regarding individual objectives. However, since multivariate models suffered from explanatory power regarding 9. Regardless, in light of the above limits, monitoring processes should be carried out under a more data-informed than the data-driven approach in which external training indicators are monitored along with internal markers, and environmental factors55.Finally, when considering re-scaled MAPE, prediction errors varied between 7% and 10%. We believe this is an acceptable accuracy since the A-V profile depends on unmeasured and uncontrolled factors, namely the opponent activity, then any psychological, environmental, or nutritional aspects. Therefore, GNSS wearable sensors could stand of value, though limited, for prediction purposes and more generally included in athlete monitoring systems while estimating external training loadscontrol task, which benefits from the relationship between the commercial features and the modelled profiles of a given game. However, a deeper analysis of the A-V estimator noise and heteroscedasticity of the outcome variables should be carried out in a future study.In our study, we provided a simple estimation of the A-V predictability through the i.e. through the profile intercept) and the capacity to produce a significant level of horizontal force at high velocities (i.e. according to the slope of A-V relationship). These mechanical factors may be key determinants of soft-tissue injury occurrence56, short sprint performances57 and could guide individual training prescription.Beyond predictive applications, A-V profiles provide relevant insights regarding the theoretical maximal isometric force of hip extensors (e.g. IMU and motion capture systems) as compared to GNSS devices, intended for discriminating exercise and its demand in ecological conditions. A physiological representation of the responses to exercise may be therefore extracted. Besides, going through raw data recorded by these systems may contribute to solving the enigma of injury occurrence, which remains a hot research area in sports science with major economic repercussions59.The technological rise provides higher sampling frequency systems  and a neural network architecture (RNN-LSTM). Two distinct functions were employed to aggregate training sessions predictors; a Global navigation satellite systems are valuable for modeling in-situ A-V profiles. However, its predictability using GNSS-derived features from training sessions remains limited.e.g. IMUs).Multivariate modeling highlights key performance indicators of A-V changes among commercial, training-related features. Alternatively, signal processing methods pave the way to new modeling perspectives of performance and injury modeling, mainly if applied to measurement systems with higher sampling rates (A-V time-derived features are likely as relevant as GNSS-based features for explaining changes in A-V profiles. It emphasizes the necessity for multidimensional modeling while considering the opponent\u2019s activity, psychological and environmental factors.Supplementary Information."}
+{"text": "Moral courage means courage to act according to individual\u2019s own ethicalvalues and principles despite the risk of negative consequences for them.Research about the moral courage of whistle-blowers in health care isscarce, although whistleblowing involves a significant risk for thewhistle-blower.To analyse the moral courage of potential whistle-blowers and its associationwith their background variables in health care.\u00a9, a video vignette of the wrongdoingsituation with an open question about the vignette, and several backgroundvariables. Data were analysed statistically and inductive content analysiswas used for the narratives.Was a descriptive-correlational study using a questionnaire, containingNurses Moral Courage ScaleNurses as healthcare professionals  were recruited from themembership register of the Nurses\u2019 Association via email in 2019. A total of454 nurses responded. The research context was simulated using avignette.\u00a9 was obtained from the copyrightholder. The ethical approval and permission to conduct the study wereobtained from the participating university and the Nurses\u2019 Association.Good scientific inquiry guidelines were followed. Permission to use theNurses\u2019 Moral Courage ScaleThe mean value of potential whistle-blowers\u2019 moral courage on a VisualAnalogue Scale (0\u201310) was 8.55 and the mean score was 4.34 on a 5-pointLikert scale. Potential whistle-blowers\u2019 moral courage was associated withtheir socio-demographics, education, work, personality and socialresponsibility related background variables.In health care, potential whistle-blowers seem to be quite morally courageousactors. The results offer opportunities for developing interventions,practices and education to support and encourage healthcare professionals intheir whistleblowing. Research is needed for developing a theoreticalconstruction to eventually increase whistleblowing and decrease and preventwrongdoing. Healthcare professionals becomewhistle-blowers if they (2) address the whistleblowing act inside the organisationto a person or party capable of ending the wrongdoing such as managers or outside topolice, health authorities or media. Whistle-blower may suffer (3) consequences suchas retaliation or discrimination after performing the whistleblowing act.19 Within this definition, thewhistleblowing act is considered to include internal whistleblowing acts as speakingup or raising concerns to the wrongdoer, colleagues or the closestmanager.20 Whistle-blowers are generally seen as brave as they act despitepotential risks for themselves,21 such as retribution orostracism, after performing the act of whistleblowing.24 The threat of negativeconsequences is prevailing, even when the act of whistleblowing is addressedanonymously.26 Whistle-blowers may repeatedly perform whistleblowing actswhich are ignored and turn into ineffective efforts to end the wrongdoing. Even when a healthcare professional is aware of a morally appropriate actionrequired when observing wrongdoing, one could be unable to act due to organisationalor personal obstacles such as punitive workplace culture or lack ofcourage.27 Particularly in health care context, these increase healthcareprofessionals\u2019 moral distress and may eventually lead to turnover or leaving theprofession,28 thereby worsening an already poor work force situation andshortage of professionals. Moral courage is considered as an empowering and positive way to reduce moraldistress.27In this study, whistleblowing is defined as a three-phased process where: (1)wrongdoing such as malpractice, workplace bullying or stealing medicine30 that can bestrengthened and developed through ethics education, educational interventions, and ethicaldiscussions.33 Moral courage is the ability to overcome fears of the potentialnegative consequences to oneself and to defend professional and personal ethicalprinciples and values. It is about having an inner will to do the right thing, forthe good of others. In addition, expressing moral courage means acting withoutexternal force by sacrificing oneself and still feeling a sense of peace. Moralcourage is \u201cthe golden mean\u201d between cowardice and rashness, with the formerindicating irrational fear and the latter thoughtlessness.34 In the health care context,research about moral courage has been scarce, although it has increased during thepast decade, particularly among nurses37 and nurse students.41 Thecharacteristics of a morally courageous nurse42 and the background variablesassociated with moral courage have been described in previous literature.41 Morallycourageous nurses are ethically sensitive, willing to risk themselves for thebenefit of others and to act according to their own conscience.45 They feelresponsibility for others45 and possess confidence in situations requiring moralcourage43 and are confident in their own ethical principles and value base.Moral courage is value-oriented, determined through individual -value base. It can beconsidered as an individual\u2019s inner strength and as an appreciated human virtue.Moral courage is said to be part of an individual\u2019s ethical competence and gender. However, some studies indicate that in terms of moral courage, gender is nota significant variable.45 In addition, education related variables \u2013 additional ethicseducation, continuing education,46 higher level of education andprevious degree in health care \u2013 are associated with moral courage and beneficial for strengthening it. Among work-related variables as working in management position and having longer work experience41 are associated with moralcourage. Moreover, associations were described between moral courage and thefrequency of encountering situations requiring moral courage at work, having nursing career plans as well as dissatisfaction with nursing profession.Studies describe associations between moral courage and socio-demographics: age and differences in the level of moral courage could explain why someindividuals are willing to become whistle-blowers while others are not.48 However,there appears to be a gap in the empirical research about whistle-blowers and theirmoral courage in health care. Thus, identifying whistle-blowers and their moralcourage provides evidence-based knowledge that potentially enhances ourunderstanding of whistle-blowers\u2019 moral courage and how to support and encouragehealthcare professionals in their whistleblowing.Moral courage is expected to increase the probability of whistleblowing,The aim of this study was to analyse the moral courage of potentialwhistle-blowers (PW-Bs) and its association with their background variables inhealth care. The goal of the study was to identify PW-Bs to support andencourage healthcare professionals in their whistleblowing.1. Who are the potential whistle-blowers in health care?2. What is, the potential whistle-blowers\u2019 self-assessed level ofmoral courage?3. What potential whistle-blowers\u2019 background variables, if any, areassociated with their self-assessed moral courage?The following research questions were addressed:\u00a9 (NMSC), a video vignette ofthe wrongdoing situation with an open question about the vignette, and severalbackground variables. A scripted video vignette was used as the whistleblowingphenomenon is challenging to capture in real-life. Additionally, video vignetteshave been acknowledged as a suitable research method for exploring individuals\u2019decision-making in situations such as whistleblowing. They enable participantsto respond to the same wrongdoing, in the same context, under the sameconditions, thereby decreasing situational factors and allowing researchers tofocus on individual-related variables.50The research design was a descriptive-correlational study using a questionnaire,containing Nurses Moral Courage Scale with a significance level of p < 0.05 and 95%confidence level, the sample size estimation was 380 nurses.A random sampling on national level was used. Data were collected between 16August and 5 September 2019 from the membership register of the Nurses\u2019Association . An email containing an invitation to participate in the studyand a link to an electronic questionnaire was sent to 30,000 nurses, in themembership register, by the membership coordinator of the association. Based onstatistical power analysisAltogether 1461 participants returned the questionnaire. Of them, 706 provided anarrative in response to an open question about the video vignette. However,only 454 participants were included in this study, as they described both (1)observing the wrongdoing in the video vignette, and (2) acting as blowing thewhistle, in their narratives, giving a response rate of 31%. The participantswere working in various health care organisations across the country andrepresentative of an average nurse on a national level.\u00a9. The development and validation studies show good reliability, validity,and internal consistency for the NMCS\u00a9.34 The NMCS\u00a9consists of 21 items measuring nurses\u2019 moral courage within four dimensions: 1)compassion and true presence (5 items), 2) moralresponsibility (4 items), 3) moral integrity (7items), and 4) commitment to good care (5 items). The responsesare given on a 5-point Likert-scale between 1 = \u201cDoes not describe me at all\u201d to5 = \u201cDescribes me very well\u201d. Lower scores indicate lower self-assessed level ofmoral courage and vice versa. In addition, one question measured nurses\u2019 overallmoral courage using a Visual Analogue Scale (VAS 1\u201310) where 1 = \u201cI never actmorally courageously even though the care situation would require it\u201d and 10 =\u201cI always act morally courageously when the care situation requiresit\u201d.34The questionnaire for data collection comprised three parts. Firstly, nurses\u2019self-assessed level of moral courage was measured using the NMCS The dimension of \u201cCompassion and true presence\u201d requirescourage on the part of the nurse courage to overcome their own fears andvulnerability to encounter the suffering and vulnerability of the patient.\u201cMoral responsibility\u201d means taking responsibility andacting courageously in situations where wrongdoing occurs despite the possiblepowerlessness caused by internal or external circumstances such as hierarchy.\u201cMoral integrity\u201d focuses on nurse\u2019s adherence to theethical principles and values of the profession in situations where there is arisk for potential negative consequences. \u201cCommitment to goodcare\u201d focuses on situations where good care is under threat due topoor care or wrongdoing, for example. The nurse needs courage to advocate forthe patient and defend the moral goal of nursing, the patient\u2019s ultimategood.33The dimensions In NMCS\u00a9 are based on a concept analysis.Secondly, the questionnaire included a video vignette of a wrongdoing situationand an open question about the wrongdoing visible in the vignette: \u201cHow wouldyou act in the situation and why?\u201d The video vignette took place in home care.In the video vignette, two nurses are visiting an elderly patient with memorydisorder, and while the other nurse is measuring the patient\u2019s blood pressureand blood sugar, the other nurse distributes the patient\u2019s medicines and puts apackage of medicine into her own pocket. The participants were instructed andable to watch the video once. Lastly, several background variables were asked:(1) socio-demographics, (2) education, (3) work, (4) personality, and (5) socialresponsibility related variables , potenti and the narratives were analysed according to the steps of inductiveanalysis technique. The narratives were analysed to identify the participants who describedobserving the wrongdoing in the video vignette and their potentialwhistleblowing acts regarding it. Descriptive statistics  were used to describe theparticipants\u2019 background variables .The data were analysed statisticallyariables , potentiariables ,particiariables  and easiariables . The fol55 andpublication ethics. Ethical approval was obtained from the Ethics Committee of the University(10/2019) and other permissions from the Nurses\u2019 Association for data collection andfrom the developer of the instrument for using NMCS\u00a9,. All potential participants received information about the study and had theopportunity to obtain additional information. Confidentiality and anonymity wereguaranteed and participation was voluntary. Returning the completed questionnairewas considered as consent to participate.55This study was conducted following good scientific standardsA total of 454 nurses (31%) returned the questionnaire with a narrative andcompleted NMCS\u00a9. Most of the PW-Bs were female (95%) with a mean age of 47\u00a0yearsand mean length of work experience of nearly 20\u00a0years. The majority wereregistered nurses (90%) with a polytechnic degree (53%) and were currentlyworking as employees (63%). PW-Bs mostly viewed their own personality as betweenan introvert and extrovert (50%). Other personality and social responsibilityrelated variables were measured using a VAS scale 0\u2013100. Out of personalityrelated variables, PW-Bs assessed the level of respecting their own professionas the highest (mean 85.2) and allowing others to influence their own opinions(44.8) as the lowest. In addition, the level of social responsibility forpatients or their next of kin was assessed as the highest (84.6). . ThemajThe majority of the PW-Bs would address the whistleblowing act inside theorganisation (98%) and only two percent externally, outside the organisation. Inaddition, they considered it would be easiest to act morally courageously whenfacing co-workers  and the easiest way would be opendiscussion among the working team members  .Compassion and true presence was assessed as the highestwith a mean of 4.55 (SD 0.46) while Commitment to good care wasthe lowest, with the mean of 4.21 (0.56). Mean of their self-assessed overallmoral courage (VAS 1\u201310) was 8.55 (0.84). . Of sum variables, (0.84). .p = 0.032). In addition, thosewith the highest degrees assessed themselves as more morally courageous thanstudents (p = 0.031) and those having a polytechnic degree(p = 0.034) and showed higher moral responsibility thanthose with a vocational (p = 0.048) or polytechnic(p = 0.039) degree. . Thehighest correlations concerning personality related variables were betweenrespecting one\u2019s own profession and total moral courage (r = 0.35). In addition,the highest correlations concerning social responsibility were between thewell-being of the work community and total moral courage and moral integrity(both r = 0.4). Negative correlations were identified between allowing others toinfluence one\u2019s own opinions and moral courage. The highest correlation was seenbetween ease of acting morally courageously when facing colleague and moralintegrity (r = 0.43). .36 The resultsare encouraging, as it is suggested that high level of moral courage entails higherprobability of whistleblowing. However, it needs to be acknowledged that there is a bias for sociallydesirable responses when using the video-vignette method and self-assessment instrument.This study produced novel evidence by identifying potential whistle-blowers in healthcare, their moral courage and background variables associated with it for the use offuture decision-making and to support and encourage healthcare professionals intheir whistleblowing. The PW-Bs assessed their moral courage as rather high,slightly higher than previous studies describing nurses\u2019 moral courage. increasing the external validity of the study. The findings indicate that relying often on ethical guidelines and previous experiences of encountering situations requiring moral couragecould enhance moral decision-making and acting morally courageously if facing actsof wrongdoing such as the one presented in the video vignette. In addition, relyingon ethical guidelines entails PW-Bs\u2019 professionalism as healthcare professionals.In this study, the PW-B was identified. The PW-B represents an average nurse on anational level in terms of socio-demographics, education and work related variables,62 The findingsof this study are encouraging as previous studies suggest that high extraversion, adequate self-esteem and high level of internal locus of control predictprosocial behaviour in the form of whistleblowing.PW-Bs considered their personality type to be between an introvert and extrovert,assessed their self-esteem as quite good, and allowed others to influence their ownopinions to a moderate degree. These are individual personality variables which theyprobably had even before entering the health care profession. Furthermore, PW-Bsassessed their internal locus of control on an average level, indicating that theydo not believe in being in total control of the events around them. One plausibleexplanation for this could be the hierarchical nature of health care.64 It is encouraging thatwhistleblowing is potentially increased by both a strong role and group identity.The findings indicate that PW-Bs have a strong role and group identity as nurses asthey respected their own profession highly and described high levels of socialresponsibility for others. Responsibility for patients was assessed as the highest,which is part of being a nurse, nurses\u2019 accountability, and a fundamental issue ofbeing the patient\u2019s advocate.36 The difference might be caused by the fact that here,participants were potential whistle-blowers and therefore, they could be moremorally courageous. In addition, moral courage, as a highly appreciated humancharacteristic, may increase socially desirable responses due to individuals\u2019 desireto present themselves in a socially or professionally acceptable light.According to the results of this study, PW-Bs assessed their overall moral couragerather high in all the dimensions of the NMCS\u00a9, which is consistent with previousresearch among nurses. However, in this study, moral courage was assessed higherthan in previous studies.34 The resultssuggest that PW-Bs possess empathy and that being patients\u2019 advocate is a crucialpart of their professional role and important for them. Furthermore, being patients\u2019advocate is one of the main facilitators for whistleblowing in healthcare.64 The dimensionof commitment to good care was assessed the lowest, which is both consistent and inconsistent with previous studies, with the latter reporting moral responsibility as thelowest dimension. However, even the lowest dimension was assessed on a quite highlevel, indicating courage to act in situations where wrongdoing occurs or good careis threatened for other reasons.Of the dimensions of moral courage, compassion and true presence was assessed as thehighest, with similar results presented in previous research. In addition, the findings are supported by earlier suggestions that moralcourage as part of ethical and professional competence could be developed andstrengthened with education.65 It is encouraging that thehighest degree was associated with moral courage, as earlier literature indicates itas one of the predictors of whistleblowing. Inconsistent findings report that the highest degree was not associated withmoral courage. Parallel results on the current work role were presented as managers assessedtheir level of moral courage higher than employees. The findings indicate that higher position in professional hierarchycontributes to moral courage, which is supported by earlier literature.41 In addition,managers have responsibility to act as courageous examples and support employees intheir moral courage and their whistleblowing.The findings of this study confirm particularly the associations between PW-Bs\u2019 moralcourage and education related variables as well as the work role as manager, whichis usually achieved through education. Consistent with the Aristotelian view,virtues such as moral courage can be developed through practice and education. Nevertheless, the findings are encouraging as the personality variablesextroversion, self-esteem, locus of control and social responsibility support moralcourage and its manifestation as a form of whistleblowing.70Personality related variables were associated with moral courage, implying thatmorally courageous PW-Bs possess certain personality traits which they assessed at arather high-level. However, further research is needed to compare whether there is adifference between healthcare professionals\u2019 and whistle-blowers\u2019 moral courage andtheir personality traits or whether these traits are something that all thehealthcare professionals possess, as it is suggested that individuals who havechosen the health care profession are usually willing to help others and to actmorally courageously and put themselves at risk for the benefit of others. such as the pandemic, the type of observed wrongdoing, ethical climate, management, organisational culture,72 support received from others,and hierarchical power structures in health care.73 To get an extensive andbroader understanding, these variables and their associations with moral courage andwhistleblowing as a courageous action require further research.The COVID-19 pandemic has emphasised healthcare professionals\u2019 need for moral courageand acting morally courageously as they have exposed themselves to a severe risk ofa contagious and potentially deadly virus for the goal of doing good to others.Moral courage and courageous action are not only dependent on individual variables,but also on situational and contextual factors However, there is evidence that vignettes may predict participantsresponding similarly to real-life situations as to simulated vignettes. In addition, vignettes enable distancing participants from potentiallydifficult and sensitive research topics, such as whistleblowing. Video vignettes are considered more realisticthan written ones, enabling observers to immerse themselves in the situation. Therefore, the video vignette method was chosen, and the video vignettewas pre-tested. Secondly, the sample was recruited from one national nurses\u2019association with a relatively low response rate (31%), although it includednurses who formed the target population of this study. Based on the statisticalpower analysis, the number of participants was adequate. In addition, theparticipants were working in various organisations, representing an averagenurse. Thirdly, data were collected from one country with a culturally specifichealth care system and prevailing social norms. However, nurses share globallyuniversal values and similar ideology with other healthcare professionals.Fourthly, the reliability and validity of the NMCS\u00a9 has been provenand it has been developed specifically for the nursing context.34 Lastly, asocial desirability bias exists as the video vignette method was used and moral courage was measured using a self-assessment instrument. However, the vignette method potentially reduces socially desirable responses.There are some methodological considerations and limitations in this study.Firstly, a simulated video vignette method was used, and one of the theoreticallimitations described is the gap between social reality and the vignette.36 indicate that nurses ashealthcare professionals, assess their moral courage at a rather high level.However, earlier literature suggests that only around half of the healthcareprofessionals observing wrongdoing actually blow the whistle. Therefore, it should be considered whether the barrier for whistleblowingis lack of courage or lack of knowledge of the manifestation of moral courage in differentways such as whistleblowing. Ethics training and ethics interventions could bebeneficial for effective manifestation of moral courage as moral courage can bestrengthened with education.74 Further research isrequired on whether the low whistleblowing rates and performing whistleblowingacts in real-life are associated with moral courage. In addition, research isneeded to explore whether moral courage and the background variables identifiedhere predict actual whistleblowing. Exploring social, situational, andindividual predictors for whistleblowing is also needed in the future. Forgeneralising the results of this study, international research needs to beconducted in different countries, cultures, and health care services.This and previous studies as whistleblowing can be seen as an effective tool for developing theorganisation.76 Therefore, organisational structures, processes andmanagement should support whistleblowing and create whistleblowing opportunitiesby providing whistleblowing channels. In addition, employees should beencouraged to blow the whistle. Moral courage and whistleblowing are associated with professional andorganisational hierarchy.20 It could be beneficial toreduce professional hierarchy in health care organisations with differentmanagement styles such as involved management. Furthermore, the workplaceculture should be open and transparent so that it is easy to raise concerns andspeak up about wrongdoing.There is a growing interest in developing legislation for whistle-blower protectionPotential whistle-blowers seem to be quite morally courageous actors when respondingto a simulated wrongdoing. The identification of potential whistle-blowers, theirmoral courage and the variables associated with it offers opportunities fordeveloping interventions, practices, and education to support and encouragehealthcare professionals in their whistleblowing. Research could focus on developinga theoretical construction aimed at eventually increasing whistleblowing anddecreasing and preventing wrongdoing."}
+{"text": "This paper outlines challenges and opportunities in operating underwater robots  on a seaweed farm. The need is driven by an emerging aquaculture industry on the Swedish west coast where large-scale seaweed farms are being developed. In this paper, the operational challenges are described and key technologies in using autonomous systems as a core part of the operation are developed and demonstrated. The paper presents a system and methods for operating an AUV in the seaweed farm, including initial localization of the farm based on a prior estimate and dead-reckoning navigation, and the subsequent scanning of the entire farm. Critical data from sidescan sonars for algorithm development are collected from real environments at a test site in the ocean, and the results are demonstrated in a simulated seaweed farm setup. We will outline the challenges and opportunities in operating autonomous underwater vehicles (AUVs) on seaweed farms. We propose a system for using AUVs for inspection. We have built a prototype of the AUV. We have tested parts of the system in simulation and using real data collected from the AUV in a seaweed farm. The motivation for using AUVs in this application is mainly one of reducing cost and risk. At the same time, seaweed farming promises to be very profitable while it helps to feed the world in a sustainable way.According to the UN\u2019s medium-variant projection, the human population will likely increase from 7.7 billion people in 2019 to 9.7 billion by 2050 and reach 10.9 billion by 2100 [2 may be captured for a longer period of time and also work as a temporary carbon sink [Compared to 2012, the total food, feed, and biofuel demand is projected to increase by 40\u201354% by 2050 , such asbon sink . Furtherbon sink , so reguThe sustainable future is \u201cBlue\u201d and we need to unlock the potential of the ocean for energy systems e.g., wind and wave energy as well as increase aquaculture in order to achieve a prosperous future for humanity. At the same time, the ocean environment is a harsh and demanding environment to work in and very expensive ships and diving is needed. Large-scale macroalgae cultivation can benefit from the automation of the inspection task. In particular, replacing current divers and data collection from boats with fully automated inspection systems using AUVs could substantially reduce risks and costs . UnderwaOperations with AUVs at sea, however, pose their own challenges regarding autonomous vehicle navigation, perception, control, and planning. This is due to the limited sensing capabilities available underwater, where electromagnetic-based sensors are quickly rendered ineffective, together with the unpredictable dynamics of the open-water domain. Some of these challenges can be overcome with high-grade navigation solutions and sensors. However, for the automation of the inspection tasks to be cost-effective at a large scale, an expensive AUV with powerful thrust and high-end navigation and sensing is not attractive. We propose a system centered around an affordable, small AUV with a compact sensor suite. Such a system then implies increased challenges for navigation especially when underwater with no GPS and a dynamically varying structure like the seaweed farm. The reduction in size also entails a more careful design of the electrical layout of the vehicle in order to avoid interference, since the various sensors, motors, and computers need to be placed in near proximity to one another. Regarding the data collection, cameras are essential to provide information about the state of the crops. Although cameras can also be used for autonomous navigation in aquaculture farms as in , sidescaSaccharina latissima) cultivation. We propose a system for kelp farm inspection including bespoke components such as the vehicle and sensors, as well as the software parts. These include a graphical user interface (GUI) for planning an inspection mission, and a behavior tree (BT) framework for executing inspection plans, control, sensing, and navigation solutions. This paper is organized as follows: in In this paper, we describe a new generation of AUVs that could be used as inspection robots in the coming blue revolution and particularly for the new generation of sea farmers in the expanding field of kelp  offer unique opportunities for steering the vehicle. Navigation and payload sensors include an inertial measurement unit (IMU), a compass, a GPS (for surface operations), a Doppler velocity logger, cameras, sidescan sonar, and environmental sensors. The AUV is designed to be very slender with no external antennas, fins, or rudders that can get entangled in cluttered spaces. SAM uses these unique maneuvering features to enable operations in the confined environments of the seaweed farm.Mission planning through Neptus: A graphical interface for the operator to plan out the mission on a world map and monitor the current status of the vehicle while it works.Robust mission execution using behavior trees: A behavior tree ensures the safety and compliance requirements while the mission executes, disallowing unsafe behaviors autonomously.Path planning using spline-fitting: Given a set of waypoints as the mission, a spline that is tuned to the vehicle is fit on the waypoints to ensure that the vehicle can reach each waypoint.Feedback control using cascaded PID controllers: PID controllers that generate the required signals for the actuators are controlled by other PID controllers that generate signals for the desired actuator set-points which helps keep the actuators from getting damaged while keeping control of the vehicle.Dead reckoning with an extended Kalman filter: An extended Kalman filter is used to fuse together the external and inertial measurements the vehicle collects to estimate the current position and orientation of the vehicle when GPS is not available underwater.Software-wise, the autonomy stack on SAM runs on the robot operating system (ROS) environment and its sub-components:All the software subsystems within ROS can be validated using the Stonefish simulator environment , thus enVehicle is launched, and a GPS fix is acquired.The AUV mission is started and the vehicle follows a plan to reach the farm\u2019s vicinity.The AUV circles around the farm, using sidescan sonar to detect buoys and construct a relative-to-AUV map of the farm.Move to the beginning of the seaweed line, using the constructed map.Start surveying the line according to the previously constructed map.Transition to line-following once some of the lines have been detected using the sidescan sonar.At end of the line execute a 180 turn to position the AUV on the other side of the line (between two lines now).Follow the line using the previous detections of the first line seen from the first side and known prior line spacing until sufficient detections of the next line are found.Repeat from (6) until complete.Once all lines are surveyed, finish the planned mission and return to base.The proposed system is designed to allow the operator to quickly set up an inspection plan with only partial information about the seaweed farm. Then she would launch the AUV and have it find its way to the farm, carry out the inspection and return with the data. The steps of a typical plan set up by the operator prior to launch are:https://github.com/LSTS/neptus (accessed on 27 June 2022)) open source software package from Laborat\u00f3rio de Sistemas e Tecnologia Subaqu\u00e1tica (LSTS) (https://lsts.fe.up.pt/ (accessed on 27 June 2022)), which has been integrated into the ROS ecosystem. The integration of Neptus into ROS is accomplished through a message bridge that can do two-way translations of intermodule communication (IMC) (https://github.com/LSTS/imc (accessed on 27 June 2022)) messages between Neptus and ROS. The Neptus GUI interfaces with the behavior tree (BT) to set up and monitor the mission in a user-friendly manner. The mission is planned on Neptus as a list of waypoints, where each waypoint contains specific requirements like depth, speed, and which sensors enable. The BT then receives this plan and uses Neptus to display the progress, current maneuvers, and any other feedback relating to the mission being executed. When a communication link is established, an operator can use Neptus to stop, pause, replace, and resume a running mission plan.Mission planning is done using the Neptus )) has a small seaweed test site. The testbed is equivalent to the seaweed farm built up in the simulator described in this paper and consists of two lines of seaweed ropes attached to supporting buoys at the surface and anchored to the sea bed by concrete blocks. The farm is approximately 15 by 15 m and the ropes are situated at about 2 m depth. Here we have been able to test the rope detection scenario in a real setting so that the algorithms we are developing are trained and tested on real data. Data have been collected during a number of visits to the farm in different conditions during the year April, June, and November).Preliminary field tests were carried out at the marine field station Kristineberg on the Swedish west coast, The relative navigation in the seaweed farm with respect to the ropes is a critical step in the operation. Direct detection of the rows of seaweed to be inspected is important as the AUV navigation while underwater is subject to substantial drift and the layout of the farm is subject to change over time. Thus, detection is needed for both finding the farm and for control along the rows.Although cameras will be needed to provide the actual inspection data on the seaweed condition, the need to potentially look for the farm using sensor information renders underwater cameras less useful in this scenario, as cameras can only obtain interpretable images at short range, given clear water conditions and sufficient lights. Sidescan sonar, an acoustic sensor that provides long-range observations without constraints on the water quality, is instead employed for control feedback sensing. Apart from being able to provide high-resolution observations of ropes and buoys, sidescan sonar has been shown to provide useful signals for seaweed growth rate, making it an ideal sensor for seaweed farm monitoring . An examThe presence of ropes and buoys are seen as change points in the 1D sidescan signal, and a window-sliding segmentation algorithm is used for change point detection. A window of fixed size is sliced across a 1D sidescan signal. Within that window, a mean and the variance from the mean are computed. The difference in variance between two adjacent windows is recorded as the score . The higs denote the 1D sidescan signal with total length of T and t starting at index i. For each window, a least square deviation from the window mean can be computed as follows:More specifically, let The discrepancy between two adjacent windows can be computed using the following formula:t is slid across the signal, and the index with the highest discrepancy is selected as the change point.This discrepancy indicates the cost gained by splitting the sub-signal The nadir is the first bottom-hitting acoustic return received by the sidescan. By definition, the change in signal strength is likely to be high. To avoid misdetecting nadir as objects, the detection algorithm runs two window-sliding sessions, first identifying the nadir and then identifying any objects in the water column. The same algorithm can be used to detect both ropes and buoys by changing the sliding window size and signal change ratio. An example rope detection procedure is shown in Before the actual inspection can begin, the AUV must localize the farm relative to its position and determine the overall layout of the farm, that is, the position of the outer lines and buoys. It is only expected that a rough layout and position of the buoys on the farm are known at the start of a mission. This is very much in line with a real scenario where the buoy locations may not be exactly known in the first place and may also have drifted during e.g., rough weather conditions. The AUV begins by navigating on the surface to the approximate farm location given by GPS coordinates. It then circumnavigates the farm according to its navigation and prior information on the farm layout. During this phase, the AUV stays relatively far from the farm and attempts to detect the buoys in its vicinity.To handle potential misdetections of buoys, the raw detection results described in The ropes that the seaweed hangs from do not lie perfectly straight. In order to hold the AUV at the proper distance for visual inspection, a line following control behavior was used. The idea is to use the last few detections of the rope to project a straight line and then to control the vehicle to a path offset from that by an appropriate amount. This is similar to a wall following the behavior of an indoor robot navigating a corridor. After moving along the initial route long enough to collect some detections of the rope, a random sample consensus (RANSAC) regressor  is used e between the current AUV position and the desired path, and ensuring that the AUV intersects with the path within a specified look ahead distance In order to follow the straight line path effectively, a line-of-sight guidance law based on look-ahead steering (see ) is usedce \u03b4 see .https://kristinebergcenter.com (accessed on 27 June 2022)) is built up in a mission simulator (https://github.com/smarc-project/smarc_stonefish_sims (accessed on 27 June 2022)) using the open source Stonefish simulation tool [The test site for the seaweed farm at the marine field station Kristineberg ; operations in a tightly spaced farm where multiple lines can be seen on both sides. Further, sensor and in particular sidescan data robustness in a small AUV with many advanced sub-systems operating in a tight encapsulated space is also an additional challenge. Noise and high-frequency disturbances from e.g., motors and servos on board are easily propagated through the powering system of the AUV into sensor data. Finally, as the main sensors on board the AUV for mapping, navigation, and underwater communication are based on acoustics, it is also essential that these systems are robust enough to operate in parallel without interfering with each other.In order for there to be any benefits from this system for the farm operator there will need to be actual data on the status of the seaweed farm collected by sensors on the vehicle. These would be video but could also include chemical and water quality sensors. Further processing to get numerical assessments of, for example, biomass estimates from the video and sonar are needed but that is not part of our work so far.Ultimately these systems could be extended with an underwater base into which an AUV could dock for recharging and data communication. Resident AUVs would have a long-term presence and allow near continuous data collections on the condition of the farm. With a robust base system in place, additional functionality can easily be added, e.g., new sensors that could also monitor the water quality.Beyond the capabilities developed and demonstrated specifically for kelp farms in this paper, such technologies could have numerous other applications. While automated aquaculture monitoring has been proposed in ,26, thesWe have proposed a system for automatic inspection of seaweed farms. A realistic simulation environment has been built and used for the validation of the system. Individual parts have been validated on data collected by the AUV in a seaweed farm. Other parts of the system have been validated at sea.The system has been realized on a working prototype. The software architecture is tested both in a realistic simulation and on the AUV. The sidescan sonar is used for the feedback control of the inspection action. The detection needed for that control has been tested on real data collected from the AUV in the seaweed farm. The control action has been tested in the simulator.https://www.nordicseafarm.com (accessed on 27 June 2022)), on the need for underwater robots as essential tools in seaweed farming. The numerical evaluation of the health and growth status of the seaweed farm from sensor data including cameras is also future work.Future work will include collaborations with local initiatives in Sweden, such as Nordic SeaFarm AB ("}
+{"text": "Medication-wide association studies (MWAS) have been applied to assess the risk of individual prescription use and a wide range of health outcomes, including cancer, acute myocardial infarction, acute liver failure, acute renal failure, and upper gastrointestinal ulcers. Current literature on the use of preconception and periconception medication and its association with the risk of multiple gestation pregnancies  is largely based on assisted reproductive technology (ART) cohorts. However, among non-ART pregnancies, it is unknown whether other medications increase the risk of multifetal pregnancies., twins and triplets) following preconception and periconception exposure to prescription medications in patients who delivered at Penn Medicine.This study aimed to investigate the risk of multiple gestational births ; model 2 ; and model 3\u2014our final logistic regression model . In all models, multiple births (MBs) were our outcome of interest (binary outcome), and each medication was assessed separately as a binary variable. To assess our MWAS model performance, we defined ART medications as our gold standard, given that these medications are known to increase the risk of MB.P values) and 10 (8.1%) medications associated with MB (using Bonferroni adjustment) in fully adjusted model 3. We found that our model 3 algorithm had an accuracy of 85%  and 89% .Of the 63,334 distinct deliveries in our cohort, only 1877 pregnancies (2.96%) were prescribed any medication during the preconception and first trimester period. Of the 123 medications prescribed, we found 26 (21.1%) medications associated with MB (using nominal Our work demonstrates the opportunities in applying the MWAS approach with electronic health record data to explore associations between preconception and periconception medication exposure and the risk of MB while identifying novel candidate medications for further study. Overall, we found 3 novel medications linked with MB that could be explored in further work; this demonstrates the potential of our method to be used for hypothesis generation. Multifetal pregnancies are at a high risk for obstetric complications, including anemia, preterm labor, pregnancy-induced hypertension, placental previa, and fetal malformations ,2. TheseART is a widely accepted treatment for infertile couples, referring to all treatments that include the handling of eggs, sperm, and embryos. Outside the scope of ART, hormonal medications for the purpose of facilitating a successful pregnancy are referred to as fertility treatment. Increased rates of monozygotic twinning have been observed in pregnancies due to ART use  -8. OvulaThe wealth of information from electronic health record (EHR) data can allow for hypothesis-driven research on the associations between medications and pregnancy outcomes. Ryan et al  proposedExcept for research conducted on nationwide health care data registries ,12,14, MThis study illustrates a proof-of-concept MWAS approach for hypothesis-driven pharmacovigilance research on EHR data, with a particular focus on MB.We used EHR data obtained from 4 different hospitals within the Penn Medicine system: the Hospital of the University of Pennsylvania, the Pennsylvania Hospital, Penn Presbyterian Hospital, and Chester County Hospital. The deliveries were identified using a previously developed algorithm called Method to Acquire Delivery Date Information from Electronic Health Records (MADDIE) . The MADThis study was approved by the institutional review board of the University of Pennsylvania (#828000).Although a majority of twin births result from natural conception, the incidence of twins and other higher order multifetal pregnancy resulting from superovulation and ART is 20 times greater than the incidence from natural conception . Therefopreconception/first trimester exposure as any medication prescription occurring from 275 days before delivery to 215 days before delivery to capture medications slightly before conception and the first trimester of pregnancy. As ART and fertility medications are often prescribed around the time of conception, we chose this window. Most multifetal pregnancies result in preterm birth and are often completed in <270 days after conception. Therefore, we chose the window of 275-215 days before birth to capture the preconception and periconception window where ART and fertility medications are likely to be used.We mapped all inpatient and outpatient medications from Epic and other EHR systems to RxNorm using a previously described method . In shorWe manually annotated the complete list of medications, adding the following elements: generic name, medication type, specific medication type, US Federal Drug Agency pregnancy category, associated comorbidities, and associations with pregnancy outcome treatment. We manually annotated this list because many drugs used in fertility treatments are used off-label; therefore, standardized medical terminology systems would be ineffective in capturing those use cases . We refeP<.05; Bonferroni-adjusted P<.05) given the multiple testing that we were performing and calculated odds ratios (ORs) with 95% CIs.We constructed 3 logistic regression models with MB as our outcome of interest , and the effect of each medication on the outcome was assessed separately . The analysis was performed using the general linear model function in R. The control group for each medication comprised all patients without exposure to the target medication (coded as 0), including patients who had no exposure to medications in the EHR data. Consequently, each target medication had its own control group. We adjusted for 3 known confounders of MB: maternal age (encounter age), ART-resulting pregnancy diagnoses (0 or 1), and infertility diagnoses 0 or 1; . A totalP<.05) with nominal P values and Bonferroni-adjusted P values were evaluated on performance to capture medications used in ART and infertility treatment with binary classification. As previously stated, ART use is an established factor in multifetal pregnancy; therefore, these medications are likely to be associated with MB. The analysis was limited to the medications captured within the defined medication exposure window. Using confusion matrices, we calculated precision, sensitivity, specificity, accuracy, and F1 score  fertility medications used in ART, (2) medications used for comorbidities associated with MB, and (3) medications not associated with MB in the current literature.We categorized medications with significant nominal We obtained EHR data from 1,060,100 female patients treated at Penn Medicine, with inpatient and outpatient visits between 2010 and 2017. A previously developed algorithm called MADDIE identified 50,560 patients who delivered a baby at Penn Medicine having 63,334 distinct deliveries . Figure We manually annotated 123 medications that were prescribed during the preconception period and the first trimester period of 1877 pregnancies of the 63,334 (2.96%) total distinct deliveries in our cohort . These 1Aside from fertility medications, the list contained several types of pain , antibiotic , and antihistamine medications . Most of the extracted medications were not formally assigned , followed by category C  and category B  medications. As expected, fewer medications were categorized as category A  and category D . We found 9.8% (12/123) of medications were categorized as category X, contraindicated in pregnancy, medications\u2014all of which are medications indicated for fertility treatment, contraception, or other indications in obstetrics and gynecology practice.P<.001 to P=.04) from our fully adjusted model  are shown with ORs (95% CIs) in a forest plot. The results for all 3 models are presented in P<.001 to P=.04) was fertility treatment  prescriptions. The forest plot in P<.001 to P=.04) and nonsignificant (P=.5 to P=.98) medications.In P<.001 to P=.04) and 11 (8.9%) medications associated with MB using the Bonferroni adjustment (P<.001 to P=.04). P value . Of the 123 medications extracted, we found 26 (21.1%) medications nominally associated with MB  were potential fertility treatment medications; 12 (46%) were associated with infertility and ART use or complications associated with multifetal pregnancy; and 3 (12%) were not previously associated with MB, ART, or fertility-related problems , oxytocin (P=.02), and lorazepam and ativan (P=.02).Prescription medications associated with comorbidities of fertility and ART treatment were found, as well as medications that may be used for obstetric complications related to multifetal pregnancy care. Of the 26 significant medications using nominal We applied 3 logistic regression models to retrospective EHR data of a cohort of patients who delivered at Penn Medicine between 2010 and 2017  to explore potential associations between the medications prescribed during the preconception and first trimester period (binary variable) and the occurrence of MB (binary outcome). We discuss the results of our MWAS from our fully adjusted model that was adjusted for age and ART and infertility diagnosis (model 3) on MB for all associations revealed using nominal.The application of an MWAS approach to MB allows the analysis of medications used outside the scope of obstetric treatment, capturing comorbidities that may increase the risk of the outcome. Not all of this is known, as MB is more commonly used as an adjustment for analysis of other pregnancy outcomes of interest. Off-label use is common in pregnancy and infertility treatment ,35. Our The graph in MB outcomes can also be observed in ART cohort databases, such as the SART Clinical Outcomes Reporting System. However, fertility medication treatment without the intention of egg retrieval will not necessarily be captured within such databases, as they are beyond the scope of ART. Moreover, not all multifetal pregnancies result from infertility treatment and ART. Finally, such databases are reported from ART clinics and are not necessarily representative of all the medications prescribed during pregnancy. An ART cohort database may have a wealth of data elements specific to ART treatment; however, these data are reported using inconsistent methods, often from a variety of reporting services . In contWe found that 5.51% (86/1562) MB pregnancies had prescription medication exposure during pregnancy. Therefore, pregnancies resulting in MB were more likely to have recorded prescription medication during the preconception and first trimester period. This is consistent with (1) the fact that ART often uses medications early on to induce pregnancy  and (2) P values , which is comforting. We observed drugs used in fertility treatment  and drugs known to be associated with multifetal pregnancy , along with 3 (11%) novel associations. Associated comorbidities of infertility overlap with obstetric complications during multifetal pregnancy, including diabetes mellitus, cardiovascular disease, thyroid dysregulation, and liver dysregulation.To assess the ability of our MWAS to capture medications that increase the risk of MB, we used medications that are known to increase an individual\u2019s chance of conceiving and have been implicated in increasing the risk of MB in the literature . We knowMedications used in fertility treatment themselves may be captured solely because of reverse causation, although they do not have a truly strong association with multifetal pregnancy. Several medications may be prescribed during IVF treatment cycles for preventive care or other indications, including antibiotics (doxycycline and amoxicillin), a corticosteroid (methylprednisolone), pain management (EMLA), progestin-induced menstruation (medroxyprogesterone acetate), and conscious sedation (diazepam) ,39.ART and ovulation induction procedures are used for fertility treatment. A comprehensive review of infertility comorbidities in women suggests that infertility is a complex health care issue, and women with infertility are at a higher risk of psychiatric disorders and endometrial cancer . InfertiMedications associated with comorbidities of infertility were identified, including treatments for asthma and IBD. Research shows that women with asthma have higher pregnancy losses  and a prMedications identified by the MWAS may be prescribed for obstetric complications associated with multifetal pregnancy. These pregnancies are at an increased risk of obstetric complications, such as preterm birth, placental problems, gestational diabetes mellitus, anemia, and preeclampsia. Owing to the time exposure range, medications typically used to treat complications typically past the first trimester of pregnancy were not captured by the MWAS. One antidiabetic medication  was identified; however, other forms of insulin and the insulin sensitizer metformin were not identified as significant. A single antithrombotic medication, heparin, was identified, but other anticoagulants and cardiovascular-related medications were not identified in our models.Migraines have a high incidence in obstetrics; one migraine pharmacological treatment (sumatriptan) was found to be associated with MB. An association between migraine history and development of ovarian hyperstimulation syndrome may indicate the risk of multifetal pregnancy , as ovarAlthough the World Health Organization\u2019s anatomical therapeutic chemical classification system is applicable, the proportion of RxNorm drugs mapped to anatomical therapeutic chemicals would result in fewer medications being included in the analysis. However, the therapeutic use of the medications has not been explicitly determined. Medications classified as fertility related are based on the SART references; however, without discrete indications, they potentially underpower performance in the validation process. In addition, a major limitation of using standard pharmacology and drug-related terminologies is that approximately 11% of medications used in women\u2019s health are off-label . This inP values and Bonferroni-adjusted P values, respectively. Sensitivity and F1 score were improved using nominal P values in comparison with Bonferroni-adjusted P values, indicating the applicability of nominal P values in exploratory MWAS studies. A total of 6 novel agents were linked to MB, with the remaining 20 medications potentially linked to the comorbidities of infertility, ART use, and obstetric complications during multifetal pregnancy. The MWAS approach can facilitate hypothesis-driven data exploration, informing the adjustments needed in the models in further research. Our approach also highlights the importance of exploring medication histories, as many patients receiving ART and fertility treatments do not have corresponding diagnosis codes indicating treatment. If medication information was not used, these patients were mistakenly labeled as having not received ART and infertility treatment. This underscores the importance of multidata modalities in retrospective EHR studies, especially for those exploring the effects and outcomes related to pregnancy.Our research demonstrates opportunities in using an MWAS approach with EHR data to explore agents previously unknown to be associated with MB outcomes. The results indicated that a number of medications used in ART and infertility treatment were associated with an increased incidence of MB likely due to multifetal pregnancy, as expected. Using these medications as our gold standard, we found that our algorithm had an accuracy of 85% and 89%, using nominal"}
+{"text": "Vaccination coverage against COVID-19 among health care workers (HCWs) of the University Health Agency Giuliano-Isontina (ASUGI) of Trieste  by 1 January 2022 was 90.4% with at least one vaccine dose, 84.9% with at least 2 doses, and 75.1% with 3 doses, 98.2% with Comirnaty  versus 1.8% with Spikevax . From 1 October 2020 to 7 February 2022, 1652 SARS-CoV-2 infections were notified in HCWs of ASUGI Trieste. Although the overall risk of SARS-CoV-2 contagion increased over time, the rate of occupational infections progressively declined, from 42.5% during the second COVID-19 wave to 15.6% in the fifth. Between 1 January\u20137 February 2022 (a period dominated by the Omicron variant), albeit no COVID-19-associated hospitalizations were recorded in HCWs of ASUGI Trieste, 669 SARS-CoV-2 infections were counted against 367 cases observed from 1 October to 31 December 2020, the 3 months preceding the implementation of the vaccination campaign against COVID-19. Job tasks and health care settings turned out to be the most significant risk factors for SARS-CoV-2 infection. However, the effect of workplace prevailed over job task on the biological risk, with greater rates of SARS-CoV-2 infections observed among HCWs operating in areas with higher levels of circulation of the virus, particularly COVID-19 dedicated units. The novel coronavirus disease COVID-19) was declared a pandemic on 11 March 2020 by the World Health Organization 9 was dec,4,5. PatAlthough males are more likely to develop the severe form of the disease, occupational COVID-19 among the health care force is reportedly more frequent among females, an epidemiological figure coherent with their higher employment prevalence among HCWs ,8. WhilsIn view of the above, this study aimed to estimate the risk of SARS-CoV-2 infections and associated factors in HCWs of the University Health Agency Giuliano-Isontina (ASUGI) of Trieste  from 1 October 2020 to 7 February 2022.N = 5019), including two major teaching hospitals, primary care services, health districts, public health services, and administrative/management services have been systematically screened for COVID-19 since the start of the pandemic, becoming an ideal population to estimate the infection risk, the associated factors, and the effectiveness of the respective vaccines [HCWs of ASUGI Trieste N = 501, includivaccines ,13,14,15Since COVID-19 vaccination was mandatory for all HCWs of ASUGI by Italian law, clinical and non-clinical HCWs unvaccinated against COVID-19 have been suspended from work, whereas those exempted from vaccination for health reasons were re-assigned to job tasks not entailing patient contact. These suspensions and job task reassignments were mainly enforced in Trieste from September/October 2021 onwards. HCWs with only one or two doses of COVID-19 vaccines by 1 January 2022 had at least one previous confirmed infection by SARS-CoV-2.The surveillance system adopted by the Occupational Medicine Unit of ASUGI Trieste required routine nasopharyngeal swabs for all HCWs with patient contact to detect SARS-CoV-2 infection by real-time polymerase chain reaction (RT-PCR) on a weekly or monthly basis depending on their job task and risk assessment, re-testing in case of symptoms or history of close contact with confirmed COVID-19 cases. In particular, all HCWs employed in department more likely to host COVID-19 patients  were considered at high risk for COVID-19 and screened weekly, regardless of the job task. Additionally, HCWs in contact with immunocompromised patients were also considered at high risk for COVID-19 and screened weekly. For all other HCWs, a monthly screening schedule was followed.N = 534), undergraduate medical/nurse students (N = 780), blue collars (N = 870), and academic staff/tutors (N = 72).The latter surveillance system was extended also to postgraduate specialist medical trainees . Hospital inpatients were routinely tested every 3 days.Occupational SARS-CoV-2 infections were defined as those developed following exposure to confirmed COVID-19 cases (patients or colleagues) in health care setting, in absence of housemates or household members positive for SARS-CoV-2. By contrast, non-occupational cases were defined as health care associated SARS-CoV-2 infections without previous exposure to confirmed COVID-19 cases among colleagues or patients. Re-infections were defined as secondary infections in the same HCW, occurring at least 90 days following a previous SARS-CoV-2 infection.with COVID-19 patients or colleagues positive for SARS-CoV-2 in the workplace; orwith close friends, housemates, or household members positive for SARS-CoV-2 outside the workplace.All HCWs testing positive for SARS-CoV-2 were interviewed over the phone for contact tracing, following international guidelines . A strucQuantitative variables were expressed as mean \u00b1 standard deviation; categorical data were reported as frequencies and percentages. Differences between continuous data and categorical data were estimated by T-Student and chi-square test, respectively. Risk factors of occupational infections were investigated by multivariable logistic regression, selecting terms significant at univariable analysis. Results were expressed as odds ratio unadjusted (OR) as well as adjusted (aOR), with 95% confidence intervals (95%CI).N = 7241; Descriptive statistics on vaccination coverage were restricted to 5019 HCWs employed by ASUGI Trieste. Logistic regression analysis on risk of occupational SARS-CoV-2 infections included also staff not employed by ASUGI Trieste, hence undergraduate medical/nursing students, post-graduate medical trainees, blue collars, and academic staff/tutors  was employed for the analysis.Our study analyzed the risk of SARS-CoV-2 infection in a population of individuals almost fully vaccinated with m-RNA vaccines (98.2% Comirnaty vs. 1.8 % Spikevax). In Italy, the vaccination campaign against COVID-19 started on 27 December 2020. Vaccine coverage against COVID-19 among 5019 HCWs employed at ASUGI Trieste by 1 January 2022 was 90.4% (=4538/5019) with at least one dose, 84.9% (=4529/5019) with at least 2 doses and 75.1% (=3770/5019) with 3 .As can be seen from During the entire study period (1 October 2020\u20137 February 2022), 129,335 swab tests were totally performed in the study population.The trend of infections in the study population mirrored the temporal distribution of incident cases in the general population of Trieste during the same time-period .Between 1 January\u20137 February 2022 (a period dominated by the Omicron variant), albeit no COVID-19 associated hospitalizations were recorded in HCWs of ASUGI Trieste, 669 SARS-CoV-2 infections were counted against 367 notified from 1 October to 31 December 2020, the 3 months preceding the implementation of the COVID-19 vaccination campaign in Italy . Out of The trend of SARS-CoV-2 re-infections over time from 1 April 2021 until 7 February 2022 can also be visualized in N = 7241), hence including also external contractors of ASUGI Trieste. Two multivariable logistic model were fitted:Model I: adjusting for patient\u2019 age; sex; COVID-19 wave, job task; worksite;Model II: adjusting for COVID-19 wave, job task; worksiteUsing the second COVID-19 wave as reference, we identified a decreased trend of occupational risk of SARS-CoV-2 infections in the subsequent waves. Non-clinical workers, typically less exposed to biological risk in the worksite, were used as reference category in the risk assessment by job task and hospital ward. Job task and health care setting turned out to be the most significant risk factors for SARS-CoV-2 infection. As can be seen from multivariable model I, patient-facing tasks were the most hazardous for occupational SARS-CoV-2 infection among HCWs of ASUGI Trieste, especially in COVID-19 dedicated units  and A&E . Health care areas less affected by SARS-CoV-2 infection were those not involving patient contact, as administrative services . Multiple model II (removing age and sex) confirmed the overall results, although the effect size and stratum specific confidence intervals of worksite shrank, whereas the effect size of job task increased, widening also the respective confidence intervals.During the entire study period (1 October 2020 to 7 February 2022) a total number of 1652 SARS-CoV-2 infections were notified in HCWs of ASUGI Trieste. The effectiveness of m-RNA vaccines seemingly reduced during the fifth COVID_19 wave, with the spread of the Omicron variant. After the second wave, the majority of SARS-CoV-2 infections among HCWs of Trieste occurred outside the workplace, predominantly among females. Patient-facing tasks were the riskiest for occupational SARS-CoV-2 infections, with nurses and nurses\u2019 aides being the professional categories more likely to become infected in the worksite. Nevertheless, the infection risk associated with the workplace prevailed over job tasks. In particular, the risk of contagion increased in A&E and COVID-19 dedicated units, whereas it was lower in health care areas not involving patient contact, such as administrative services.Risk reduction rules need to be strictly observed in Italian health care settings in order to benefit from social compensation schemes for sickness absence due to COVID-19. The massive number of SARS-CoV-2 infections among HCWs of ASUGI Trieste during 1 January\u20137 February 2022 was by far acquired outside health care premises, where health protection measures\u2014such as social distancing, hand washing, and especially the use of personal protection equipment (PPE)\u2014were less stringently enforced. In a survey on 1266 HCWs recruited from a cancer center in Catalunia (Spain) during the first pandemic wave (21 May 2020\u201326 June 2020), whilst no difference in seroprevalence was observed between onsite workers and teleworkers, living with a person with COVID-19 significantly enhanced the risk of seropositivity to SARS-CoV-2 .Although effective to prevent the severe form of COVID-19, the effectiveness of m-RNA vaccines against the spread of SARS-CoV-2 infection seemingly declined from January 2022 onward (with the spread of the Omicron variant). Human coronaviruses are known to cause respiratory re-infections, regardless of pre-existing humoral immunity ,17 and OFollowing the initial stage of the pandemic, HCWs started to familiarize themselves with PPE  and the Since occupational risk of SARS-CoV-2 infection increases with frequency, duration and intensity of patient contacts, patient-facing tasks inevitably pose the highest risk of contagion. Nurses and nurses\u2019 aides were in fact professional categories more at risk of occupational SARS-CoV-2 infection in the present study. Likewise, in a retrospective study on 2,199,745 individuals recruited from Catalunia (Spain) between 21 March 2020 and 16 September 2021, the age and sex adjusted cumulative rate of SARS-CoV-2 infection was highest in HCWs (27.7%), particularly among nurses\u2019 aides (29.4%), doctors (27.3%) and nurses (26.3%) .However, whilst healthcare settings are among the most hazardous contexts for SARS-CoV-2 infection , the preNevertheless, the very high adjusted risk estimates for occupational SARS-CoV-2 infections associated with worksite were accompanied by very wide confidence intervals, likely reflecting the low numbers involved.The present study relies on data from a relatively large population routinely and stringently tested for SARS-CoV-2, thus providing reliable estimates of infection rates. Moreover, the long study period, stretching up to 16 months, enabled to monitor SARS-CoV-2 infections in the same population over time, contrasting the number of infections in the same population before the implementation of the COVID-19 vaccination campaign and after full immunization . Lastly, this study distinguished occupational versus non-occupational SARS-CoV-2 infections by contact tracing, as recommended by the Italian occupational compensation scheme.Nevertheless, this study has also some limitations. Contact tracing by questionnaire is intrinsically affected by potential recall bias. Furthermore, some HCWs might have intentionally neglected some sources of SARS-CoV-2 infections outside the workplace  in order to benefit from the occupational compensation scheme, which is more convenient than the non-occupational scheme. However, in the case of coexistent exposures , the approach adopted by the Occupational Medicine Unit in ASUGI Trieste was to consider all these infections as occupational.A weekly/monthly testing schedule might have skipped some asymptomatic/pre-symptomatic COVID-19 cases, thus potentially underestimating to some extent the true incidence of SARS-CoV-2 infections. However, although endorsed by some authors to monitor SARS-CoV-2 infectious state in HCWs, daily screening by RT-PCR was beyond the capacity of the laboratory service of ASUGI Trieste . MoreoveLastly, due to Italian privacy law, out of 481 unvaccinated HCWs the exact number of those suspended from work or exempted from COVID-19 vaccination for health conditions and re-assigned to job tasks not entailing patient contact was not available. However, the latter group contributed to the total number of SARS-CoV-2 notifications during the entire study period, maintaining the original job task until re-assignment. Likewise, suspended HCWs contributed to the number of SARS-CoV-2 infections until work suspension, maintaining their original job task up until then.Whilst effective to prevent severe forms of COVID-19, the effectiveness of m-RNA vaccines against the spread of SARS-CoV-2 infection seemingly declined from January 2022 onward, with the fifth wave and the spread of the Omicron variant. Compliance with risk reduction norms was confirmed to be the main preventative factor against SARS-CoV-2 contagion in health care settings. Consistent with the open literature, nurses and nurses\u2019 aides were professional categories more at risk of occupational SARS-CoV-2 infection, stressing the importance of patient-facing contact as a critical risk factor for the contagion. However, the effect of the workplace prevailed over job tasks on the biological risk, with greater rates of SARS-CoV-2 infections observed among HCWs operating in areas with higher levels of exposure to the virus, particularly COVID-19 dedicated units and A&E."}
+{"text": "Linguatula serrata, have been reported from several animals in Romania, including some domestic dogs translocated to other parts of Europe. In this study, gray wolves , golden jackals , red foxes , and roe deer  were examined for pentastomes. Overall, 17.5% of wolves were found to be infected with specimens of Linguatula, with a range of infections of one to five individuals per animal. Golden jackals and foxes had much lower infection levels, with 1.73% of golden jackals and 1.69% of foxes infected; both host species were found to be infected with one or two individual pentastomes per animal. The single deer specimen was infected with three individual pentastomes. The pentastomes collected from the wolves and golden jackals were determined to be immature and mature adult specimens of L. serrata based on morphological examination and molecular analysis using the 18S rRNA gene. No pentastomes collected from the red foxes were available for identification. The pentastomes collected from the roe deer were expected to be L. arctica but determined to be mature adult male specimens of an unknown Linguatula, herein, referred to as Linguatula sp. based on its morphology; the results of molecular sequencing for the Linguatula specimen collected from the deer were inconclusive, preventing a final species identification. This study presents the first report of L. serrata in any hosts from Romania through both morphological and molecular characterization, and also presents the first report of a Linguatula sp. in Ca. capreolus, utilizing morphological characterization. Issues of morphological variability are discussed, including the presence of spines in the hook pit of specimens of Linguatula. This study highlights the need to examine all specimens of Linguatula to confirm the stage of development. Despite the inconclusive molecular result for some specimens, the authors still urge future researchers to incorporate a combined molecular and morphological approach in identifying specimens of Linguatula.Specimens of the pentastomid parasite,  Linguatula Fr\u00f6lich, 1789 have a long and problematic taxonomic history ) in Norway and were immediately referred to as L. serrata, the cosmopolitan species, until Riley et al.  (n\u2009=\u200980) (collected 1998\u20132010), golden jackals  (n\u2009=\u2009115), red foxes ) (n\u2009=\u2009236), and roe deer ) (n\u2009=\u20091) . All animals were lawfully collected by local hunters in accordance with the relevant guidelines and regulations and the carcasses were made available for dissection. The nasal cavities of the various carnivores could not be accessed by the method utilized in Shamsi et al. ; the head was not examined.The following hosts were examined for pentastomes: gray wolves  for further molecular and morphological examination; one specimen was preserved in 10% buffered neutral formalin for morphological examination only.Eight pentastome specimens were sent to the Parasitology Laboratory, Charles Sturt University, Australia, for identification. Collection details of these specimens are provided in Table Important characteristics of pentastomes\u2019 features were studied/measured (in mm). For details see Shamsi et al.  and Bart2 in a Tousimis\u00ae Autosamdri-931 critical point dryer (USA). A carbon tab was used to attach the sample to a stub before gold coating (25\u00a0mA for 2\u00a0min) in an Emitech K550X Sputter Coater . The sample was examined in a scanning electron microscope  with accelerating voltage set at 10\u201315\u00a0kV.Four pentastome specimens were selected for scanning electron microscopy (SEM), including two from wolves  and two from deer . The female specimen was initially preserved in formalin, while all male specimens were initially preserved in ethanol. To obtain SEM images, the specimens were washed in 70% ethanol and dehydrated using a graded series of ethanol. Excess 70% ethanol was removed from around the sample using a transfer pipette before being replaced by 80% ethanol overnight. This step was repeated with 90%, 95%, and 100% ethanol and followed by 100% anhydrous ethanol dehydration three times. The sample was then dried in liquid COA small piece of tissue was collected from each of two pentastome specimens for genomic DNA extractions: one collected from a wolf (SPL 1098) and one from a deer (SPL 1165). Molecular analysis was completed using DNeasy Blood & Tissue Kits  according to the modified version of the manufacturer\u2019s protocol after elution into 45\u00a0\u03bcl of water  golden jackals and four (1.69%) foxes infected; both species were found to be infected with one or two individuals per animal. The single deer specimen was infected with three individuals.Overall, 14 (17.5%) wolves were found to be infected with specimens of L. serrata based on morphological and molecular analysis. No specimens collected from the red fox were sent for identification. Measurements of individual specimens are provided in Table L. serrata collected from wild dogs in Australia. The specimen from the wolf was a mature adult female, whereas the specimen from the golden jackal was an immature adult female. Both specimens had much larger hook measurements than those presented by Shamsi et al. , the position of the male genital opening on the second annulus (compared to L. serrata where it is located on the fifth annulus), and the presence of fine spines on the proximal part of the hook blades and the lining of the hook pits (compared to L. serrata where these have not been reported previously). Variations in the measurements of the specimens collected from deer, compared to those of L. arctica from reindeer, in combination with the inconclusive molecular results (see below), prevent identification of these specimens to species level. These specimens are designated as Linguatula sp.The specimens collected from roe deer were determined to be mature adult male specimens of a species of gy Figs. , 4. MeasL. serrata sequenced by Gjerde  between the 18S sequences of L. serrata and L. arctica which led Gjerde  in other studies  and/or increased urbanization may have impacted transmission of the parasite to dogs. About 5% of domestic dogs were found to be infected in Romania in 2002; Ioni\u021b\u0103 et al. (2016) reported infection of L. serrata in a rescued dog within the city of Bucharest, Romania. Over recent years, many stray and rescued dogs have been exported from Romania to other parts of Europe, with almost half of the 44,500 dogs imported into the UK in 2019 originating from Romania  were unable to detect infection due to this. This suggests that surveillance of imported dogs for infection of L. serrata needs to be undertaken across a number of fecal examinations over a period.y Gjerde  originaty Gjerde  found thLinguatula arctica, reported as an adult from an ungulate, was originally described by Riley et al. , which is sharply delineated between the broad anterior section and the thin \u201ctail,\u201d the low number of annuli , and the extensive patch of minute spines covering the posterior hook. For the male specimens described by Riley et al.  infected and decreasing in prevalence with no reindeer aged over 28\u00a0months infected. Riley et al. (L. arctica from reindeer, and its various subspecies, from around the Arctic Circle, which had originally been ascribed to infection with L. serrata. Further studies have reported L. arctica in reindeer from Norway (Haugerud et al. 1990; Gjerde Riley et al.  found iny et al.  also lisDictyocaulus capreolus Gibbons and H\u00f6glund, 2002 in the lungs of 26% of 73 roe deer. However, no mention of other parasites in the lungs was reported. Shimalov (Linguatula.Roe deer are native to Romania (Ionescu et al. L. serrata; subsequent morphological examination determined that they were in fact adult male Linguatula sp. Adult male pentastomes are similar to nymphs in morphology, being much smaller than the female. Also, the finding of them not in a nasal sinus and in a host that has not been recorded as a definitive host previously, could suggest that the specimens were nymphs. Dependent on how long the host animal had been dead, the pentastomes may have migrated from the nasal cavities to the lungs or, given the smaller overall size of the Linguatula sp. specimens in this study compared to the measurements provided by Riley et al. (L. serrata, before migration to the nasal sinus, given the truncated life cycle as postulated by Haugerud (Linguatula to ensure that they are, in fact, nymphs, not adults, and not to disregard the fact that adult Linguatula species occur in hosts other than canids. Linguatula arctica, itself, was mistakenly identified as L. serrata, despite being found in a number of ungulates by many authors (Riley et al. Linguatula collected from various ungulates and emphasized the need to carefully examine wild ungulates in other parts of the world. Until this study, there have been no other reports of Linguatula infections in other groups of ungulates. However, a list of the early reports of L. serrata, under its various names, Poore (L. serrata were found in the frontal sinus of horses, mules, and sheep, while Christoffersen and De Assis (L. serrata infections, including reports from the lungs of a bull and the sinus of sheep. Haugerud (Linguatula or a facultative life cycle strategy. Either way, although nymphal L. serrata have been reported from the lungs of various animals (Liatis et al. L. serrata without morphological verification.At the time of collection of the pentastomes from the lungs of the deer, due to their location, it was thought that they were nymphs of y et al. , it may Haugerud  and Haugs, Poore , 2012b ds, Poore  with spes, Poore  also staDe Assis  list allHaugerud  postulat"}
+{"text": "Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.In yeast  In the budding yeast Saccharomyces cerevisiae, rapamycin binds to the peptidyl-prolyl isomerase Fpr1 and this combination inhibits the Target of Rapamycin Complex 1 (TORC1) consisting of either one of the two yeast kinases, Tor1 or Tor2, as well as three additional proteins Kog1, Lst8, and Tco895. In response to nutrients, Torc1 controls growth by regulating many macromolecular processes that include cell cycle progression, translation, and transcription8. Exposure to rapamycin leads to the inhibition of Torc1, which causes multiple physiological changes such as alteration in the pattern of gene expression, the phosphorylation status of many factors, cessation of cell growth, degradation of proteins, and finally G1 cell cycle arrest11. Despite many years of research, the precise molecular mechanism whereby rapamycin leads to G1 arrest in yeast remains unclear. However, it is believed to be the result of a combination of events including the degradation of translational initiation factors14, downregulation of the G1 cyclin genes10, and stabilization of Sic1 that inhibits the B-type cyclin-dependent protein kinase17.Rapamycin or its related derivatives such as sirolimus is an immunosuppressant used for treating various diseases that include kidney carcinomas, diabetes, ageing, and neurodegenerative disorders21. We recently screened an entire collection consisting of 442 histone mutants and reported the identification of nine mutants that affect S. cerevisiae response towards rapamycin23. Eight of the nine histone mutants displayed varying resistance to rapamycin and not to other drugs, while the remaining one mutant H2B E65A exhibited severe sensitivity to rapamycin as well as to other drugs when compared to several other histone mutants in the collection, and with the respective wild-type (WT) background23.We have previously shown that isomerization of the C-terminal domain of the large subunit of RNAPII by the peptidyl-prolyl isomerase Rrd1 plays a prominent role in the redistribution of the polymerase along the genome in response to rapamycin, and this is associated with nucleosomal changes and transcriptional activation implying that histone modifications would govern these processes1 arrest23. STE5 is a key gene of the pheromone pathway that is drastically downregulated in the H2B R95A mutant23. It encodes the scaffold protein Ste5 that performs multiple functions and serves as an essential component for the sequential activation by phosphorylation of the MAPKs, Kss1 and Fus3, required for \u03b1 factor-induced G1 arrest during signalling via the pheromone pathway. Ste5 possesses at least eight phosphorylated sites on the N-terminal, which can be regulated by the G1 cyclin Cln2 and its target cell cycle-dependent kinase Cdc28 through a Cln2 docking site located on Ste524. Ste5 can also autoregulate the levels of Cln224. We have shown that cells lacking Ste5 display nearly identical resistance as the H2B R95A mutant towards rapamycin, suggesting that Ste5 is a critical target that responds to the effects of the drug23. However, unlike the Ste5 response to \u03b1-factor by sequentially phosphorylating the MAPKs, this phenomenon does not appear to occur following rapamycin exposure23. In fact, there is a sharp induction of the G1 cyclin Cln2 (~\u20093- to 4-fold) in the ste5\u0394 mutant within 30\u00a0min of exposure to rapamycin as compared to the parent where the induction process is delayed, raising the possibility that rapamycin signalling via the TORC1 is a complex process that involves an interplay with Ste5 leading to cell cycle arrest in the G1 phase23.One of the resistant mutants, in particular, the H2B R95A  exhibited remarkable resistance to rapamycin due to its inability to arrest the cell cycle in response to the drug. The H2B R95A mutant lost the ability to express 26 genes of the pheromone response pathway, which is required to mediate \u03b1 factor-induced GSTE5 target gene. We exclude the possibility that the regulation would involve epigenetic modification(s) such as methylation on H2B arginine 95 since mass spectrometry data from our studies and that of another group revealed that there is no alteration on this residue in particular towards rapamycin response25. Herein, we conducted immunoprecipitation analysis coupled with mass spectrometry to search for unique proteins that would interact with FLAG-tagged H2B from the parent, but differ from the H2B R95A mutant. The analysis revealed the Spt16 protein, a component of the FACT complex involved in nucleosomal disassembly and reassembly, interacted weakly with the FLAG-tagged H2B R95A mutant as compared to the H2B WT. Reciprocal immunoprecipitation analysis revealed that a GFP tagged version of Spt16, Spt16-GFP, pulled down significantly less of the FLAG-tagged H2B R95A mutant as compared to the FLAG-H2B WT, indicating that the arginine 95 residue of H2B may play a role in recruiting Spt16. We showed that two variant forms of Spt16, spt16 E857K and spt16-11, exhibited sensitivity to rapamycin. Interestingly, these two spt16 mutants caused the expression of abnormal forms of Ste5-GFP. While the spt16 E857K mutant weakly expressed a lower molecular weight Ste5-GFP as compared to the parent, the spt16-11 expressed a normal level of Ste5-GFP, but of a higher molecular weight form. Our data indicate that defects in the Spt16 function can produce altered forms of Ste5 that are unable to mediate normal parental resistance to rapamycin.We set out to gain insights on how H2B R95A might regulate the expression of the 23. The wild-type WT-1 strain and the indicated isogenic mutants were derived from S288C, and the wild-type WT-2 and the isogenic mutants were derived from W30327. Epitope-tagging of strains at the endogenous gene locus was performed as previously described28. Cells were grown at 30\u00a0\u00b0C for 24\u00a0h in either Yeast Peptone Dextrose  or SD minimal media. The single-copy plasmid pSTE5-GFP carrying the entire STE5 gene under its promoter and tagged with GFP was kindly provided by Dr. Peter M. Pryciak . All chemical reagents including rapamycin were purchased from Sigma, St Louis, USA.The yeast strains and the isogenic mutants used in this work are listed in the Supplementary Table S1SDS gels were stained with the SilverQuest Staining kit according to the manufacturer (Life Technologies).Polypeptide bands were excised from the silver-stained gels and subjected to micro-capillary LC/MS/MS analysis mass spectrometry for identification and or modifications .29 was adapted for studies with yeast cells. Cells were grown in 1\u00a0ml of YPD media and incubated at 30\u00a0\u00b0C overnight. The next day, the cells were sub-cultured for 2\u20133\u00a0h. An aliquot of 100\u00a0\u00b5l of the sub-cultured cells was pelleted and resuspended in 150\u00a0\u00b5l of 100\u00a0mM PIPES/KOH pH 9.4 containing 10\u00a0mM of DTT. The cells were incubated for 10\u00a0min at 30\u00a0\u00b0C, pelleted and resuspended in 250\u00a0\u00b5l of YPD containing 0.6\u00a0M sorbitol, 25\u00a0mM Tris\u2013HCl pH 7.5 and 50\u00a0\u00b5l of lyticase (5\u00a0mg/ml). The cells were incubated for 30\u00a0min at 30\u00a0\u00b0C, then washed twice with 150\u00a0\u00b5l of YPD containing 0.6\u00a0M sorbitol and 25\u00a0mM of Tris\u2013HCl pH 7.5. To the cell pellets, 250\u00a0\u00b5l of MB , and 1% dithiothreitol) and 125\u00a0\u00b5l of ethanol 100% was added, then stored at \u2212\u200920\u00a0\u00b0C for 5\u00a0min, centrifuged for 15\u00a0min at max speed and the recovered pellet containing the DNA and bound proteins was washed twice with 200\u00a0\u00b5l of EtOH 75%, each time with centrifugation at max speed for 10\u00a0min. The protein-bound DNA pellet was resuspended in 200\u00a0\u00b5l 8\u00a0mM of NaOH and an aliquot of 100\u00a0\u00b5l was diluted in 200\u00a0\u00b5l of TBS (50\u00a0mM Tris\u2013HCl pH 7.5 and 150\u00a0mM NaCl). A sample of 10\u00a0\u00b5l was used for DNA quantification using Syber Green I Dye and different amounts of the DNA was loaded onto a slot blot equipped with nitrocellulose membrane. Empty wells were loaded with TBS containing bromophenol blue and a gentle vacuum was applied to the slot blot apparatus and once the wells were empty, each well was washed once with 200\u00a0\u00b5l of TBS and the recovered membrane was cut and each piece was processed by Western blot analysis with the appropriate antibodies29.This assayThe strains were grown in YPD broth media at 30\u00a0\u00b0C for 24\u00a0h then the OD 600\u00a0nm was adjusted to 1.0. The adjusted OD was serially diluted to 1:10, 1:50, 1:100, 1:500, and 1:1000 in a 96-well plate. Four microliters of each dilution were inoculated on YPD agar without and with 2\u00a0ng/ml and 2.5\u00a0ng/ml of rapamycin. The plates were incubated at 30\u00a0\u00b0C for 48\u00a0h and  was used to document the results.Cell extracts from H2B WT and H2B R95A were extracted in yeast suspension buffer (YSB) supplemented with protease inhibitors. Protein extracts were incubated with anti-GFP antibodies (Roche Antibodies) at 4\u00a0\u00b0C overnight and then the immune complex was conjugated to pierce protein A magnetic beads (ThermoFisher Scientific) for 2\u00a0h at room temperature. After IP, the beads were washed 4 times thoroughly with TRIS buffered saline containing 0.01% Tween 20 and protease inhibitors. Immunoprecipitated proteins were eluted using 2\u2009\u00d7 SDS loading buffer and then boiled at 95\u00a0\u00b0C for 5\u00a0min. Denatured proteins were subsequently separated on SDS PAGE gels and immunoblotted against anti-FLAG antibodies (Sigma-Aldrich). For SPT16-GFP tagged strains, cell extracts were incubated with Chromotek-GFP trap magnetic agarose beads (Proteintech) for 2\u00a0h at 4\u00a0\u00b0C. after IP, the beads were washed three times with the wash buffer  supplemented with protease inhibitors. Proteins were eluted in 2\u2009\u00d7 SDS loading buffer and then boiled at 95\u00a0\u00b0C for 5\u00a0min. Samples were loaded on SDS PAGE gels and blotted against anti-FLAG monoclonal antibody , MA1-91878, Invitrogen, USA).g for 3\u00a0min. Pellets were washed once with sterile water and then resuspended in 20% trichloroacetic acid (TCA). Pellets were transferred to 1.5\u00a0ml tubes containing equal volumes of yeast cells extraction glass beads . Cells were lysed using a bead mill homogenizer  at 5\u00a0m/s for 5\u00a0s and repeated 10 times. Supernatants containing protein extracts were collected in fresh tubes. Beads were topped with 150\u00a0\u00b5l of 5% TCA and subjected to the bead mill homogenizer at 5\u00a0m/s for 5\u00a0s, 10 times. Supernatants were collected and combined with the first extracts then centrifuged for 10\u00a0min at 10,000\u00a0rpm at 4\u00a0\u00b0C. The supernatants were discarded, and existing pellets were washed once in 70% ethanol to remove residual TCA. The pellets were resuspended in 100\u00a0\u00b5l of 1\u2009\u00d7 SDS loading buffer. PH was adjusted by adding 5\u00a0\u00b5l of 1\u00a0M Tris base solution. Proteins were then separated on SDS PAGE gels then transferred onto 0.22\u00a0\u00b5m nitrocellulose membranes. Membranes were immunoblotted against primary antibodies overnight at 4\u00a0\u00b0C. Antibodies used are anti-GFP IgG (Roche Antibodies), anti-Phospho-p44/42 MAPK (Erk1/2) Thr202/Tyr204  generously provided by Dr. Essam Abdelalim . Membranes were visualized using Pierce\u2122 ECL Western Blotting Substrate (ThermoFisher Scientific).Exponentially growing yeast strains were pelleted at 3220\u00d7Yeast overnight cultures were collected the following day by centrifugation at 4000\u00a0rpm for 5\u00a0min. The pellets were washed once with sterile water, before proceeding with the RNA extraction protocol using RiboPure\u2122 yeast RNA purification kit (Qiagen) as per the supplier protocol. RNA concentration and purity were measured by Nanodrop 2000. cDNA was prepared from the total RNA (0.5\u00a0\u00b5g) using high-capacity cDNA reverse transcription kit (ThermoFisher Scientific) according to the recommended protocol. Real-time PCR (qPCR) was performed using SYBR Green Master Mix (ThermoFisher Scientific). Samples were run on Quanti Studio 6 Flex qPCR machine. ACT1 was used as a loading control. Primers used in this study were as follows: ACT1-F, 5\u02b9-TGGGTATCCAAGCACATCAA; ACT1-R, 5\u02b9-TGATAAACCCGCTGAA CACA; STE5-F, 5\u02b9-CGTCCGGAGCAAACTCTATC; and STE5-R, 5\u02b9-ATGACCTTAACAGC GGCAAC.The quantification analysis was conducted using ImageJ software.23, we examined whether this could be reflected by the proteins differentially associated with H2B R95A as compared to the H2B WT. In the histone mutant collection used in our recent study, the H2B WT and its variants all carried a FLAG epitope tag useful for pulldown assays23. Total protein extracts were prepared from exponentially growing cells and the H2B WT-FLAG and H2B R95A-FLAG were pulled down from equal amounts of total protein extracts using protein A beads carrying anti-FLAG antibodies followed by immunoblotting probed with anti-FLAG. Both the H2B WT and H2B R95A were equally pulled down with the anti-FLAG beads  was sharply reduced from the H2B R95A-FLAG strain, as compared to the H2B WT-FLAG , and spt16-D776G, thus their responses towards rapamycin cannot be properly assessed as compared to the isogenic parental strain WT-1(SPT16), derived from the S288C genetic background   in the parent strain WT-1  and no other detectable specie, as compared to the parent strain WT-1  as compared to its parent (~\u2009170\u00a0kDa) strain WT-2  or treated with rapamycin and cultures harvested at the indicated time (0.5\u20134\u00a0h) and processed for immunoblot analysis probed with a peroxidase-antiperoxidase complex , which detects protein A of the TAP tag. As shown in Fig.\u00a0spt16-11 mutant already maintained a very high level of Cln2-TAP in the absence of rapamycin treatment  complex that plays multiple roles including transcription, replication, and chromatin remodelling50. In the latter case, mutating the glutamic acid residue H2B E57 to alanine diminished its interaction with Spt1650. While the H2B E57A mutant shared phenotypes in common with spt16 mutants, such as sensitivity to hydroxyurea and a propensity to engage in cryptic transcription, this mutant exhibited parental response towards rapamycin50. However, we have not yet examined if this particular mutant is expressing an altered molecular weight form of Ste5-GFP. Thus, our recent report demonstrating that the H2B R95A mutant is unable to express several genes, including genes of the pheromone response pathway, might be explained by its inability to properly recruit Spt16 which ultimately affects the FACT complex to promote gene expression23. In our experiments, we did not examine whether the Pob3 component of the FACT complex was also weakly pulled down by H2B R95A-FLAG. Since both Spt16 and Pob3 are in a complex that interacts with the H2A-H2B dimer, we anticipate that Pob3 would also be weakly pulled down by H2B R95A-FLAG. However, we cannot exclude the possibility that the native H2B R95 may only recruit Spt16, and not Pob3, as highlighted by recent studies showing that Spt16 can act independently of the FACT complex to modulate transcription52. It would be interesting to explore in the future whether yeast mutants, such as san1\u0394, with the ability to maintain higher levels of Spt16 while keeping the Pob3 level constant can activate STE5 gene expression51.Herein, we provide evidence that the substitution of Arg95 with alanine in histone H2B weakened the interaction with Spt16, and thus this would likely impede FACT function. Other H2B mutants have been previously reported that interrupted FACT binding to the H2A-H2B dimer and these include amino acid substitution at the essential histone residues H2B L109 and H2B M62 and the non-essential residue H2B E57STE5, and both H2B R95A and the ste5 mutants displayed nearly similar hyperresistance phenotype to rapamycin prompted us to check whether spt16 mutants would engender similar resistance to rapamycin and thus interfere with the expression of STE523. From a small collection of spt16 mutants, we found two mutants, spt16 E857K and spt16-11, that showed sensitivity to rapamycin, but none of the spt16 mutants alone displayed resistance to the drug. Indeed these two spt16 mutants were unable to express the native form of Ste5-GFP, in particular, the spt16 E857K expressed a lower molecular weight specie and the spt16-11 expressed a higher molecular weight specie. Although the lower molecular weight Ste5-GFP polypeptide ~\u2009140\u00a0kDa found in the spt16 E857K mutant can be explained by proteolytic cleavage of the native Ste5-GFP, the most likely explanation for this polypeptide specie is that transcription starts from a cryptic promoter within the ORF of the STE5 gene. The spt16 E857K substitution mutation was identified through its inability to prevent transcription from a cryptic internal promoter located within the open reading frame of the FLO8 gene, which is normally repressed by the native nucleosomal structure in the parent SPT16 strain47. If indeed the Ste5-GFP polypeptide is the result of an internal transcription start site, this shorter protein may be responsible for sensitizing the spt16 E857K mutant to rapamycin. The observation that the spt16 E857K mutant showed induced activation of the Fus3 MAP kinase as compared to the parent is consistent with a defect in the scaffold function of the shorter Ste5-GFP protein in controlling the activation of Fus3 2 dimer: tetramer interface to promote transcription34. Herein, we observed that the spt16-11 mutant expressed nearly the same amount of Ste5-GFP as the WT, but the expressed protein displayed a higher molecular weight (Fig.\u00a01 cyclins23. Unexpectedly, the spt16-11 mutant maintained substantially higher levels of the Cln2 protein, nearly 15-fold higher than the parent strain (Fig.\u00a01 cyclins. Based on our findings and the known functions of FACT, it would appear that the spt16-11 mutation is unable to reestablish the repressive chromatin thereby allowing continuous expression of Cln2.The -H2B:H3\u2013H2 dimer: spt16-11 mutant23. The newly synthesized Cln2 is required to activate Cdc28 to promote G1 to S phase transition which would commit some cells to proliferate in the presence of rapamycin before growth ceases. However, the spt16-11 mutation failed to induce Cln2 in response to rapamycin and the high levels of preexisting Cln2 in this mutant began to drastically disappear (Fig.\u00a0spt16-11 mutant failed to induce Cln2, although this observation is consistent with the mutant showing sensitivity to rapamycin; mutants that are resistant to rapamycin such as ste5\u0394 show rapid induction of Cln223. However, we offer two possible explanations for the failed Cln2 induction by the spt16-11 mutant. One possibility is that the modified Ste5 in the spt16-11 mutant may no longer allow docking of Cln2 onto its Leucine-Proline rich sequence blocking Cln2 from maintaining the transcriptional repressor Whi5 in the hyperphosphorylated form\u00a0and setting Whi5 free from inhibiting the SBF transcription factor that activates, for example, CLN2 gene expression54. Alternatively, the failed Cln2 induction may be a consequence of the spt16-11 mutation causing a defective FACT complex that is unable to recruit the transcription factors MBF and SBF to the promoter elements on the CLN2 gene55.We have recently shown that rapamycin treatment induced the synthesis of Cln2 within 30\u00a0min, and this observation is reproduced in the present work with a different genetic background strain, W303, and the parent of the spt16-11 mutant would restore to this strain parental resistance to rapamycin since this H2B mutation has been shown to correct the hydroxyurea and temperature sensitivities of the spt16-11 mutant34. Surprisingly, the H2B A84D suppressor mutation completely blocked the expression of Ste5-GFP and conferred upon the spt16-11 mutant striking resistance to rapamycin (Fig.\u00a0spt16-11; H2B A84D double mutant could be due to the proximity of the negatively charged Ala84Asp substitution relative to the positively charged Arg95 residue, and this would likely interfere with the recruitment of an already defective spt16-11 preventing the expression of the Ste5-GFP protein. It is noteworthy that the H2B A84D mutation alone did not affect rapamycin response or hydroxyurea sensitivity34. However, we cannot exclude other possibilities that the spt16-11; H2B A84D double mutant may use a downstream cryptic promoter that produces unstable mRNA or the altered nucleosome may facilitate the expression of noncoding miRNAs that block the expression of Ste5-GFP56. In support of this latter possibility, the spt16-11 mutant shared similar phenotypes with another spt16 mutant, spt16-G132D, that exhibits increased cryptic gene transcription and cryptic noncoding transcription associated with nucleosomal changes56.We also chose to examine whether the H2B A84D suppressor mutation of the 23. The H2B Arg95 residue appears to play a vital role in recruiting Spt16, a component of the FACT complex, required to promote the disassembly and reassembly of the nucleosomes to allow efficient gene expression. So far, we and others have not been able to identify any modification on H2B Arg9523. As such, it remains unclear how the H2B Arg95 residue performs its role to recruit Spt16 to promote the expression of specific subsets of genes such as genes of the pheromone response pathway and not genes belonging to another physiological pathway.In short, we have provided evidence to explain the defective gene expression caused by the H2B R95A mutantSupplementary Information."}
+{"text": "Prader-Willi syndrome (PWS) is associated with hypothalamic dysfunction. It has been reported that the HPA axis might show a delayed response during acute stress, and it is unknown whether the response of the HPA-axis during acute stress changes with age in children with PWS.To investigate the HPA-axis response during an overnight single-dose metyrapone (MTP) test in children with PWS and to assess if the response changes with age, whether it is delayed and if it changes with repeated testing over time. In addition, we evaluated different cut-off points of ACTH and 11-DOC levels to assess stress-related central adrenal insufficiency (CAI).An overnight single-dose MTP test was performed in 93 children with PWS. Over time, 30 children had a second test and 11 children a third one. Children were divided into age groups .Most children did not have their lowest cortisol level at 7.30h, but at 04.00h. Their ACTH and 11-DOC peaks appeared several hours later, suggesting a delayed response. When evaluated according to a subnormal ACTH peak (13-33 pmol/L) more children had an subnormal response compared to evaluation based on a subnormal 11-doc peak (< 200 nmol/L). The percentage of children with a subnormal ACTH response ranged from 22.2 to 70.0% between the age groups, while the percentage of a subnormal 11-DOC response ranged from 7.7 to 20.6%. When using the ACTH peak for diagnosing acute-stress-related CAI, differences between age groups and with repeated testing over time were found, whereas there was no age difference when using the 11-DOC peak.Early morning ACTH or 11-DOC levels are not appropriate to determine acute stress-related CAI in children with PWS, thus multiple measurements throughout the night are needed for an accurate interpretation. Our data suggest a delayed response of the HPA-axis during acute stress. Using the 11-DOC peak for the test interpretation is less age-dependent than the ACTH peak. Repeated testing of the HPA-axis over time is not required, unless clinically indicated. ClinicaThe mortality rate in subjects with PWS is high (3% per year until the age of 30 years) . A largeIn literature, there is no agreement on the prevalence of CAI during acute stress in PWS . SeveralThe clinical consequences of under- or over diagnosing acute stress-related CAI are significant. Underdiagnosing can lead to potentially life-threatening situations such as an adrenal crisis during acute stress, and over diagnosing can lead to overuse of hydrocortisone, which might lead to unnecessary side effects. It is, therefore, important to expand the knowledge about acute stress-related adrenal insufficiency in PWS and to create recommendations on the necessity, frequency and repeating the diagnostic procedures for CAI in PWS . It has We analyzed the ACTH, 11-deoxycortisol (11-DOC) and cortisol levels at different time-points during an overnight single-dose MTP test in 93 Dutch children with PWS and investigated whether the response to the single-dose MTP test changes with age, using two different cut-off points of acute stress-related CAI based on either the ACTH or 11-DOC peak. In addition, we investigated whether the response of ACTH and 11-DOC after a single-dose of MTP is normal or delayed in (young) children with PWS. With this study, we aimed to increase our insights in the response of the HPA axis during acute stress in children with PWS and whether this would have consequences for clinical practice. We hypothesized that the hormonal responses after a single-dose of MTP are different between children and adults with PWS because these changes are associated with age. In addition, we hypothesized that only few children with PWS have CAI requiring daily hydrocortisone treatment or an increased dose during mild to moderate stress, but that the response of the HPA-axis during severe acute stress as tested during the single-dose MTP test, would be delayed in children with PWS.22.1All participants were diagnosed with PWS, confirmed by methylation analysis of the PWS region, and participated in the Dutch PWS Cohort study , 27.2.2In this prospective study, all children underwent at least one single-dose MTP test during an overnight stay at the Pediatric Intensive Care Unit of the Erasmus Medical Center/Sophia Children\u2019s Hospital . MTP tests were performed between March 2006 and June 2022. When a child was ill at the time of the MTP test, the test was not performed. All children visited the Dutch PWS Reference Center in Rotterdam and received multidisciplinary care by the PWS team in collaboration with pediatric endocrinologists and pediatricians in other Dutch hospitals. The Dutch PWS Cohort study was approved by Medical Ethics Committee of the Erasmus University Medical Center. Written informed consent was obtained from parents and children older than 12 years. Assent was obtained from children younger than 12 years. The study was conducted according to the guidelines of the Declaration of Helsinki II .2.3Metyrapone was administered at 23.30h, in a dose of 30mg/kg (maximum dose of 3\u00a0g) at the Pediatric Intensive Care Unit of the Erasmus Medical Center/Sophia Children\u2019s Hospital . The GH dose was given around 19.00-20.00h in the evening prior to the start of the MTP test, thus approximately 3-4 hours before the first blood draw. Metyrapone blocks the synthesis of cortisol by inhibiting 11-\u03b2-hydroxylase type 1, which converts 11-deoxycortisol to cortisol. The decline in plasma cortisol stimulates ACTH production, which causes 11-deoxycortisol to accumulate before the enzyme blockade. In four healthy adults, the maximal decrease in cortisol levels was found at 2 hours after oral administration of MTP and maintained for 7 hours post-dose (40mg/kg) . After o2.4Tubes for adrenocorticotropic hormone (ACTH) determination were immediately stored on ice after the blood draw. Blood samples were measured in the Biochemical and Endocrine laboratories of the Erasmus University Medical Center, Rotterdam. ACTH and cortisol levels were analyzed with Siemens Immulite 2000XPi and 11-deoxycortisol (11-DOC) with UPLC-MSMS .2.5Statistical analyses were performed using IBM SPSS Statistics 25. Categorical variables were reported using frequencies (N (%)) and continuous variables were expressed as median \u00b1 interquartile range (IQR), as not all variables had a normal distribution. Correlation between age and the results of the MTP test were analyzed with Spearman\u2019s Rho. Differences between the different age-categories were analyzed by Kruskall-Wallis tests. Categorical data were compared by Chi-Square tests. The longitudinal changes in patients who were tested multiple times were analyzed with Wilxocon signed rank test, McNemar test, Friedman test or the Cochran\u2019s Q test. Level of significance was set at a p-value of 0.05.3We performed 166 tests, of which 13 (7.8%) had incomplete lab results. Of the 153 complete tests, 17 (10.9%) were inconclusive, due to inadequate suppression of the cortisol levels (<200 nmol/L) at 04.00h, 06.00h or 07.30h. In total, 136 tests were included in this study. In 93 children, we performed one test, in 30 children we also performed a second test, in 11, a third one and in 2, a fourth one.3.13.2r=-0.246, p=0.004), but there was no correlation between age and 11-DOC levels at any time point. An inverse correlation was also found between age and cortisol levels at time points 04.00h, 06.00h and 07.30h . Based on the correlation between age and the results of the MTP test, we decided to present our results per age group.An inverse correlation between age and serum levels of ACTH was found at time point 06.00h 3.3p = 0.641).3.4At the first test, 18 children were still prior to the start of GH treatment, but all children started GH before the second test. A 11-DOC peak < 200 nmol/L was found in one of these eighteen patients at the first MTP test and in three patients at the second MTP test (p=0.625). An ACTH peak of < 13 nmol/L was found in none of the patients at the first test and in one patient at the second MTP test (p=0.999).4This is the largest study to date investigating acute stress-related CAI in young children with PWS with the overnight single-dose MTP test. Most children had their lowest cortisol levels at 04.00h, but their ACTH and 11-DOC peak levels appeared several hours later at 06.00h or 07.30h, which might indicate a delayed response of the hypothalamic\u2013pituitary\u2013adrenal axis to acute stress in children with PWS. As most (young) children with PWS did not have their ACTH and 11-DOC peak levels and lowest cortisol levels at 07.30h, multiple measurements during the overnight, single-dose MTP test appear to be needed in children with PWS for an adequate interpretation of the HPA-axis response to a single-dose of MTP. The prevalence of a subnormal ACTH peak (13 - 33 pmol/L) during the overnight single-dose MTP was different between the age groups (ranged from 22.2 to 70.0%), but the prevalence of a subnormal 11-DOC peak (< 200 nmol/L) did not significantly change with age (ranged from 7.7 to 20.6%). In addition, the prevalence of a subnormal stress response based on the 11-DOC peak did not change with repeated MTP testing over time. We, therefore, conclude that the 11-DOC peak provides more consistent information than the ACTH peak to interpret the single-dose MTP test. We also showed that repeated MTP testing over time provides similar results.Our study gives insight in the prevalence of acute stress-related CAI in children with PWS. Steiner et\u00a0al. showed that an ACTH plasma level < 33 pmol/L as well as 11-DOC levels < 200 pmol/L are cut-off points with a high sensitivity of diagnosing CAI . HoweverIn other studies, serum levels of ACTH, 11-DOC and cortisol were only measured at 7.30h in the morning \u201333 and tAlmost all children had their lowest cortisol level measured at 04.00h, but almost none of them had their ACTH or 11-DOC peak at that same time. Metyrapone is quickly absorbed after oral administration , and maxvia the tube, these very young children had a guaranteed intake of MTP. We recommend to perform multiple measurements during the night, when the overnight single-dose MTP test is performed to study acute stress-related CAI in young children, as the HPA axis might be restored in the early morning in young children with PWS.Notably, although all children included in our study had at least one cortisol measurement < 200 nmol/L, the early morning cortisol levels were higher, especially in the very young children. According to the interpretation of the MTP test in adults, results are inconclusive when cortisol levels are not suppressed at 07.30h. We measured the hormone levels multiple times during the night, to provide more insight in the suppression of cortisol levels and the peak ACTH and 11-DOC levels. We found that cortisol levels were lowest at 04.00h in almost all children. Interestingly, although all children had a sufficient suppression of cortisol after the single-dose of metyrapone, cortisol levels increased again after 04.00h in the very young children, which resulted in cortisol levels > 200 nmol/L at 07.30h. It might be that the very young children have a faster metabolism of metyrapone and therefore a faster elimination of metyrapone and metyrapol, and that the HPA axis is already restored in the early morning. Another explanation might be that the dose of 30mg/kg is too low for very young children to maintain a sufficiently long blockade of 11-\u03b2-hydroxylase type 1. Current dosing is calculated as mg/kg, which results in a relatively low dose in very young children , compared to older children. For example, a young child of 5\u00a0kg with a body surface area (BSA) of 0.3 m2 receives 150 mg metyrapone, which corresponds to 500 mg/m2, while an older child of 50\u00a0kg with a BSA of 1.6 m2 receives 1500 mg metyrapone, corresponding with a much higher dose of 938 mg/m2. Dosing metyrapone in mg/m2 might solve this. However, this needs to be investigated further and the dose should not increase the maximum of 3000mg. As most infants with PWS were tube fed and received the MTP This is the first study describing the results of repeated overnight single-dose MTP tests over time in children with PWS. As a negative correlation between age and peak cortisol levels to the low dose ACTH test was previously described , 21, theThere are, to our knowledge, no overnight, singe-dose MTP test results reported in healthy young children and thus no reference values of peak ACTH and 11-DOC levels for the pediatric population. Although our results suggest a delayed response of the HPA-axis during acute stress, in line with Oto et\u00a0al. , we coulIn our previous study, the prevalence of acute stress-related CAI was 60% . This prUntreated growth hormone deficiency may mask symptoms of CAI, as low serum insulin-like growth factor-1 levels lead to higher levels of the enzyme that converts cortisone to cortisol . In 18 pIn conclusion, sing 11-DOC for the interpretation of the MTP test seems more accurate and less age-dependent than ACTH peak levels. The percentage of a subnormal ACTH response ranged from 22.2 to 70.0% between the age groups and the percentage of a subnormal 11-DOC response from 7.7 to 20.6%. Multiple laboratory measurements during the night after MTP administration are needed, as most younger children had their lowest cortisol level at 04.00h and their ACTH and 11-DOC peak at 06.00h. epeated testing for CAI should not be standard procedure, but should only be performed when there is a clinical suspicion of CAI. The increase in ACTH and 11-DOC during MTP test was found several hours after the lowest cortisol levels, suggesting a delayed response of ACTH during the overnight MTP test. As this delayed response of ACTH might lead to symptoms of CAI during acute stress, it seems safer to treat children with PWS with hydrocortisone during surgical procedures and severe acute illness (for example when the child is admitted to the Intensive Care Unit), while standard use of hydrocortisone during mild to moderate stressful conditions in daily life might not be indicated.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medical Ethics Review Committee of the Erasmus MC. Written informed consent to participate in this study was provided by the participants\u2019 legal guardian/next of kin.LG and AH-K were responsible for conceptualization, analysis was performed by LG, GK and AH-K were responsible for the methodology, supervision was done by GK and AH-K. Writing \u2013 original draft performed by LG, review and editing performed by LG, GK, AJ, DT-T AND AH-K. All authors contributed to the article and approved the submitted version."}
+{"text": "To date, a large number of functional magnetic resonance imaging (fMRI) studies have been conducted on psychosis. However, little is known about changes in brain functioning in psychotic patients using an emotional auditory paradigm at different stages of the disease. Such knowledge is important for advancing our understanding of the disorder and thus creating more targeted interventions. This study aimed to investigate whether individuals with first-episode psychosis (FEP) and chronic schizophrenia show abnormal brain responses to emotional auditory processing and to compare the responses between FEP and chronic schizophrenia.Patients with FEP (n = 31) or chronic schizophrenia (n = 23) and healthy controls  underwent an fMRI scan while presented with both emotional and nonemotional words.Using HC as a reference, patients with FEP showed decreased right temporal activation, while patients with chronic schizophrenia showed increased bilateral temporal activation. When comparing the patient groups, individuals with FEP showed lower frontal lobe activation.To the best of our knowledge, this is the first study with an emotional auditory paradigm used in psychotic patients at different stages of the disease. Our results suggested that the temporal lobe might be a key issue in the physiopathology of psychosis, although abnormal activation could also be derived from a connectivity problem. There is lower activation in the early stage and evolution to greater activation when patients become chronic. This study highlights the relevance of using emotional paradigms to better understand brain activation at different stages of psychosis. Psychosis is a major social burden due to the tendency to chronicity, the impact on physical health, and the reduction of life expectancy by a mean of 14.5\u00a0years . A cruciAnother way to study the brain is with functional MRI (fMRI), which could provide an important means of understanding what happens in the brain during the processing of different stimuli. In a previous systematic review about this issue, we showed great heterogeneity among different studies . Most stOur group developed an emotional auditory paradigm based on some of the most common symptoms reported by FEP patients , 16. UndPatients were recruited from the outpatient psychiatry clinics. People who experienced psychotic symptoms for the first time in life were submitted to the Valencia Clinic Hospital First-Episode Unit for evaluation and treatment. Patients diagnosed with schizophrenia and persistent hallucinations  were recruited from the Valencia Clinic Hospital and considered chronic patients. All patients and control participants gave written informed consent to participate in this research, which was approved by the local ethics committee. All subjects, patients, and controls were older than 18\u00a0years old.The inclusion criteria for the patients were as follows: (a) first-episode samples, individuals who had been diagnosed with a psychotic disorder for the first time in their lives within the first 18\u00a0months after symptom onset; (b) chronic patient samples, a diagnosis of chronic auditory hallucinations that met the selection criteria for persistent hallucinations ; (c) theThe exclusion criteria for the patients were as follows: (a) individuals who had previous contact with mental health services for psychosis, (b) evidence of psychotic symptoms precipitated by an organic cause, (c) transient psychotic symptoms resulting from acute intoxication, (d) substance abuse (except tobacco), (e) a metal prosthesis, or (f) claustrophobia. Two weeks after their diagnosis, all of the patients underwent a semistructured interview including sociological information and clinical scales. After the clinical evaluation, they were subjected to an fMRI scan, with a minimum interval of 6\u00a0months after their diagnosis.A sample of HCs matched for age, sex, education level, and work status was recruited from the same geographic areas as the patients. The inclusion criteria for the HCs were the same as those for the patients, except for past or present psychotic symptoms. The exclusion criteria were the same as those for the patients but also included (a) past or present psychotic symptoms or past criteria for psychoactive drug abuse or dependence (except for tobacco) and (b) having a first-degree relative with a history of a diagnosed psychotic disorder. No individual in either group suffered from hearing loss.The patients\u2019 sociodemographic data are shown in The final sample consisted of 85 subjects, which included 31 FEP patients with nonaffective psychotic disorders , 23 chronic patients with schizophrenia and persistent auditory hallucinations, and 31 HCs.Patients were assessed by personnel trained in FEP evaluation. Interviews were conducted during the first episode and a few days after the start of treatment (JS). All FEP patients underwent several psychopathological measurements, including the Clinical Global Impression , to provAn emotional auditory fMRI stimulation paradigm designed by our group , 16 was The mean time between clinical treatment and fMRI acquisition was 4\u00a0weeks (sd\u00a0+/\u2212\u00a06). A 3-T magnet  was used to acquire the fMRI images. A 32-channel head coil and a dynamic echo-planar imaging T2*-weighted sequence were used , covering the whole brain with 40 contiguous slices. Additionally, a 3-dimensional spoiled gradient-echo pulse T1 image was also acquired for anatomic assessment . Each fMRI session  had a total duration of 160\u00a0s. After the acquisition, a radiologist and a computer engineer checked the images to ensure data quality. During the acquisition, the patients were under direct observation and they asked about their experiences immediately after the fMRI procedure. Data were then anonymized and transferred to a workstation for postprocessing analysis.x, y, z) >1.80\u00a0mm (voxel size) were discarded, resulting in a final sample of 31 HCs, 31 FEPs, and 23 chronic patients. Afterwards, a slice timing correction to adjust for temporal delays between the first and the last slices in each fMRI dynamic was applied. Then, a normalization algorithm to standardize the brains to a referenced template  was used. Finally, the normalized images were smoothed with a 6-mm FWHM Gaussian smoothing kernel. These analyses were conducted using the Statistical Parametric Mapping SPM software  on MATLAB R2017a .Functional images were realigned to correct for involuntary movement of the patient\u2019s head during the test. Those subjects with translation movements (t-tests. To assess whether patients with FEP (or with chronic schizophrenia) showed abnormal fMRI responses, we used two-sample t-tests . Finally, to compare the fMRI responses between FEP and chronic schizophrenia, we also used two-sample t-tests. We did not use ANOVAs because we were not interested in knowing if there were differences between the three groups per se; we only used the HC data to provide a reference of normality. To correct for multiple testing, the familywise error (FWE) was applied. Only uncorrected tests for exploratory purposes were applied when no clusters survived the FWE rate correction. Additionally, an extent threshold was applied to consider only those clusters with the minimum number (k) of contiguous voxels as the expected number of voxels per cluster provided by SPM. Finally, the Automatic Area Labeling [To describe the fMRI response in each group of patients (or in HCs), we used one-sample Labeling  atlas waPatients with FEP showed activation during the emotional word task in the bilateral middle temporal (BA 21), bilateral hippocampus , right inferior orbitofrontal (BA 47), and left cerebellum .Table 3.HCs showed a similar pattern of activation, including the bilateral middle temporal (BA 21), left inferior temporal (BA 20), bilateral cerebellum, left precentral (BA 6), right supplementary motor area (BA 6), left superior frontal (BA 32), left putamen (BA 48), and right hippocampus (BA 20) see .Chronic patients mainly activated the right middle temporal (BA 21), left superior temporal (BA 21), bilateral inferior frontal , bilateral cerebellum, bilateral precentral , left insula (BA 47), right hippocampus (BA 20), and right frontal inferior gyrus (BA 38) see .p\u00a0<\u00a00.001), they showed hypoactivation in the right temporal lobe (BA 21). Similarly, the inferior frontal gyrus  showed a significant reduction in activation in FEP patients when compared with chronic patients (uncorrected p\u00a0<\u00a00.001)   .Three main findings resulted from the present study. First, this study provides an image comparison of FEP patients to HCs and found reduced activity in the bilateral temporal cortex (mainly on the right side). Functional studies about emotional processing in HCs have differentiated successive steps  of cerebral processing involving auditory analysis within the temporal lobe , 26.Second, interestingly, the CHrP group showed the opposite, greater activation in the bilateral superior temporal lobe than HCs and FEP patients during the processing of emotional words. These results suggested that the temporal lobe plays a critical but complex role in the pathogenesis of psychosis. The auditory cortex has been reported to play a pivotal role in the processing of affective prosody , 27.We observed a decrease in activation in the right middle temporal lobe in the FEP group. This finding is in accordance with the assumption that this area subserves the representation of meaningful prosodic sequences in the processing of emotional words and suggests that abnormal processing of emotional words in FEP patients was conducted in the limbic system rather than in the auditory cortex .Interestingly, our previous systematic review of longitudinal fMRI studies in FEP  showed aIn this sense, our results confirm that middle temporal functional alterations in FEP patients represent one key neural factor in the pathophysiology of psychosis. Considering that when HCs\u2019 enhanced brain activity is involved, it is predominantly the temporal cortex, and passive listening to speech with prosody-caused activation relies on the right temporal cortex , 30.Activation in the temporal lobes is shown , correspThe fact that the temporal lobe and limbic regions were the most clearly affected areas in our previous studies using the same paradigm in chronic patients  shows hoThe most likely explanation for understanding the differences between our results and previous studies is that the alterations in the emotion fMRI auditory paradigm in psychotic patients are different in specific stages of the disorder and might be more noticeable during certain stages . Thus, iAlthough neuroimaging studies have revealed alterations in emotional processing  and sociThird, FEP patients showed lower activation in the bilateral inferior frontal area than chronic patients during the processing of emotional words. Although differences in brain activation patterns between FEP and chronic psychotic stages have been widely assessed, the results have been controversial. In a systematic review, Mwansisya et al.  found a p\u00a0=\u00a00.04), which could also affect the fMRI results.This study has some limitations. Although most fMRI studies involve <30 patients, it is a limitation of the field, and this study must be interpreted wisely due to the relatively small sample size recruited. Differences between sample sizes in the HC and FEP groups and gender distribution should also be considered. Although the results in each clinical group were reported using the more restrictive statistical correction for multiple comparisons (FWE), the between-group test did not show any surviving cluster, and the results were presented in an exploratory way, uncorrected. Most likely, the relative number of samples in each group may cause that situation. Additional studies with more samples are needed to confirm these findings. The second limitation was that all FEP patients were on medication, mostly first-generation antipsychotics, at the time of the study, and there was no standardization of medications or doses, which could clearly affect the results. Thus, it cannot be omitted that medication dose had an impact on brain activity; however, dysfunctions in emotional processing can be unaffected by antipsychotics in FEP patients , 37, andConversely, this study has some strengths that should be noted. The main strength is that this is the first cross-sectional study investigating fMRI changes in FEP and CHrP samples with an emotional auditory paradigm. Paradigm selection is a key question in neuroimaging, and there is a lack of studies using noncognitive tasks during scans. This study provides relevant information to understand the dysfunction of the emotional response in FEP patients compared with CHrP. The study used a rigorous approach to analyze brain activity during the emotional fMRI auditory paradigm.To the best of our knowledge, this is the first study to identify fMRI activity involved in emotional auditory processing in FEP and CHrP patients. Our results suggested that the temporal lobe might be a key issue in the physiopathology of psychosis, although abnormal activation could also be derived from a connectivity problem. This role of the temporal lobe changes depending on the stage of the illness. Lower activation in the early stage might progress to greater activation when patients become chronic. This study highlights the relevance of using not only cognitive tasks but also emotional paradigms to better understand brain activation at different stages of psychosis and the potential of this method as a biomarker for the prediction of FEP outcomes."}
+{"text": "It is undeniable that our brain remembers and responds to a good story better than an isolated collection of facts . Hence, i.e., generate your narrative.Good non-academic examples for this can be found in sports journalism about professional baseball. In one story, the author describes how a new technical rule considering the dimensions of baseball infields does not have the same impact throughout the league . In anotWater Research X was launched as an independent journal, separate from Water Research, in January 2023, and in their editorial, Editor-in-chief Eberhard Morgenroth of Water Research and Editor-in-chief Zhiguo Yuan of Water Research X describe the new journal (Water Research X shall write Leading-Edge research papers with a compelling narrative. journal . As a keThere are various high-level recipes and discussions available for writing narrative style papers that authors can refer to, such as Water Research X is also aimed at supporting the narrative writing style (The structure of Leading-Edge manuscripts in ng style . Directli.e., readability of letter-style papers and completeness of traditional full-length research papers such as published in Water Research.Leading-edge manuscripts are limited to 3000 words excluding Materials and Methods, References, figures and tables and their captions, and may contain up to 6 figures and tables. If you compare this to other journals, you will notice that the paper structure is intended to be comprehensive yet concise trying to pick the best of both worlds, Water Research X.We hope that by now, we have convinced you that adopting narrative style writing will enhance your research impact. As editors we think that by providing you with the above-described manuscript structure we gave you a good tool in hand. So now over to you as here we remain, eager to read and hopefully publish your research stories in The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "Visual inspection with acetic acid (VIA) is one of the methods recommended by the World Health Organization for cervical cancer screening. VIA is simple and low-cost; it, however, presents high subjectivity. We conducted a systematic literature search in PubMed, Google Scholar and Scopus to identify automated algorithms for classifying images taken during VIA as negative  or precancerous/cancerous. Of the 2608 studies identified, 11 met the inclusion criteria. The algorithm with the highest accuracy in each study was selected, and some of its key features were analyzed. Data analysis and comparison between the algorithms were conducted, in terms of sensitivity and specificity, ranging from 0.22 to 0.93 and 0.67 to 0.95, respectively. The quality and risk of each study were assessed following the QUADAS-2 guidelines. Artificial intelligence-based cervical cancer screening algorithms have the potential to become a key tool for supporting cervical cancer screening, especially in settings where there is a lack of healthcare infrastructure and trained personnel. The presented studies, however, assess their algorithms using small datasets of highly selected images, not reflecting whole screened populations. Large-scale testing in real conditions is required to assess the feasibility of integrating those algorithms in clinical settings. In 2020, cervical cancer was the fourth leading cause of death in women due to cancer and the main one in 36 countries, mostly low- and medium-income countries (LMICs) . The gloIn high-income countries (HICs), VIA is performed with the assistance of a colposcope, i.e., a low-power microscope, to magnify the view of the cervix. However, those devices are rarely available for screening in LMICs due to limited financial resources, poor infrastructure and a lack of healthcare professionals. In such conditions, the assessment is usually performed with the naked eye. Visual inspection\u2014with the naked eye or with a colposcope\u2014is highly subjective as it entirely relies on the healthcare providers\u2019 training and experience. Several studies suggest a high intra- and inter-observer variability with sensitivities ranging from 25.0% to 94.4% for VIA ,4,5 and Recent advances in artificial intelligence (AI) show great potential for a more objective automated detection of cervical precancer and cancer. Indeed, AI-based methods using smartphones and colposcopes images have been introduced, and yielded very promising results ,16,17,18The protocol has been registered in the international prospective register of systematic reviews (PROSPERO CRD42021270745) and reported in compliance with the recommendation of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) .A systematic literature search for publications between January 2015 and July 2022 was performed in PubMed, Google Scholar and Scopus. The search strategy included three different concepts combined using logical operators: (i) the disease , (ii) the use of artificial intelligence-based algorithm  and (iii) the acquisition technique . The exact searches and results are reported in the The studies should assess an automated artificial intelligence-based algorithm aimed at distinguishing CIN2+ (positive) from normal and benign conditions (negative), relying only on images taken during VIA. Were excluded: studies considering CIN1 as positive or not containing healthy cases.Were excluded: studies not mentioning their gold standards or with unclear gold standards.For precancerous and cancerous lesions, the gold standard used to study the accuracy of the algorithms should be histopathology results, while histopathology or normal cytology and normal colposcopy were considered sufficient for negative cases. This criterion is motivated by the fact that even though histopathology results remain the most reliable method for diagnosis, some screening procedures only conduct biopsies when there is a suspicion of precancerous lesion after cytology and colposcopy. The studies should be written in English and published between January 2015 and July 2022.Were excluded: review articles, conference abstracts articles and pre-prints.The studies should be original research articles and peer-reviewed. In order to select studies that could be further compared, the inclusion criteria were the following:The literature search was performed by one reviewer (RV) in PubMed and Google Scholar and by another reviewer (MC) in Scopus. The software \u2018Publish or Perish\u2019  was usedFor all included studies, the following characteristics were reported: publication details, algorithms, acquisition device, gold standard, number of patients included, dataset size and partition  and performance of the algorithms.The quality of the included studies was assessed with the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool . Both riThe studies are highly heterogenic in terms of data acquisition method, technical approach and performance reporting. The main challenge for data analysis is raised by performance reporting: while using cross validation hinders direct comparison, most of the studies report an average accuracy, sensitivity and specificity without confidence intervals. Due to this performance reporting discrepancy, no meta-analysis could be performed.In cases where performance metrics were not provided, but confusion matrices were, sensitivity and specificity were computed from those. When only the receiver operating characteristic (ROC) curve was provided, the following equation was used to compute the accuracy in each sensitivity and specificity measurement pair:Most of the included studies describe and compare several algorithms based on the same dataset. We thus selected the algorithm with the highest accuracy of each study for the data analysis. Sensitivity and specificity of the selected algorithms (one per included study) were reported in scattered charts. Several scattered plots were generated according to the following features: year of publication, acquisition device, dataset characteristics, algorithm family and cross validation type. Those charts directly convey visual information about the size of the dataset used  and the feature of interest . The plots were then analyzed qualitatively.In addition to classification techniques, the preprocessing steps of each study are briefly described and compared qualitatively.We identified 482, 533 and 1979 studies in PubMed, Scopus and Google Scholar, respectively. After removal of the duplicates, 2608 publications were screened by title and abstract, which resulted in 111 studies. Full-text review of those publications led to the inclusion of 11 studies. The other 100 were mainly excluded due to mismatch between the inclusion criteria and the gold standard (n = 37), the task (n = 33), the input data (n = 27) or the absence of peer-review process (n = 3). Indeed, in some studies, the gold standard was unclear or not mentioned, not fulfilling the inclusion criteria. In others, the classification task was not the detection of cervical cancer lesions but classification of transformation zones or automatization of preprocessing steps. The classification categories could also be different , or the methodology was unclear. While the algorithms of some studies relied on more than only VIA images , others did not use VIA images. The process of study selection is illustrated in Through a cohort in Costa Rica led by the National Cancer Institute (NCI) of the United States, some HPV and cervical cancer-related data were collected for seven years, including pictures taken during VIA. The resulting dataset (later referred to as the \u2018Guanacaste dataset\u2019) was made publicly available . The imaCho et al. , Peng etXue et al. , Vi\u00f1als Characteristics of the images, such as brightness, sharpness and colorfulness, were only described by Zhang et al. . The focHistopathology was used as the gold standard in ,16,17,18Even though all studies used histopathology as ground truth for positive cases, and some also for negative, most of the studies do not detail the methodology for biopsy collection, i.e., number, location and interpretation of biopsies ,16,17,18The number of patients varies considerably between studies. Three of the eleven included studies were conducted by the same research group ,9,10 andIn , 9406 paAlyafeai et al.  used a sCho et al.  selectedXue et al.  used a dPeng et al.  used a bVi\u00f1als et al.  relied oZhang et al.  presenteFinally, Li et al.  used a dMost of the included studies use single images as input to their algorithms. For instance, Cho et al.  acquiredIn the included studies, two preprocessing aspects seem to be specific to the use of VIA images: the handling of reflections caused by the speculum and mostly the detection of the region of interest (ROI), i.e., the cervix. Li et al.  handled Most of the detailed studies detect the region of interest as a preprocessing step of their algorithm. The methods to achieve that purpose, however, differ from manual selection to CNN-based techniques through data-driven ones. While isolation of the cervix was performed manually in Vi\u00f1als et al.  and Li eMethods for preprocessing steps are highly heterogenous. Those features were thus not included in the performance analysis of the various algorithms.Most of the studies present several algorithms, which are summarized in All the studies reported the accuracy, sensitivity and specificity of their algorithms, except , which pIn order to allow further qualitative analysis, The first criterion under which the various methods are compared is their year of publication c. The elThe various approaches are then separated according to the device used to acquire data d. Three Regarding the input to the algorithm, almost all methods rely on single images for the classification task e. One stThe algorithms used in the studies are separated into four families f. Six stAlgorithms are then compared regarding their use of a cross validation technique g. One stFinally, for the studies that provided a comparison of the AI algorithms with respect to the medical experts, both the algorithms and the experts\u2019 performances are illustrated h. The AIThe quality of the included studies has been assessed using QUADAS-2 . We analThe risk related to flow and timing and index test is low for all the included studies. Nevertheless, in ten ,16,17,18Out of the eleven studies, only ,13 have Among the algorithms with high specificity, three were recently published in 2020 and 2021 ,15,16 Fc. NevertMost algorithms are device-specific and are usually evaluated in very controlled settings. In some of the included studies, cross validation has been used to enhance the estimation of algorithm performance and limits overfitting. This is, however, insufficient to provide reliable generalizability for which training and testing on larger datasets are required, as well as screening populations in real conditions. Screening should indeed be conducted without high selection of the images, using different devices in different acquisition settings. The diversity of data in real conditions will raise many challenges that no one has yet considered and that are key to ensuring the success of AI algorithms.Different screening approaches were used to collect the data. For instance, each screening visit from the Guanacaste dataset includes cytology, HPV testing, visual inspection with acetic acid and biopsy in some cases, resulting in a large number of patients. Hu et al.  used mosCho et al. , Xue et Two out of the three algorithms with the highest specificity use more than one image per patient: Peng et al.  relied oQuality and preprocessing of images can play a major role in the performance of classification algorithms. In the context of diagnosis based on VIA images, ROI detection and quality assessment of the images are essential. In addition to the different visual aspects of the cervix , the VIA images may vary in terms of light, focus, position, zoom, motion, reflections or even obstruction. All those elements influence the quality of the images, and no standardized methodology considering several of these aspects was found. Also, even though the methods to isolate the cervix from the rest of the image differ among studies, the importance of detecting the region of interest is indisputable.The algorithms\u2019 performances of the included studies are presented in many ways, which complicate the comparison. Most of the studies do not mention confidence intervals nor the number of false positives, false negatives, true positives and true negatives. Those metrics can be computed from the accuracy, sensitivity and specificity values. Nevertheless, the use of cross validation without reporting the confidence intervals nor the confusion matrix prevents this computation ,12,15,17Only three of the included studies\u2014Hu et al. , Vi\u00f1als Beyond the reliability and acceptability of computer-aided diagnostics tools, two technical key features are essential for their use in LMICs: the acquisition device and the size of the algorithm. For LMICs, portable devices seem to be more appropriate for acquiring and analyzing images than expensive tools such as colposcopes. As shown in The use of simpler algorithms facilitates the integration into mid-range smartphones, allowing offline use of the tool. By contrast, more sophisticated algorithms might require the use of external servers for performing the classification. Scant evidence is available on both the usability and impact of all those studies. The assessment of the clinical effectiveness of such tools for the automated detection of cervical cancer is necessary, especially in resource-limited settings with a severe shortage of experts.Our inclusion criteria aimed at selecting comparable studies, but as a result, also restricted the scope of the analysis and introduced a selection bias. Indeed, interesting studies in terms of technical aspects might have been discarded due to the nature of their input data and ground truth.We decided to focus on VIA only, while many research groups work on multimodal inputs. This choice could be disputed, but we consider the goal of such tools to be implemented in LMICs, which means to rely on the least information necessary in order to limit restrictions on the usage of the technology.Also, we decided to rely on studies using only histopathology as ground truth or histopathology for positive cases and normal colposcopy and normal cytology for negative cases. However, this is comforted by the fact that histopathology remains the most widely used clinical gold standard for cervical cancer diagnosis. However, even when using histopathology as the gold standard, most studies are not precise in their methodology for biopsies collection and interpretation. This methodology is of high importance to ensure the truthfulness of the ground truth on which the automated algorithms rely. Ideally, multiple biopsies should be performed and interpreted by pathologists specialized in gynecology. CIN lesions should be reviewed independently by more than one pathologist, and the use of a two-tiered terminology (LAST) is recommended . P16 immAs aforementioned, other limitations include the variety in performance reporting, the limited number of patients or images, the high selection of patients used to train and test the algorithms, or the lack of large-scale tests. As the details about the screening and recruitment methods above transpire, the risk of bias due to patient selection was high for ,16,17,18Automated algorithms to detect precancerous and cancerous lesions using images acquired during VIA are promising, especially for countries lacking health infrastructures and where most of the deaths caused by cervical cancer occur. These images can be taken by portable devices such as smartphones or cameras, which are more accessible in LMICs than colposcopes, with no observed decrease in the performance of the algorithms.The performance of AI algorithms using single or sequential cervical images may be as accurate as\u2014or even better than\u2014the human interpretation of the same images. Nevertheless, numerous limitations and constraints remain to be overcome. Most of the included studies have assessed their algorithms using small datasets, with highly selected images and without reflecting the incidence rate of the disease in screened populations. Furthermore, most of the studies do not report confidence intervals, hindering objective comparison and analysis.In the near future, AI algorithms will probably be an essential tool in cervical cancer screening. Nevertheless, algorithms must first be generalizable, preprocessing steps optimized, and above all, tested in real-life conditions. Algorithms should be trained on larger samples with reliable histological ground truths and various populations with different acquisition procedures, including devices and settings. Proper statistical analysis of their results, demonstrating their generalizability to other populations and settings, is required to assess their potential to become the future of cervical cancer screening."
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