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+{"text": "Using a cell line derived from the VM spontaneous murine astrocytoma, a reliable in vitro-in vivo model of human malignant glioma has been developed. In this paper we examine the effects of cytotoxic drugs with known activity against other animal brain tumour models and human disease on the in vivo VM model. The drugs BCNU, CCNU and vincristine produced significant volume reduction in tumours growing at a subcutaneous location in syngeneic animals. Procarbazine was ineffective. Similarly, BCNU, CCNU and vincristine produced small but statistically significant increases in survival of VM mice bearing the intracerebral tumour, but procarbazine was again ineffective. The modest, but significant, response of the VM model to the nitrosoureas mimics the human situation more closely than previously described animal models."}
+{"text": "In Greek mythology, Lyssa was the goddess of rage, fury, and rabies, known for driving mad the dogs of the hunter Acteon and causing them to kill their master. Aristotle (4th century BCE) said, \u201cDogs suffer from the madness. This causes them to become irritable and all animals they bite to become diseased.\u201d The disease in humans was characterized by hydrophobia, in which the sick person was simultaneously tormented with thirst and with fear of water. Hippocrates is believed to refer to rabies when he said that persons in a frenzy drink very little, are disturbed and frightened, tremble at the least noise, or are seized with convulsions.From the Greek Lyssavirus is a genus of the family Rhabdoviridae, which includes rabies virus and other related viruses that infect mammals and arthropods ."}
+{"text": "Mt1 expression by GnRH occurs directly in gonadotroph cells, can be reversed in adulthood by blockade of GnRH receptors, and requires EGR-1. We first confirmed the endogenous expression of Mt1 mRNA in the \u03b1T3-1 gonadotroph cell line. Stimulation of these cells with a GnRH agonist resulted in a rapid increase of Egr-1 mRNA expression, which peaked after 30\u201360 minutes, and a more prolonged elevation of nuclear EGR-1 immunoreactivity. Moreover, the GnRH agonist significantly decreased Mt1 mRNA. We then treated adult male rats with the GnRH antagonist cetrorelix or saline. After 4 weeks of daily injections, cetrorelix significantly reduced serum LH concentration and testis weight, with histological analysis confirming absence of spermatogenesis. Despite the successful inhibition of GnRH signalling, pituitary Mt1 expression was unchanged. Next we studied the proximal region of the rat Mt1 promoter. Consistent with previous work, over-expression of the transcription factor PITX-1 increased Mt1-luciferase reporter activity; this effect was dependent on the presence of consensus PITX-1 promoter binding regions. Over-expression of EGR-1 inhibited PITX-1-stimulated activity, even following mutation of the consensus EGR-1 binding site. Finally, we studied Egr1\u2212/\u2212 mice and observed no difference in pituitary Mt1 expression between Egr1\u2212/\u2212 and wild-type litter mates. This work demonstrates that GnRH receptor activation directly down-regulates Mt1 expression in gonadotroph cells. However, pituitary Mt1 expression in adults is unaltered by blockade of GnRH signalling or absence of EGR-1. Our data therefore suggest that melatonin receptor regulation by GnRH is not reversible in adulthood and doesn't require EGR-1.Melatonin receptor expression exhibits profound developmental changes through poorly understood mechanisms. In mammals, a current model suggests that pubertal reactivation of gonadotrophin-releasing hormone (GnRH) secretion down-regulates MT1 melatonin receptors in pituitary gonadotroph cells, via the induction of early growth response factor-1 (EGR-1). Here we have examined this model by testing the hypotheses that inhibition of  The hormone melatonin is implicated in multiple diverse aspects of physiology Pineal melatonin production is driven by the master circadian clock in the suprachiasmatic nuclei of the hypothalamus and thus exhibits a robust daily rhythm. This rhythm varies in proportion to the length of the night and so melatonin encodes both daily and seasonal time Mt1 promoter activity is stimulated by the transcription factor pituitary homeobox-1 (PITX-1) Mt1 is thought to be inhibited by factors involved in Rathke's Pouch proliferation, such as MSX-1 Msx-1 coincides with the onset of Mt1 expression in the foetal rat pituitary. Following a period of melatonin sensitivity, it is proposed that the pubertal reactivation of GnRH secretion then finally down-regulates Mt1 expression, likely via induction of early growth response factor-1  We have previously studied the regulation of MT1 melatonin receptors in the pituitary gland and suggested a mechanism controlling MT1 expression during reproductive development. In our model, hypogonadal mice, which are unable to synthesise GnRH, exhibit elevated levels of Mt1 expression than their wild type controls Mt1 are most extensively characterised. Due to the availability of suitable gonadotroph cell lines and transgenic \u2018knockout\u2019 animals, other parts of the study have used mouse tissue. Such an approach takes advantage of the benefits of each system and has been used successfully before, e.g. This model received preliminary support from the observation that adult 2 in growth medium: DMEM  supplemented with 10% fetal bovine serum (Invitrogen), antibiotic/antimycotic (Invitrogen), and sodium pyruvate . Data shown are from a representative of at least three independent experiments.Unless otherwise specified, all cells were cultured at 37\u00b0C and 5% CO10, D-ala6 ]-LH-RH ethylamide acetate salt hydrate; Sigma-Aldrich) at final concentration of 100 nM. After the required treatment time(s), cells were harvested for analysis of mRNA by TaqMan real-time PCR or protein by western blot, as described below.For studies of GnRH signalling, \u03b1T3-1 cells Mt1 promoter activity, COS-7 cells  were seeded in 96 well plates at a density of 3,500 cells per well. After 24 hours, cells were transfected using FuGene6 reagent , according to the manufacturer's protocol. Each well received DNA containing 5 ng of MT1-luciferase reporter plasmid, and appropriate expression vectors, made up to a total of 50 ng with pcDNA3. Forty-eight hours after transfection, reporter gene activity was measured using the Dual-Glo system . Each treatment was performed in quadruplicate wells per experiment. Rat Mt1-luciferase plasmids were based on the \u2212445 bp vector described previously TCATCC to TGGCGC; the proximal PITX-1 site (P2) was modified from TAATCC to TGGCGC; the EGR-1 site was modified from AGGCGCGGGAGG to AGGCTCTTTAGG.For studies of rat 2, cervical dislocation).Experiments using rats were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986, under licence from the UK Home Office (PPL 70/7059). Experiments were also approved by the University of Surrey's Animal Welfare Ethical Review Board. All experimental work with mice was conducted in accordance with the European Communities Council Directive 86/609/EEC and the French National Committee (87/848). No surgical procedures were undertaken in this study. Animal suffering was minimised by sacrificing animals according to approved procedures  or saline control , according to the manufacturer's instructions. In brief, 50 \u00b5l of sample or standard was mixed with 100 \u00b5l of enzyme conjugate and incubated at 37\u00b0C for 2 hours. Assay plate wells were rinsed before 100 \u00b5l of TMB solution was added and incubated at room temperature for 20 mins, in the dark. Finally the reaction was stopped by adding 50 \u00b5l of 2N HCl and the optical density was measured at 450 nm using a microtiter well reader. Concentration of LH was calculated from the standard curve.Sections of frozen testis (7 \u00b5m) were prepared and post-fixed with ice-cold 4% paraformaldehyde for 10 mins then processed for hematoxylin and eosin staining. Sections were examined for general morphology using light microscopy.Mt1 mRNA expression in brain/pituitary sections was performed using a well validated assay, as described previously 35S-labelled riboprobe corresponding to nucleotides 30\u2013466 of GenBank accession number U14409. Hybridisation signal was quantified against optical density standards on each autoradiography film.Analysis of Mt1, Egr-1 and Gapdh was measured using TaqMan RT-PCR Mt1 forward primer: 5\u2032-TCTGCTACGTGTTCCTGATATGGAT-3\u2032; Mt1 reverse primer: 5\u2032-TGGAGTGTTCCGGTTTGCA-3\u2032; Mt1 probe: 5\u2032(FAM)-CTGACACTCATCGCCATCATGCCC-3\u2032(TAM); Egr-1forward primer: 5\u2032-CCTTTTCTGACATCGCTCTGAA-3\u2032; Egr-1 reverse primer: 5\u2032-GGCAACCGAGTCGTTTGG-3\u2032; Egr-1 probe: 5\u2032(FAM)-CTCGTCTCCACCATCGCCTTCTCATT-3\u2032(TAM).After treatment, cells were washed twice with warmed 1\u00d7 PBS. Total RNA extraction and cDNA synthesis were performed as described previously Cytoplasmic and nuclear-enriched protein was extracted from cells using the NE-PER kit . From each sample, 30 \u00b5g of total protein was separated on a 10% polyacrylamide gel. Protein was transferred from gels to methanol-activated PVDF membranes, which were then incubated in wash buffer , blocking buffer , and wash buffer (3\u00d75 minutes at room temperature).Membranes were incubated with 1\u2236200 dilution of anti-EGR-1 antibody (Egr-1 (588); Santa Cruz Biotechnology Inc, Heidelberg, Germany) in blocking buffer for 60 minutes at room temperature. After rinsing in wash buffer , membranes were then incubated with a 1\u22365000 dilution of horseradish peroxidase-coupled anti-rabbit secondary antibody (Sigma-Aldrich) in blocking buffer for 60 minutes at room temperature. Next, membranes were rinsed in wash buffer  and protein expression detected using the enhanced chemiluminescence  system according to the manufacturer's protocol.Following detection of EGR-1 protein, membranes were briefly rinsed in ddH2O and wash buffer, before being incubated in strip buffer  and wash buffer . Non-specific binding was blocked as described above and membranes were then incubated with 1\u22362000 dilution of anti-actin antibody (Sigma-Aldrich) in blocking buffer for 60 minutes at room temperature. Washing, secondary antibody incubation and ECL detection were then performed as described above.Quantitative data are presented as mean \u00b1 SEM and were analysed by either unpaired t-test or one-way ANOVA with Bonferroni post-hoc test, as appropriate. Units of analysis were data from either one animal or one well of cells. Statistical significance was defined as p<0.05.Egr-1 mRNA expression (Mt1 mRNA expression (Mt1 mRNA expression was observed at earlier time points (data not shown).Treatment of \u03b1T3-1 cells with GnRH agonist induced a significant  induction of pression . Maximalpression . Followipression . No signDaily injection of rats with cetrorelix impaired reproductive function, as revealed by a significant  reduction of both serum LH concentration  in densitometry between the two treatment groups  modified by experimental conditions  difference in promoter activity between control and PITX-1-stimulated groups  in densitometry between the two genotypes  and functional GnRH receptors, but not the LH beta subunit Mt1 mRNA, making them an ideal model to study the interaction between GnRH and endogenous melatonin receptors. As described previously, stimulation of \u03b1T3-1 cells with a GnRH agonist rapidly induces transient expression of Egr-1 mRNA Mt1 mRNA. Allowing for a delay between Mt1 transcriptional inhibition and decrease in steady state mRNA levels, the relative time course of these events may be consistent with a functional relationship between EGR-1 and Mt1 in perinatal gonadotroph cells. The half life of Mt1 mRNA is estimated to be 2\u20133 hours in ovine pars tuberalis cells Mt1 inhibition is not inconsistent with its estimated half life. However, attempts to demonstrate a causal relationship between these events were prevented by an inability to transfect the \u03b1T3-1 cells with inhibitors of EGR-1 expression or function.Our previous studies led us to hypothesise that the perinatal decline in pituitary MT1 melatonin receptor expression is due to the pubertal reactivation of GnRH secretion from the hypothalamus. We therefore first studied Mt1 expression Mt1 by GnRH signalling in adulthood is unknown. We therefore next investigated the effect of a GnRH receptor antagonist, cetrorelix, on Mt1 expression in the adult rat pituitary. Daily intra-peritoneal injections of cetrorelix successfully shut-down the rats' reproductive system, as demonstrated by analysis of serum LH concentration and testis morphology. However, despite this physiological effect, there was surprisingly no change in pituitary Mt1 expression. This finding contrasts with the ability of cetrorelix to induce MT1 receptor expression in the GT1-7 neuronal cell line Mt1 mRNA, despite the lack of GnRH signalling.Our previous in vivo data demonstrated that adult rodents unable to synthesise GnRH throughout development exhibit elevated pituitary Mt1 mRNA caused by the treatment would have to be mirrored by an equal decrease in Mt1 expression within other cell types. Moreover, the increased Mt1 mRNA observed in hypogonadal mice was readily detectable by the same in situ hybridisation protocol Mt1 expression in the adult rat pituitary. It also remains possible that adult mice treated with cetrorelix may exhibit a similar increase in pituitary Mt1 mRNA expression as we previously observed in hypogonadal mice. However the species-specific mechanisms that could cause such a difference are unclear.A limitation of the current study is that our in situ hybridisation protocol measured gene expression in all cell types present in the tissue sections and not just gonadotroph cells. However, to explain our cetrorelix data, any elevation of gonodotroph Mt1 promoter activity in vitro. As shown previously Mt1-luciferase construct and this PITX-1-stimulated activity is strongly inhibited by co-transfection with an EGR-1 expression vector. The ability of PITX-1 to stimulate Mt1 promoter activity was inhibited by mutagenesis of either of its consensus sequences, indicating that both are required for successful promoter activation. However, EGR-1 retained its ability to inhibit PITX-1-stimulated promoter activity even after mutation of its consensus binding sequence. This finding suggested that, in our in vitro system, EGR-1 is able to inhibit Mt1 promoter activity without binding to DNA and thus presumably via protein-protein interactions. Such a mechanism would be consistent with reports of functional interactions between EGR-1 and other proteins involved in transcriptional regulation We next extended previous analyses of rat Mt1 expression in the pituitary of Egr-1\u2212/\u2212 mice. As observed previously Mt1 expression. In contrast to the upregulation of Mt1 in hypogonadal mice that are unable to synthesise GnRH, and despite inhibition of Mt1 promoter activity by EGR-1 in vitro, there was no difference in pituitary Mt1 expression between Egr-1\u2212/\u2212 mice and wild type litter mates. Thus, despite the ability of EGR-1 over-expression to inhibit Mt1 promoter activity in vitro, EGR-1 is not necessary for GnRH to regulate Mt1 in vivo. One possible explanation for this finding is that there is developmental compensation in the knock-out model Egr-1\u2212/\u2212 mice remain infertile due to a lack of LH synthesis Mt1 regulation. A second and perhaps more likely explanation for the absence of an effect of genotype is that additional pathway(s) link GnRH signalling to Mt1 expression, thus providing functional redundancy of signal transduction mechanisms. At present we are unable to distinguish between these possibilities.Finally, in order to investigate the role of EGR-1 in melatonin receptor regulation in vivo, we examined Mt1 melatonin receptor mRNA, both in vivo and in vitro. Although underlying signal transduction mechanisms are unclear, our current data expand upon previous work and reveal a direct ability of GnRH signalling to down-regulate melatonin receptor expression in \u03b1T3-1 gonadotroph cells. Despite this effect and the elevated Mt1 previously observed in hypogonadal mice, blockade of GnRH signalling in adult animals didn't increase pituitary Mt1 mRNA. Together these findings suggest that GnRH may induce a long-term change in melatonin sensitivity, rather than merely a tonic inhibition of gene transcription. As discussed elsewhere Mt1 promoters possess some common regulatory elements In summary, we have provided novel information describing the regulation of pituitary"}
+{"text": "PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PRIP Interacting protein with Methyl Transferase domain) was first isolated as a transcriptional coactivator PRIP/NCoA6 interacting protein (NCoA6IP) in a yeast two hybrid screen . In addition to the conserved phosphorylation site, PIMT also contained putative MAPK docking sites; which frequently mediate high level interaction between MAPK kinase and its substrate. PIMT has one D domain (119RNKVKIKKK) and one DEF motif  and PIMT-C  in the presence or absence of RAF-BXB in 293T cells. Post transfection, PIMT was immunoprecipitated using Anti-PIMT and was probed with ERK substrate antibody (Anti-MPM2) followed by re-probing with Anti-PIMT antibody. As shown in 298Ala) abolished RAF-BXB dependent phosphorylation of PIMT (We next examined whether PIMT is phosphorylated at Ser of PIMT . Taken tp <0.05) in the presence of PIMTS298A  on Med1 mediated PPAR\u03b3 driven transcriptional activity as assessed by luciferase activity . This inIMTS298A  revealinWe previously reported that PIMT enhances the transcriptional activity of PPAR\u03b3 and RXR\u03b1 utilizing 3XPPRE-luc reporter assays ,6. It is298 increases the transactivation ability of PPAR\u03b3 and that PIMT is a part of PEPCK transcriptional apparatus. We hypothesized that ERK dependent phosphorylation of PIMT may also modulate the expression of PEPCK. To address this possibility, we used a PEPCK promoter-luciferase system. We transiently transfected 293T cells with the full length PEPCK promoter-luciferase gene along with other indicated constructs  in transcriptional activity was observed. Above experiments demonstrated that ERK2 dependent phosphorylation of PIMT at Sernstructs . In absenstructs . Phosphonstructs  suggesti298 to Asp (a phosphomimetic mutation) and studied its effect on PIMT mediated transcriptional activation activity using promoter-reporter assays. Ser298Ala mutation (PIMTS298A) significantly reduced the promoter activity while the phosphomimetic mutation of PIMT (PIMTS298D) induced reporter activity to the levels comparable to that of WT PIMT observed in the presence of MAPK inducers. However, in the absence of RAF-BXB there was no change in the reporter activity upon overexpression of either PIMT or its mutants. Based on the above observations, it is reasonable to conclude that phosphorylated PIMT enhances its ability to promote PEPCK promoter activity and therefore PIMT may potentially play an important regulatory role in gluconeogenesis. Mutation of serine to aspartate creates a negative charge in the residue that in part mimics the effects of the phosphorylated serine. Thus, we mutated Ser298 mutants of PIMT in primary rat hepatocytes along with Med1. Ad/eGFP was used as the control. Infection of cells with Ad/PIMT or Ad/Med1 increased the glucose output compared to Ad/eGFP infected cells  and G6Pase (G6Pc), the rate limiting enzymes of gluconeogenesis, were observed to increase compared to that of Ad/eGFP supporting our observation that PIMT enhances hepatic glucose output  and PGC1\u03b1 (the master regulator of metabolism)  . Furtherabolism) . Taken trd day or 5th day of injection. Consistent with the above observation, upon overexpression of PIMT, PEPCK mRNA levels were increased post 5th day of injection when compared to that of Ad/LacZ . Importantly, in PIMT\u0394LivKO mice, these genes were not increased at all (see G6P and HNF4\u03b1) or increased only to modest levels (PCK1 and PGC1\u03b1). To firmly establish the role of PIMT in gluconeogenesis in liver and to complement the PIMT overexpression results described above, we have used mice in which PIMT gene was selectively deleted. We recently showed that PIMT is essential for normal development and that disruption of PIMT results in embryonic lethality at day 3.5 . To studvKO mice) . These fl/fl and PIMT\u0394LivKO mice, incubated with medium containing sodium lactate and sodium pyruvate and measured the release of glucose into medium after 6 h. In agreement with the in vivo data described above, glucose release was reduced significantly in hepatocytes derived from PIMT\u0394LivKO as compared to PIMTfl/fl livers (\u0394LivKO livers (shown above) and our overexpression data presented in earlier sections of this manuscript unambiguously show that PIMT plays a critical role in gluconeogenesis.To determine whether the gene changes involved in gluconeogenesis reflect in glucose release from liver, we prepared hepatocytes from PIMTl livers . These d3 enhanced the levels of pERK1/2  which is located in the context of ERK1/2 consensus sequence, PXS/TP (PSSP) [298 of PIMT markedly reduced ERK2-dependent modification, substantiating the authenticity of this site. ERK is required for its nuclear receptors transcriptional coactivator activity, as determined by transient-transcription assays  . Mutation assays . We obseS298A) significantly reduced the expression of PEPCK. In contrast to phosphodeficient mutation (PIMTS298A), phosphomimetic mutation (PIMTS298D) of PIMT restored its ability to enhance PEPCK expression. Consistently, we noted that glucose output was also enhanced in PIMT or PIMTS298D but not PIMTS298A overexpressing primary hepatocytes. Further, we showed that PIMT and its interacting partner Med1, a key component of mediator complex, additively enhanced glucose production. Moreover, overexpression of PIMTS298D but not PIMTS298A along with Med1 enhanced hepatic glucose output. Taken together, our data suggest that phosphorylation of PIMT at Ser298 leads to enhanced hepatic gluconeogenesis via modulating the expression of PEPCK and other gluconeogenic genes. PEPCK is a critical regulator of glucose homeostasis under nutritional depletion condition and/or hormonal changes. Numerous hepatic nuclear receptors including PPAR, TR\u03b21, HNF4\u03b1, FOXO1 and co-activators like PGC1\u03b1, CBP/Ep300, PRIP/NCoA6 and SRC1/2 are bound to PEPCK promoter either directly or indirectly and are essential for gluconeogenic response ,47-51. SWe also observed that the pro-gluconeogenic action of PIMT or Med1 requires the intact MEK1/ERK signaling. PIMT or Med1 dependent increase in hepatic glucose output was inhibited upon the pharmacological blockade of signaling through MEK1/ERK pathway. This was consistent with our current and previous findings that phosphorylation of PIMT and Med1 augmented their transcriptional activating potential . Our stu\u0394Liv KO mice and analyzed the consequences on the expression of the gluconeogenic genes upon PIMT deletion. The expression of PEPCK and G6P, the two rate limiting enzymes of hepatic gluconeogenesis and PGC1\u03b1, the master regulator of metabolism, were noted to be significantly decreased upon PIMT deletion. Also hepatocytes isolated from liver of PIMT\u0394Liv KO mice showed dwindled glucose production. Thus, our data strongly implicate the pivotal role of PIMT in regulating the hepatic gluconeogenesis.To unmask the impact of PIMT in context of hepatic gluconeogenesis, we used PIMTet al [Data obtained in this study are in striking contrast with the observations of Xuet al . Overexpet al . The obset al . 298 of PIMT augments its activity. However, the degree of association between PIMT and Med1 was observed to be independent of their phosphorylation status PIMT harbors consensus MAPK/ERK phosphoacceptor site and docking domains  and the two putative conserved MAPK/ERK docking sites are also indicated. (B) Alignment of PIMT amino acid sequences across different mammalian species. SP site (MAPK/ERK target site) is conserved across the species. D-domain (hydrophobic amino acid rich domain) and DEF motif  are also observed to be highly conserved.(TIF)Click here for additional data file.Figure S2Overexpression of PIMT or Med1 enhances hepatic glucose output in MEK1/ERK dependent manner (A) Primary rat hepatocytes were infected with control Ad/LacZ or Ad/PIMT eGFP and/or Ad/Med1. After 24 h of infection, the cells were cultured in glucose production medium for 6 h in presence of DMSO or UO126 (25\u03bcM) and amount of glucose released in cell supernatants were measured. The values were normalized to corresponding total protein content and expressed relative to Ad/LacZ (without inhibitor) control which was considered as 1. Data are representative of 3 independent experiments. Statistical analysis was performed using one way ANOVA followed by Bonferroeni\u2019s post-hoc test.*p<0.001, # p<0.001 vs Ad/LacZ (column 1) and @p<0.0001vs Ad/PIMT eGFP and Ad/Med1 . (B) DMSO or U0126 treated rat hepatocytes were lysed and analyzed by western blotting with phospho-ERK1/2 and total ERK1/2 antibodies. (C) Rat hepatocytes infected with Ad/PIMT eGFP were treated with DMSO or U0126 (25 \u00b5M) and the number of GFP positive cells (minimum 150 cells for each treatment) was counted using fluorescent microscope. (D) Rat hepatocytes were infected with Ad/LacZ or Ad/PIMT eGFP and expression of PIMT was analyzed by western blotting with anti-GFP antibody.(TIF)Click here for additional data file.Figure S3Overexpression of PIMT enhanced expression of gluconeogenic genes in primary hepatocytes in MEK1/ERK dependent manner Primary rat hepatocytes were infected with Ad/PIMT eGFP and Ad/Med1 or Ad/LacZ (control). Post 6 h of infection cells were cultured either in DMSO or treated with 25\u00b5M of UO126. After 24 h of infection, RNA was isolated and expression of (A) PCK1, (B) G6Pc (C) HNF4\u03b1, (D) PGC1\u03b1, (E) PIMT and (F) Med1 was measured using qPCR. The values were normalized with the 18S rRNA and expressed relative to Ad/LacZ (DMSO) as 1. Statistical analysis was performed using one way ANOVA followed Bonferroni's post hoc test to determine the difference between the test result. *p<0.05, **p<0.005, ***p<0.001vs Ad/LacZ.(TIF)Click here for additional data file.Figure S4Overexpression of PIMT and Med1 in mouse liver qPCR analysis to confirm the over-expression of (A) PIMT and (B) Med1 in the liver of wild type mice injected with Ad/PIMT eGFP and Ad/Med1 respectively.(TIF)Click here for additional data file.Figure S5A)PIMT- Med1 interaction is independent of their phosphorylation state . FITC labeled anti FLAG and Cy3 labeled secondary antibody against Med1 were used to visualize the localization of PIMT and Med1, respectively using Deltavision deconvulation microscopy. (B) 35S labeled invitro translated PIMT-N was and incubated for 2 h at 4\u00b0C with GST-Med1-C either unmodified or phosphorylated by purified ERK2. Following GST pull-down samples were resolved by SDS-PAGE and signals were visualized by autoradiography. (C) Invitro translated 35S labeled full length Med1 (Med1-FL) was incubated for 2 h at 4\u00b0C with GST-PIMT-N either unmodified or phosphorylated by purified ERK2. After incubation, PIMT-N was precipitated using glutathione sepharose beads followed by SDS-PAGE and autoradiography.(TIF)Click here for additional data file.Methods S1Supporting Materials and Methods.(DOCX)Click here for additional data file.Table S1Primer sequences. Sequences of the primers used in this study are provided. Nucleotides in bold represent mutations and primers prefixed with letter \u201cQ\u201d were used for qPCR analysis.(DOCX)Click here for additional data file."}
+{"text": "Candida abundance of 82 Dutch adults aged 58\u201380 years was established relative to the bacterial load by quantitative PCR analysis of the Internal Transcribed (ITS) region (Candida) and 16S rDNA gene (bacteria). The salivary microbiome was assessed using barcoded pyrosequencing of the bacterial hypervariable regions V5\u2013V7 of 16S rDNA. Sequencing data was preprocessed by denoising and chimera removal, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. Both OTU-based  and phylogeny-based analyses  were performed. Saliva of Dutch older adults contained 0\u20134 \u00d7 108 CFU/mL Candida with a median Candida load of 0.06%. With increased Candida load the diversity of the salivary microbiome decreased significantly (p<0.001). Increase in the Candida load correlated positively with class Bacilli, and negatively with class Fusobacteria, Flavobacteria, and Bacteroidia. Microbiomes with high Candida load were less diverse and had a distinct microbial composition towards dominance by saccharolytic and acidogenic bacteria - streptococci. The control of the acidification of the oral environment may be a potential preventive measure for Candida outgrowth that should be evaluated in longitudinal clinical intervention trials.Currently there are no evidence-based ecological measures for prevention of overgrowth and subsequent infection by fungi in the oral cavity. The aim of this study was to increase our knowledge on fungal\u2013bacterial ecological interactions. Salivary  Secondly, there are extrinsic factors such as polypathologies , polymedications and malnutrition e.g., Candida) may cause oral infectious disease.In the next decades healthcare services are faced with an aging population. In the European Union the proportion of 65 year and older is predicted to reach 53% of the total population by the year 2025 Candida species are a part of the commensal oral microbiota of healthy individuals at all ages with a reported prevalence between 15\u201375% i.e. mycoses Candida spp. being the fourth leading cause of all cases vice versain vitro at oversimplified conditions and with only a few species involved. No efforts so far have been made to assess the ecology and commensal relation of Candida with the oral microbiota at the breadth of the oral microbiome. There is an acute need to enhance our knowledge on the ecology of fungal \u2013 bacterial interactions in search for potential targets for a guided ecological balance towards a healthy oral ecosystem.Recent advances in research on bacterial\u2013fungal inter-kingdom communication have shown that bacteria are capable of interfering with or supporting the surrounding fungal community and Candida and the diversity and composition of the oral bacterial microbiome. For this we obtained oral bacterial pyrosequencing profiles of healthy Dutch older adults with various levels of oral Candida carriage and established that there is a relationship between the oral Candida load and the bacterial microbiome.The specific aim of this study was to assess the relationship between the load of oral Streptococcus (34% of reads), Rothia (12%), Veillonella (11%), Prevotella (10.5%), Gemella (6.9%), Actinomyces (6.7%), Neisseria (4.5%), Porphyromonas (2.6%), Haemophilus (1.8%), Lactobacillus (1.2%) and Leptotrichia (1% of reads) , 90% passed the quality control, 73% passed denoising and 59% reads  remained after removing the reads with chimeric sequences. The 601 unique sequences clustered into 425 OTUs of which 334 OTUs contained at least 5 reads. On average, a sample contained 87  OTUs. In order to avoid the effect of variable sample size on the diversity analyses, a randomly picked subset of 800 reads per sample was created and used for all subsequent analyses. After random picking of the subset of 800 reads per sample, 300 OTUs remained in the dataset with an average 43  OTUs per sample . The subf reads) .Candida as a proportion of Candida specific ITS gene over bacterial DNA (16S rRNA gene), with a median Candida load of 0.06% . The microbial profiles of these samples showed a high interindividual variation already at the bacterial class level , while class Bacteroidia , Flavobacteria  and Fusobacteria  correlated negatively with the Candida load (Lactobacillus (81% of reads) and Streptococcus (16% of reads). This particular sample was the abovementioned outlier also regarding the Candida load.Saliva of Dutch older adults contained between 0 and 14.5% of 0.06% . The excss level . Increasida load . Sample Candida load correlated positively with the Dominance Index  and negatively with the Shannon Diversity Index  . To assess the relation of the Candida load with the individual microbiome, the samples were divided into three categories - low (<0.01% Candida), medium (0.01\u20130.1%) and high Candida load (>0.1%) groups. The samples with a high Candida load were less diverse than samples with low Candida (p<0.005) (Diversity statistics showed that the  \u22120.403)  and the p<0.005) .Candida load. Rather than that, a continuous gradient from low to high Candida load samples was observed . There was no clear separation into sample clusters by the observed , arrow. observed , while wobserved  showed aCandida load sample direction in the PC1 were classified as Prevotella (4 OTUs), Actinomyces, Veillonella, Megasphaera, Leptotrichia (2 OTUs) and in the PC2\u2013 Porphyromonas, Neisseria, Haemophilus and Prevotella. The OTUs that contributed towards high Candida load sample direction were classified as Streptococcus (2 OTUs), Lactobacillus (2 OTUs), Rothia and Gemella. Prevalence of OTUs classified as Lactobacillus (2 OTUs) and Scardovia were associated with the high Candida load . Abundance of OTUs classified as candidate division TM7, Leptotrichia (2 OTUs), Prevotella (3 OTUs), Peptostreptococcus, Capnocytophaga and Johnsonella were associated with the low Candida load .Based on the loadings of the PCA, the OTUs that contributed most towards the low From the information obtained by the questionnaire we could split the subjects into a dentate (N\u200a=\u200a34) and an edentate (N\u200a=\u200a20) group . The edep\u200a=\u200a0.001, Mann-Whitney test) and had higher sample diversity  than subjects with full dentures  (p\u200a=\u200a0.021)  than samples from the edentate group  (=\u200a0.021)  D\u2013F.Candida load  than edentate subjects , there was no statistically significant difference in the Candida load between these groups .Although dentate subjects showed a trend for a lower Prevotella (4 OTUs), Actinomyces, Leptotrichia and Neisseria contributed towards the dentate group, while Rothia, Lactobacillus (3 OTUs) and Streptococcus (3 OTUs) contributed to the edentate group.The three data reduction approaches - the OTU-based PCA and the PCoA of the unweighted and the weighted UniFrac distances  - all stParvimonas, Peptostreptococcus, unclassified Micrococcales, Prevotella, Solobacterium, Neisseria and Abiotropia  and a higher abundance of OTUs belonging to genus Prevotella, Leptotrichia, Peptostreptococcus, Parvimonas, Solobacterium, Dialister and Porphyromonas  after Bonferroni correction). Edentate subjects had a higher prevalence of Lactobacillus  and a higher abundance of an OTU classified as Rothia .Subjects with a dentition had a higher prevalence of OTUs classified as genus Candida load with the microbiome profiles, we used the dentate group (N\u200a=\u200a34) alone. The diversity statistics (Candida load (N\u200a=\u200a14) were less diverse  than samples with low Candida (N\u200a=\u200a8)  . Additionally, the Dominance Index was significantly higher in the high Candida load samples (p\u200a=\u200a0.012).To exclude the covariance of dentures on the relation of the atistics  showed tCandida load samples and some medium load samples in the upper right quadrant of the PCA distant from the high Candida load samples (Candida load were less dispersed than medium and high Candida load samples (Candida samples in a similar way to the PCA (Streptococcus (4 OTUs) contributed to samples from the high Candida load, while Prevotella (3 OTUs), Leptotrichia (2 OTUs), Neisseria and Porphyromonas determined the position of the low Candida load samples. The low Candida load was associated with higher abundance of OTUs classified as Prevotella, Porphyromonas and Haemophilus .The PC 1 (18% of variance) and PC 2 (13% of variance) of the OTU-based PCA positioned most of the low  samples . The PCo samples . Weighte samples  separate the PCA . LoadingCandida load and the bacterial profiles of saliva of Dutch older adults. With increased Candida load the diversity of the salivary microbiome decreased and the composition changed towards dominance by Bacilli (streptococci and lactobacilli) and disappearance of genera within class Fusobacteria and Bacteroidia. The microbiome of dentate subjects differed from that of the fully edentate, but this difference was not significantly associated with the Candida load.In this study we assessed the relation between the Candida load: 0\u20134\u00d7108 CFU/ml with a median Candida load of 0.06%. Studies on comparable population report about 0.02% Candida in saliva, detected by culturing Candida-ITS gene positive. Studies on a similar age group, though using a culturing approach, have reported around 80% Candida prevalence Candida-specific internal transcriber spacer (ITS) region, used here.Saliva samples of our study group showed a large variation in Due to the study logistics it was not possible to collect samples from defined intraoral habitats such as plaque, tongue or denture surfaces. Although different intraoral niches select for a habitat-specific microbiome and salivary microbial profiles show higher similarity to mucosal sites than to dental surfaces Candida frequently leading to denture stomatitis Candida load in edentulous full prosthesis wearers. The lack of statistical significance could be explained by the small sample size  and thus lack of statistical power, as well as by good oral and general health of the study participants. It has been shown that denture stomatitis is a multifactorial disease where poorly fitting dentures, continuously worn dentures, poor oral hygiene and reduced salivary flow all increase the ability of Candida to colonize the dentures and contribute to the etiology of the disease Dentures, especially in edentate individuals are known to be one of the main predisposing factors for outgrowth of i.e. hospital-acquired pathogens. However, in two cases relatively high proportions of pathogenic species were found: staphylococci in a single sample (LASA161) contributed to 9% of the reads, and two Pseudomonadacea OTUs contributed to 11% of the reads in an another sample (LASA137). Interestingly, both of these cases belonged to high Candida load group. Candida has been previously co-associated with Staphylococcus aureus and Pseudomonas in human infections C. albicans physically interacts with S. aureus and differentially regulates specific virulence factors of S. aureus in vitroSaliva profiles of Dutch older adults were composed of commensal bacteria commonly found in human salivary microbiomes Candida load and the diversity of the salivary microbiome. We also showed that absence of teeth and presence of full dentures as such already were related to decreased microbiome diversity, irrespective of the Candida load. Therefore the effects of Candida load were assessed also on the dentate subgroup. Decreased diversity was seen both in the entire study sample and in the subgroup of dentate subjects. Decreased bacterial diversity is associated with a disbalanced community, e.g., the salivary microbiome from caries-active individuals was shown to be less diverse than microbiome from caries-free individuals We observed a negative correlation between the Candida load. These taxa are generally less acid-tolerant than Bacilli, which was the only class that increased with the increasing Candida load. This suggests that the oral environment of a high Candida load samples was acidified, i.e. exposed to prolonged low pH episodes. Oral Candida isolates have been shown to be highly acid tolerant and acidogenic The proportion of certain bacterial classes such as Fusobacteria, Bacteroidia, and Flavobacteria correlated negatively with the Candida load. The interactions between fungi and streptococci appear to be synergistic Candida albicans is enhanced if co-cultured with oral streptococci in the presence of sucrose Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis and Streptococcus anginosus are known to coaggregate with C. albicans, especially if the yeast cells are subjected to glucose starvation C. albicans adhesion S. gordonii is mediated through streptococcal cell surface polysaccharide receptors and polypeptide adhesins Candida, on the other hand, in addition to reducing the oxygen tension to levels preferred by streptococci, may provide growth stimulatory factors for the bacteria as a result of nutrient metabolism Candida load. While most lactobacilli are recognized as probiotics regarding Candida control Lactobacillus casei was shown to stimulate germ tube growth \u2013 an important C. albicans virulence factor Within the class Bacilli, streptococci increased most with increased Candida load was associated with among others, genus Prevotella, Porphyromonas and Peptostreptococcus. There are no comparable clinical reports regarding any relationships of these bacteria with fungi. In in vitro studies however, two putative periodontal pathogens \u2013 Prevotella nigrescens and Porphyromonas gingivalis \u2013 have been shown to exert a suppressive effect on C. albicans viability P. gingivalis and Prevotella intermedia inhibited C. albicans germ tube formation in vitroCandida or is affected by it directly. Our finding of Peptostreptococcus together with Prevotella in low Candida load samples might be due to a symbiotic relationship between these two taxa. For instance, it has been shown that Peptostreptococcus anaerobius is nutritionally dependant from Prevotella bovia , where P. bovia provides amino acids required for the growth of P. anaerobiusCandida or are affecting the presence of Candida. The exact mechanisms should be investigated further, both in longitudinal clinical trials and in fundamental experimental research in vitro.A low Candida and is the major ecological factor that perturbs the oral commensal microbiome leading to the shift towards an increase of aciduric microbiota and reduction of natural diversity of the bacterial microbiome. The actual sequential order of the events  can only be elucidated in well-planned longitudinal studies.The current findings strongly suggest that acidification of the environment coincides with the high load of Candida infection. Second, the finding of inverse relationship of certain taxa with the Candida load suggests a potential in search for and the use of natural, with oral health-associated probiotic strains, in fungi-bacteria warfare. These efforts will eventually bring us closer to developing strategies for prevention of microbial shifts and Candida overgrowth and to unraveling of and interfering with the interkingdom signaling processes.Our results bring us to two potential preventive strategies. First, the pH-lowering potential as well as duration of the low pH episode after carbohydrate consumption could be diminished by enrichment of base-producing bacteria. This could be achieved by modifying a diet from a carbohydrate- to an arginine-rich protein diet that could enhance the proportion of alkali-producing microbiota. Supplementation with prebiotics such as arginine or urea Candida load and the bacterial microbiome profile in older adults. Microbiomes with a high Candida load are less diverse and have a distinct microbial composition predominated by streptococci. The actual role of Candida as opportunistic pathogen in shaping the microbiome should be determined in further research including fungal-microbial interactions in complex microcosm models in vitro and following natural interactions in healthy and diseased populations during longitudinal clinical trials.There is a relation between oral salivary http://www.lasa-vu.nl), a prospective study on predictors and consequences of changes in autonomy and well-being in the aging population in the Netherlands. The study was approved by the ethical review board of the VU University Medical Center , and all participants gave written informed consent. Subjects were chosen from three culturally distinct geographical areas in the West, Northeast, and South of the Netherlands. Details on the sampling and data collection procedures have been published elsewhere Study subjects ; min 58, max 80 years; 50% females) were participants of the Longitudinal Aging Study Amsterdam (LASA)  of TTCTCCGCTTATTGATAT) modified from Candida concentration. Primers and probe specific for the prokaryotic 16S rDNA gene . For 16S, 7.5 pmol primers and 3.8 pmol probe, for PhHV 1.8 pmol primers and 0.4 pmol probe and for ITS 24 pmol of each primer was used. The conditions for the qPCR reaction were: an activation step of 10 min at 95\u00b0C followed by 50 cycles consisting of a denaturation step at 95\u00b0C for 30 sec, an annealing step at 60\u00b0C for 30 sec and an extension step at 72\u00b0C for 30 sec. Standard curves obtained from the overnight cultures of Candida dubliniensis and Escherichia coli were used to extrapolate DNA concentrations (CFU/ml) of the Candida (ITS) and bacterial (16S) concentration (CFU/ml) in the saliva samples, respectively. The CFU numbers were validated in saliva samples spiked with six different Candida overnight cultures  and performing both, culturing of Candida and performing Candida specific qPCR.6FAM-CGTATTACCGCGGCTGCTGGCAC-BBQ) Amplicon libraries of the small subunit ribosomal RNA gene V5\u2013V7 hypervariable region were generated for each of the individual samples. PCR was performed using the forward primer 785F (GGATTAGATACCCBRGTAGTC) and the reverse primer 1175R (ACGTCRTCCCCDCCTTCCTC). The primers included the 454 Life Sciences  Adapter A (for forward primers) and B (for reverse primers) fused to the 5\u2032 end of the 16S rDNA bacterial primer sequence and a unique 10 nt sample identification key.2 (Stratagene), 240 \u00b5M dNTP  0.5 \u00b5M of each primer. To each reaction 100 pg of DNA template was added. After denaturation , 9 cycles were performed consisting of denaturation , annealing , and extension , followed by 23 cycles of denaturation , annealing , and extension . The amplicons were purified by means of the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit . The quality and the size of the amplicons were analyzed on the Agilent 2100 Bioanalyser with the DNA 1000 Chip kit . The amplicon libraries were pooled in equimolar amounts and sequenced unidirectionally in the reverse direction (B-adaptor) by means of the Genome Sequencer FLX Titanium system .The amplification mix contained 2 units of Pfu Ultra II Fusion HS DNA polymerase , 1 unit Buffer Pfu Ultra II [10x], including 2.0 mM MgClde novo and the reference-based approach were combined and reads marked as chimeric were removed.The sequencing data were processed using QIIME  The cleaned reads were clustered into Operational Taxonomic Units (OTUs) at a minimal sequence similarity of 97% using UCLUST Reference Optimal To allow comparisons among different samples, a randomly subsampled dataset of 800 reads per sample (the minimum nr of reads per sample was 881) was created. Data reduction by principal component analysis (PCA) on log2 transformed OTU data and the diversity statistics  was performed using PAST software n samples in (n \u20131)-dimensional space, was used to compare groups of samples based on unweighted and weighted UniFrac distance metrics. PCoA is analogous to a PCA. The distinction is that PCA begins with a table of the number of times each OTU was observed in each environment, whereas PCoA begins with a table of distances between each pair of environments For phylogenetic measures of community \u00df diversity, unweighted UniFrac  and weighted UniFrac (a quantitative measure) Candida (% ITS/16S) in saliva and the abundance of individual bacterial classes, the Mann-Whitney test was used to compare diversity statistics between sample groups  using SPSS version 17.0. A G-test of Independence and ANOVA (implemented in QIIME 1.4.0.) were used to identify OTUs that are differentially represented among the groups of samples. The G-test of independence determines whether presence/absence of an OTU is associated with a category. ANOVA determines whether the relative abundance of an OTU is different between categories. The categorical variables used were: high versus low Candida load; dentate versus edentate subjects; and within the dentate group \u2013 high and low Candida load. The medium Candida load was arbitrarily set at an ITS/16S gene proportion between 0.01% and 0.1%. All values below 0.01% were considered low, values above 0.1% \u2013 high Candida load. The probability after multiple comparisons was corrected using Bonferroni correction. In this correction the p value is multiplied by the number of comparisons (the number of OTUs remaining after applying the filter). The filter that was applied required that a minimum of 10 samples contained the OTU for the OTU to be included in the analysis.Spearman\u2019s correlation was performed to assess the correlation between the proportion of Figure S1Relative abundance of four bacterial classes that showed correlation with Candida load plotted against the Candida load. The abundance (nr of reads/sample) of (A) Bacilli, (B) Bacteroidia, (C) Fusobacteria and (D) Flavobacteria by Candida load. Candida load was measured as the proportion of ITS gene over 16S gene abundance by qPCR. The values were normalized by log10 transformation.(TIF)Click here for additional data file.Figure S2Diversity statistics of salivary microbiomes by Candida load. Diversity statistics of salivary microbiomes by Candida load (A\u2013C) in saliva and by Dentition of the subjects (D\u2013F) as boxplots of  OTUs per sample,  Dominance Index and  Shannon Diversity Index. Each box shows the median, quartiles, and outliers (circles). Connector connects statistically significantly different groups .(TIF)Click here for additional data file.Figure S3Salivary microbiome UniFrac distance data by Candida load. Salivary microbiome UniFrac distance data plotted by Candida load in all individuals  and in dentate individuals only  as unweighted  and weighted  UniFrac distance PCoA plots. Samples are colored by Candida load: green - low, blue - medium and red - high Candida load.(TIF)Click here for additional data file.Figure S4Salivary microbiome data plotted by dentition. Salivary microbiome data plotted by dentition of the subjects: A) PCA of OTU-based distances, B) PCoA of unweighted  UniFrac distances and C) PCoA of weighted (quantitative) UniFrac distances. Samples are color-coded by the dentition status of the subjects: dentate (N\u200a=\u200a34) \u2013 blue, edentate (N\u200a=\u200a20) \u2013 red.(TIF)Click here for additional data file.Table S1Relative abundance of reads and number of OTUs per phylum.(DOCX)Click here for additional data file.Table S2Candida (ITS gene) in saliva samples.Relative and absolute abundance of (DOCX)Click here for additional data file.Dataset S1OTU-table of salivary microbiome data.(XLSX)Click here for additional data file.Dataset S2Distribution of higher taxa (genus or higher taxon) in saliva samples.(XLSX)Click here for additional data file."}
+{"text": "Blended care, a combination of online and face-to-face care, is seen as a promising treatment option. However, actual use of blended interventions in practice is disappointing.The objective of this study was two folded. The first aim was to develop a blended exercise therapy intervention for patients with knee and hip osteoarthritis that matches the values of the users and that can be implemented in the daily routine of physical therapists. The second aim was to investigate the feasibility through interviews and a pilot study.In this paper, we employed the first 3 steps of the CeHRes road map to develop a blended intervention for patients with knee and hip osteoarthritis. We used interviews, a focus group and discussions with stakeholders to explore the needs, values, and requirements with respect to our to-be-developed blended intervention, which we called e-Exercise. The first version of e-Exercise was tested in a pilot study. Feasibility outcomes, including recruitment rates within each practice, website usage (assignments completed and website visits), and user satisfaction, were measured. In addition, therapists and patients from the pilot study were interviewed to investigate users\u2019 experiences.The study captured important information about stakeholders\u2019 needs and perspectives. Based on our findings, we created a first version and attuned the application\u2019s content, functionality, and structure. Patients and, to lesser extent, physical therapists were satisfied with the e-Exercise intervention. Eight patients were recruited by 8 physical therapists. Of the 8 patients, 6 completed more than 7 of 12 modules.This study outlines the development and feasibility of a blended exercise therapy intervention for patients with knee and hip osteoarthritis. E-Exercise offers an alternative approach in the physical therapy treatment of knee and hip osteoarthritis. This study provides valuable information to conduct a further trial to evaluate the (cost) effectiveness of e-Exercise compared to usual physical therapy.Netherlands Trial Register Number: NTR4224; www.trialregister.nl/trialreg/admin/rctview.asp?TC=4224 (Archived by WebCite at http://www.webcitation.org/6fOK4lrTO). Knee and hip osteoarthritis (OA) are leading causes of disability in older people . In the Although patients with knee and hip OA generally tend to avoid physical activity , physicaThere is a clear need for more feasible and easily accessible strategies in order to regulate therapeutic costs and make exercise therapy attainable for a broader range of OA patients. This can be accomplished through self-management support. Self-management implies individuals\u2019 ability to manage the symptoms, treatment, physical and psychosocial consequences, and lifestyle changes inherent in living with a chronic disease . The useAlthough promising in terms of evidence and accessibility, the adoption of eHealth technologies is disappointing . EmbeddiThe success of eHealth is hampered by insufficient attention paid to abovementioned determinants during the development process. The majority of eHealth technologies is created through ad-hoc procedures without a thoughtful approach . High raThe Centre for eHealth Research and Disease Management (CeHRes) road map is a development approach in which co-creation plays a central role . This CeIn order to enhance clarity and optimize the execution of each step, we have chosen to present the methods and results sections together. In the following section, we describe the first 3 steps of the CeHRes road map, namely the contextual inquiry, value specification, and design. We present a pilot study on the feasibility of the blended intervention. The first 3 stages of the CeHRes road map and pilot study provide the basis for steps 4  and 5 , which will be conducted in a later phase of the project. The study has been approved by the Medical Ethical Committee of the St. Elisabeth hospital Tilburg, the Netherlands .During the contextual inquiry and value specification we aimed to establish stakeholders\u2019 most important needs, values, and requirements with respect to our to-be-developed blended intervention. The input of this phase was mainly based on another project, executed by the same authors . In thisPhysical therapists in the focus group indicated that a blended intervention will be a useful instrument in the treatment of OA patients. The 24/7 availability of information and exercises, the possibility to extend the physical therapy treatment in the home environment of the patient, and the potential to enhance the adherence of home exercises were mentioned as possible advantages. On the other hand, the fact that the proposed blended intervention aims to substitute conventional visits may lead to reduced revenues per patient. According to physical therapists, this lack of financial incentive was seen as a potential barrier to use the proposed intervention in practice. The results in the matrix, which represent the stakeholders\u2019 needs and perspectives with respect to Join2move, showed positive attitudes toward the to-be-developed blended intervention. As a stakeholder from a rehabilitation institute stated: \u201cPatients will benefit from the blended intervention because it is cheap, independent of time or place, and promotes self-management in the home environment of OA patients.\u201d Another facilitator for implementation is the potential to reduce treatment costs. An employee of a health insurance company summarized this by saying: \u201cThe proposed blended intervention will possibly result in lower costs since the average number of sessions will be decreased. This will lead to a cost reduction of the OA treatment.\u201d The patients were also positive. They had a positive attitude toward the idea that eHealth will be an integrated part of their treatment, especially for information and education purposes.E-Exercise is a combination of (1) visits with a physical therapist, and (2) a Web-based physical activity intervention. The technical functionality of the Web-based part is based on a previously developed physical activity intervention . This inOver the course of a half year, a team of experts from NIVEL developed the e-Exercise program. The starting point of the development process was a previously developed Web-based exercise intervention  and the This pilot study employed a multicenter 1-group design. The purpose was to evaluate the feasibility of the e-Exercise treatment in the daily practice of physical therapists.Physical therapists working in a private practice were recruited through the website of the Royal Dutch Society for Physical Therapy and the social network of the authors. Eventually, 8 physical therapists were included in the pilot study. All participating physical therapists received a half day of training about the study procedures and how to use e-Exercise in their practice. Eligible patients, who visited a participating center during the study period, were enrolled by the physical therapists. Enrollment started on March 3, 2014, and ended May 6, 2014. Participants were suitable for inclusion if they (1) were aged 40 to 80 years and (2) had the diagnosis OA of the knee and/or hip according to the clinical criteria of the American College of Rheumatology . ParticiFeasibility measures included website usage, user satisfaction with the website, and recruitment rates of participants within each practice. Program use was measured by the number of modules completed. Based on a previous study , we consA total of 8 eligible OA patients were included in the pilot study by the 9 participating physical therapists. Patients were on average 62 years old, had 1 or more comorbidities (88%), and most of them were female (75%). None of the participants withdrew from the study. An overview of the sample characteristics is presented in Overall, interviewees were satisfied with the intervention. One patient summarized this sentiment by saying: \u201cI have told many friends and family that this is a great program because the program motivates you to perform exercises in your own time. I would therefore definitely recommend e-Exercise to others.\u201d Physical therapists also expressed positive feedback regarding the content of e-Exercise. To cite 1 therapist: \u201cI am especially pleased with the information about osteoarthritis provided by the videos. More insight into the disease and the role of pain is important prerequisite to encourage a physically active lifestyle.\u201d Although physical therapists were generally satisfied, they stressed that e-Exercise must be adapted for suitable integration into practice. As 1 physical therapist commented: \u201cThe program provides no insight [into] which modules patients receive. This was truly a downside of the program because I had little or no control over patients\u2019 progress.\u201d It was also reported by some patients that they liked the effective approach of e-Exercise. One patient commented: \u201cI liked the effective approach of the intervention. You need only a few face-to-face treatments to get on track. The provision of weekly physical therapy sessions is not useful because you have to exercise yourself.\u201dProvision of blended care requires a harmonious integration of technology into practice, combining complementary face-to-face treatments with eHealth technology. Implementing a blended intervention into health care is a complex process that changes existing routines, relationships, and budgets. Developers and researchers have to anticipate these implementation difficulties. While research supports the effectiveness of health technology, health care professionals often lack the time, skills, and resources to integrate eHealth into their daily practice. Input of end-users and other stakeholders throughout the development process is a prerequisite for the successful implementation of blended interventions into practice . The aimThe involvement of patients, physical therapists, and other stakeholders was extremely valuable throughout the development process. The first 3 phases of the CeHRes road map yielded unique insights into different needs and values of end-users and various stakeholders. Steps from the CeHRes model were not purely sequentially executed but involved a continuous process. For instance, the identification of needs and problems was mainly derived from experiences with a previous eHealth project, rather than a separate phase in the current project. The results from the post-pilot interviews demonstrated that e-Exercise is feasible in the treatment of patients with knee and hip OA. In line with the findings from the study by Pietrzak et al , particiThe visit-based method of recruitment was challenging. Over the 10-week enrollment period, we intended to recruit 2 patients per participating physical therapist. However, only 8 eligible patients were recruited by 8 physical therapists. Others have reported similar challenges with the recruitment of patients -32. The The findings of the pilot study need to be interpreted in light of several limitations.\u00a0The small number of participants and the absence of a control group are major limitations of the current study. Moreover, the generalizability might be limited by the self-selected sample in this study. Obviously, included physical therapists are techno-enthusiasts who are more willing to adopt technology in their practices than are others.Results from this study are valuable to set up a follow-up study to compare e-Exercise with usual physical therapy. We plan to conduct a larger, adequately powered, randomized controlled trial to investigate the effectiveness (including cost effectiveness) of e-Exercise . The rec"}
+{"text": "Exercise therapy in patients with hip and/or knee osteoarthritis is effective in reducing pain, increasing physical activity and physical functioning, but costly and a burden for the health care budget. A web-based intervention is cheap in comparison to face-to-face exercise therapy and has the advantage of supporting in home exercises because of the 24/7 accessibility. However, the lack of face-to-face contact with a professional is a disadvantage of web-based interventions and is probably one of the reasons for low adherence rates. In order to combine the best of two worlds, we have developed the intervention e-Exercise. In this blended intervention face-to-face contacts with a physical therapist are partially replaced by a web-based exercise intervention. The aim of this study is to investigate the short- (3\u00a0months) and long-term (12\u00a0months) (cost)-effectiveness of e-Exercise compared to usual care physical therapy. Our hypothesis is that e-Exercise is more effective and cost-effective in increasing physical functioning and physical activity compared to usual care.This paper presents the protocol of a prospective, single-blinded, multicenter cluster randomized controlled trial. In total, 200 patients with OA of the hip and/or knee will be randomly allocated into either e-Exercise or usual care . E-Exercise is a 12-week intervention, consisting of maximum five face-to-face physical therapy contacts supplemented with a web-based program. The web-based program contains assignments to gradually increase patients\u2019 physical activity, strength and stability exercises and information about OA related topics. Primary outcomes are physical activity and physical functioning. Secondary outcomes are health related quality of life, self-perceived effect, pain, tiredness and self-efficacy. All measurements will be performed at baseline, 3 and 12\u00a0months after inclusion. Retrospective cost questionnaires will be sent at 3, 6, 9 and 12\u00a0months and used for the cost-effectiveness and cost-utility analysis.This study is the first randomized controlled trial in the (cost)-effectiveness of a blended exercise intervention for patients with osteoarthritis of the hip and/or knee. The findings will help to improve the treatment of patients with osteoarthritis.NTR4224. Osteoarthritis (OA) is worldwide one of the leading causes of pain and disability. Most common affected sites are the hip and knee joints . In the Exercise therapy is the widely recommended non-pharmacological intervention in patients with hip and/or knee OA \u201313. TherThe internet has created new possibilities to combine face-to-face care with online care, called blended healthcare . The parUp till now, previous research in web-based interventions has focused on interventions without human support. Unfortunately, the effects of these interventions are small, especially in the long-term \u201324. ThesA prospective, single-blinded, multicenter cluster randomized controlled trial (RCT) will be conducted. The study has been approved by the Medical Ethical Committee of the St. Elisabeth hospital Tilburg, the Netherlands . The e-Exercise intervention will be compared with usual care . A flow diagram of the study protocol is shown in Figure\u00a0A stratified random sample of 800 physical therapy practices in three provinces of the Netherlands  will be invited by letter to participate in the study. Contact information of physical therapy practices will be obtained from the national database for physical therapists of the Netherlands Institute for Health Services Research (NIVEL). Additionally, a recruitment advertisement will be placed in the online newsletter of The Royal Dutch Society for Physical Therapy (KNGF). Each participating physical therapy practice will be asked to enroll one or two physical therapists. The researchers will recruit 100 physical therapists. Inclusion criteria for physical therapists will concern: (i) practicing in primary care, (ii) treating at least six patients with OA of the hip and/or knee each year. Physical therapists practicing in another physical therapy practice participating in the study will be excluded.In order to include 200 participants, each physical therapist is requested to recruit about two patients. Since the study of Veenhof et al.  showed tPhysical therapists willing to participate in the study will be screened on in- and exclusion criteria by a researcher (CK). Cluster randomization will be performed at the level of the participating physical therapy practices that will randomly be assigned to the intervention (e-Exercise) or the control group  by means of a computer-generated random sequence table. Physical therapists will receive a half day training about e-Exercise and the study procedure (intervention group) or about practicing according to the \u201cKNGF guideline OA hip-knee\u201d  and the In this single-blind study , the physical therapists are not blinded since they will treat patients according to the randomization. The researchers will be blinded to group allocation until completion of the statistical analyses. Participants will be assigned to a unique digital trial code to ensure that treatment outcome measurement and statistical analysis will be performed blind to treatment allocation. Patient information will be stored in a separate database.The 3-month program e-Exercise is based on the Dutch guideline for physical therapists 10) and is a combination of (i) maximum five face-to-face sessions with a physical therapist, and (ii) a web-based PA intervention. Table\u00a00 and is During the first face-to-face session (week 1), physical therapists will provide information about OA, the importance of PA and the relation of PA with pain. Together with their physical therapist, patients choose one physical activity, for example, walking, cycling or swimming. Physical therapists select and instruct four strength & stability exercises. Patients are instructed to perform the first module of the web-based part of the intervention. In this module, the patients will be asked to determine their physical load ability based on a 3-day self-test. The second assignment is the execution of strength & stability exercises. During the second face-to-face session (week 2), patients\u2019 physical load ability will be discussed and personal short and long-term goals will be formulated according to the principles of Goal Setting, which is based on the idea that goals can affect action . The strThe web-based part of e-Exercise is based on the web-based intervention Join2move  and consPatients in the control group will receive usual care. For the current study, usual care is defined as any treatment provided by physical therapists. Physical therapists will be encouraged to practice according to the \u201cKNGF guideline OA Hip-Knee\u201d . AccordiThree online questionnaires  will be used for data collection. Participants will receive an accelerometer for the measurement of objective PA . The physical therapists will measure physical functioning objectively at baseline and post-treatment (3\u00a0months). In addition, online cost questionnaires will be sent . We offer no financial incentives to complete questionnaires or to wear accelerometers. Table\u00a0Physical functioning will be assessed subjectively with the subscale \u2018function in daily living\u2019 of the Hip OA Outcome Score (HOOS) [e (HOOS)  and/or te (HOOS) , dependie (HOOS) . In thisPhysical activity will be measured subjectively with the SQUASH [e SQUASH . The quee SQUASH , except e SQUASH  will be Information on the patients\u2019 healthcare utilization, (unpaid) productivity losses, and sports costs due to OA will be gathered with four retrospective 3-month cost questionnaires that cover the full 12-months of the program. Healthcare utilization due to OA comprises of visits to a physical therapist, general practitioner, massage therapist, alternative therapist, medical specialist, as well as informal care, hospital care, the use of both prescribed and over the counter drugs and medical devices. Healthcare utilization will be valued using Dutch standards costs [ds costs . If thesds costs . Unpaid ds costs . Paid prds costs . The FCAds costs . The pards costs , 43\u201345. ds costs .Health Related Quality of Life will be measured with the EuroQol-5D (EQ-5D) [ (EQ-5D) . This qu (EQ-5D) . UtilitiSelf-perceived effect will be assessed by a single question about the degree of change in osteoarthritis symptoms since their previous assessment. Patients will score this effect on a 7-point Likert scale . A higher score indicates a better self-perceived effect.Pain and tiredness will be measured with a numeric rating scale . Furthermore, pain will be assessed with the pain subscale of the HOOS and/or the KOOS [the KOOS , 35.Self-efficacy will be measured by the Arthritis Self-efficacy Scale (ASES) [e (ASES) . SubscalAdherence will be measured objectively by quantitative data about usage which is automatically stored on the backend of the website. Usage is defined as completed week modules. Subjective adherence is measured by a questionnaire about patients\u2019 adherence to the Graded Activity modules and Strength & Stability exercises (frequency and intensity).Content of physical therapy sessions will be measured trough registrations forms, developed by the researchers. The registrations forms collect information about the adherence and content of the sessions.Patient characteristics i.e. age, sex, height, weight, educational level, location of OA, disease duration and the presence of comorbidities will be assessed at baseline.The power calculation is based on a previous multicenter cluster RCT study among patients with hip and/or knee OA  and perfDescriptive statistics will be used to describe the main characteristics of the study population and to explore baseline comparability . Primary baseline variables between the response and the non-response group will be performed in order to investigate selective attrition. The primary analysis will be performed according to the intention-to-treat principle. In addition, per-protocol analyses that include only adherent patients of the intervention group and the entire control group will be performed. For all analyses, a two-tailed significance level of p <\u20090.05 is considered to be statistically significant. All analyses will be carried out with the statistical package STATA.To determine the short (baseline-3\u00a0months) and long term (baseline \u221212\u00a0months) effectiveness of e-Exercise on primary and secondary outcomes, multi-level modelling of repeated measures will be performed controlling for baseline values and relevant confounders such as age, OA location and gender. With multilevel modelling of repeated measures it is possible to correct on one side for dependency of observations within subjects and, on the other side, to take into account the variation between physical therapists , 51. TheA cost-utility analysis (CUA) and a cost-effectiveness analysis (CEA) will be performed from the societal and the healthcare perspective. From the societal perspective all costs will be taken into account irrespective of who pays or benefits, whereas solely those borne by the healthcare sector will be included when the healthcare perspective is applied . For theRecruitment of physical therapy practices begun in May 2014. The trial will start in September 2014. Until December 2014 patients are able to enrol the program. The follow-up will last until December 2015. Analysis of the data will start in January 2016.Scarce health resources and a growing number of patients with OA of the hip and/or knee require cost-effective treatment strategies in patients with OA. The presented RCT will study the (cost)-effectiveness of e-Exercise, an intervention in which face-to-face exercise therapy sessions are partly replaced by a web-based PA intervention. This study is, as far as we know, the first RCT that investigates the (cost)-effectiveness of a blended intervention in patients with knee and hip OA. Therefore, this RCT will provide internationally relevant results regarding the short- and long-term (cost)-effectiveness of an exercise therapy intervention that incorporates modern technologies.The primary goal of e-Exercise is to improve levels of PA and physical functioning in a cost-effective manner. In addition to our outcome measurements, e-Exercise might have several other benefits beyond the primary scope of this study. First, a number of studies showed that exercise therapy may help to postpone joint replacement surgery \u201357. For Although the study is well-considered, we take into account potential operational issues. First challenge is the recruitment of sufficient numbers of physical therapists. Since e-Exercise is characterized by fewer physical therapy sessions, physical therapists will receive less reimbursement from health insurances compared to usual care. To deal with this challenge, accreditation points for participating physical therapists will be supplied in order to make study participation more attractive. Another incentive is that physical therapists keep their access to the website after the study is finished. The second challenge is the non-usage of the web-based part of e-Exercise. Previous studies have indicated that participants in online interventions are less motivated and feel less pressure to continue compared to traditional face-to-face interventions . HoweverThere are several strengths in the design of this study. First, we elaborate on the study results of Joint2Move . This in"}
+{"text": "This study aims to examine the patterns and socio-demographic predictors of health and environmental co-benefit behaviours that support climate change mitigation in a densely populated Asian metropolis\u2014Hong Kong.A population-based, stratified and cross-sectional random digit dialling telephone survey study was conducted between January and February 2016, among the Cantonese-speaking population aged 15 and above in Hong Kong. Socio-demographic data and the self-reported practice of 10 different co-benefit behaviours were solicited. Ethics approval and participant\u2019s verbal consent were sought.The study sample consisted of 1,017 respondents (response rate: 63.6%) were comparable to the age, gender and geographical distributions of the Hong Kong population found in the latest 2011 Hong Kong Population Census. Among the co-benefit behaviours, using less packaging and disposable shopping bags were practiced in the highest frequency (70.1%). However, four behaviours were found to have never been practiced by more than half of the respondents, including bringing personal eating utensils when dining in restaurants or small eateries, showering less than five minutes, having one vegetarian meal a week, and buying more organic food. Results of multivariable logistic regression showed that frequency of practicing co-benefit behaviours were consistently associated with gender and age.Urban residents in Hong Kong do not engage in the practice of co-benefit behaviours in a uniform way. In general, females and older people are more likely to adopt co-benefit behaviours in their daily lives. Further research to assess the knowledge and attitudes of the population towards these co-benefit behaviours will provide support to relevant climate change mitigation policies and education programmes. Climate change is known to pose risks to human health ,2. ThrouClimate change and human health are so closely linked that many mitigation measures naturally promote public health and environmental benefits at the same time, as captured in the concept of co-benefits. As defined by Intergovernmental Panel on Climate Change (IPCC) and World Health Organization (WHO), co-benefits involve \u201ca climate change adaptation or mitigation strategy which has additional, positive effects on health or other areas\u201d  and \u201cheaThe significant effect of individual lifestyles on both environmental change and health has drawn rapidly increasing attention in the research community recently . For insA population-based, stratified and cross-sectional random digit dialling telephone survey was conducted between 28 January and 4 February 2016. The survey was conducted in Cantonese language, as 95.8% of the Hong Kong population are Cantonese speakers or able to use Cantonese , which eFor the sample size calculation, we assumed the prevalence of co-benefit behaviours is 50%, which was a conservative hypothesis. A sample size of 784 participants was calculated with a 3.5% margin of error and 95% confidence interval. To account for potential missing data and increase the modelling flexibility, 1,017 participants were recruited. In addition, quota sampling was used to ensure the demographic representation of the general population in Hong Kong in terms of age, gender, and district of residence.Ethics approval and consent procedure of the study was reviewed and obtained from the Survey and Behavioural Research Ethics Committee at The Chinese University of Hong Kong (dated on 13 January 2016). Verbal consent was obtained from each participant in the beginning of the study. Each participant was required to indicate their willingness to participate in the survey and have the interview audially recorded. The interview was stopped immediately when the participant required to exit the survey. Similar to previous research studies with the same telephone study methodology that target general population \u201325, no aA self-reported questionnaire was designed for data collection, which included information on the general socio-demographic background and practices of 10 different co-benefit behaviours of a respondent, as seen in The survey questionnaire was pilot-tested and revised in December 2015. Fifty-two samples were collected through a population-based, stratified and cross-sectional telephone survey, using random digit dialling method. The pilot study also adopted a quota sampling method to ensure the sample\u2019s representativeness of the general population in terms of age, gender and district of residence. Quota sampling was also used in the main study. After the pilot test, additional questions related to the behaviours of showering less than five minutes every day, consuming less meat, and having one vegetarian meal a week were included to capture a more comprehensive picture of practicing health and environmental co-benefits behaviours. A binary scale of practicing co-benefit behaviours or not was modified into a 5-point Likert scale in the main survey tool to obtain a more finely differentiated frequency of practicing these behaviours.The telephone survey was conducted from 28 January to 4 February 2016. The Random Digit Dialling (RDD) method was used for each of Hong Kong\u2019s 18 districts to generate a randomly selected representative sample. Telephone calls were made during weekday evenings  and during daytime on the weekends (Saturday and Sunday) to avoid an under-representation of the working population. A participant was selected from each of the contacted households through the \u201clast birthday method\u201d. An eligible family member (defined as a Cantonese-speaking Hong Kong resident over the age of 15 without hearing impairment) who has passed the birthday most recently was invited to participate in the study. At least five attempts were made in different time slots to reach a household by dialling a number before that number was considered invalid. All telephone interviews were conducted by trained interviewers in Cantonese. Each interview took approximately 15\u201320 minutes to complete.Descriptive statistics on the socio-demographic variables and frequencies of practicing the 10 co-benefit behaviours were reported. A backward-stepwise (likelihood ratio) multivariable logistic regression was performed to investigate the association between socio-demographic variables and the adoption of co-benefit behaviours. Respondents were excluded from a regression analysis if they had refused to answer the pertaining co-benefit behaviour question. All statistical analyses were performed using IBM SPSS Statistics 21 for Windows at a significance level of \u0251 = 0.05.A total of 3,500 telephone numbers were dialled, of which 1,598 calls were responded to by eligible persons. Among them, 1,125 eligible respondents agreed to participate and gave verbal consent, and 1,017 respondents successfully completed the questionnaire, with a response rate of 63.6% (1017/1598). As shown in The participants comprised of 437 males and 580 females. Over half of the participants were 45 years old or above (56.7%). Around half of the participants were from the New Territories district. Nearly two-thirds of them had received only a secondary education or below (62.9%). Approximately 60% of the participants were married and one-third of them had a household income of less than HKD 20,000 per month . While two-thirds owned their residence (62.9%), one-third lived in public housing (33.2%) see .As shown in The results of multivariable logistic regression with backward stepwise elimination indicated that the adoption of different types of co-benefit behaviours were associated with different socio-demographic variables, as indicated in Older people, both those between 45 and 64 and those 65 and above, were more likely to practice co-benefit behaviours daily when compared with younger people. Associations with older age were found in the following co-benefit behaviours: Buy more organic food , Consume less meat , Have one vegetarian meal a week , Use less electricity , Shower less than five minutes every day , and Separate household waste .Among the 10 co-benefit behaviours, education level was significantly associated with only bringing personal eating utensils when dining in restaurants or small eateries. The participants with post-secondary education level or above had 139% higher frequency of bringing their personal eating utensils than those who obtained primary or below education .Regarding the other socio-demographic predictors, marital status was significantly associated with active travel and having one vegetarian meal a week. Married participants were 96% more inclined to walk/cycle more in daily life  but 35% less intended to have one vegetarian meal a week . Household income was significantly associated only with buying more organic food. Participants with household incomes of HKD 40,000 or above per month were at least 66% more likely to buy more organic food daily . Compared with those living in public housing, respondents who lived in private permanent housing were less likely to use less AC  but more willing to separate household waste , whereas those who lived in subsidized home ownership housing were also more willing to separate household waste .In summary, among the 10 co-benefit behaviours, using less packaging and disposable shopping bags was practiced daily by the highest proportion of people (70.1%). However, four behaviours were found to have been practiced by less than half of the population, including bringing personal eating utensils when dining in restaurants or small eateries, showering less than five minutes, having one vegetarian meal a week and buying more organic food. Multivariable logistic regression results showed that practicing co-benefit behaviours were consistently associated with gender and age.2(4) = 27.681, p<0.001). This may be attributed to the perception and ease of the behaviour among the respondents, as it may be easier to reduce practicing a behaviour than it is to schedule or regulate a specific dietary change. A similar situation can be found for the behaviours of using less packaging and fewer disposable shopping bags and bringing personal eating utensils when dining in restaurants or small eateries, whereby reducing the practice of a current behaviour  was found among a significantly higher proportion of people (70.1%) than instilling the practice of a non-normative behaviour (4.0%) (\u03c72(4) = 140.685, p<0.001). Interestingly, in both cases, the patterns of socio-demographic associations were similar between the two behaviours despite differences in proportion of daily practice. In the case of buying organic food, the low proportion of daily practice (4.3%) can be attributed to a smaller share in the overall food market, complications around certification and labelling, and higher prices [As indicated in the findings, urban population in Asia showed a diverse pattern in the practice of health and environmental co-benefit behaviours in their daily life. Using less packaging and fewer disposable shopping bags received the highest frequency in daily practice (70.1%), reflecting success of the 2015 Plastic Shopping Bag Levy Scheme, a regulatory policy that requires a charge of HKD 0.5 (USD 0.06) for a plastic shopping bag . Howeverr prices , as suppFemales consistently performed better than males across different co-benefit behaviours, which is consistent with the findings of previous studies on the gender associations of environmental friendly behaviours . This stContrary to a popular belief, our study findings demonstrated that younger generations do not necessarily perform better in behaviours that benefit health and the environment. Instead, older people (age 45 or above) were more likely than younger people (age 44 or below) to practice co-benefit behaviours daily, especially for all dietary-related behaviours, reducing electricity and water usage, as well as separating household waste. These findings reveal a pattern similar to a meta-analysis of the relationship between age and pro-environmental variables, where older age was associated with more conservation behaviours  . This maIncome and real estate ownership had diverse associations with the practice of different co-benefit behaviours. Those with higher household income were more likely to buy more organic food. A possible explanation could be that those with lower household income had a lower proportion of disposable income available for purchasing higher-priced organic food. On the other hand, respondents who lived in private permanent housing were less likely to use less AC, while both they and those living in subsidized housing were more likely to separate household waste when compared with public housing residents. Generally, the residents in subsidized housing have higher monthly income than the group who live in public housing, but lower than the group in private housing.This paper used a population-based, stratified and cross-sectional random digit dialling telephone survey to collect data on co-benefit behaviours in the general population of an urban Asian metropolis. The cross-sectional study design can only demonstrate associations between patterns and social-demographic predictors, as causation cannot be attributed to the findings. In addition, data collection through the telephone survey was based on self-reported information, which might be subjected to reporting bias as well as imprecisions in the measurement of each co-benefit behaviour of interest. Moreover, with the finite amount of time in each telephone interview, this study could only collect data based on a limited number of questions and this restricted our ability to examine each of the behavioural patterns in detail. Furthermore, although the scientific relationships between health and environmental co-benefits of the studied health messages in this paper are based on sound theoretical deduction, and various published studies have demonstrated associations between the behaviour patterns and health outcomes, more scientific investigations are needed to quantify the relationship between the health outcomes and co-benefit behaviours, and to explore what might be other non-knowledge-based factors  and if the level of behavioural adoption might affect actual health outcomes.The local government has previously implemented a series of measures to promote co-benefit behaviours among the Hong Kong population. However, as supported by our study findings, the impact of these measures varied. For example, the Plastic Shopping Bag Levy Scheme implemented in 2015, which charges HKD 0.50 (USD0.06) per plastic shopping bag , was demThis is the first study done in an Asian metropolis on the community practice of health and environmental co-benefit behaviours. The study findings indicated a great variation in the frequency of practicing co-benefit behaviours among an Asian urban population. Although over 70% of respondents reported to use less packaging and disposable shopping bags daily, four behaviours were found to have never been practiced by more than half of the population, including bringing personal eating utensils when dining in restaurants or small eateries, showering less than five minutes every day, eating one vegetarian meal a week, and buying more organic food. More advocacy and policy implementation could be carried out to encourage the practice of these co-benefit behaviours, since these behaviours provide benefits for both health and the environment.Overall, women and older people were more inclined to practice co-benefit behaviours frequently in their daily lives, compared with men and younger people. Income and real estate ownership had mixed associations with co-benefit behaviours, since those more affluent were more likely to buy more organic food, separate household waste, and engage in active travel, but less likely to use less AC.The demographical trends found in this study are useful for targeted promotion, especially by multidisciplinary stakeholders of the health sector, government and other civil society organizations in the community. Policy makers should incorporate concepts of co-benefits into account to ensure an optimal outcome, not only in terms of climate change and the environment, but also for the health of the population. In addition, this study provides a baseline of behaviour frequency, useful for further research on practice of co-benefit behaviours. Future research should examine if other factors  might be associated with the uptake of these co-benefit behaviours. Focus group study on community subgroups will also help to provide qualitative based insights to examine the potential enablers and barriers for promotion of related health programs, policies and strategies. To maximize limited resources for supporting both health protection and environmental sustainability, improving our understanding of causal pathways and predictors that might promote health and environmental co-benefit behaviours should also be a high priority for further research in the coming decades.S1 File(PDF)Click here for additional data file."}
+{"text": "We evaluated whether the simultaneous inhibition of GLUT-1 and HIF-1\u03b1 expression improved the radiosensitivity of laryngeal carcinoma. We explored whether the expression of HIF-1\u03b1 and GLUT-1 was correlated with 2\u2032-deoxy-2\u2019-[18F]fluoro-D-glucose (in vivo model was applied by subcutaneously injecting Hep-2 (2 \u00d7 107/mL \u00d7 0.2 mL) and Tu212 cells (2 \u00d7 107/mL \u00d7 0.2 mL) into nude mice. The effects of HIF-1\u03b1 antisense oligodeoxynucleotides (AS-ODNs) (100 \u03bcg) and GLUT-1 AS-ODNs (100 \u03bcg) on the radiosensitivity of laryngeal carcinoma were assessed by tumor volume and weight, microvessel density (MVD), apoptosis index (AI) and necrosis in vivo based on a full factorial (23) design. 18F-FDG-PET/CT was taken before and after the treatment of xenografts. The relationships between HIF-1\u03b1 and GLUT-1 expression and 18F-FDG uptake in xenografts were estimated and the value of 18F-FDG-PET/CT was assessed after treating the xenografts.To verify the above hypotheses, an p < 0.05) and in Tu212 xenografts 12 days after treatment (p < 0.001). Standardized uptake value ( SUVmaxT/N) did not show a statistically significant correlation with GLUT1 and HIF-1\u03b1 expression and therapeutic effect .10 Gy X-ray irradiation decreased the weight of Hep-2 xenografts 8 and 12 days after treatment, and the weights of Tu212 xenografts 8 days after treatment. GLUT-1 AS-ODNs decreased the weight of Tu212 xenografts 12 days after treatment. There was a synergistic interaction among the three treatments  in increasing apoptosis, decreasing MVD, and increasing necrosis in Hep-2 xenografts 8 days after treatment (18F-FDG-PET/CT wasn't useful in evaluating the therapeutic effect on laryngeal cancer in this animal study.Simultaneous inhibition of HIF-1\u03b1 and GLUT-1 expression might increase the radiosensitivity of laryngeal carcinoma, decreasing MVD, and promoting apoptosis and necrosis.  Although promising therapeutic strategies have been described, the poor overall survival rate of patients with laryngeal carcinoma remains unchanged . One posin vitro and in vivo  fluorothymidine (18F-FLT) may better reflect the proliferative capacity of tumor cells and may be useful in evaluating the therapeutic response  \u00d7 BW, where RTA is the measured radiotracer tissue activity (mCi), RID is the radiotracer injected dose (mCi), and BW is the mouse body weight (g). The maximum SUV (SUVmax) of the tumor (SUVmaxT) and opposite normal subcutaneous tissue (SUVmaxN) were recorded. SUVmaxT/N=SUVmaxT/SUVmaxN, SUVmax T/N on day 0, day8 and day 12 were referred as SUVmax T/N0, SUVmax T/N1, SUVmax T/N2, respectively. The change in 18F-FDG uptake for each tumor was determined using the following equations:\u0394SUVmaxT/N1=(SUVmaxT/N1-SUVmaxT/N0)/SUVmaxT/N0, \u0394SUVmaxT/N2=(SUVmaxT/N2-SUVmaxT/N0)/SUVmaxT/N0, \u0394SUVmax T/N12= (SUVmax T/N2- SUVmax T/N1)/ SUVmax T/N1.2O (RNase free) was added up to 14.5 \u03bcL. Reactions were incubated at 70\u00b0C for 5 min and kept on ice after centrifuging briefly, and then 5 \u00d7 RT buffer (4 \u03bcl), HRP(RRI)/RNase inhibitor (0.5 \u03bcl), and M-MLV (1 \u03bcL) were added. After gentle mixing, the tubes were incubated at 42\u00b0C for 60 min, 95\u00b0C for 5 min. The PCR reagents included 10 \u03bcL 2\u00d7 real-time PCR master mix (SYBR\u00ae-Green), 2 \u03bcL dNTP (2.5 mM), 0.25 \u03bcL Ex Taq, 2.5 \u03bcL 10 \u00d7 Ex Taq E buffer, 1 \u03bcL cDNA, 9.25 \u03bcL ddH2O, in a total volume of 25 \u03bcL. The PCR primers used were as follows:Tumor tissue homogenates were collected and added Trizol according to the manufacturer's protocol, RNA was isolated and reverse transcribed. First,4.2 \u03bcg RNA, 2 \u03bcL Oligo(dT) (10 \u03bcM), 2 \u03bcL dNTP (2.5 mM) were mixed and ddHGAPDH sense, 5\u2032-TGTTGCCATCAATGACCCCTT-3\u2032, GAPDH antisense, 5\u2032-CTCCACGACGTACTCAGCG-3\u2032 (202 bp), GLUT-1 sense, 5\u2032-GTCAACACGGCCTTCACTG-3\u2032, GLUT-1 antisense, 5\u2032-GGTCATGAGTATGGCACAACC-3\u2032 (111 bp), HIF-1\u03b1 sense, 5\u2032-TTACAGCAGCCAGACGATCA-3\u2032, HIF-1\u03b1 antisense, 5\u2032-CCCTGCAGTAGGTTTCTGCT-3\u2032 (233 bp). To calculation differential gene expression, the 2\u2212\u0394\u0394Ct formula was used.Tumor tissue homogenates were lysed in Radio Immunoprecipitation Assay (RIPA) lysis solution and were separated by gel electrophoresis and transferred to membranes. The membranes were blocked with 5% non-fat dry milk in TBST and then soaked in the primary antibody buffer, overnight at 4\u00b0C , . The membranes were soaked in secondary antibody buffer and incubated for 2 h at room temperature. The proteins were visualized using enhanced chemiluminescence and exposed to X-ray film. Protein expression was analyzed semi-quantitatively using the Gel Logic analysis system .Sections were cut at 5 \u03bcm and placed on a Fisher Superfrost slide and dried for 1\u20132 h at room temperature. Slides were fixed with 4% polyoxymethylene and incubated with PBS containing 0.5% Triton X-100 successively to improve the penetration of the antibody. Primary antibody(CD34) was dropped on the coverslips and incubated overnight at 4\u00b0C according to the manufacturer's protocol. The slides were observed using a microscope, positive signals were pale brown or brown. To quantify the MVD, an area with maximum concentration of vessels was identified at low magnification (\u00d7100) and the three most intense fields were chosen for blood vessel counts at \u00d7200 magnification. The mean of the three counts was calculated.In Situ Cell Death Detection Kit-POD . Tissue sections were fixed using fixation solution and incubated with blocking solution. TUNEL reaction mixture was added according to the manufacturer's protocol. Staining was visualized under an optical microscope. Cells in which the nuclei were brown or brown-yellow were considered as positive. The total number of apoptotic cells at \u00d7400 magnification in five randomly selected fields was counted. The AI was calculated as the percentage of positively stained cells, AI=number of apoptotic cells \u00d7 100/total number of nucleated cells.Tumor sections were assessed using the 2O for 1 min, and stained with 1% eosin Y solution for 10\u201330s. Sections were dehydrated using two changes of 95% alcohol and two changes of 100% alcohol for 30s each. Each slice was assessed in three random microscopic fields (\u00d7400). The necrosis rate = necrosis area/total area in the field.Microscope slides with rehydrated tumor sections were prepared and dipped into a Coplin jar containing Mayer's hematoxylin for 30s, rinsed in H3 factorial design was adopted in this study concerning with the effects of formulation variables and their interactions on response variables to obtain the optimized formulation. Pearson's analysis was used to evaluate the relationships among GLUT-1 and HIF-1\u03b1 expression, 18F-FDG accumulation and therapeutic effects. P values < 0.05 were considered to indicate statistical significance.Data were analyzed using SPSS software (ver 22.0). A 2All procedures performed in studies involving mice were in accordance with in accordance with institutional guidelines of the First Affiliated Hospital, College of Medicine, Zhejiang University and with appropriate institutional certification.Informed consent was obtained from all individual participants included in the study."}
+{"text": "Rhizopus oryzae to produce fumaric acid in the presence of glycerol and/or various monosaccharides as carbon sources was examined for seventeen different strains of this fungi. These strains were tested in shake-flask cultures on media containing glycerol and seven different carbohydrates, including glucose, fructose, galactose, mannose, xylose, arabinose, and rhamnose. An interesting and applicationally useful phenomenon was observed. This work presents a new approach to the conventional microbiological method of producing fumaric acid. In the presence of 40\u00a0g/l glycerol as the sole carbon source, fumaric acid production reached 0.16\u20136.1\u00a0g/l after 192\u00a0h. When monosaccharides were used as a single carbon source, the maximum fumaric acid concentration was much higher; for example, 19.8\u00a0g/l was achieved when 40\u00a0g/l xylose was used. In the co-fermentation of xylose (40\u00a0g/l) and glycerol (20\u00a0g/l), post-culture broth contained approx. 28.0\u00a0g/l of fumaric acid with a process yield of 0.90\u00a0g/g after 168\u00a0h. The production of fumaric acid by Rhizopus oryzae was also increased in the dual presence of glycerol and monosaccharides like fructose, galactose, and mannose. However, results obtained on glucose-glycerol-based medium did not follow this trend, showing instead complete utilization of glucose with significant glycerol consumption, but unexpectedly low final amounts of fumaric acid and process yields. Understanding how Rhizopus oryzae utilize various carbon sources may provide alternative avenues of fumaric acid fermentation.The ability of  Therefore, it is important to find new applications for crude glycerol. It has been reported that glycerol is a good carbon source for many microorganisms, such as fungi and bacteria involved in the production of value-added fuels and chemicals . This glycerol utilization phenomenon can be seen from archaebacteria  is an important regulatory mechanism utilized by microorganisms, oftentimes appearing as sequential utilization of substrates present in the medium. Microorganisms can quickly adapt to utilization of a readily available and energy-efficient carbon source over relatively less easily accessible carbon sources. In industrial production, the occurrence of such a phenomenon significantly extends the length of the process and decreases the productivity Gawand .E. coli, Saccharomyces cerevisiae, and Zymomonas mobilis engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway .Data from the literature demonstrate that co\u2013fermentation of glucose with glycerol leads to better growth of lactobacilli , NITE Biological Research Center , Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH  and Institute of Agricultural and Food Biotechnology . Rhizopus sp. 29, 30 and 60 were isolated from cereal grains harvested in Lublin Province. All studied fungi were grown on potato dextrose agar (Difco) slants at 30\u00a0\u00b0C for 4\u00a0days and stored at 4\u00a0\u00b0C. To prepare inocula, agar plates containing sporulated fungi were washed with sterile water to obtain a spore suspension, adjusted to a concentration of 105 spores/ml.2PO4\u00a0g/l, MgSO4\u2009\u00d7\u20097H2O 0.5\u00a0g/l, (NH4)2SO4 1.4\u00a0g/l, CaCl2 0.3\u00a0g/l and 0.5\u00a0ml of microelements solution  and distilled water added to make a final volume of 1 l. All media were sterilized by autoclaving at 121\u00a0\u00b0C. Carbohydrates  were prepared and sterilized separately, then added to the medium. 1\u00a0g of CaCO3 was added to glass tubes and sterilized in a drying oven at 150\u00a0\u00b0C for 3\u00a0h (to avoid contamination when added separately to each flask).The culture medium contained: carbohydrates 40\u00a0g/l or carbohydrates 40\u00a0g/l and glycerol 20\u00a0g/l, KH3 was added to cultures when the pH level was below 4.5 (measured after 24 and 48\u00a0h of culture).10\u00a0ml spores were inoculated into 100-ml Erlenmeyer flasks containing 40\u00a0ml of culture medium. Cultivation was performed at 35\u00a0\u00b0C, 200\u00a0rpm in rotary shakers  for 5\u00a0days (sole carbohydrates), 7\u00a0days (carbohydrates with glycerol) or 8\u00a0days (glycerol). 2%\u00a0(g/v) CaCO2SO4 to neutralize excess CaCO3. Next, the post-culture broth was sterilized at 100\u00a0\u00b0C for 10\u00a0min. Then samples were collected and centrifuged at 10,000\u00a0rpm for 10\u00a0min. The supernatant was analyzed by high performance liquid chromatography (HPLC). Fumaric acid and carbohydrates were determined and quantified by HPLC using Aminex HPX87H organic acid column coupled to UV\u2013Vis diode array  at 210\u00a0nm and a refractive index detector . The column was eluted at 65\u00a0\u00b0C with 5\u00a0mM H2SO4, at a flow rate of 0.5\u00a0ml/min. Fumaric acid and carbohydrate standard solutions of known concentrations were used for calibration.The final culture broth was acidified with 1.5\u00a0M HAll experiments were performed in triplicate and the means are indicated in the text. The yield of fumaric acid (g/g) is expressed as the amount of product synthesized divided by the amount of carbohydrate consumed by fungi.Rhizopus sp. strains/isolates were screened for their growth on media containing different pentoses/hexoses or glycerol as sole carbon source. The highest production of fumaric acid and the process yield was observed for six strains after culture in the medium containing 40\u00a0g/l of xylose (Table\u00a0d-mannose was the carbon source (15.0\u00a0g/l and 0.38\u00a0g/g). Compared to d-xylose and d-mannose, fumaric acid production on d-glucose and d-fructose for all studied fungi were significantly lower. Among strains tested on medium containing galactose, markedly higher concentrations of fumaric acid (14.8\u00a0g/l) and its yield (0.37\u00a0g/g) were found in the filtrate after culture of R. oryzae NRRL-1526  was obtained in the culture of Rhizopus oryzae strains with xylose and glycerol co-fermentation compared to the culture with xylose alone. Moreover, in the presence of fructose, higher glycerol utilization was observed than for other saccharides after the culture of the majority of Rhizopus oryzae strains.Thus far, only a few works have been published on the production of fumaric acid by It is possible that glycerol as a co-substrate may also play a protective role under stress conditions caused by high initial concentrations of the substrates and/or product. This mechanism is common amongst fungi. Counteracting the stress caused by dehydration consists of polyols accumulation, primarily glycerol, in the outer layers of fungal cell membranes, in order to protect the interior from environmental factors that can stun the action of essential enzymes (Blomberg and Adler Rhizopus (Liu et al. Rhizopus species metabolism under specific co-utilization conditions. Promising results of fumaric acid production give an opportunity for microbiological production to assist or even replace the chemical synthesis of this acid.Effective glycerol utilization observed in some cases of the co-fermentation strategy in this study can be explained by the fact that the fungus has time to adapt its metabolism to consume a second, more complicated, source of carbon like glycerol is. The acceleration of monosaccharide consumption, even without the utilization of glycerol from the co-fermentation media, seems to be a very positive aspect of the strategy suggested in this paper. Although the transportation mechanism of glycerol molecules into fungal cells had already been described (Fakas et al."}
+{"text": "Optical means for modulating and monitoring neuronal activity, have provided substantial insights to neurophysiology and toward our understanding of how the brain works. Optogenetic actuators, calcium or voltage imaging probes and other molecular tools, combined with advanced microscopies have allowed an \u201call-optical\u201d readout and modulation of neural circuits. Completion of this remarkable work is evolving toward a three-dimensional (3D) manipulation of neural ensembles at a high spatiotemporal resolution. Recently, original optical methods have been proposed for both activating and monitoring neurons in a 3D space, mainly through optogenetic compounds. Here, we review these methods and anticipate possible combinations among them. Controlling and monitoring neuronal activity with a light has become a common practice in many neurobiological studies, throughout the last decade. The continuously expanding toolbox of molecular probes that activate/inhibit  or imagein vivo by light-stimulation at near-infrared to minimize scattering effects and optimize spatial resolution via non-linear multiphoton absorption processes. Ideally, these studies also demand three-dimensional (3D) accessibility both for activation and imaging at physiological time scales . 3D imaging approaches enable the use of complementary strategies to access volumes extending up to a few hundred \u03bcms in the axial direction, by proposing the use of piezo scanners to scan the objectives in specific trajectories  . Althougtemporal precision in optogenetic activation, i.e., the degree of reproducibility of the occurrence timing of a photo-evoked AP   Figure  and tempe Figure . Minimize Figure  can easie Figure . Later se Figure .With regards to 3D spatial resolution, scanning methods using 3D-CGH show an intrinsically good spatial resolution, thanks to the small spot size of the excitation beam (close to diffraction limit). Nevertheless, resolution can dramatically decrease when using intensities close to the saturation levels for the opsin, which results in out-of-focus excitation . ParalleHere we review the methods proposed so far for 3D photoactivation and present the possibilities for combination with 3D imaging modalities, to establish precise and flexible microscopy methods for all-optical manipulation of neural circuits. The methods we present here have mostly been developed for optogenetic photostimulation but in principle, any other photoactivation technique, such as uncaging of caged neurotransmitters, or activation of photoactivable proteins, could benefit from them.2+ imaging  uncaging of MNI-glutamate with multiple beamlets generated with a diffractive optical element, or one single beam multiplexed in time, and 2P Ca imaging . Similarlutamate . In , or beams created with more flexible light-patterning methods such as CGH, generalized phase contrast (GPC) or amplitude modulation. As already mentioned, the limit of extended light patterns is the deterioration of the axial confinement, an issue that can be solved by using temporal focusing. Common experimental configurations of TF make use of a diffraction grating in a plane conjugate to the focal plane of the microscope objective (image formation plane), separating the spectral frequencies of the laser femtosecond pulses (dispersion of different wavelengths in different angles) . In othe3 with the axial confinement varying from 5 \u03bcm Full Width Half Maximum (FWHM) at the center of the field of excitation (FOE) to 10 \u03bcm at the edges of it, tested with spots of 20 \u03bcm in diameter. The number of regions equal to the number of planes to be addressed. The system was used for selective 2P 3D photoconversion of the Kaede protein (2 depending the illumination duration) and for 2D optogenetic activation of ChR2 in zebrafish spinal cord neurons co-expressing GCaMP5G (excitation at 900 nm with 0.6 mW/\u03bcm2). Monitoring of Ca2+ traces in that case was performed with visible illumination and two-color HiLo imaging (2). protein  in the b imaging   . Althoug3.   .3 and an average axial PSF of 11 \u03bcm FWHM. The theoretical FOE for the optical parameters they used was 750 \u00d7 750 \u00d7 990 \u03bcm3, but was experimentally limited by the size of the optics used.Higher flexibility and better axial resolution was demonstrated by Accanto et al. who presented a system for multiplexed temporally focused-light shaping (MTF-light shaping), where the beam-shaping part was either 2D-CGH or GPC  Figure . For MTF3), as expected for TF-GPC patterns  and to photoconvert Kaede in the zebrafish larva hindbrain . Parallel illumination of neurons allowed fast photoconversion in both cases, with minimal photoactivation of untargeted neighboring cells. Especially in the case of paGCaMP, neuronal processes of the targeted cells could be clearly distinguished from the background, allowing the possibility to precisely track neuronal morphology (MTF-CGH was used in a multi-cell excitation of photoactivatable GCaMP (paGCaMP) in the central nervous system of the drosophila larvae  .Although the use of TF in neuronal photoactivation with parallel methods has offered the possibility to locally confine the excitation volume, such as to preserve single-cell resolution, this might not be necessary for low-scattering samples, excitation of small size cells or sparse staining. In that case, 3D-CGH alone can be used for the projection of extended light patterns in different planes . Thus 2Piameter) . MoreoveFinally, as a general comment we should note that 3D-TF methods using CGH for multiplexing the excitation spot, can produce powerful experimental configurations in volumetric FOEs, with the quality of all spots being the same across the whole FOE, since the spots multiplexed are replicas of the same single original spot. However, for volumes reaching mm-range, care should be taken to homogenize the excitation properties of the projected spots, by taking into account factors such as scattering with increasing depth, SLM-diffraction-efficiency corrections, optical aberrations due to the large defocusing of spots (objective used at its limits) or projection near the borders of the FOE, and spectral aberrations for TF occurring by cropping spectral frequencies in the optics when using large defocus .Scanning methods are alternative approaches for neuronal stimulation and have been widely used in 2P optogenetics , mainly 2, since the surface of the illumination spot in that case is about 1 \u03bcm2, illumination for 2.8 s with 175 continuously repeated spirals, each lasting \u223c16 ms).Studies using hybrid methods in all-optical configurations have so far used C1V1 as optogenetic actuator excited at 1040 nm . The autAn estimate of the maximum possible number of targets to address with each approach in the framework of an all-optical experiment, presumes knowledge of the total light losses of an optical system from the laser source to the objective output, which can significantly vary from one system to another. Moreover, the power necessary per target used, can vary according to opsin type, expression level, cell health and activation depth. In general, power losses for parallel illumination methods mainly consist of losses on the SLM(s) and the diffraction grating used for TF. For hybrid methods losses are approximately 2\u20133 times less than those of parallel ones, since they are mainly due to the use of a single SLM. It has also been reported that parallel approaches need about twice the power used by a spiral scanned beam, to induce a neuronal response with the same properties in both cases . Thus, i2 independently on the opsin type, see also  than scanning approaches (2) focused beams of low total average power   the average rise was 1 K, while for a focused beam in a spiral trajectory  the mean temperature rise was <0.5 K, and the local rise at the center of the spiral was again \u223c1 K . Otherwise, the heat load starts to significantly increase locally  . Specifiain \u223c1 K . For mul0 \u03bcm2/ms , and t t locally . Moreove locally .Notably, the considerations of the study by In order to combine photostimulation and functional imaging over large neuronal populations in extended volumes, it is necessary to elaborate strategies to decouple and independently control the photostimulation and the imaging plane. An exhaustive presentation of the existing imaging techniques have recently been presented in literature . Here weAdoption of approaches for 3D imaging, involving fast mechanical axial movements of the objective lens with piezoelectric positioners , in comblens-effect in an upstream location of the imaging path, possibly in a plane conjugated to the objective back aperture to obtain a telecentric system. Clearly, the control of laser divergence must be fast enough to be compatible with functional imaging rates. Few technologies are commercially available for high-speed focus control through lenses. They are based either on the curvature change of a flexible-membrane electrically controlled lens   resulting in high-rate volume imaging ranging between 14 Hz (375 \u00d7 112 \u00d7 130 \u03bcm3) and 56 Hz (60 \u00d7 4 \u00d7 30 \u03bcm3).Strategies involving the fast repositioning of the imaging focus by modulating the imaging beam divergence Figure , appear ns; ETL)  or on ulns; ETL) . ETLs hans; ETL) . They hans; ETL)  and on fns; ETL) . In TAG ns; ETL) . Volume in vivo on a CCD camera  and z axial displacement (determined by the amount of chirp) to the illumination beam. Being completely inertia-free, AOD systems can achieve very short commutation (24.5 \u03bcs for 3D random access) and dwell (0.05\u20131.0 \u03bcs) times , it is possible to impress a precise s) times . This mas) times  the auth example , researcp to 75%  and requp to 75% . Howeverp to 75% .Remote focusing is another approach enabling fast sequential imaging of axially separated planes. It allows remote axial shifting the imaging beam by integrating a classical raster scanning system with an axial scan unit, which comprises of an objective lens and a lightweight mirror , 2012. Sad hoc read-out demultiplexing strategies are adopted, to distinguish the signal coming from those planes. One possible strategy involves temporally multiplexed beams. Pulsed beams can be sent at different foci with different time delays. If the delays are superior to the fluorescence lifetime decay (in the range of few ns) and inferior to the inter-pulse interval, the fluorescence signal originating from different locations, can be distinguished by temporally demultiplexing the detected signal  imaging, where the illumination PSF elongates axially . Since so 160 \u03bcm .a priori information of the origin of the fluorescence signal, through targeted excitation with CGH spots, can remove any ambiguity arising from imaging unknown objects with extended axial features . Imaging can then be performed with a CCD and computational tools can be used to recover image information over the entire extended depth of field, as in the case of the elongated excitation PSF. Researchers have shown that the use of cubic phase masks in such configurations, in combination with CGH-based target illumination, allows for simultaneous imaging of fluorescence signals arising from different 3D targeted points . Moreovefeatures . Here, tin vivo in the brains of mammals   Figure . A serie mammals . If coupFinally, light-sheet microscopy (LSM) represents another volumetric imaging approach Figure  particulFrom this overview of the methods for 3D photoactivation and imaging, it is evident that these domains have tremendously advanced over the last few years. However, the combination of all-optical approaches is so far limited to use 2P scanning imaging modalities with an ETL , which iNevertheless, the first steps have now been completed and we are entering an era where there will be an increasing demand for high-performance all-optical methods to tackle more complex biological questions. This will certainly prompt further developments for all-optical strategies in large excitation volumes and multi-area microscopes, where it will be possible, for instance, to activate a population of neurons in one area while monitoring the effects in another area of the brain . FurtherEP conceived and organized the structure of the manuscript. EP and ER wrote the manuscript and prepared the figures. VE revised and contributed in writing the manuscript.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. The handling Editor declared a past co-authorship with the authors."}
+{"text": "Dear Editor,A 34-year-old female patient presented to the breast diagnostic clinic with a palpablenodule in the lower outer quadrant of the left breast. Ultrasound showed a solid,hypoechoic, well-circumscribed nodule, measuring 12 \u00d7 8 mm, in the lower outerquadrant of the left breast . The nod,2.Tubular adenoma of the breast is a rare benign epithelial tumor of the breast that hasnot been widely studied; the World Health Organization defines it as a \u201cbenign, usuallyround, nodules formed by a compact proliferation of tubular structures composed oftypical epithelial and myoepithelial cell layers\u201d. Tubular adenoma accounts for 0.13-1.7%of all benign neoplasms of the breast. Although the size of the tumor ranges from 1 cm to 7.5 cm, itrarely exceeds 5 cm. The vastmajority of cases are in young women, and the disease is much more rare inpostmenopausal women, 90% of the patients being under 40 years of age .Nevertheless, there have been reports of its occurrence in males. It has not been foundto be associated with oral contraceptive use or pregnancy,5. It isconsidered a variant of fibroadenoma, appearing in the same clinical context and withoverlapping imaging characteristics.It can be difficult to make the histological differentiation between tubular adenoma andfibroadenoma if the tubular adenoma has a relatively abundant stromal component or thefibroadenoma shows significant proliferation of small ducts.Although four cases of malignant transformation of tubular adenoma have been reported inthe literature, studies indicate that there is no high risk for carcinoma. On mammographyand ultrasound, these tumors have the appearance of noncalcifiedfibroadenomas. Onmammography, the lesions typically appear as well-circumscribed nodules, with noevidence of calcifications. However, in older patients, punctate or irregularcalcifications can be observed, findings that justify a biopsy to exclude malignantneoplasm of the breast. Occasionally, mammography shows lesions with ill-definedmargins. On ultrasound, the tumors are generally described as hypoechoic,well-circumscribed nodules. Noncalcified tubular adenomas generally have a relativelyhomogeneous internal texture and may have posterior acoustic reinforcement. Other differential diagnoses thatshould be included are ductal adenomas, lactating adenoma, gestational hyperplasia, andductal carcinoma. Sengupta etal., analyzing 32 confirmedcases of tubular adenoma, concluded that, although radiological and cytological studiescan distinguish between benign and malignant lesions, the final diagnosis depends on thehistopathology.Clinically, tubular adenomas of the breast can be asymptomatic, occasionally beingdetected on mammography or physical examination as a palpable nodule that graduallyincreases in size"}
+{"text": "Rhipicephalus sanguineussensu lato. By developing a multiplex allele-specific PCR assay targeting a fragment of the 12S rRNA ribosomal region of the mtDNA, we showed the occurrence of paternal leakage and mtDNA heteroplasmy in R. sanguineuss.l. ticks originated from experimental crosses, as well as in individuals collected from the field. Our results add a new evidence of paternal leakage in arthropods and document for the first time this phenomenon in ticks. Furthermore, they suggest the importance of using allele-specific assays when searching for paternal leakage and/or heteroplasmy, as standard sequencing methods may fail to detect the rare mtDNA molecules.Paternal leakage of mitochondrial DNA (mtDNA) and heteroplasmy have been recently described in several animal species. In arthropods, by searching in the Scopus database, we found only 23 documented cases of paternal leakage. Therefore, although arthropods represent a large fraction of animal biodiversity, this phenomenon has been investigated only in a paucity of species in this phylum, thus preventing a reliable estimate of its frequency. Here, we investigated the occurrence of paternal leakage and mtDNA heteroplasmy in ticks belonging to one of the most significant tick species complexes, the so-called  A fragment of the gene encoding for the cytochrome oxidase subunit 1 (cox1) has been even used for the barcoding of animal species5. The success of mtDNA as a molecular marker has been associated to some features generally attributed to it, such as high mutation rate, maternal inheritance and absence of recombination1.Mitochondrial DNA (mtDNA) has long been considered an optimal marker in population genetics, phylogenetics and phylogeographic studies for both vertebrates and invertebrates10. However, the transmission of the male parent\u2019s mitochondria to the offspring  has been observed in an increasing number of taxa11. An outcome of paternal leakage is a state of heteroplasmy in the progeny, where both paternal and maternal mtDNA are present within an individual10. Cases of heteroplasmy due to paternal leakage have been revealed in several animals, such as fishes, reptiles, birds and mammals11, which is calling into question the assumption of strict maternal inheritance. Notably, mtDNA heteroplasmy could be higher than currently recognized, since the commonly used techniques, such as standard PCR-amplification and Sanger sequencing, are not always able to reveal mtDNA molecules occurring at low frequencies12. Furthemore, mtDNA leakage might be more frequent than currently observed because it is only detectable when the parental mtDNA genomes are so different to be recognized. As consequence, although it appears to be more common in interspecific crosses, paternal leakage may be common after crosses between conspecific individuals belonging to genetically divergent lineages13. Undoubtedly, additional studies about the frequency of paternal leakage among different species would be valuable, as this phenomenon could eventually affect the inferences about the evolutionary history of species or populations based on mtDNA data14.Maternal inheritance of mtDNA has been deemed as a rule in animal species and several stochastic and deterministic molecular mechanisms preventing the transmission of paternal mtDNA to the zygote have been described19,\u00a0to date the occurrence of paternal leakage and mtDNA heteroplasmy has been documented only in a paucity of species . Fontaine et al.13 reviewed the cases of paternal leakage in animals up to 2007 and revealed that only 11 cases were documented in arthropod species. By searching for \u201cpaternal leakage AND animals\u201d in the Scopus database since 2007, we found 31 papers and, among them, 10 new cases in arthropod species (up to 30th October 2018) , as crossbreeding experiments between two temperate lineages of R. sanguineuss.l., namely Rhipicephalus sp. I (R. sp. I) and Rhipicephalus sp. II , showed offspring individuals harbouring paternal mtDNA20. Here, we assessed whether paternal leakage and heteroplasmy occur in R. sanguineuss.l., by analysing R. sanguineus s.s\u00a0and R. sp. I individuals originated from experimental crosses20, as well as wild-caught ticks coming from the same areas where parental individuals used in the aforementioned crossbreeding experiments were collected. The occurrence of heteroplasmy in R. sanguineuss.l. ticks was screened by a newly developed multiplex allele-specific PCR assay (MAS-PCR) targeting a fragment of the 12S rRNA ribosomal region in the mtDNA.In this paper, we aimed to contribute to fill this gap of knowledge. In ticks, the occurrence of paternal leakage has been recently hypothesized in the brown dog ticks . The sensitivity of the MAS-PCR, assessed by amplifying serially diluted mixed DNA of both lineages at known concentrations, showed a detectable amount as low as 0.05\u2009ng   and three individuals showed both the 160 and 270\u2009bp bands   Fig.\u00a0. In the A) Table\u00a0.Table 21R. sp. I individuals analysed from southern Italy showed bands of both sizes (160\u2009bp and 270\u2009bp)  Table\u00a0.In all heteroplasmic individuals the two parental alleles were found in a similar ratio Fig.\u00a0. No bandR. sp. I haplotype_h4 KC243794.1, and the 160\u2009bp PCR product identical to R. sanguineus s.s. haplotype_h1KC243802.1).Sequencing of the PCR products from a subset of parental individuals , from all individuals with the paternal mtDNA type and from all heteroplasmic individuals, confirmed the genetic identity of ticks 11. Alternatively, it simply did not occur in this cross by chance or it could not be detected by our MAS-PCR assay. Interestingly, in the cross \u201c\u2640 R. sp. I\u2009\u00d7\u2009\u2642 R. sanguineus s.s.\u201d, three out of the F1 individuals analysed carried only paternal mtDNA, with no evidence of maternal mtDNA , and stochastic processes due to bottleneck effect or random assortment of parental mtDNA during the early developmental stages22. In R. sanguineuss.l. ticks, a role of the environmental factors, such as temperature can be excluded, as it was kept constant in our experiments. Considering that the inheritance of paternal mtDNA could impact not only on the evolution of the molecule but also affect species or population fitness24, specific studies addressing the individual fitness will allow us to assess the relative roles of selective and stochastic processes in the observed pattern.Paternal leakage was observed in the cross \u201c\u2640 NA Table\u00a0, Fig.\u00a03.R. sp. I individuals from Putignano. This result could suggest the possible occurrence of hybridization between R. sp. I and R. sanguineus s.s. in nature with subsequent paternal leakage-driven mtDNA heteroplasmy. Hybrid zones, where divergent genetic lineages meet and exchange genes31, have been often associated with paternal leakage and mtDNA heteroplasmy, as the molecular mechanisms preventing paternal mtDNA inheritance may not be efficient between hybridizing species or populations, due to the high genetic divergence between parental lineages11. Interestingly, sympatric areas have been reported between R. sp. I and R. sanguineus s.s. ticks, since both lineages were found in Serbia, Algeria, and in southern and central Italy34. In this paper, we analysed one population from southern Italy, that allowed us to detect the occurrence of mtDNA heteroplasmy in Rhipicephalus spp. ticks. A more intense sampling of the Italian populations, as well as of the other sympatric areas will allow us to estimate how frequent this phenomenon is in nature.The results from the crossbreeding experiments corroborated those obtained with wild-caught ticks, indeed, mtDNA heteroplasmy was found in 36. As far as R. sanguineuss.l. ticks, the finding of paternal leakage suggests that multiple approaches are recommended, such as the use of both nuclear and mitochondrial genetic markers and of specific assays to detect the potential occurrence of heteroplasmy.The occurrence of paternal leakage and mtDNA heteroplasmy may affect taxonomy and systematic inferences based on mtDNA data alone, because it may lead to misleading identification of individuals. This may be particularly relevant in areas where different species coexist, such as secondary contact zones, or areas of recent colonization. Furthermore, heteroplasmy can lead to recombination between heterologous mtDNA molecules, which, if unaccounted for, can affect the interpretation of phylogenetic reconstruction as well as the population demographic histories inferred by mtDNA data43. On the contrary, mtDNA heteroplasmy due to paternal leakage has been documented only in a paucity of arthropod taxa, and at our best knowledge, it has never been described in ticks to date. Our finding, therefore, represents the first evidence of this phenomenon in ticks and adds to the few examples of paternal leakage and mtDNA heteroplasmy described in arthropods45. Notably, as the commonly used PCR-Sanger sequencing approach can fail to detect mitochondrial heteroplasmy45, studies aimed to search for paternal leakage and mtDNA heteroplasmy in hybrid zones using specific assays are desirable to really appreciate how frequent they are in nature, as well as to assess their evolutionary relevance.MtDNA heteroplasmy due to mutation in the mitochondrial genome sequence has been described in different arthropod species, including ticks21. Reference sequences of the 12S rRNA gene fragment (347\u2009bp) of R. sp. I and R. sanguineus s.s. were used to design the primers for our MAS-PCR assay21. The software Primer3Plus, that identifies a list of possible primer pairs, and Beacon designer, that allows checking for cross homologies and template structures during the primer design, were used. We designed: i) a common forward primer for R. sp. I and R. sanguineus s.s. in a conserved region of the 12S gene ; ii) an allele-specific reverse primer for R. sp. I (270\u2009bp amplicon) ; iii) an allele-specific reverse primer for R. sanguineus s.s. (160\u2009bp amplicon)  . Negative controls containing all reagents but DNA were included in each PCR reaction to check for contaminations. PCR cycling conditions were as follow: 95\u2009\u00b0C for 5\u2009min followed by 34 cycles at 93\u2009\u00b0C for 1\u2009min, 55\u2009\u00b0C for 1\u2009min, 72\u2009\u00b0C for 1\u2009min 30\u2009s, and a single final step at 72\u2009\u00b0C for 10\u2009min. The PCR products were then separated by electrophoresis run on 2% agarose and visualized by staining with Gelred . The sizes of the DNA fragments were assessed using a 100\u2009bp DNA ladder  run on the same gel. The reproducibility of the MAS-PCR assay was tested by performing three technical replicates of each reaction. To further support the specificity of our approach, the PCR products, after gel purification by NucleoSpin gel and PCR Clean-up purification kit , were double strand sequenced (https://www.gatc-biotech.com).To test the specificity of the assay, we performed multiplex PCRs using as a template the genomic DNA of known R. sp. I and R. sanguineus s.s. was mixed at different ratios. A total of 5\u2009ng of DNA was used in each PCR reaction and the proportion of R. sp. I (or R. sanguineus s.s.) was serially diluted to 0.5, 0.1, 0.05 and 0.005\u2009ng to obtain ratios of R. sp. I vs R. sanguineus s.s. DNA of 1:10, 1:50, 1:100, 1:1000.To assess the sensitivity of the MAS-PCR assay, the DNA of et al.20 realized pure and crossed lines using R. sp. I individuals from Italy  and R. sanguineus s.s. individuals from Portugal (Faro)46. Briefly, ten unfed female and male ticks of the same lineage and of both lineages were placed on na\u00efve rabbits to originate four experimental crosses: i) \u2640 R. sp. I\u2009\u00d7\u2009\u2642 R. sp. I; ii) \u2640 R. sanguineus s.s.\u2009\u00d7\u2009\u2642 R. sanguineus s.s.; iii) \u2640 R. sp. I\u2009\u00d7\u2009\u2642 R. sanguineus s.s. and iv) \u2640 R. sanguineus s.s.\u2009\u00d7\u2009\u2642 R. sp. I20.In a previous study, Dantas-Torres In this paper, we developed a specific MAS-PCR assay and screened a total of 240 individuals from the above crossbreeding experiments, that included: all parental individuals  originating the crosses; forty F1 offspring from each cross, including larvae, nymphs, adult females and males.et al.20, quantified using Nanodrop and diluted with sterile water to have a final concentration of 5\u2009ng/\u00b5l. Genomic DNA (1\u2009\u00b5l) from each individual was used as template for MAS-PCR following the amplification protocol and conditions described above.Genomic DNA was extracted from single ticks following Dantas-Torres R. sp. I and R. sanguineus s.s.\u00a0were also analysed , whereas R. sanguineus s.s. ticks were collected from privately owned dogs living in Faro . All ticks were morphologically identified following Dantas-Torres et al.21. Genomic DNA was extracted from each individual and MAS-PCR assay was performed as described above. To check for consistency with MAS-PCR assay results a subset of parental and F1 ticks as well as wild-caught individuals were sequenced.Individuals from natural populations of ed Table\u00a0. A totalOriginal photos of the gels in Figures 2 and 3"}
+{"text": "Additionally, bilateral cochlea ablated animals were scanned. 3D image data were transferred into a stereotaxic standard space and evaluated using volume of interest (VOI) analyses and statistical parametric mapping (SPM). In normal hearing rats alongside the auditory pathway consistent activations of the nucleus cochlearis (NC), olivary complex (OC) and inferior colliculus (IC) were seen comparing stimuli with background. In this respect, no increased activation could be detected in the auditory cortex (AC), which even showed deactivation with white noise stimulation. Nevertheless, higher activity in the AC in normal hearing rats was observed for all 3 auditory conditions against the cochlea ablated status. Vice versa, in ablated status activity in the olfactory nucleus (ON) was higher compared to all auditory conditions in normal hearing rats. Our results indicate that activations can be demonstrated in normal hearing animals based on 18F-FDG PET in nuclei along the central auditory pathway with different types of noise stimuli. However, in the AC missing activation with respect to the background advises the need for more rigorous background noise attenuation for non-invasive reference conditions. Finally, our data suggest cross-modal activation of the olfactory system following cochlea ablation\u2013underlining, that 18F-FDG PET appears to be well suited to study plasticity in rat models for cochlear implantation.Activation studies with positron emission tomography (PET) in auditory implant users explained some of the mechanisms underlying the variability of achieved speech comprehension. Since future developments of auditory implants will include studies in rodents, we aimed to inversely translate functional PET imaging to rats. In normal hearing rats, activity in auditory and non-auditory regions was studied using  Auditory and visual stimuli as well as cochlea ablation and tinnitus condition have been investigated 3 and were used to validate coregistration.All rats were imaged using a hybrid Inveon PET/CT system . A dose of 18.2\u00b10.7 MBq of the radiotracer 18F-FDG image template [3 as given by the template. In line with clinical methods, the image values were normalized to the observed average whole brain activity for further analysis.For a regional statistical analysis on group level, brain regions were extracted using a VOI atlas for rats  adapted template  via corrtemplate . The ima18F-FDG templates bregma coordinates [max-values and the ratio of Tmax to T-value at p<0.001 for assessment of extent and peak height of differences between different auditory conditions.We evaluated the activities in VOIs of the nucleus cochlearis (NC), the olivary complex (OC), the inferior colliculus (IC), the medial geniculate body (MGB) and the auditory cortex (AC) as auditory target regions. The somatosensory cortex (SC), cerebellum (CB) and olfactory nucleus (ON) were used as non-auditory control regions. All VOIs were analyzed in individually paired t-tests of two respective auditory conditions for all animals. All positions of coronal slides are given with reference to the rdinates . AdditioThe used stimuli and applied reference condition determined which sub-regions of the auditory system revealed activation respectively. For individually analyzed animals activations were only observable in up to two regions, while group analyses revealed activations in up to 4 of 5 evaluated auditory regions.18F-FDG uptake could be visually assessed for RN(95dB) stimulation in IC and AC, while no distinct difference to other conditions was detected in MGB, NC or OC. In individual difference images of RN(95dB) and BG(55dB) which had the largest sound level difference in normal hearing animals in our experiment, visual activation evidences were limited to the IC (images not shown). Therefore group analyses were required. In individual images, higher normalized In Using BG(55dB) as a reference, stimulation with either RN(95dB) or WN(65dB) resulted into significant activation in IC, OC and NC. The highest activation by (15.6\u00b13.6) % occurred in the IC. No increase in activation could be seen with the BG(55dB) reference in the AC. Instead, stimulation by WN(65dB) induced a low but significant deactivation compared to BG(55dB) (-2.8\u00b11.6) %, which formally introduced an increased activation, if RN(95dB) is compared to WN(65dB). Moreover, MGB activation was only induced by RN(95dB) but not with WN(65dB) stimulation. In normal hearing animals (i.e. without cochlea ablation) neither activation nor deactivation could be observed in non-auditory regions .Employing ABL as reference condition, significant activations were observed with all other conditions (RN(95dB), WN(65dB) and BG(55dB)) in IC, OC and AC. Activations were considerably higher for all three regions\u2013e.g. (35.0\u00b13.8) % in the mean with RN(95dB) in IC\u2013in comparison to those obtained with BG(55dB) as reference (15.6\u00b13.6) %. Moreover, no activation was seen consistently in MGB\u2013and in NC activation was only observed with RN(95dB). With respect to non-auditory regions, activity in ON was significantly higher in ABL condition compared to all acoustic conditions in healthy rats .Corresponding to the comparisons carried out at a VOI level, pairwise voxel-based analyses were performed using SPM8.0. The results are given in max being 2.66 times of the Tp = 0.001 threshold. The nearly total coverage/activation of the IC due to RN(95dB) stimulation vs. BG(55dB) is illustrated in st column, 1st and 2nd row). Moreover, as in VOI analyses, SPM showed a deactivation of the AC under WN(65dB) stimulation compared to BG(55dB) (coverage 3%) and MGB activation induced by RN(95dB) but not with WN(65dB) stimulation. Bilateral deactivation in the AC (due to WN(65dB) stimulus against BG(55dB)) can also be spotted in rd column, 2nd row). Furthermore in line with VOI analyses, neither activation nor deactivation could be in SPM analyses for non-auditory regions  of normal hearing animals.With BG(55dB) as a reference, SPM revealed in healthy animals like VOI analyses for RN or WN as stimulation condition significant activations in IC, OC and NC. Up to 89% of the IC-VOI were covered with Tmax-fractions) with ABL compared to BG(55dB) reference\u2013e.g. for the IC and RN(95dB) stimulation 4.70 vs. 2.66.Employing ABL as reference condition in SPM analyses nearly identically to VOI analyses significant activations could be observed with all other conditions (RN(95dB), WN(65dB) and BG(55dB)) in IC, OC and AC. The only exception was a lack of activation in OC due to BG(55) condition. This can also be extracted from max-fractions -1.86 to -2.73). SPMs displayed in Moreover, SPM analyses with ABL reference did not reveal any activation in MGB or NC. With respect to non-auditory regions\u2014again in line with VOI analyses\u2014SPM detected significantly higher activity in ON in ABL condition compared to all acoustic conditions in healthy rats  and 100 dB (strongest stimulus) were applied. This type of stimulus is different compared to the pulsed rippled noise used as strongest stimulus in our study. We chose a lesser sound pressure level (95 dB) as prolonged 100 dB stimulation can induce noise trauma in rats [Using g et al.  employed in rats , 21 and  in rats , 23. Nev in rats .Different results with strongest stimuli were observed for AC with no change in our study (\u00b10%) and a significant decrease of more than -5% in the mean in Jang\u2019s study . Nonethe18F-FDG uptake phase has been demonstrated [On the other side, the lack of AC activation in rats might, in addition, have species-specific reasons. In humans, activation of the AC due to different and not specifically adaptation-avoiding stimuli presented during nstrated \u201328. Furtnstrated , 29, 30.nstrated . Vice venstrated , 29, 30.nstrated . In factnstrated , which instrated .14C-2-Deoxyglucose (2-DG) ex-vivo autoradiography [18F-FDG PET [18F-FDG uptake in AC and IC together with no significant change in MGB four weeks after ablation compared to measurements during background noise condition preoperatively correspond to the changes observed in these regions by Ahn et al. [Effects of cochlea ablation on activity in the central auditory pathway and cortex have been previously studied using iography  and 18F--FDG PET  performen et al. . In our n et al.  and Hsu n et al. , 35. Witn et al. . Furthern et al.  showed t18F-FDG PET is performed as an integrative measure over 30min, detection probability of cortical activation of small regions, such as single cells, or with low frequency of activation is only limited. Visualization of information transmission by sparse coding as discussed for the AC [18F-FDG-PET.As r the AC  might noFurthermore, use of standard VOI atlases in analyses might result in misalignment and low coverage due to the variability among animals even of the same breed. For small regions, this could cause loss of detected activations which are identifiable in SPM by visual assessment. In our study, the SPM results displayed in We decided to selected hypothesis-driven representative control regions\u2013ON and CB as non-cortical control and SC as cortical control\u2013instead of data-driven VOI regions.Even though constant acoustic, climatic and olfactory conditions have been maintained, a small possibility remains that changes in the olfactory system have been influenced by hidden changes in the environment over the duration from January to May for example due to seasonal clothing. We expect any hidden impact to be negligible compared to the effect caused by ablation.18F-FDG to image auditory system activations along nuclei of the central auditory pathway in normal hearing rats. For the strongest, non-adaptive stimulus  against background noise (55 dB) activations were consistently seen in NC, OC, MGB and IC with VOI and voxel-based analyses. A functional deactivation of the AC was measurable comparing a continuous white noise to both previous mentioned auditory conditions. Nevertheless, our data indicate, that sound deprivation to the greatest possible extent is necessary for the reference condition to allow for detection of increased activation in the AC even when a strong non-adaptive stimulus is used for stimulation. Furthermore, we found evidence for neuronal plasticity following continuous complete sound deprivation (cochlea ablation) in the form of increase (compensatory) activity in the olfactory system. The specificity of auditory and cross-modal compensatory activations is supported by the fact that no activations were detected in any reference non-auditory region . In summary, small animal 18F-FDG PET appears to be very promising to evaluate the pathophysiology of hearing loss in rats and the process of hearing restoration following auditory / cochlear implantation. Due to the minimal invasive procedures of PET imaging, the combinability of 18F-FDG imaging with other additional PET tracers, e.g. for different neurotransmitters [This study demonstrates the usefulness of small animal PET with smitters , furtherS1 TableResults of ANCOVA with the condition, the order of condition and animals as well as anatomical area as fixed effects\u2013uptake in the PET scan as dependent variable and animal as a random effect (performed using JMP10 software). ANCOVA was performed without the ablation data as stimulation conditions are only possible before ablation.(DOCX)Click here for additional data file."}
+{"text": "The aim of this work was to assess the effect of liraglutide on ectopic fat accumulation in individuals with type 2 diabetes mellitus.2) who were randomly assigned  to receive liraglutide 1.8\u00a0mg once daily or placebo for 26\u00a0weeks, added to standard care. Participants, study personnel and outcome assessors were blinded to treatment allocation. The secondary endpoints of visceral adipose tissue (VAT), abdominal subcutaneous adipose tissue (SAT) and epicardial fat were measured with MRI. Hepatic triacylglycerol content (HTGC) and myocardial triacylglycerol content (MTGC) were quantified with proton MR spectroscopy. Between-group differences (change from baseline) were tested for significance using ANCOVA. Mean differences with 95% CIs were reported.This study is a pre-specified subanalysis of the MAGNetic resonance Assessment of VICTOza efficacy in the Regression of cardiovascular dysfunction In type 2 diAbetes mellitus (MAGNA VICTORIA) study, with primary endpoints being the effects of liraglutide on left ventricular diastolic and systolic function. The MAGNA VICTORIA study was a single-centre, parallel-group trial in 50 individuals with type 2 diabetes mellitus (BMI >25\u00a0kg/mn\u2009=\u200923) vs placebo (n\u2009=\u200926) significantly reduced body weight . HbA1c declined in both groups without a significant treatment effect of liraglutide vs placebo . VAT did not change significantly between groups , while SAT was reduced by a significantly greater extent with liraglutide than with placebo . Epicardial fat did not change significantly between groups . Change in HTGC was not different between groups . MTGC was not different after treatment with liraglutide (1.5\u2009\u00b1\u20090.6% to 1.2\u2009\u00b1\u20090.6%) vs placebo (1.3\u2009\u00b1\u20090.5% to 1.2\u2009\u00b1\u20090.6%), with an estimated treatment effect of \u22120.1 %. There were no adjudicated serious adverse events.The trial was completed in 2016. Twenty-four participants were randomised to receive liraglutide and 26 to receive placebo. One patient in the liraglutide group withdrew consent before having received the study drug and was not included in the intention-to-treat analysis. Liraglutide .The online version of this article (10.1007/s00125-019-05021-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Insulin resistance and type 2 diabetes mellitus are hallmarked by excess fat storage in visceral adipose tissue (VAT), liver, skeletal muscle, myocardial tissue and epicardial fat . VAT is Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist (GLP-1RA) commonly used to achieve glycaemic control in individuals with type 2 diabetes. Liraglutide\u2019s actions include stimulation of glucose-dependent insulin release from beta cells, and promotion of satiety resulting in reduced energy intake and modest weight loss. Since modest weight loss, at least by energy restriction, is associated with a reduction in ectopic fat accumulation , liraglu1H-MRS) [Recent technical developments have enabled non-invasive direct quantification of ectopic fat depots in humans with high accuracy using MRI and proton magnetic resonance spectroscopy (1H-MRS) . Using t1H-MRS) . The priClinicalTrials.gov registered trial endpoints that have already been published, those reported in the present manuscript, and endpoints that will be published in future manuscripts. In short, the study aimed to include 50 participants (men and women) aged 18\u201369\u00a0years with BMI 25\u00a0kg/m2 or above and HbA1c level of 53\u201386\u00a0mmol/mol (7.0\u201310.0%) despite use of metformin, and/or sulfonylurea derivative (SUD) and/or insulin. Main exclusion criteria were as follows: use of other glucose-lowering therapy; renal, hepatic or cardiovascular disease; gastric bypass surgery; chronic pancreatitis or previous acute pancreatitis; pregnancy or lactation; and MRI contra-indications. The trial was approved by the local ethics committee and performed in accordance with the principles of the revised Declaration of Helsinki. Written informed consent was obtained from all participants before study. The trial was conducted at the Leiden University Medical Center (LUMC), Leiden, the Netherlands and was registered at ClinicalTrials.gov (registration no. NCT01761318).This study was part of the MAGNA VICTORIA study, which was an investigator-initiated randomised, double-blind, assessor-blinded, placebo-controlled, single-centre clinical trial with 26\u00a0weeks follow-up . ElectroIncluded participants were randomised  to receive liraglutide  or placebo (provided by Novo Nordisk). The study drug was up-titrated to 1.8\u00a0mg once daily from week 3 onwards. The dose was reduced if necessitated by adverse events. During the study, blood-glucose-lowering drugs were titrated according to clinical practice guidelines by means of dose adjustment of SUD and/or insulin.1c was measured using boronate affinity high-performance liquid chromatography  throughout the first part of the study, and changed to measurement using ion-exchange high-performance liquid chromatography  for subsequent measurements. HbA1c values assessed by the boronate affinity method were corrected on the basis of the correlation coefficient derived from a validation experiment that used data of 196 samples measured on both analysers. All other blood samples were processed and analysed as described previously [At study entry and at 26\u00a0weeks, blood examinations were performed after participants had fasted for at least 6\u00a0h. HbAeviously . Adipone1H-MRS protocol using a clinical 3 Tesla Ingenia whole-body MR system  at baseline and follow-up. Participants were scanned in the supine position after they had fasted for at least 6\u00a0h. The body coil was used for transmission, and reception was achieved with a 16-element anterior array and 12-element posterior array. A 3D breath-hold dual-echo mDIXON sequence of the abdomen was performed  with transverse slice orientation. Using MASS software , post-processing involved generation of three 10\u00a0mm transverse slices with 2\u00a0mm slice gap at the level of the fourth and fifth lumbar vertebral bodies. Semi-automated segmentation of VAT and abdominal SAT was depicted by threshold-based inclusion of fat, with manual correction. VAT and SAT were calculated as mean area of fat in three slices. 1H-MRS of the liver was performed using a 20\u2009\u00d7\u200920\u2009\u00d7\u200920\u00a0mm voxel of interest, which was localised using a Point Resolved Spectroscopy Sequence . The voxel was placed preferably in liver segment V, VI, VII or VIII. The position of the voxel at baseline was used to guide placement of the voxel at follow-up MRI in the same liver segment. Four signal averages were acquired without, and 32 with, water suppression using the Multiply Optimized Insensitive Suppression Train sequence. Spectra were acquired during free-breathing at end-expiration with pencil beam navigator-based respiratory triggering [1H-MRS of the heart was assessed as described previously [All participants underwent an MRI and iggering . 1H-MRS eviously . The 15\u2009eviously , before 1c, serum triacylglycerols, NEFA, total cholesterol, HDL-cholesterol, LDL-cholesterol, adiponectin and liver enzymes. Endpoints that were not predefined were paracardial and pericardial fat.We previously reported the primary endpoints of the MAGNA VICTORIA study that involved left ventricular diastolic and systolic function . The stu1c was performed. First, correlation between change in HbA1c and HTGC was estimated using Pearson\u2019s correlation. Subsequently, a stepwise multiple linear regression analysis was performed to assess the following: (1) unadjusted association of the independent variables change in HbA1c, sex, age, treatment group allocation and weight loss with the dependent variable change in HTGC; (2) adjusted association for the independent variables mentioned above. All statistical analyses were performed as described previously [p value of <0.05 statistically significant.Data are shown as mean \u00b1 SD when normally distributed, or as median (interquartile range) when not normally distributed. For all presented study endpoints, we performed an ANCOVA of between-group differences of change from baseline, with randomisation arm as fixed effect and baseline measurement of dependent variable as covariate. In addition, a sensitivity analysis was performed that also included sex and insulin use as covariates in the ANCOVA model, since randomisation was stratified by sex and insulin use. Mean changes from baseline \u00b1 SD are reported for within group changes, and estimated treatment effect with 95% CIs are displayed for between-group differences. In addition to these pre-specified analyses, an assessment of associations between observed change in HTGC and change in HbAeviously . We cons1c values. This resulted in decreased total use of SUDs and insulin in liraglutide-treated participants and an increase in placebo-treated participants. An overview of concomitant drug use was described previously [Participants were enrolled between December 2013 and September 2015, with the final participant visit taking place in March 2016. The trial flow chart was published previously [1c differences from baseline were not different between groups. In liraglutide-treated participants, HbA1c decreased from 66.7\u2009\u00b1\u200911.5\u00a0mmol/mol to 55.0\u2009\u00b1\u200913.2\u00a0mmol/mol (8.4\u2009\u00b1\u20091.1% to 7.3\u2009\u00b1\u20091.2%), and in placebo-treated participants HbA1c decreased from 64.7\u2009\u00b1\u200910.2\u00a0mmol/mol to 56.9\u2009\u00b1\u20096.9\u00a0mmol/mol (8.2\u2009\u00b1\u20091.0% to 7.5\u2009\u00b1\u20090.7%).Changes in anthropometric and laboratory measures are displayed in Table p\u2009=\u20090.43). 1H-MRS of the liver was technically not successful on two occasions , and one participant in the placebo group displayed a biologically implausible rise in HTGC from 1.7% at baseline to 39.5% at follow-up without changes in serum liver enzymes. This measurement was therefore excluded from analysis . 1H-MRS of the heart was successful on all but four occasions: one at baseline due to low signal-to-noise ratio and three at follow-up due to low signal-to-noise ratio or incorrect peak frequency. With regard to MTGC, there was also no significant treatment effect of liraglutide vs placebo. With regard to epicardial, paracardial and pericardial fat, between-group differences were not significant. None of the ectopic fat variables showed significant correlation with indices of left ventricular function that had been published previously [The results of the MRI and MRS analysis of ectopic fat are summarised in Table eviously  . Figure 1c and change in HTGC between baseline and follow-up. The regression line had an unadjusted slope of 0.28 . Stepwise multiple linear regression analysis revealed that, of the independent variables change in HbA1c, sex, age, treatment group allocation and weight loss, only change in HbA1c significantly correlated with change in HTGC (ESM Table 1c was 0.50 (p\u2009=\u20090.001).Reduction of HbAThis study shows that, compared with placebo, liraglutide reduced body weight and subcutaneous fat but not visceral fat, hepatic steatosis, myocardial steatosis, epicardial fat, paracardial fat or pericardial fat. Despite a significant 4\u00a0kg weight loss in liraglutide-treated participants over 6\u00a0months, there was no reduction of ectopic fat accumulation.In addition to blood-glucose lowering, liraglutide decreases energy intake and lowers body weight. In view of the preferential loss of VAT by modest weight loss induced by diet , one wouIt is interesting to speculate on the mechanism by which liraglutide could preferentially diminish SAT. First, it can be hypothesised that liraglutide directly affects the lipogenesis:lipolysis ratio in adipose tissue by binding to GLP-1Rs in adipocytes. However, studies using validated specific methods have not been able to detect the canonical GLP-1R in adipocytes , so othe1c goal <53\u00a0mmol/mol (7%). Therefore, our study is best compared with studies using active comparators against GLP-1RA treatment. In their open-label trial, Tang et al. treated individuals with type 2 diabetes with liraglutide vs insulin, resulting in comparable improvement in glycaemic control among treatment arms, and no between-group difference in liver fat fraction [p\u2009=\u20090.007) [1c reductions in these studies were comparable with those reported in our study, an important difference was that individuals already using insulin were excluded in these studies. Whether that explains the discrepancy with our study is unclear because randomised studies comparing the effect of add-on GLP-1RA on liver fat in insulin-treated individuals are currently lacking. In a single-arm study by Petit et al., 68 individuals with type 2 diabetes (21% using insulin at baseline) were treated with liraglutide 1.2\u00a0mg once daily, resulting in a 31% RR reduction of hepatic steatosis [n\u2009=\u200916) who underwent intensification of the glucose-lowering regimen with insulin and who showed a non-significant decline of liver fat fraction. Apart from the non-randomised design, the results of this study could have been influenced by the fact that patients also received instructions on healthy diet and exercise. The only study evaluating the effect of liraglutide on histological resolution of NASH was the Liraglutide Safety and Efficacy in Patients with Non-alcoholic steatohepatitis (LEAN) trial [1c in the liraglutide vs control group. In keeping with the hypothesis that reduction in HbA1c is associated with reduction in HTGC, we and others [1c reduction and HTGC reduction. One theory could be that improved glycaemic control directly reduced HTGC  . Yan et teatosis . This coN) trial . This pld others , 15, 31  protein ). Howeve1H-MRS of the heart. The abundance of intracellular triacylglycerols is associated with increased deposition of toxic lipids that interfere with cardiac energy metabolism and cell survival [Myocardial steatosis is characterised by an increased triacylglycerol content in cardiomyocytes, as assessed by survival . In indisurvival , 33. Revsurvival . Based osurvival , 35 and survival , liraglusurvival . These rsurvival .Excess epicardial fat accumulation is hypothesised to exert local effects by leading to secretion of inflammatory and metabolic peptides by tissues that might contribute to coronary artery disease. Epicardial adiposity has been linked to visceral obesity  and coroLiraglutide has been shown to have a safe cardiovascular endpoint profile, with less major cardiovascular events compared with placebo added to standard care . There aThe primary limitation of this study was that the presented outcome measures were not the primary outcomes of the MAGNA VICTORIA study. This could imply that the study was underpowered with regard to evaluation of the treatment effect on ectopic fat. In that regard, the 95% CIs of between-group changes from baseline are crucial to the interpretation of this study . In line2 and/or more severe hepatic steatosis.In conclusion, this pre-specified secondary study showed that liraglutide did not reduce ectopic fat accumulation in individuals with type 2 diabetes, compared with placebo treatment added to standard care. Tight glycaemic control in both treatment groups was associated with reduced hepatic steatosis, with no added effect of liraglutide. From a clinical perspective, weight loss caused by liraglutide therapy might not be crucial for its beneficial cardiovascular actions, wherein other mechanisms such as inflammation and lipid metabolism are probably involved . Future ESM(PDF 319 kb)"}
+{"text": "After application of AHSC, the soldier\u2019s wound closed in nine weeks with pliable, sensate skin. The patient retained function without contractures limiting ankle motion or adhesions limiting tendon gliding. The successful treatment of this complex war zone injury with AHSC has allowed the soldier to quickly participate in unrestricted physical therapy and is on a trajectory for near-term return to active duty.Extremity injuries are common in contemporary combat and have become more prevalent as fatality rates have dropped to historic lows. Traumatic extremity wounds, especially those sustained in theater, often present with exposed structures such as tendon, bone, and joint, preventing the use of split-thickness skin grafts (STSG) for coverage. Traditional reconstructive options for these complex wounds include skin substitute with delayed STSG, local flaps, debridement of tendons, pedicled distant flaps (such as cross-leg flap), free tissue transfer, and amputation. STSG, whether on top of skin substitutes or after tendon debridement, can result in contracture and functional limitations in the extremities. Flap reconstructions require prolonged procedures, hospital stays, and periods of immobility. As an alternative to traditional reconstructive options, an autologous homologous skin construct (AHSC) uses a small full-thickness elliptical skin harvest from the patient, which is sent to a biomedical manufacturing facility, processed into AHSC, and can be returned and applied to a wound bed as soon as 48 hours after harvest and used up to 14 days after harvest. We present in this case report the treatment of a 42 cm Advances in military medicine and the introduction of sophisticated armor for individuals and vehicles have significantly reduced the rate of battlefield case fatalities in the post-9/11 conflicts in Afghanistan and Iraq 9.4%) compared to World War II (19.1%) and Vietnam 15.8%) [% compare.8% % co. More brTraumatic extremity wounds, especially those sustained in a combat theater, are often complicated due to contamination and exposure of tendons, ligaments, and bone, which eliminates the feasibility of an early split-thickness skin graft (STSG). More complex reconstructions are typically needed for traumatic extremity wounds, such as free tissue transfer, but can result in outcomes that prevent warfighters from returning to duty. Complication rates for free tissue transfer are also high for the treatment of injuries sustained in combat environments due to an extensive zone of injury and in some cases limited available donor sites .TM, PolarityTE MD, Inc., Salt Lake City, UT) has shown promise in the treatment of complex wounds [An FDA-registered and commercially available autologous homologous skin construct  right dorsolateral ankle wound with multiple exposed extensor tendons.A 38-year-old Caucasian male with a past medical history significant for right plantar fasciitis suffered multiple lower extremity injuries following a motor vehicle rollover accident (MVA) while an active duty army non-commissioned officer serving in Iraq. Of note, he was an active, long-time cigarette smoker at the time of injury. Injuries to bilateral lower extremities were sustained from the MVA, including of the right lower leg, right foot and ankle, left knee, and left lower leg. Open joints were suspected in the right ankle and left knee. A left torus fibular fracture and a right malleolus fracture were confirmed on a plain radiograph. The patient underwent initial debridement and two-incision, four-compartment fasciotomies for compartment syndrome. Screening for traumatic brain injury was negative. Following stabilization and evacuation, first to Germany and then to the United States, the acute injuries were treated with serial debridements, delayed primary closures, and open reduction and internal fixation of fractures with plates and screws. Eventually the last remaining untreated injury was a 7 x 6 cm  one week post-injury. The patient was pessimistic about all traditional treatments options, such as STSG, cross-leg flap, and free-tissue transfer for both donor site morbidity and functional outcome reasons. His goal was to regain normal ankle, foot, and toe function with minimal downtime and a quick return to active duty. Following a comprehensive discussion of treatment options, he chose to pursue treatment with AHSC for coverage of the remaining exposed tendons and epithelial closure due to the minimal donor site impact (5 cm linear groin incision) and the potential for functionally normal skin.Approximately six weeks post-injury and five weeks following application of bilayer collagen-GAG matrix, a 5 x 2 cm elliptical harvest of full-thickness skin, including a small amount of subcutaneous fat, was taken from the right groin Figure  and shipAHSC was returned six days after harvest and applied evenly across the wound seven days after harvest Figure  followin2 wound, there was only one small 1 cm2  central area with mild thickening and scar formation, which did not result in functional limitations. The remainder of the wound was covered with pliable, sensate skin that had normal capillary refill. There was no restriction in tendon gliding  less desirable, as long-term contracture or tendon adhesions could impair ankle range of motion and require operative revision. Cross-leg flap was rejected as first-line reconstruction because of the need for prolonged bedrest, immobility, and need for coverage of the flap donor site.Free tissue transfer was considered, but disadvantages included a prolonged operation, need for specialized equipment and hospital support, intensive care unit (ICU) postoperative monitoring, a long hospital stay, and prolonged immobilization and non-weight-bearing. Free flaps in the lower extremity, especially with an extensive zone of injury from the talus up to the knee, have a relatively high failure rate . Even ifAmputation would have been considered an extreme option and considered only for catastrophic treatment failure, but certainly was a possibility given the constellation of compartment syndrome, bony injury, nerve injury, and implanted hardware.2 area of central mild scarring that caused no functional limitations, the regenerated skin is soft, supple, sensate, and with good vascularization. The patient, surgeon (O.N.J.), and medical care team were all extremely satisfied with the result achieved. After this experience using AHSC to treat a complex wound, we now have another tool in our reconstructive armamentarium. Although AHSC may not be the best choice for all wounds, we believe patient scenarios like the one in this case report may benefit from its low-risk profile and potential for return of normal function with two outpatient procedures.AHSC was chosen by the patient after comprehensive discussion, and in the view of the surgical team it provided a low-risk option that would not preclude the use of any traditional reconstructive options if it were to fail. Notably, both the full-thickness skin harvest and AHSC application were performed as outpatient procedures. The donor site was minimal and AHSC allowed for a 4.2-fold expansion of the harvested skin surface area. The main goal of treatment was expeditious return of function and ultimately returning the patient back to active duty, which is expected to occur by nine months post-injury after beginning unrestricted physical therapy following wound closure. Aside from the small 1 cmThe potential benefits of AHSC include minimal donor site morbidity, simple procedure, coverage of exposed structures, wound closure with functional skin that avoids contracture, contour matching to the surrounding wound bed, and fast return to pre-injury activity. These characteristics of AHSC make it a low rung on the reconstructive ladder.Military personnel deployed to a combat theater of operations sustain complex extremity wounds, both in battle and non-battle scenarios. Traditional treatments have certain limitations, due to patient factors, surgeon and facility support requirements, and post-treatment disability profile. In this case, AHSC was used to successfully treat a high-risk patient with a high-risk ankle injury. Stable wound closure, normal foot and ankle function, and hardware and tendon coverage were obtained. Additional studies are needed to characterize the effectiveness in treating combat trauma and measuring the potential contribution to readiness goals that AHSC may enable."}
+{"text": "Terrestrial animal communities are largely shaped by vegetation and climate. With climate also shaping vegetation, can we attribute animal patterns solely to climate? Our study observes ant community changes along climatic gradients  within different habitat types  on the Colorado Plateau in the southwestern United States. We sampled ants and vegetation along two elevational gradients spanning 1,132 m with average annual temperature and precipitation differences of 5.7\u00b0C and 645mm, respectively. We used regression analyses and structural equation modeling to compare the explanatory powers and effect sizes of climate and vegetation variables on ants. Climate variables had the strongest correlations and the largest effect sizes on ant communities, while vegetation composition, richness, and primary productivity had relatively small effects. Precipitation was the strongest predictor for most ant community metrics. Ant richness and abundance had a negative relationship with precipitation in forested habitats, and positive in open habitats. Our results show strong direct climate effects on ants with little or no effects of vegetation composition or primary productivity, but contrasting patterns between vegetation type  with precipitation. This indicates vegetation structure can modulate climate responses of ant communities. Our study demonstrates climate\u2010animal relationships may vary among vegetation types which can impact both findings from elevational studies and how communities will react to changes in climate. Animal communities are largely shaped by climate and vegetation. Comparing these effects on ant communities along an arid elevational gradient, we show that climate effects outweigh vegetation. In this arid system, we expected precipitation to have the strongest effects on ants.22.1https://www.sega.nau.edu/). Productivity and vegetation composition were distinctly different within seven of our 12 sites (located in pinyon/juniper and ponderosa life zones), with structurally complex and high\u2010biomass forests adjacent to open (meadow) habitats. In these seven sites , plots were paired as forested or open habitats. Single plots were established at the three cool desert sites which had only open habitat available, and two mixed\u2010conifer sites which had only forest habitat available, leading to a total of 19 plots (10 open and 9 forested). Plots were 30\u00a0\u00d7\u00a030\u00a0m (900\u00a0m2) in size and located at least 100\u00a0m from disturbed areas such as roads. Within each 900\u00a0m2 plot, five subplots were used for ant community sampling and vegetation cover measurements. These five (1\u00a0m2) subplots were located as follows: One in the center and four located halfway between the center subplot and the corners of the large plot such that all subplots were separated at least 10\u00a0m from one another and from the edge of the 900\u00a0m2 plot . PRISM data were downloaded at a spatial resolution of 800 meters and extracted using R version 3.2.3 (2015\u201012\u201010) \u00a9 2015 The r Foundation for Statistical Computing Platform: x86_64\u2010apple\u2010darwin13.4.0 (64\u2010bit) and the package \u201craster\u201d version 2.5\u20102 based on observed latitude and longitude of each site.Percent cover of herbaceous vegetation was estimated using a point intercept method with 25 points on a grid pattern within a 1\u00a0m2.4R)\u00a0=\u00a017.06) \u00a0=\u00a012.16), which then represented vegetation composition in regression and ordination analyses .To represent ant and vegetation composition, nonmetric multidimensional scaling (NMDS) ordination  in this case. All variables were checked for normality via Shapiro\u2010Wilk tests and transformed when necessary. To test explanatory variables of climate and vegetation, stepwise multiple regressions with backwards elimination (p\u00a0=\u00a0.05) were used on ant richness and abundance. Ordinations were used to examine ant composition relatedness between life zones and habitats, and to test correlations of climate, weather, and vegetation. To determine if the visual separation of groups (life zone/habitats) was significant between ant communities, a one\u2010factor permutational multivariate analysis of variance (perMANOVA) , weather (precipitation and temperature during sampling), and vegetation  were used as explanatory variables for ant community metrics . We used average  richness because of uneven sampling driven by a flooding event. However, average richness correlated with total richness (2.5amos 5.0 (2003 spss Inc.). For each endogenous variable, error terms are assigned to represent unexplained variance. The model suggested a correlation between the error terms of vegetation NDMS axis two and productivity which was added post hoc. The final models were evaluated with Joreskog's goodness\u2010of\u2010fit (GIF) and X2 tests. Contrary to most tests, a high p value indicates a good probability that a model fits the data and is thus desired. GIF\u00a0>\u00a00.95 are considered a good fit ]. One species (Monomorium cyaneum) and one species group (Solenopsis fugax group) were ubiquitous (Table Tapinoma sessile (r\u00a0=\u00a0.3), Dorymyrmex insanus (r\u00a0=\u00a0\u2212.32), Forelius mccooki (r\u00a0=\u00a0\u2212.31), F. pruinosus (r\u00a0=\u00a0\u2212.49), Pheidole bicarinata (r\u00a0=\u00a0\u2212.35), and Pheidole ceres (r\u00a0=\u00a0\u2212.43), while axis two correlated with Formica aserva (r\u00a0=\u00a0.42), Formica neogagates (r\u00a0=\u00a0.31), Camponotus modoc (r\u00a0=\u00a0\u2212.44), Pogonomyrmex rugosus (r\u00a0=\u00a0.38), Monomorium cyaneum (r\u00a0=\u00a0\u2212.30), and Crematogaster punctulata (r\u00a0=\u00a0\u2212.42). Therefore, the patterns of NMDS axis one and two are indicative of two different species sets.A total of 5,300 ants were collected from the two sampling periods in 2015, with 36 identifiable taxa [34 species including one morpho\u2010species, two species complexes, and one genus with multiple unidentified species .Vegetation, and vegetation\u2010mediated effects of climate, had weak effects on ants compared with direct climate effects  had contrasting relationships with precipitation. This indicates the influence of vegetation on ant communities along our elevational gradients is primarily through vegetative structure rather than plant composition or productivity. Our approach is correlative, and caution must be used when assigning causality. However, within our life zones and habitats, the model fit well, with strong explanatory variables for most ant community metrics. Our results are therefore indicative of how large\u2010scale climate and vegetation mechanisms shape animal communities along elevational gradients.Hypothesized mechanisms shaping elevational distributions of animals are largely climate and geography based, mainly considering vegetation a by\u2010product  which all responded differently to climate and vegetation. Community metrics typically respond differently to environmental variables ; data curation (lead); formal analysis (lead); investigation ; methodology ; visualization (lead); writing \u2013 original draft (lead); writing \u2013 review and editing (lead). Richard W. Hofstetter: Conceptualization ; data curation ; funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision ; writing \u2013 review and editing . Michael Remke: Conceptualization (supporting); formal analysis (supporting); methodology (supporting); writing \u2013 review and editing . Sneha Vissa: Visualization ; writing \u2013 original draft ; writing \u2013 review and editing . Karen A. Haubensak: Conceptualization ; data curation ; funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision ; writing \u2013 review and editing .Appendix S1Click here for additional data file."}
+{"text": "Synapses play a central role for the processing of information in the brain and have been analyzed in countless biochemical, electrophysiological, imaging, and computational studies. The functionality and plasticity of synapses are nevertheless still difficult to predict, and conflicting hypotheses have been proposed for many synaptic processes. In this review, we argue that the cause of these problems is a lack of understanding of the spatiotemporal dynamics of key synaptic components. Fortunately, a number of emerging imaging approaches, going beyond super-resolution, should be able to provide required protein positions in space at different points in time. Mathematical models can then integrate the resulting information to allow the prediction of the spatiotemporal dynamics. We argue that these models, to deal with the complexity of synaptic processes, need to be designed in a sufficiently abstract way. Taken together, we suggest that a well-designed combination of imaging and modelling approaches will result in a far more complete understanding of synaptic function than currently possible. Synaptic efficacy and plasticity are key determinants of all brain functions, and of the corresponding behavioral output. Conversely, aberrant synapse transmission is the cause of many neurological and psychiatric disorders. It is therefore not surprising that synapses have been the focus of substantial numbers of quantitative analyses, providing information on virtually all aspects of their structure and function. Nevertheless, we are still unable to understand synaptic function on a sufficient level of detail to predict it with reasonable precision. This problem is deepened by the fact that individual processes, such as synaptic vesicle exocytosis or endocytosis, can be explained by a multitude of hypotheses, all of which are typically plausible, and therefore could, in principle, fulfill the respective functions. Such hypotheses led to arguments that still persist, decades after their inception . HoMany types of quantitative synaptic analyses have been performed, from detailed electrophysiological investigations ,9, to imA subsequent analysis of the composition of the presynaptic bouton  providedThis final aspect, the dependence of endocytosis kinetics on stimulus strength, could be explained by an analysis of the copy numbers of the cofactors in the clathrin pathway. Most such cofactors are present at about 2000\u20134000 copies for the average brain synapse (obtained from mixed rat cortex and cerebellum synaptosomes), an amount that is only sufficient for the recycling of a few vesicles at one time, since many copies of each cofactor are needed for recycling one vesicle (please note that these average synapses contained some 400 synaptic vesicles). For example, clathrin forms during endocytosis a spherical lattice that contains up to ~300 copies of the clathrin heavy and light chains , implyinOverall, this overview confirms our suggestion that copy numbers, as well as locations, are an important issue in synaptic analyses, as we discuss in further detail below.A major problem with static information on protein copy numbers is that the used methods have a low temporal resolution, or can even measure the state of the system at only one point in time. By contrast, the composition of the synapse is likely to change rapidly, as proteins diffuse across the limited synaptic space, especially as vesicles move and are exo- and endocytosed . Howeve elegans ,23,24,48 elegans . This ev elegans ,50,51). Taken together, the discussed example shows that the consideration of quantitative knowledge of copy numbers and of mobility data of proteins, as well as the usage of mathematical models to integrate such data from different methods, considerably advances the understanding of the dynamics of synaptic processes. Much of how we understand synaptic function comes from our perception of the synapse organization, and of the arrangements of its proteins. However, in spite of almost 15 years of synaptic investigations by super-resolution microscopy ,52, subs2+ sensor synaptotagmin-1 has been proposed to form a ring-like arrangement at the interface between docked synaptic vesicles and the plasma membrane, which helps in modulating the exocytosis kinetics [To showcase this, in the following we turn again to synaptic vesicle proteins, as an example. Synaptic exocytosis needs to take place with sub-millisecond kinetics, for accurate neurotransmission. These kinetics are difficult to understand, because the SNARE proteins responsible for exocytosis \u201cintrinsically operate on a timescale of about a second\u201d . The solkinetics . Other vkinetics . Vesiclekinetics . The clukinetics ,55,56. TLow imaging resolution is not the only difficulty encountered here. Sample preparation and the identification of the molecules using different types of probes has long been a concern, and has only become more so over the last decade, as the improving microscopy precision implies that the tolerance for artifacts is increasingly lower. The large majority of microscopy studies rely on chemically fixed samples, in which particular proteins are then revealed by the use of immunolabeling probes . The typical protocol involves fixation using an aldehyde solution, with the 4% paraformaldehyde (PFA) being the preferred condition, for periods ranging from 10 to 60 min. The sample is then permeabilized using a detergent , to enable the penetration of antibodies. The sample is then incubated with primary antibodies, to reveal the protein of interest, and then with secondary antibodies, to reveal the primary ones. Every one of these steps suffers from substantial difficulties. First, PFA fixation results in changes to the morphology of the samples and it induces the mislocalization of many target proteins, in an unpredictable fashion, due to the fact that it penetrates into the samples with difficulty and fixes them slowly . Following fixation, a permeabilization step is required for allowing epitope recognition by the antibodies. Various detergents are used to disrupt the cell membranes, removing lipids from the membranes, and causing hydrophobic patches to be exposed. This results in membrane collapse on the nanoscale and may be an important cause of membrane patterning, as no continuous labeling can be obtained in a discontinuous membrane. Fortunately, experiments with non-permeabilized membranes also demonstrated the nano-patterning of large numbers of proteins (for example ), but thThe subsequent antibody application may be the least precise step in the entire procedure. IgGs are among the most commonly employed tools for protein imaging ,69, but 15 sequences, is incubated with the protein of interest. The few sequences that can bind the protein of interest are retained, while all others are washed off. This procedure is then repeated several times, under increasingly harsher binding conditions, until sequences of suitably high affinity are selected. The resulting aptamers are directly coupled to the desired fluorescent dyes and are used in microscopy studies, where, being much smaller than antibodies , they penetrate better into the tissue and identify with more ease the epitopes [Current research still employs antibodies for high-resolution imaging, but smaller, monovalent probes are replacing them slowly. Two prominent categories are the aptamers and nanobodies, which we explain here in brief. Aptamers are small DNA or RNA oligonucleotides , which aepitopes . The samepitopes . Nanobodepitopes . The useepitopes . The maiepitopes ,80.These improvements in protein labeling are currently being mirrored by improvements in imaging resolution. It is generally known that imaging resolution has been limited by diffraction to about half of the wavelength of the imaging light . Two main strategies have been generated to overcome this issue. First, the so-called coordinate-targeted approach, in which a beam of light is patterned and is applied onto the specimen in a fashion that enables reading the coordinates from which specific fluorophores are allowed to emit. Examples for this approach come from the stimulated emission depletion microscopy group of technologies  and froImportantly, major advances in cryo-electron tomography (cryo-ET) methods have recently enabled the description of particular molecular assemblies in cells, such as proteasomes or polyglutamine inclusions ,93. AlthThus, experimental data and insights are continuously changed by new technological developments, but also by diverse sources of errors; although recent technology advances offer exciting potential solutions.The perspective we argue for, that a quantitative analysis of the synapse would result in data that would enable a better understanding of synaptic function, derived through synaptic modelling, is only valid as far as data from different studies can be combined. While in general lines synapses seem to employ the same protein machineries, presumably for similar functions ,3,49, itDrosophila, and C. elegans, along with small synaptic boutons from the rodent hippocampus, the large calyx of Held synapse from the auditory pathway, and the highly-specialized lamprey reticulospinal giant synapses. These synapses contain widely different levels of synaptic vesicles, from ~200 vesicles in the hippocampal synapses and in the C. elegans boutons, to thousands of vesicles in the lamprey, tens of thousands in Drosophila, and hundreds of thousands at the frog neuromuscular junction and in the calyx of Held . In eld . Neverteld . In general, computational models of a synapse will be far more complex, formalizing a large number of processes and their relations  and systematic parameter studies, while field-theoretic models that only capture the universal features of lipid architecture, allow straightforward access to free energies ,110. TheMuch of the recent success of coarse-grained models stems from the increasing computational power, model developments, e.g., extension of coarse-grained force fields to describe proteins , and advA detailed molecular model of the whole synapse could be used to investigate the molecular underpinnings of certain diseases and the mode of action of medicines. However, atomistically detailed modelling of all processes in an entire synapse, and even coarse-grained particle-based models, will remain computationally unfeasible in the next decade(s); the time and length scales  are simply too large for direct particle-based simulations. Furthermore, due to its detailedness, the susceptibility of such a model parameterization to experimental inaccuracies or variations, as discussed above, needs to be managed carefully.Alternatively, abstract computational models summarize the essential dynamics of the synapse in rather simple mathematical formulations using coupled differential equations ,124. SucAlthough there is a gap between molecular models and the more abstract computational models employed in network neuroscience in terms of time and length scales (and therefore in terms of investigated phenomena), there are also multiple points of contact. On the one hand, the qualitative correlations between determinants that underlie synaptic processes on the molecular scale may suggest relevant degrees of freedom in more abstract neuroscience models, and inform functional dependencies employed in these approaches. On the other hand, the abstract neuroscience models, in turn, provide information about the spatiotemporal changes of the local variables at the site of fusion and fission in the course of synaptic function, such as concentration of proteins or signaling molecules. This information can set the boundary conditions for coarse-grained molecular models and thereby focus the exploration of parameters on relevant regions. Of course, the above-discussed examples describe two extremes of possible abstraction level to describe synaptic functioning. Between the discussed molecular and network level is a wide range of possible levels of abstraction, some being covered by computational models. On each level of abstraction, to derive the general principles via carefully designed, parameterized and devised computational models, quantitative data from different methods is required. For instance, data of polymerization and depolymerization of actin filaments, imaging data of the spatial distribution of actin in the post-synapse, and a model of membrane dynamics have been integrated to investigate the spatial-temporal dynamics of a dendritic spine . This moThus, to advance the understanding of synaptic function, we think that a set of models across different abstraction levels is required. For this, the synapse has to be subdivided into loosely dependent processes. This distinction can be made based on physical space, known function, involved proteins, etc. The functioning of these individual processes, happening on a rather detailed level, can already be investigated using quantitative experiments and molecular models. On a higher level of abstraction, several of these processes can be combined and integrated into more abstract computational models. This allows the investigation of their interplay and the validation of the results by additional experiments. These abstract computational models of different processes can again be combined and integrated on the next level of abstraction. For instance, the presynaptic processes of vesicle supply, inactivation, and recovery have been integrated into the Tsodyks and Markram model of short-term synaptic plasticity ,127, whiOverall, we argue here that future research will benefit from sub-dividing the synaptic space and the synaptic functionality in compartments that can be investigated with molecular precision, independently of one another. Such processes may include movement and organization in the vesicle clusters, or active zone organization and function during stimulation, on the presynaptic side, as well as receptor movement and clustering, or PSD dynamics on the postsynaptic side. As long as sufficiently precise data can be obtained, covering multiple proteins in each compartment, this would enable computational models that allow us to predict the spatiotemporal dynamics of each compartment. These could then be integrated into larger-scale models, ultimately resulting in reasonably precise models of synaptic function, which would be able to offer new hypotheses for experimental testing, in synaptic function and dysfunction. While some gaps are still apparent, especially concerning the ability to image synapses at molecular resolution, or the understanding of synaptic protein kinetics, these are increasingly covered by the advancing research in the fields of both fluorescence and electron microscopy, thus offering the hope that functional synaptic models will become available in the next decade."}
+{"text": "Skeletal muscle is a highly heterogeneous tissue that plays a crucial role in mammalian metabolism and motion maintenance. Myogenesis is a complex biological process that includes embryonic and postnatal development, which is regulated by specific signaling pathways and transcription factors. Various non-coding RNAs (ncRNAs) account for the majority of total RNA in cells and have an important regulatory role in myogenesis. In this review, we introduced the research progress in miRNAs, circRNAs, and lncRNAs related to embryonic and postnatal muscle development. We mainly focused on ncRNAs that regulate myoblast proliferation, differentiation, and postnatal muscle development through multiple mechanisms. Finally, challenges and future perspectives related to the identification and verification of functional ncRNAs are discussed. The identification and elucidation of ncRNAs related to myogenesis will enrich the myogenic regulatory network, and the effective application of ncRNAs will enhance the function of skeletal muscle. This inMyf-5 resulted in a loss of myomiR expression in developing somites. Their results indicated that myomiRs regulate myogenesis through MRFs. Additional gain- and loss-of-function experiments are needed to illustrate the role of myomiRs during myogenesis by affecting MRF expression. In Xenopus laevis, Vergara et al. examined the expression of miR-206 accompanying somitogenesis. Both knockdown and overexpression of miR-206 resulted in abnormal somite formation   in the mouse embryo , are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation  sequencing, especially next-generation sequencing (NGS). The limitations of NGS on the identification of ncRNAs and its regulatory mechanisms need to be addressed as non-poly (A) forms of ncRNAs such as circRNAs are often ignored. Furthermore, there are a variety of ncRNA databases available, such as miRBase, TargetScan, ribosomal RNA-depleted RNA-seq datasets, lncRNA Database v2.0, and Linc2GO. We need to determine a systematic identification approach to combine ncRNA data generated from different methodologies.Most known miRNAs and circRNAs demonstrate cytoplasmic localization, whereas lncRNAs are present in both the nucleoli and cytoplasm. Furthermore, nuclear miRNA-directed gene regulation constitutes a departure from the prevailing view of miRNA functions Roberts, , which rCurrent experiments need to be based on the million-cell scale, and their accuracy does not support the single-cell level experiment. The current omics research field has been transformed from mixed sample research to single-cell level research. Single-cell sequencing technology has revealed the existence of cellular heterogeneity and related molecular mechanisms in detail. For some precious cell samples or embryonic samples, rigorous validation experiments are needed.via RIP, ChIRP, CHART, or GRID. One limitation of these probe approaches is that their efficacy is based on the size and location of ncRNAs. In recent years, many studies have reported that ncRNAs play biological functions through exosomes mediating intercellular communication (Romancino et al., in vitro (Nakamura et al., The mechanism of ncRNAs has been assessed The functional interaction maps for a majority of characteristic ncRNAs with DNA or transcription factors, except for some well-studied cases, remain largely unknown. One crucial issue hindering the progression of this field is the limitations of RNA-DNA binding interaction technology and the lack of an ncRNAs-DNA-protein international database. Addressing these issues would significantly enhance our understanding of mechanisms that dictate ncRNAs association with myogenesis.in vitro and in vivo systems to illustrate the integrated network of myogenesis.Furthermore, the identification of functional ncRNAs to improve muscle development remains a challenge that needs to be resolved. A major hindrance for most applications is the tissue delivery and distribution of ncRNAs for efficacy (Kaemmerer, HL and WL wrote the manuscript and selected the literature. BZ and ZX proposed the topic, wrote the manuscript, and corrected and gave suggestions to improve the manuscript. HL, WL, QT, JJ, ZX, and BZ reviewed the manuscript and prepared tables and figures. All authors read and approved the final manuscript.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": "We report the case of a 51-year-old patient who underwent the implantation of a bi-ventricular implantable cardioverter defibrillator (ICD) complicated by a sub-acute right ventricular minimal perforation with pericardial effusion and echocardiographic signs of tamponade. A new echocardiographic plane orientation allowed us to diagnose this condition in emergency and to make the right decision without delay, which consisting in unscrewing the active fixation screw under fluoroscopy guidance, while the pericardiocentesis was postponed. Thanks to the intervention focused on eliminating the cause of the postcardiac injury syndrome, the patient recovered rapidly and ultimately avoided the pericardiocentesis procedure. Cardiac perforation  by pacemaker and cardioverter defibrillator leads is, unfortunately, an escalating phenomenon , with anThis implies lead dislodgements and worrisome consequences like cardiac tamponade, which is handled with urgent pericardiocentesis . It is eWe report a case of minimal right ventricular apex perforation of the tip (screw) of a ventricular active-fixation lead, with consequent pericardial irritation and pericardial effusion with impending tamponade  . EchocarA 51 year-old man with ischemic dilated cardiomyopathy (DCM), already revascularized with a PTCA procedure on the left anterior descending (LAD) coronary artery, underwent the implantation of a three-chamber biventricular implantable-cardioverter-defibrillator (ICD)  due to the persistence of a significative symptomatic (NYHA class III) left ventricular systolic dysfunction  associated with left bundle branch block (LBBB). The procedure was performed under local anaesthesia. The device was implanted in the sub-clavicular area and connected to the three leads. The right atrial bipolar passive-fixation lead  was positioned via puncturing the left subclavian vein and positioned in the right atrial appendage. The right ventricular (RV) bipolar screw-in (active fixation) silicone lead  was inserted via the cephalic vein and positioned in the right ventricular apex. The left ventricular passive fixation lead  was inserted via the jugular vein and positioned in the coronary sinus. Atrial and ventricular sensing and pacing thresholds were satisfactory. The device programming was : Capture threshold: left ventricular voltage 0.4 \u00d7 0.5 ms; atrial lead voltage 0.2 \u00d7 0.5, right ventricular voltage 0.3 \u00d7 0.5 ms. Stimulation threshold: left ventricular lead voltage 5 \u00d7 0.5 ms; atrial lead 5 \u00d7 0.5 ms; right ventricular lead voltage 5 \u00d7 0.5 ms.Impedance: Left ventricular lead 879 \u2126; atrial lead 532 \u2126; right ventricular lead 589 \u2126.Implantation was apparently uncomplicated. After 48 h from the ICD implantation, the patient was discharged asymptomatic; during the chest radiography and echocardiography performed after implantation, no complication was evident. Some hours after discharge, the patient began to complain of atypical chest pain. An ECG performed in the emergency department showed left ventricular hypertrophy and new negative T waves in D1, aVl, V5 and V6. A chest-x ray revealed a lung hilum enlarged with a possible left pleural effusion. The echocardiogram showed a moderate pericardial effusion . Laboratory markers were: Hb 10.1 g/dl, rbc 3.330.000, wbc 7580., and Hct 29.3%. The patient was therefore re-admitted to our unit, and in the ensuing days showed a clinical improvement, with a pericardial effusion reduction , and he was then discharged. A few days later, he was admitted once again for an ICD inappropriate shock, due to an atrial flutter with high ventricular response; the echocardiographic examination showed little increase in pericardial effusion . A cardiac injury syndrome by lead irritation was hypothesized, and the patient was discharged with the suggestion of a subsequent elective control. A few days after discharge, the patient returned to the emergency department complaining of atypical chest pain, shortness of breath, and asthenia. Clinical evaluation revealed regular tachycardia at 145 b/m, blood pressure at 110\u201370 mmHg, no recognizable jugular waves, and jugular engorgement  with positive markedly positive jugular reflux, mild pulsus paradox (5 mmHg), and no palpable apex beat. An EKG showed atrial flutter, with an atrio-ventricular conduction ratio of 2:1. The pacing parameters were within a normal range value, and substantially unchanged with respect to the baseline value. Echocardiography showed a further worsening of the pericardial effusion , with echocardiographic signs of tamponade . The patient was thus scheduled for urgent pericardiocentesis; before the procedure, a new echocardiographic examination, specifically aimed at best visualizing the tip of the catheters, was performed . A four-As the cause of the pericardial effusion with impending tamponade was quite clear, we decided to cancel the pericardiocentesis procedure and first of all remove the cause of the pericardial irritating stimulus and in the meantime, temporarily treat the congestion symptoms with diuretic therapy; this was simply attained by carefully unscrewing the fixating screw , along The major finding in our case is that a precise diagnosis with echocardiography allowed pericardiocentesis to be avoided. As a general and golden rule in medicine, the elimination of the cause(s) brings about the healing process . Thus, iEchocardiography played a pivotal role in our case. In our view, echocardiography has great potential for handling patients with possible lead complications. This technique has the advantage of being rapidly available at the bedside, a quality which is maximally appreciated in emergencies like our case; it is the principal tool in diagnosing effusion and cardiac tamponade, which are major complications of lead perforation ; in addiIn other cases in the literature, late cardiac perforations were diagnosed with echocardiography: in one case (one month after implantation), a passive tined lead caused an important perforation, with the tip moving freely in the mediastinum, crossing the myocardium ; in anotConsidering that most patients could be initially asymptomatic , TTE couOther techniques are available for lead perforation, but they are scarcely appealing when minimal perforation is suspected. In fact, chest radiography and fluoroscopy can be useful only when the lead migrates quite far from the heart, but in cases with minimal perforation of the heart, these tests are often non-diagnostic . CT of tLastly, regarding the factors contributing to this complication, we think that the major factor was the use of an active fixation lead ,15,16,17Our case shows that an optimized echocardiographic approach, with the use of non-conventional tomographic planes, can promptly detect, at the bedside, minimal cardiac catheter perforation and pericardial effusion with impending tamponade, thus driving an optimal non-invasive therapeutic strategy with fast clinical recovery."}
+{"text": "Pediatric penile skin grafting is rarely performed. We present a case series of four pediatric patients receiving skin grafting due to the loss of penile skin. The four boys were followed up for 1 to 5 years. One full-thickness skin graft and three split-thickness skin grafts (STSGs) survived well with low Vancouver scar scale scores. One boy gradually developed lymphedema of the distal foreskin and underwent a second preputioplasty. He presented with normal erectile function and did not experience any pain. We propose thick STSGs as the most appropriate choice for pediatric penile skin reconstruction. Lymphedema of the foreskin is an important long-term complication of penile skin grafting. Penile congenital abnormalities or traumas require adequate skin coverage for reconstruction.A 4.11-year-old boy and an 8-year-old boy suffered a high fall-related penile injury. The 4.11-year-old boy was treated 8\u2009hours after injury. The defect area measured 3.0\u2009\u00d7\u20092.5\u2009cm. He received a FTSG after debridement because the time after injury was relatively short and the penile wound surface was relatively clean . Full-tThe 8-year-old boy was admitted to the hospital 24\u2009hours after injury. The defect penile skin area measured 2.0\u2009\u00d7\u20092.8\u2009cm, and the defect scrotal skin area measured 3.0\u2009\u00d7\u20093.5\u2009cm. He received debridement and an artificial dermis implant  during the primary surgery, followed by a thick STSG 14 days after surgery .A 6.6-year-old boy, who sustained an electric cautery circumcision-related penile injury, was transferred from the urology department to the burns and plastic surgery department 10 days after injury. The defect area measured 1.5\u2009\u00d7\u20092.5\u2009cm. After debridement, a thick STSG was performed .A 12.5-year-old boy, who suffered a traffic accident-related penile injury, received primary suturing at another hospital. The penile skin necrosis area measured 2.2\u2009\u00d7\u20093.1\u2009cm, and the left thigh necrosis skin area measured 38\u2009\u00d7\u200915\u2009cm. He was transferred to our department 10 days after injury, and he received debridement and an artificial dermis implant  during the primary surgery. After 14 days, the acellular dermal matrix was removed, and a 0.2-mm STSG was placed on the granulation tissue wound .This STSG was harvested from the scalp using a Zimmer electric dermatome. The donor site was covered with antibiotic dressing and healed naturally after 2 weeks. The penile dressing and the catheter were removed 7 days after STSG placement.A Foley catheter was inserted and retained throughout the treatment. We used the dressing for hypospadias surgery as a reference to design the skin graft dressing. On the inner side, a mesh-like lipid hydrogel dressing  was evenly applied to the grafted skin so as to form a moderately tight sheath and to avoid dressing adhesion. An antibiotic ointment (mupirocin) was applied to the second layer, and the outer layer was wrapped with an elastic dressing. The graft was applied tightly to the wound surface, and the absence of dissolution or scabbing was considered to indicate good skin graft survival. The Vancouver scar scale  was used to evaluate the scar quality in all patients. The scale consists of the following variables: vascularity, pliability, height, and pigmentation .The three thick STSGs survived completely, with no signs of dissolution or scabbing . The FTSome adult diseases lead to severe penis tissue loss and require skin grafting, for example, infection, trauma, burns, malignancy, skin disorders, and primary lymphedema.An ongoing debate exists about the optimal skin type to be used for penile skin grafts. A greater amount of dermis is included in FTSG than in STSG; therefore, some important distinctions in the nature and potential uses of the two types of grafts should be borne in mind.As the penile skin is thin, hairless, and flexible, and it undergoes a substantial change in size with penile erection, some researchers suggested FTSG as the preferred choice for penile skin graft.Long-term complications of penile skin grafting should be borne in mind. The 12.5-year-old boy who received an artificial dermis implant  and a thick STSG gradually developed lymphedema of the distal penile skin  and reqIn conclusion, pediatric penile skin grafting is a rare procedure. Based on our experience and observations published in the scientific literature, thick STSG appears to be the most appropriate choice for pediatric penile skin reconstruction. Lymphedema of the foreskin is an important long-term complication of penile skin grafting."}
+{"text": "Deferasirox is an orally active, lipophilic iron chelating drug used on thousands of patients worldwide for the treatment of transfusional iron overload. The essential transition metals iron and copper are the primary catalysts of reactive oxygen species and oxidative damage in biological systems. The redox effects of deferasirox and its metal complexes with iron, copper and other metals are of pharmacological, toxicological, biological and physiological importance. Several molecular model systems of oxidative damage caused by iron and copper catalysis including the oxidation of ascorbic acid, the peroxidation of linoleic acid micelles and the oxidation of dihydropyridine have been investigated in the presence of deferasirox using UV-visible and NMR spectroscopy. Deferasirox has shown antioxidant activity in all three model systems, causing substantial reduction in the rate of oxidation and oxidative damage. Deferasirox showed the greatest antioxidant activity in the oxidation of ascorbic acid with the participation of iron ions and reduced the reaction rate by about a 100 times. Overall, deferasirox appears to have lower affinity for copper in comparison to iron. Comparative studies of the antioxidant activity of deferasirox and the hydrophilic oral iron chelating drug deferiprone in the peroxidation of linoleic acid micelles showed lower efficiency of deferasirox in comparison to deferiprone. There are hundreds of thousands of iron overloaded patients belonging to different categories of inherited and other diseases, who receive regular red blood cell transfusions for the treatment of their refractory anemia ,2,3. IroThere are currently three regulatory approved iron chelating drugs which are used daily for the treatment of transfusional iron overload, namely deferoxamine, deferiprone (L1) and deferasirox (DFRA) ,6,7,8,12Investigations on the affinity and other interactions of the chelating drugs with essential and redox active metal ions are crucial for determining their therapeutic activity and toxicity potential ,14,15. OThe molecular interactions with other metal ions in addition to iron and copper, such as zinc, and with nutrients such as ascorbic acid (Asc) and also many other natural or xenobiotic molecules, are also expected to interfere with the therapeutic and toxicity potential of iron chelating drugs as well as other drugs ,20,21,22The study of the pro-oxidant/antioxidant effects of the iron chelating drugs and their iron and copper complexes is important for pharmacological/toxicological parameters affecting their mode of action and possibly their efficacy in vivo. However, the determination of the antioxidant potential of chelating drugs is also of major pharmacological interest because of possible therapeutic applications in many diseases associated with oxidative stress toxicity such as cancer, neurodegenerative, cardiac, liver, renal and other diseases ,26,27,28In this work the pro-oxidant/antioxidant effects of DFRA and its iron and copper complexes have been investigated using several molecular models of oxidative damage of linoleic acid (LA), Asc and dihydropyridine (DHP). In some of the studies L1 and Asc were used for comparison.1H-NMR spectroscopy.Linoleic acid peroxidation is widely studied in different model systems, including liposomes , but in 2+ ions has stoichiometry 1:1, and the stoichiometry of DFRA complex with Fe3+ ions 2:1. In the present studies we have tried to answer the question whether these complexes are redox active or not. For this purpose, the transition metal-induced oxidation of molecular model systems, namely LA, an unsaturated lipid, Asc and DHP as analogs of the nicotinamide adenine dinucleotide (NADH) molecule  are formed, which, in turn, are capable of conversion to other types of ROS, including the peroxyl radical and superoxide anion. The next stage of these reaction processes is \u201cinitiation\u201d, in which the LA radical and the LA peroxyl radical are formed (3) and (4) as follows:In these reaction processes, OH radicals  and (6) as follows:At the \u201ctermination stage\u201d, various LA dimers and polymers are formed (7)\u2013(9) as follows:1H-NMR spectra of the initial LA in the presence of ferrous sulfate (FeSO4) and after 24 h in the absence and in the presence of DFRA. It can be seen that after 24 h, the total signal intensity of LA protons was reduced significantly. In addition, the lines at 2.7 and 2.09 ppm, corresponding to protons near the double bonds (1 and 2), disappeared. All of the above, together with the fact that an insoluble precipitate was formed during the reaction, indicates the complete consumption of LA in the solution and the formation of polymers. In the presence of DFRA, 24 h after mixing, the overall signal level was also reduced, indicating the formation of polymers, but the signals corresponding to protons near double bonds did not completely disappear. This observation suggests that there is a decrease of the initiation rate of LA oxidation in the presence of DFRA.It is also widely known that LA forms micellar solutions in an aqueous environment . In thisIt is known that the reaction of LA with OH radical starts with hydrogen atom abstraction from position 1 see  31], an, an31], This molecular model of oxidative damage monitoring using LA peroxidation has been previously used for studying the antioxidant activity of the iron chelating drug L1 in the presence of copper ions . SimilarAs can be shown in 3 protons in LA, are shown in CH3(t) is the integral intensity of the signal from CH3-groups of LA, which corresponds to the concentration of LA. The integral intensity of the initial signal of the signal from CH3-groups at 0.9 ppm (ICH3(0)) was normalized to 100. Intensity is given in arbitrary units (a.u.).The kinetics of the formation of polymeric products of LA during the oxidation reaction with ferrous ions, which are calculated from the changes in the signal intensity of CHAs can be shown in The experimental points in The results presented in An additional molecular model system for studying the antioxidant activity of DFRA was the oxidation of DHP. The reactions involving DHP are often used as models of radical processes of electron and proton transfer in living systems with the participation of NADH ,55. Dihy3OD producing \u25cfCD2OD C-centered radical [The formation of radicals for the NMR studies was initiated by the Fenton reaction using both iron and copper as catalysts and under similar conditions reported in previous studies ,56. Sinc radical . As a re radical . The mai1H-NMR experiments, which were performed in the presence of iron and copper ions. Differences were observed in the 1H-NMR spectra of different reaction mixtures. 1H-NMR spectra of pure DHP, as well as reaction mixture of DHP with Fe3+ ions in the presence and absence of DFRA, 15 h after the initiation of the reaction.The antioxidant activity of DFRA in the molecular model of the oxidation reaction of DHP was studied in a series of It appears from The results in The experimental findings of this study have several important pharmacological, biological and physiological implications, especially for understanding the influence of DFRA on various redox systems. Deferasirox manifests its antioxidant activity in all studied model systems, including oxidation of Asc, peroxidation of LA micelles, and oxidation of DHP, with reduction in the rate of oxidation by up to several tens of times. Comparative analysis of the antioxidant activity of DFRA and L1 in lipid peroxidation showed a lower efficiency of DFRA in comparison to L1. The significance of these processes is underlined below from the different experiments carried out within this study.3+ and Cu2+, which decompose to form Fe2+ or Cu+, and the ascorbate radical (11) and (12) [Taking into account that in living systems copper and iron ions are mainly in bound forms, the study of the interaction of chelating drugs with natural complexants is an important task for elucidating the mechanism of drug action. One of the main natural antioxidant with chelating properties is Asc ,43,45,58and (12) ,60:Fe3+ Subsequently, ascorbate radicals are also capable of oxidizing, forming dehydroascorbate, which, in turn, undergoes the stages of hydration and decarboxylation, forming secondary reaction products ,62. In aThe study of Asc oxidation was carried out in ethanol since DFRA and its complexes with iron and copper ions have much lower solubility in water. Based on Another, but no less important, effect of the antioxidant activity of chelators is the ability to inhibit lipid peroxidation. The study of this reaction is part of the solution to a wider problem, namely, the study of ferroptosis-programmed cell death. This mechanism is based on the oxidation of the cell membrane by ROS, formed in redox reactions with the participation of iron ions ,65. At tAt the moment, there are many different methods for studying lipid peroxidation, the vast majority of which are based on the detection of oxidation by-products of trichloroacetic and 2-thiobarbituric acids ,34,67,68In the present study it was shown that DFRA inhibits LA peroxidation in micelles; however, the antioxidant effect of this chelator is significantly lower than that of L1. This finding can be explained by two possible mechanisms. One mechanism is that L1 oxidizes faster than DFRA, ferrous to ferric iron and forms ferric iron complex, which is not facilitating LA peroxidation. The other mechanism is related to the much higher lipophilicity of DFRA , which a3+ and Cu2+ ions. It can be seen that the efficiency of the antioxidant activity of DFRA is higher in the case of Fe3+ than with Cu2+ which is explained by the higher affinity of the chelator for iron ions than for copper ions [The last part of our study was to investigate the effect of DFRA on redox active transition metal induced DHP oxidation. Dihydropyridines are widely used as analogues of NADH, which is a natural substrate in metabolic reactions of electron transfer in living cells ,72. Uncoper ions ,38.The antioxidant activity of DFRA in all three molecular model systems, confirms similar antioxidant activity observed in these and other models of oxidative damage with the other two chelating drugs L1 and deferoxamine ,73,74,753, 97%), ferrous sulphate , copper chloride  and H2O2 (35.5%) were obtained from Sigma-Aldrich, Moscow, Russia. Deferiprone  was received from LIPOMED Inc., Arlesheim, Switzerland. Deferasirox  was obtained from Shanghai Daeyeon Chemicals Co., Ltd., Shanghai, China, DHP 1,1\u2032--di(propan-1-one) (99%) was synthesized according to the classic Hantzsch scheme and obtained from prof. G. Duburs, Institute of Organic Synthesis, Riga [Ascorbic acid , ferric chloride  with Asc (0.1 mM) at room temperature and by monitoring changes at the absorption spectra. The wavelength of 262 nm, which corresponds to the absorption band of Asc, was selected based on the UV-Vis spectrometry data and the analysis of the spectra. This wavelength was selected because it also corresponds to a local minimum in this region of the absorption spectra of DFRA chelate complexes with iron and copper ions. The concentrations of the reagents used in the study were selected in the way that the optical density of the solution did not exceed the detection limit  of the UV-Vis spectrometer.UV-Vis optical density spectra and kinetics were measured in ethanol solution in 1 cm quartz cuvette using a SF-2000 spectrophotometer . The influence of DFRA on the oxidation of Asc was studied by addition of various concentrations of chelator into the mixture of iron or copper salts (CuCl2O2 (0.5 M), DFRA or L1 (1 mM), and FeSO4 or CuCl2 (0.1 mM) in deuterated phosphate-saline buffer (0.1 M) (pH 7.4). The reaction was initiated by addition of hydrogen peroxide. In the case of DFRA, LA was mixed with DFRA in chloroform, then the solvent was evaporated leaving behind a film, which was dissolved in deuterated phosphate-saline buffer (pH 7.4). Then FeSO4 or CuCl2 was added and the sample was incubated for 30 min to establish equilibrium. In the case of L1, LA was dissolved in chloroform, then the solvent was evaporated and the remaining film was dissolved in deuterated phosphate-saline buffer (pH 7.4). Then L1 and FeSO4 or CuCl2 was added and the sample was incubated for 30 min. The reaction was initiated by addition of hydrogen peroxide. The 1H-NMR spectra were recorded using a Bruker AVHD-500 spectrometer (500 MHz) .The study of lipid peroxidation was carried out using a reaction mixture consisted of LA micelles (3.5 mM), H2O2 (0.2 M), DFRA (2 mM), and FeCl3 or CuCl2 (0.2 mM) in deuterated methanol. The reaction was initiated by the addition of the metal salt into the sample. Dihydropyridines are quite sensitive to external influences, such as light [1H-NMR spectra were also recorded using a Bruker AVHD-500 spectrometer (500 MHz).The investigations of dihydropyridine were carried out using a reaction mixture which consisted of 1,1\u2032-di(propan-1-one) (DHP) (5 mM), Has light , therefoMolecular studies including the redox effects of DFRA and its metal complexes with iron and copper are of major importance for determining the general pharmacological and toxicological properties of DFRA, as well as all other drugs. Furthermore, the findings from such studies may have therapeutic implications for patients receiving DFRA and other drugs.In this study several molecular model systems of oxidative damage on Asc, LA micelles and DHP caused by iron and copper catalysis have been used for examining the effects of DFRA. The antioxidant activity of DFRA in the three molecular model systems of oxidative damage in this study, confirms previous findings in similar models and also the general antioxidant potential in biological systems of chelating drugs including DFRA, L1 and deferoxamine. However, differences remain between the chelating drugs on redox properties, affinity for metal ions including iron and copper, pharmacological and toxicological properties and also therapeutic effects.Further studies are needed to investigate the antioxidant effects of DFRA and the other iron chelating drugs in different in vitro, in vivo and clinical models of oxidative damage. It is hoped that the antioxidant properties of DFRA and other chelating drugs will increase the prospects for the development of antioxidant drugs for the treatment of many different categories of free radical pathology patients, including patients with neurodegenerative diseases, cancer and cardiac, renal, liver and other organ damage."}
+{"text": "Male rats  maintained in normoxic (21% O2) or hypoxic chambers (10% O2) for 14 days were subdivided in 4 sub-groups: placebo, l-arginine (20 mg/ml), the NO donor molsidomine (15 mg/kg in drinking water), and phoshodiesterase-5 inhibitor sildenafil . Hypoxia depressed homeostasis and increased erythropoiesis, heart and right ventricle hypertrophy, myocardial fibrosis and apoptosis inducing pulmonary remodeling. Stimulating anyone of the 3 mechanisms that enhance the NO-cGMP pathway helped rescuing the functional and morphological changes in the cardiopulmonary system leading to improvement, sometimes normalization, of the pressures. None of the treatments affected the observed parameters in normoxia. Thus, the 3 modulatory sites are essentially similar in enhancing the NO-cGMP pathway, thereby attenuating the hypoxia-related effects that lead to pulmonary hypertension.Manipulation of nitric oxide (NO) may enable control of progression and treatment of pulmonary hypertension (PH). Several approaches may modulate the NO-cGMP pathway in vivo. Here, we investigate the effectiveness of 3 modulatory sites: (i) the amount of  Many studies suggest that NO bioavailability is indeed reduced in patients and in rodent models of pulmonary vascular diseases, and that enhancement of the NO-cyclic guanosine monophosphate (cGMP) pathway  by endothelial NO synthase (eNOS). After its release in the circulation, it establishes an equilibrium with plasma nitrates and nitrites (NOx) and, to a minor extent, with circulating hemoglobin (Hb) to form nitrosylated Hb or nitrosothiolated Hb. Part of NO enters the smooth muscle cell, where it activates soluble guanylate cyclase (sGC) generating cGMP, which in turns activates protein kinase G that sequesters Ca++ thereby favoring smooth muscle relaxation. Although controversial, the same mechanism in the myocytes might contribute to decrease muscle contractility thereby acting as a negative inotropic factor. The cellular level of cGMP is kept under the control of the activity of phosphodiesterase 5 (PDE5), which converts active cGMP in inactive 5\u2032-GMP.Pharmacological modulation of nitric oxide (NO) pathway  may repr) and neonatal respiratory diseases associated with PH. Although any one of the mentioned actions to exploit the NO-cGMP pathway-based therapies could be considered effective, in the clinical practice this does not occur, and a recent Cochrane survey shows that there is currently insufficient evidence to recommend NO donors, L-Arg or eNOS inhibitors in acute stroke, and only one drug, glyceryl trinitrate is able to give measurable clinical endpoints. Although inhaled NO is approved to treat certain types of PH and PDE5 inhibitors are approved in the treatment of PAH, the overlapping of several side reactions that eventually masks the final outcome also plays a role in this disappointing result.The mentioned chain of events may be modulated at 3 levels at least, by altering: (i) the amount of L-Arg entering the chain; (ii) the amount of NO in the plasma that can stimulate sGC; or (iii) the amount of cGMP that is inactivated to 5\u2032-GMP by PDE5. Several drugs have been proposed to manipulate the NO-cGMP pathway activity through 3 broad approaches: i) by manipulating the level of L-Arg, ii) by increasing the amount of plasma NO, for example through the use of NO donors, and iii) by modulating the activity of PDE5, for example through the use of PDE5 inhibitors. The NO-cGMP pathway has become a therapeutic target and several treatments, as inhaled NO, administration of PDE5 inhibitors and soluble guanylates cyclase modulators, have been proposed to treat several cardiopulmonary diseases, such as pulmonary arterial hypertension  or hypoxic chambers (10% O2) for 14 days. The chambers, built to accommodate 4 rats each, differed only for the gas mixture flowing through them. When the hypoxic chambers needed to be opened for cleaning and monitoring, they were equipped with a compensation chamber flowed with the same gas mixture of the hypoxic chamber, thereby avoiding any contact of the rat with room air and its reoxygenation. Rats were treated with the assigned drug since day 0 of normoxia or hypoxia exposure. At day 15, rats were first transferred anaerobically to the compensation chamber, then anesthetized i.p. with a cocktail containing 10 mg/kg xylazine, 100 mg/kg ketasol and 1500 IU heparin within the compensation chamber at 10% O2, thus avoiding reoxygenation before sacrifice. In each subgroup, 2 subsets of rats were used, one for hemodynamic and morphological analyses, and one for tissue assays.Adult male Sprague-Dawley rats  were randomly assigned to 1 of 4 treatments: Control (no treatment), L-Arg (20 mg/ml in drinking water), the NO-donor molsidomine , and the PDE5 inhibitor sildenafil . In each group, rats were maintained either in normoxic . The protocol was approved by the local Institutional Animal Committee . All efforts were made to minimize animal suffering during the experiments.max). Briefly, a small traverse incision was made in the abdominal wall, the diaphragm was exposed and opened. A 24-gauge butterfly needle with tubing attached to a pressure catheter was inserted into the RV and pressure measurements were recorded with PowerLab .To examine PH development, we measured the RV systolic pressure (RVSP) and the RV contractility expressed as maximum rate of pressure development . The remaining sample was centrifuged for 15 min at 3000g. Plasma was stored at \u221280\u00b0C. For the measurements of NOx, we used the Nitric Oxide Colorimetric assay kit  following the manufacturer\u2019s protocol.The anesthetized animals were sacrificed by exsanguination, heart and lung were washed with PBS at 4\u00b0C, harvested and processed for analysis. The RV was separated from the left ventricle (LV) and septum (S), and all parts were weighed separately. The degree of RV hypertrophy was calculated as the RV/LV+S ratio.The degree of cardiac fibrosis was determined by Sirius red staining. Briefly, 10 \u00b5m-thick paraffin-embedded tissue sections were deparaffinized, rehydrated and stained in iron hematoxylin for 10 min, followed by rinsing in water for 10 min. The slides were then stained in 0.1% (w/v) Sirius Red F3B  in saturated aqueous picric acid  for 1 h. Sections were washed twice in 0.5% v/v acetic acid, dehydrated rapidly 3 times in 100% alcohol, cleared in xylene and mounted using Merckoglas medium . Each section was photographed under a light microscope  at 10x magnification. At least 10 fields were randomly selected for each rat, each of which contains at least 6 vessels. Images were analyzed using Image software. The percentage collagen was calculated as fibrosis area/total area of the tissue section. which includes lung inflation with formalin, embedding in paraffin and sectioning (8-\u00b5m thickness). Samples were then blocked with goat serum and incubated with an antibody against smooth muscle \u03b1-actin , followed by incubation with goat anti-mouse IgG secondary antibody . After developing with 3,3\u2032-diaminobenzidine and counter staining with hematoxylin and eosin, transversal cut arterioles were counted . Pulmonary arterial thickening was measured by calculating the percentual pulmonary artery thickness as described in the literature. We analyzed 10 vessels for each rat (n = 6/group) by double-blind morphological analysis.To determine the degree of muscularization in pulmonary arterioles, we used the technique described in Nydegger et al,\u22121 HCl (10% wet wt/vol) and centrifuged (2000 rpm for 10 min at 4\u00b0C). The measurement of cGMP was made by an immunoassay kit . Caspase-3 activity was tested by the Caspase-3/CPP32 colorimetric assay kit (MBL). NOx was determined by the Nitric Oxide Colorimetric assay kit  following the manufacturer\u2019s protocol.We measured cGMP in frozen tissue that was homogenized at 4\u00b0C with 0.1 mol\u00b7lP = 0.05.Data are expressed as box and whiskers plot following the Tukey method, with outliers plotted individually when present. To detect significant differences, we first ran ordinary 2-way ANOVA analysis. If significant, we proceeded to the multiple comparison tests. To test whether hypoxia has effect on the single treatments, we used the Sidak\u2019s multiple comparison test (significant comparisons are marked as # in the figures). To detect whether treatments had an effect with respect to controls within normoxia and hypoxia, we used the Dunnett\u2019s test (significant comparisons are marked as \u00a7 in the figures). Analyses were performed using GraphPad Prism version 8, San Diego, California USA. The significance level was set at None of the rats that entered this study  died nor showed signs of discomfort. Unless otherwise stated, the treatments did not have remarkable effects in normoxia rats.Fourteen-day hypoxia depressed the BW gain and increased (Hb) concentration ([Hb]) . Plasma The treatments in hypoxia did not substantially affect neither the BW gain nor [Hb], although Sild slightly improved the BW gain and Mols slightly increased [Hb]. L-Arg and Sild were unable to alter plasma NOx, but Mols further increased it by virtue of its NO-donor effect. Although Sild did not change plasma NOx with respect to untreated hypoxic rats, plasma NOx was higher than in normoxia.Myocardial hypertrophy was evaluated through the heart weight/body weight (HW/BW). Hypoxia markedly increased the HW/BW ratio . This inMyocardial fibrosis was evaluated separately in the RV and LV by Sirius Red Staining . HypoxiaThe degree of apoptosis was evaluated through the activation of Caspase-3, a hallmark of apoptosis in myocardial cells . HypoxiaAll treatments were able to normalize apoptosis in hypoxia-challenged hearts. By contrast, the myocardial cGMP level was increased only by Sild, as a result of its function as PDE5 inhibitor. All treatments increased the production of NO.max). Hypoxia did not affect the left heart function (max were markedly increased (max (Hemodynamics was assessed by left and right heart catheterization with measure of the end-diastolic (EDP) and systolic (SP) pressures, and of the rate of pressure development  antagonist, with 3-week hypoxia. This treatment causes severe PAH in rats, characterized by smooth muscle proliferation, vascular rarefaction and perivascular fibrosis that contribute to increase precapillary vascular resistance, RV afterload and hypertrophy even after return to normoxia for 5-10 weeks. Compared with rats exposed to chronic hypoxia alone, Su-Hx rats develop decompensated RV failure, as evident from elevated RV EDP, RV dilatation and decreased RV ejection fraction with maladaptative RV remodeling. A third PH model is constituted by the persistent PH model obtainable in lambs by surgical constriction of the fetal ductus arteriosus, which results in increased right-to-left extra-pulmonary shunting of deoxygenated blood and mimics with augmented vascular constriction and smooth muscle hypertrophy, which leads to persistently increased pulmonary vascular resistance and RV hypertrophy.The stimulation of anyone of the 3 broad mechanisms that enhance the NO-cGMP pathway did not produce appreciable changes in normoxia, but tended to improve, and in some instance to normalize most of the parameters that were challenged by hypoxia. This highlights the validity of the experimental model with respect to the pathological situation of PH patients. Remarkably, monocrotaline-treated rats, a common rodent model of PH, develop symptoms similar to those elicited by hypoxia only, including pulmonary vessel remodeling, RV hypertrophy and apoptosis. that in this model most of the data reported here remain constant for 4 weeks. Despite ongoing hypoxia adaption as from the steadily increasing erythropoietic response, most parameters related to the compensation to PH appear relatively constant, including the RV/(LV+S) ratio, the number of lung vessels, the wall thickness of small vessels, RV fibrosis, and myocardial NOx. Remarkably, the effect of one of the interventions described here, e.g. PDE5 inhibition, elicits similar responses after 2- and 4-week hypoxia. Noteworthily, despite constant LV SP and RV SP, LV EDP and dP/dtmax were subjected to instability on opposite directions: tendency toward normalization for dP/dtmax, and toward further deterioration for LV EDP.In this study, rats were exposed to hypoxia for 14 days, a time long enough to rise some adaptation to hypoxia or compensation to PH. Therefore, data taken at a single time point may be considered potentially unstable and not fully representative of hypoxia-induced PH. However, we previously showed Consequently, increasing the substrate for NO production (L-Arg supplementation) is thought to attenuate PH development in both monocrotaline- and hypoxia-challenged rats. In PH patients, L-Arg supplementation considerably reduces pulmonary vascular resistance. It must be noted that L-Arg supplementation can be replaced by other treatments or supplementation, as for example combining l-citrulline with tetrahydrobiopterin, which produced encouraging results in hypoxic pigs. Besides providing the substrate for NO formation in endothelial cells, L-Arg prevents eNOS uncoupling, a phenomenon whereby eNOS generates ROS rather than NO, thus serving as ROS scavenger, decreasing their formation and increasing NO release. In this study, L-Arg attenuated RV hypertrophy and muscularization of small pulmonary vessels secondary to the effect of NO in inhibiting vascular pulmonary artery smooth muscle cell proliferation and attenuating the morphological changes by improving cardiopulmonary remodeling. It should be noted that, besides providing substrate for eNOS, L-Arg also feeds the inducible form of NO synthase (iNOS), which, at odds with eNOS, generates considerable oxidative and nitrosative stress.PH patients, in particular group 3 due to hypoxia or lung disease, usually display low L-Arg levels compared to healthy subjects. Among several developed NO donor molecules, Mols, which releases NO non-enzymatically, was suggested for acute pre-ischemic treatment that improves cardiac ischemia tolerance in animals adapted to hypoxia. Several reports have shown that Mols reduces the pulmonary arterial pressure in rats with PH induced by monocrotaline injection or hypoxia and reduces both pulmonary vascular remodeling and ET-1 expression. In addition, Mols improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability. In this study, we found that Mols acts similarly to L-Arg, but, in addition, it provides a further stimulus to erythropoiesis. This is in agreement with the observation that the NO-cGMP pathway upregulates erythropoiesis at the level of gene transcription, thereby providing a novel target to stimulate erythropoiesis in vivo.NO donors were first reviewed in 1995 and classified as \u201ca new class of drugs [\u2026with\u2026] potential utility in the treatment of coronary and pulmonary arterial diseases.\u201d and rescues hypoxia-induced RV hypertrophy. This phenotype is the outcome of the increase of cGMP secondary to upregulated eNOS phosphorylation, which translates into mitigated apoptosis. Sild does not only contribute to vasodilation, but also favors the recruitment of bone marrow-derived c-kit+ cells, that improves pulmonary hemodynamic through a mechanism that persists over time, thus independent from hypoxia adaptation. Sild improves pulmonary remodeling and RV function in hypoxia by increasing NO signaling.It was reported that inhibition of PDE5 by Sild alleviates symptoms in several cardiopulmonary diseases including PH the selected dose of L-Arg (1.6 g/day/kg for 2 weeks) compares with that associated with improved resting systolic blood pressure and quality of life in PH patients with microvascular angina (0.1 g/kg/day for 4 weeks). Similar doses were also effective to improve hemodynamics and exercise capacity in patients with precapillary PH (up to 0.15 g/kg) as well as to ameliorate functional and quality of life outcomes (0.1 g/kg/day for 3 months). Likewise, the selected dose of Mols (15 mg/kg/day) compares with that reported in the MEDCOR clinical trial demonstrating beneficial effects on the myeloperoxidase activity/antigen ratio in angina patients undergoing percutaneous coronary intervention (16 mg/day for 1 year) and is identical to that reported to be effective to reduce PH in hypoxic rats. As for Sild, the typical per os dose used for PAH treatment is usually higher (25-100 mg/kg twice-daily per os) than that used in the present study. However, for consistency with previous studies, we opted for i.p. delivery of Sild in a single daily dose within the range that saturates protection in humans. Despite the plasma half-life of 4-6 h, the selected dose was observed to elevate myocardial cGMP in a rodent model of hypoxia, indicative of efficient inhibition of PDE5 activity. Other /agents such as the stimulator of sGC riociguat might soon be competitive. This consideration is further supported by the observation that deactivating by oxidation the \u03b1 subunit of protein kinase G does not change the benefits of the stimulation of sGC, thereby highlighting the combination therapy with PDE5 inhibitors and sGC stimulators. Here we show that the effect of Sild is virtually indistinguishable from those of L-Arg and Mols, with a slight improvement of body homeostasis. As expected, Sild did not affect the parameters upstream PDE5 inhibition, e.g. neither plasma nor myocardial NOx.In this study, we did not attempt dose/response assays to detect the drugs optimal concentration, but rather we approximated the dose normally used in clinical contexts. Assuming a water intake of 20 ml/day in hypoxic rats,max. In facts, in addition to the well-known effect of vasodilatation, an additional effect of NO on skeletal muscles fibers has been proposed, i.e. through nitrosation/nitrosylation of target proteins, which leads to depressed isometric force, shortened velocity of contraction, with the outcome of \u201cbraking\u201d muscle contraction and lowering force output by altering the excitation-contraction coupling. By contrast, other Authors documented lack of inotropic effect of NO in rat myocardium. administration of inorganic nitrite and nitrate, such as sodium nitrite, as well as diethylamine NONOate diethylammonium, sodium nitroprusside and the sGC stimulator riociguat. As for PDE5 inhibitors, several other drugs besides Sild have been proposed, each with different pharmacokinetics, efficiency and persistence in the circulation, but with essentially similar mechanisms of action and outcomes. Finally, in this study we did not assess any combination of these approaches in the search of synergistic effects.In this study, no attempt was made to compare directly the effectiveness of the 3 strategies to improve the NO-cGMP pathway. This goal should be preceded by complex pharmacokinetic evaluations as well as dose/response preliminary studies. Likewise, a large number of drugs and approaches were not examined in this study, but we focused in a representative drug for every site of modulation. As far as NO donors are considered, alternative drugs/treatments include inhaled NO therapy,Enhancing the NO-cGMP pathway in any form attenuates the hypoxia-related PH syndrome. Remarkably, the tested treatments appeared to be effective in hypoxia at doses that were ineffective in normoxia. Thus, the 3 strategies to enhance the NO-cGMP pathway in vivo appear equivalent in terms of efficacy and molecular mechanisms of action. The choice of the best treatment, therefore, should not be based on the mechanisms of action but rather on clinical relevance, such as tolerability, side effects and persistence in the circulation."}
+{"text": "Nature Communications 10.1038/s41467-018-03476-6, published online 13 March 2018.Correction to: The original version of this article contained an error in the author list. The author, Frederick F. Lang, was incorrectly listed as Frederick Lang. This error has been corrected in the HTML and PDF versions of the article."}
+{"text": "The use of micro- and nanoparticles in biological applications has dramatically grown during the last few decades due to the ease of protocols development and compatibility with microfluidics devices. Particles can be composed by different materials, i.e., polymers, inorganic dielectrics, and metals. Among them, silica is a suitable material for the development of biosensing applications. Depending on their final application, the surface properties of particles, including silica, are tailored by means of chemical modification or polymeric coating. The latter strategy represents a powerful tool to create a hydrophilic environment that enables the functionalization of particles with biomolecules and the further interaction with analytes. Here, the use of MCP-6, a dimethylacrylamide (DMA)-based ter-copolymer, to coat silica microspheres is presented. MCP-6 offers unprecedented ease of coating, imparting silica particles a hydrophilic coating with antifouling properties that is able to provide high-density immobilization of biological probes. Microparticles composed of different materials, i.e., polymers, inorganic dielectrics, and metals, are widely explored for varying biomedical applications ,2,3. The4)2SO4), dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS ester), phosphate buffer saline tablets (PBS), streptavidin, 3-azide-1-propylamine, bovine serum albumin (BSA), sodium dodecyl sulphate (SDS), Tris, HCl, Amicon Ultra 30 MWCO and 100 MWCO centrifugal filters, dimethylacrylamide (DMA), N-succinimidyl acrylate (NAS), 3-(trimethoxysilyl)propyl methacrylate (MAPS), and polyclonal rabbit IgG were purchased from Sigma-Aldrich . The polymer MCP-2 was purchased from Lucidant Polymer . Bradford Protein Assay Dye Concentrate was purchased from Bio-Rad . Goat antirabbit IgG was purchased from Jackson ImmunoResearch . Oligonucleotides COCU8: 5\u2032-GCCCACCTATAAGGTAAAAGTGA-3\u2032, COCU11: 5\u2032-TCACTTTTACCTTATAGGTGGGC-3\u2032, and DNA-negative: 5\u2032-ACTTAGGACTCAGTACAGGATAGACTTGATATCGGTTGGA 3\u2032 were synthesized by MWG-Biotech AG . COCU8 was modified either with DBCO-linker or biotin at 5\u2032 end, COCU11 was labeled with Cy5 at 5\u2032 end, and DNA-negative was labeled with biotin at 5\u2032 end. Oligonucleotides were freeze-dried and resuspended in de-ionized water (DI water) at a final concentration of 100 \u03bcM before use. Silica microbeads were purchased from Bangs Laboratories Inc. . Spectrofluorimetric analysis was performed using a Jasco FP-550 spectrofluorometer equipped with thermo-stated Peltier cell holder. Bradford protein assays were performed using a Thermo Labsystems Multiskan Ascent microplate spectrophotometer. \u03b6-Potential measurements were performed on a Zetasizer Nano ZS Instrument , and samples were loaded in a Zetasizer nano series Dip Cell Kit .Ammonium sulfate , filtered on a B\u00fcchner funnel, and dried under vacuum at room temperature.MCP-2 was modified by reaction with 3-azido-1-propylamine, as reported elsewhere . To intrw/v, diameter = 1 \u00b5m) were sonicated in a water bath for 10 min and vortexed 30 s to ensure proper resuspension. Then, 50 \u00b5L (=5 mg) were transferred into a 1.5 mL tube and washed twice with MQ water. After each washing or incubation step, beads were separated from supernatant by centrifugation. Beads were resuspended in 1 mL solution of MCP-6 (1% w/v in 0.9 M ammonium sulphate) and incubated 30 min at 25 \u00b0C under stirring followed by 30 min at 25 \u00b0C without stirring. Beads were washed twice with 1 mL of MQ water and used for further experiments.Silica microspheres  at pH 7. Each result was averaged from at least three measurements.g) to a final volume of around 50 \u00b5L. The same procedure was repeated on 20 mg of uncoated silica microspheres as negative control. Samples were diluted five times using water, and the concentration of BSA or lysozyme released by beads upon SDS-mediated denaturation was assessed by Bradford protein assay.Twenty mg of silica microspheres was coated with MCP-6 as described in Five mg of MCP-6 coated silica microspheres was washed in 1 mL of PBS and resuspended in 100 \u00b5L of DBCO-modified COCU8 in PBS (different concentrations ranging from 1 to 20 \u00b5M were tested) and incubated overnight at 37 \u00b0C under stirring. Beads were washed twice with 1 mL of water and once with 1 mL of PBS.Five mg of beads functionalized with COCU8 was resuspended in 100 \u00b5L of Cy5-labeled COCU11 in PBS (at the same concentration used for COCU8 during immobilization step) and incubated for 1 h at 25 \u00b0C under stirring. Beads were centrifuged and supernatant was recollected. Beads were washed twice with 100 \u00b5L of PBS; after, beads were centrifuged and supernatant recollected. Supernatants were pooled together and, only in samples where the concentration of DNA used during incubation was 5 \u00b5M or higher, diluted 1:10 using PBS. Further, 150 \u00b5L of pooled supernatants (diluted or not) was mixed with 350 \u00b5L of PBS, and the fluorescence emission intensity at 658 nm was measured using a Jasco FP-550 spectrofluorometer in 1 cm quartz cuvettes.To 1 mL of 1 mg/mL streptavidin in PBS, 9 \u00b5L of 4 mM DBCO-NHS ester were added . The solution was allowed to react 30 min at room temperature. Reaction was quenched adding 100 \u00b5L of Tris-HCl 1 M pH 8. After 5 min at room temperature, the solution was transferred to Amicon Ultra 30 MWCO centrifugal filters and the excess of DBCO-NHS ester was removed by centrifugation. The final volume was adjusted to 1 mL by adding PBS.Ten mg of MCP-6 coated silica microspheres was resuspended in 500 \u00b5L of 1 mg/mL DBCO-modified streptavidin and incubated overnight at 37 \u00b0C under stirring. Beads were then washed 3 times with 1 mL of PBS and finally resuspended in 100 \u00b5L of PBS.One mg of streptavidin-coated silica microspheres, prepared as described in To 500 \u00b5L of 1 mg/mL rabbit IgG in PBS, 12.3 \u00b5L of 4 mM DBCO-NHS ester was added . The solution was allowed to react 30 min at room temperature. Reaction was quenched adding 50 \u00b5L of Tris-HCl 1 M pH 8. After 5 min at room temperature, the solution was transferred to Amicon Ultra 100 MWCO centrifugal filters and the excess of DBCO-NHS ester was removed by centrifugation. The final volume was adjusted to 500 \u00b5L by adding PBS.Ten mg of MCP-6 coated silica microspheres was resuspended in 500 \u00b5L of 1 mg/mL DBCO-modified rabbit IgG and incubated overnight at 37 \u00b0C under stirring. Beads were then washed 3 times with 1 mL of PBS and finally resuspended in 100 \u00b5L of PBS. As a negative control, 10 mg of MCP-6 coated silica microspheres was resuspended in PBS and followed the same experimental procedure.The two aliquots of beads prepared as described in Transmission electron microscopy (TEM) images were taken by a ZEISS LIBRA 200 FE microscope that has a FEG source (200 kV of emission power) and is equipped with a second-generation column \u2126 filter. The microparticle size was measured by TEM Imaging Platform Olympus.When coating silica microspheres, choosing an appropriate polymer is crucial to determine the properties of the particles. The antifouling properties of the coating and biomolecule immobilization density are strongly influenced by surface chemistry. MCP-6, whose structure is shown in w/v). The salt acts like a salting out agent that, by limiting the polymer\u2019s solubility in water, promotes its adsorption on the surface of the beads [MCP-6 has proven to be highly efficient in coating glass and silicon microarray chips. The polymer interacts with these materials through both covalent  and non-covalent interactions . The polymer is capable of coating silica beads that expose free silanols on their surface. To obtain a stable coating, the silica microbeads were immersed into a 0.9 M ammonium sulfate solution of the polymer (1% he beads . After 12), and density (0.18 g/cm3). Since the azide molar fraction is 2%, and its molecular weight is 100.12 g/mol, we can estimate the density of the azido groups on the surface, which is around 0.04 nmol/cm2.The presence of the polymeric coating was demonstrated by zeta potential measurement. The uncoated and MCP-6 coated silica microbeads were dispersed and diluted in a NaCl solution and analyzed. The measured zeta potentials were \u221272 mV and \u221212.1 mV for the uncoated and MCP-6 coated beads, respectively. This result confirmed the successful formation of the coating. In fact, while bare silica presents a high density of negative charges on its surface, the coating masks these charges. The high difference between the zeta potential for the uncoated and coated beads (around 60 mV) suggests the presence of a uniform film on the beads. During the last 20 years, our group has acquired extensive knowledge on a family of DMA-based copolymers that includes MCP-6 ,20,25. ASubsequently, to demonstrate that MCP-6, once adsorbed onto silica beads, retains its peculiar antifouling behavior, the coated beads were incubated overnight with 50 mg/mL protein solution in PBS at 37 \u00b0C. In particular, bovine serum albumin (BSA) and lysozyme were chosen because of their tendency to interact non-specifically with surfaces. Furthermore, BSA and lysozyme possess different isoelectric points ; thuAs highlighted in Once the antifouling character of MCP-6 was demonstrated, we evaluated its ability to provide efficient immobilization of biological probes. In particular, the immobilization rate of a panel of biological molecules including oligonucleotides, streptavidin, and antibodies was assessed.The immobilization density of the different biological probes was measured using an indirect assay. The beads functionalized with capture probes were incubated with their biological counterparts. The different concentration of the target in the solution, before and after the incubation, was measured to assess the amount of analyte captured on the surface. This value allows inferring the quantity of the immobilized probe. This indirect assay could potentially underestimate the amount of probe that is bound to the surface. However, it reports the amount of probe that, most likely, takes part in the target recognition.We first evaluated the ability of MCP-6-coated silica microbeads to immobilize DBCO-modified antibodies. A polyclonal rabbit IgG was functionalized with DBCO groups and incubated overnight with the beads to promote efficient immobilization. The negative control was obtained by incubating the beads overnight with just PBS. After several washing steps, both sets of beads were incubated with a secondary antibody (goat antirabbit IgG). The secondary antibody concentration was evaluated with a Bradford protein assay see  before aSimilarly, we evaluated the immobilization of streptavidin on MCP-6-coated silica microbeads. DBCO-modified streptavidin, synthesized as described in This result is important since it demonstrates that biotinylated DNA is specifically captured by streptavidin bound to MCP-6-coated silica beads. Additionally, the binding capacity of biotinylated ssDNA is slightly higher than that of commercially available beads .Finally, the capacity of the binding ssDNA was evaluated. For this purpose, 5 mg of MCP-6 coated beads was incubated overnight with 100 \u03bcL of DBCO-modified COCU8 at different concentrations  at 37 \u00b0C. After washing, the beads were incubated with 100 \u03bcL of Cy5-labeled COCU11 (at the same concentration used for COCU8 immobilization) for 1 h at 25 \u00b0C. After incubation, the supernatant was recovered, properly diluted, and the fluorescence emission evaluated with a spectrofluorometer. The COCU11 concentration was inferred using a calibration curve. The measured concentration was subtracted to the starting concentration to assess the amount of COCU11 captured on functionalized beads, and this value was used as an indirect measure of the COCU8 bound to MCP-6 beads. The results are shown in 9 \u03bcm2, respectively), the density of the DNA probes on the surface can be easily calculated and is reported in As can be noticed, there is a linear correlation between the concentration of DBCO-modified COCU8 used for the immobilization on MCP-6 coated beads and the amount of ssDNA effectively immobilized on the surface. Since the molecular weight of DBCO-modified COCU8 and the total surface of 5 mg silica beads are known ."}
+{"text": "Herein, a highly selective synthesis of \u03b2-mannosides and \u03b2-rhamnosides from glycosyl hemi-acetals is reported, following a one-pot chlorination, iodination, glycosylation sequence employing cheap oxalyl chloride, phosphine oxide and LiI. The present protocol works excellently with a wide range of glycosyl acceptors and with armed glycosyl donors. The method doesn't require conformationally restricted donors or directing groups; it is proposed that the high \u03b2-selectivities observed are achieved  Breaking from the current paradigm, a highly selective synthesis of hard-to-make \u03b2-mannosides and \u03b2-rhamnosides from simple glycosyl hemi-acetals has been achieved without using conformational restrictions. Koenigs\u2013Knorr glycosylations employing mannosyl halides using insoluble silver salts is not in widespread use, possibly because of the outcome being quite dependent on the donor/acceptor identities . We hypothesised that if glycosyl iodides could be generated in situ from hemiacetals using a modification of the catalytic Appel conditions, then a one-pot glycosylation could be developed. During these investigations we made a serendipitous discovery of conditions that led to highly selective \u03b2-mannosylation and \u03b2-rhamnosylation using simple donors 2  proved to be excellent substrates. The MeBn group has been reported to be orthogonal to Bn, Bz, and PMB groups44 and thus donor 1c could be an efficient building block in 1,4-\u03b2-mannan synthesis. Donors 1g,h gave only moderate yields, although high to excellent selectivity was still obtained. In the case of benzoyl-protected donor 1h, the yield was lowered due to formation of elimination product, competing transesterification under glycosylation conditions and subsequent intramolecular reaction to give an anhydro sugar. Disarmed donors e.g., peracetylated mannose showed poor reactivity and gave complex mixtures under our conditions, elimination to give glycals and migration of acetyl groups to acceptor 3a were noted .Based on procedures by Dentonectivity . Thus, g/(COCl)2 , and fol2NEt was found to give better conversion to the desired glycoside (more unreacted acceptor and elimination product resulted with lower amounts of base). High \u03b2-selectivity was obtained with a wide range of glycosyl acceptors. Monosaccharide acceptors bearing primary and secondary alcohols at positions 3 and 4 were tolerated. In the case of benzylidene-protected acceptor 3d, some cleavage of the acetal was also observed which lowered the yield of 4d. The excellent selectivity with glucosamine derivative 3h enables the synthesis of a \u03b2-d-Man(1\u21924)-GlcN linkage. The lower yield in this case reflects the poor nucleophilicity of this acceptor and thus the elimination reaction on the donor competes. In addition, phenolic acceptors 3e\u2013g gave excellent selectivities and yields of the O-glycoside. 4-Nitrophenol \u03b2-mannopyranoside 4g, which is of interest for enzymatic studies was synthesised in excellent yield and selectivity.45 A limitation was that ester-substituted acceptors either gave no reaction or gave complex mixtures; we believe the latter result arises, at least in part, from transesterification and thus multiple acceptors being present in solution. We confirmed that ester migration occurred on a benzoylated acceptor with iPr2NEt in CHCl3 at 45 \u00b0C in the absence of other reagents .However, in contrast to our initial hypothesis, and to literature reports invoking these alkoxyphosphonium salts as key intermediates, we did not find that the phosphine oxide is beneficial in the glycosylation step in our system. When glycosyl chloride 7 are key intermediates and that the presence of LiI promotes the stereoselective glycosylation step. In the absence of LiI no glycosylation products were observed (entry 3). With NaI in place of LiI no stereoselectivity was obtained (entry 4) and some unreacted glycosyl chloride was present. In the absence of added alcohol, the \u03b1-glycosyl iodide 7 was formed with complete consumption of the chloride 6a.52,53 Thus glycosyl iodides can be generated directly from glycosyl hemiacetals using the combination of Appel conditions and LiI in CHCl3 \u2013 this approach may be advantageous compared to other methods in many circumstances, especially in terms of practicality.54\u201356Our evidence suggests that glycosyl iodides 57\u201360 but under in situ anomerisation conditions \u03b1-mannosides are usually obtained.61,62 We note that the \u03b2-mannosylations using cyclic ethers were conducted in the presence of MgO and that lithium and magnesium ions have similar ionic radii.57\u201360 The salts LiI, LiCl, NaI and NaCl are insoluble in CHCl3. Since glycosylations of mannosyl chlorides and bromides promoted by insoluble silver salts have been reported,1,2,63 a filtration of the reaction mixture was carried out prior to addition of alcohol to remove insoluble LiCl and LiI \u2013 this did not lead to a change in selectivity when carried out in the absence of Ph3PO (entry 5 vs. entry 6); it is possible that the excess LiI is important for preventing the erosion of \u03b2-selectivity due to the presence of Ph3PO , or that a lithium salt in solution may be activating glycosyl iodides, or that lithium alkoxides are the active nucleophiles. When TMSI was used to generate glycosyl iodide from glycosyl acetate 8a (route B), the glycosylation was slightly \u03b1-selective (entry 11), however, when LiI was added it was moderately \u03b2-selective (entry 12); the moderate selectivity might arise because TMSOAc interferes. We confirmed that the presence of 4 \u00c5 MS doesn't impact on the selectivity (entry 10). Replacing LiI with LiCl also led to poor selectivity (entry 13). Route C was used to generate the glycosyl iodide through reaction of hemiacetal 1a with an iodophosphonium salt generated in situ.55,64 As in route B, the glycosylation was slightly \u03b1-selective (entry 14) but exhibited high \u03b2-selectivity when LiI was added (entry 15). These preliminary experiments suggest LiI is important for more than one fundamental role in achieving the desired reactivity and \u03b2-selectivity during glycosylation. Investigation of the underlying mechanism and the role of LiI is the subject of ongoing work in our laboratory.Mannosyl iodides have been reported to react with cyclic ethers and thioethers to give \u03b2-selectivityN1-like mechanism as this would be expected to give \u03b1-selectivity. Although we did not observe \u03b2-iodide 7, we cannot rule out the possibility that the \u03b1/\u03b2-anomers are in rapid equilibrium under reaction conditions.65,66 The minor \u03b1-glycoside could arise from an SN2-like pathway from \u03b2-iodide 7 (which would be expected to be more reactive), whereas the major \u03b2-glycoside could come from SN2-like reaction with the major \u03b1-iodide 7 promoted by LiI (1,2 but has greater applicability, especially with respect to scope. In part, this may come from the greater reactivity of glycosyl iodides, but it also suggests that other \u03b2-glycosylation methods could be developed that do not rely on restricting the conformation of the donor.We discount an Sd by LiI . Given t38,67,68 Our new method enables \u03b2-mannosides to be accessed from the same starting material. Thus mannosyl hemiacetals or chlorides are \u2018bimodal donors\u2019,69 representing a common precursor for all mannoside linkages, reducing by half the number of building blocks required for the synthesis of a library of oligosaccharides where both \u03b1 and \u03b2-linkages are required.70Previously, it has been shown that mannosyl chlorides can be converted to \u03b1-mannosides with excellent selectivity.In summary, we have developed a highly \u03b2-selective mannosylation and rhamnosylation protocol starting from readily available glycosyl hemi-acetals. The method uses commercially available reagents and straight-forward reaction conditions. It breaks with the prevailing paradigms in that it doesn't rely on protecting groups to conformationally constrain the donor and doesn't require cryogenic conditions or molecular sieves. The reaction proceeds through glycosyl chloride and then iodide intermediates. The mechanism will be the subject of further investigations in our laboratory, as will the extension of these conditions to other glycosides. We believe the method will be of immediate practical use to the community, and anticipate that it could pave the way for the development of new glycosylation reactions.Detailed experimental procedures, characterisation, scope limitations and copies of NMR spectra are provided in the ESI.IP, DAP and JJR carried out investigations in the laboratory. EMM provided supervision. All authors contributed to the writing of the manuscript.There are no conflicts to declare.SC-012-D1SC01300A-s001SC-012-D1SC01300A-s002"}
+{"text": "Interacting with others wearing a face mask has become a regular worldwide practice since the beginning of the COVID-19 pandemic. However, the impact of face masks on cognitive mechanisms supporting social interaction is still largely unexplored. In the present work, we focused on gaze cueing of attention, a phenomenon tapping the essential ability which allows individuals to orient their attentional resources in response to eye gaze signals coming from others. Participants from both a European  and an Asian  country were involved, namely two countries in which the daily use of face masks before COVID-19 pandemic was either extremely uncommon or frequently adopted, respectively. Both samples completed a task in which a peripheral target had to be discriminated while a task irrelevant averted gaze face, wearing a mask or not, acted as a central cueing stimulus. Overall, a reliable and comparable gaze cueing emerged in both experiments, independent of the mask condition. These findings suggest that gaze cueing of attention is preserved even when the person perceived is wearing a face mask. From the COVID-19 outbreak onwards, face mask has been introduced by governments worldwide as a protective gear to stop the spread of the virus e.g., . From a From a psychological perspective, faces have a profound impact on a variety of cognitive mechanisms supporting social interaction. For instance, from the unchangeable features of others\u2019 face we can acquire information concerning their age, gender, and, more in general, identity . SimilarSocial attention has been widely studied by adopting the so-called gaze cueing task e.g., , which tTo sum up, in the present study, we conducted two experiments based on a gaze cueing task in which averted gaze faces, either wearing a face mask or not, were employed as cueing stimuli. The gaze cueing task was delivered to a sample of Italian individuals living in Italy (Experiment 1) and to a sample of Chinese individuals living in China (Experiment 2). Because ethnic membership can deeply shape gaze cueing e.g., , in bothData were collected from October 23, 2020, until November 7, 2020, during the second wave of the pandemic in Italy, which took place in the fall period. In particular, during the time window in which data were collected, the new daily cases of COVID-19 in Italy increased from 19.143 to 39.809, according to the COVID-19 data repository by N\u2009=\u200946 . Before the experiment, participants were asked to read an informed consent, which was provided by pressing a specific keyboard key. The project was conducted in line with the Declaration of Helsinki, and it was approved by the Ethics Committee for Psychological Research at the University of Padova.The sample was composed of Italian students at the University of Padova, Italy, who took part on a voluntary basis. Based on previous studies in which a whole face vs. only eyes were visible e.g., , we aimeFacial stimuli consisted of high resolution faces (300 px width\u2009\u00d7\u2009450 px height), depicting two White males and two White females with neutral expression, extracted from the MR2 database . For eacIn the gaze cueing task . Before both the gaze cueing experiment and the questionnaire, a specific text screen appeared containing the instructions to successfully complete the requested task.https://www.jamovi.org/).Data of all participants who completed the experiment were analysed. Partial data provided by participants who aborted the task before its ending were not stored by the Pavlovia platform. Data analyses were performed through JAMOVI software  were rare and therefore not further analysed. Wrong responses  were analysed separately. Outliers, namely correct trials with a latency smaller than 150\u2005ms or greater than 1500\u2005ms , were discarded from RT analyses. Data were analysed both using a frequentist approach and a Bayesian framework, in order to establish the best model fitting the available data.F\u2009=\u200912.378, p < .001, 2p\u03b7\u2009=\u2009.216, due to smaller RTs on congruent  than on incongruent  trials, as well as the main effect of SOA, F\u2009=\u2009125.698, p < .001, 2p\u03b7\u2009=\u2009.736, due to smaller RTs at the longer  than at the shorter  SOA, consistent with a foreperiod effect. No other results were significant , including the two theoretically relevant spatial congruency\u2009\u00d7\u2009mask, and spatial congruency\u2009\u00d7\u2009SOA\u2009\u00d7\u2009mask interactions  for being preferable over the null model, and strong evidence (10BF\u2009=\u200919.835) for being preferable over the first best model including the theoretically relevant interaction between spatial congruency and mask .RTs for correct trials were analysed through a repeated measures ANOVA, with spatial congruency (2: congruent vs. incongruent), SOA (2: 200 vs. 700\u2005ms), and mask (2: mask vs. no mask) as within-participants factors. The main effect of spatial congruency was significant, F\u2009=\u20095.041, p\u2009=\u2009.030, 2p\u03b7\u2009=\u2009.101, with fewer errors at the longer  than at the shorter  SOA. No other results were significant . The Bayesian ANOVA, performed identically as that used for RTs analyses, confirmed that the model including only the main effect of SOA was the best one fitting the data. This model received anecdotal evidence (10BF\u2009=\u20092.228) for being preferable over the null model, and extreme evidence (10BF > 100) for being preferable over the first best model including the theoretically relevant interaction between spatial congruency and mask .Wrong responses were analysed through an identical ANOVA as that used for RT analyses. The main effect of SOA was significant, \u03b1\u2009=\u2009.20), they were kept separated; responses to the items concerning the subjective measures (Section 2 of the questionnaire) were intercorrelated (Cronbach's \u03b1\u2009=\u2009.74), and therefore they were averaged, thus obtaining a single score. Nevertheless, no significant correlations emerged for both the objective  and the subjective  measures. For completeness, correlations were also performed with an overall index of gaze cueing  and non-significant results emerged for both the objective  and the subjective  measures.The mean scores observed for each item of the questionnaire (see Appendix A) are reported, separately for each experiment, in Data were collected from November 11, 2020, until November 13, 2020. During the time window in which data were collected, the new daily cases of COVID-19 in China were extremely rare and changed from 15 to 18, according to the COVID-19 data repository .Mean age\u2009=\u200919.13 years, SD\u2009=\u2009.86, 14 males). The informed consent was provided by participants as in Experiment 1.We aimed to collect a similar number of participants as in Experiment 1. The sample was composed of 46 Asian students at the University of Guangzhou, China, who took part on a voluntary basis. Data collection was stopped at N\u2009=\u200946 (ps > .27).Everything was identical to Experiment 1, with only one exception: Faces belonged to Asian individuals. Importantly, since the MR2 database  providesData of all participants who completed the experiment were analysed. Partial data provided by participants who aborted the task before its ending were not stored by the Pavlovia platform. Data were analysed as in Experiment 1.Missing responses  were rare and therefore not further analysed. Wrong responses  were analysed separately. Outliers  were discarded from RT analyses.F\u2009=\u200955.335, p < .001, 2p\u03b7\u2009=\u2009.552, due to smaller RTs on congruent  than on incongruent  trials, as well as the main effect of SOA, F\u2009=\u200983.247, p < .001, 2p\u03b7\u2009=\u2009.649, due to smaller RTs at the longer  than at the shorter  SOA. The spatial congruency\u2009\u00d7\u2009SOA interaction was also significant, F\u2009=\u20096.047, p\u2009=\u2009.018, 2p\u03b7\u2009=\u2009.118, and it was further explored, for each SOA, through two tailed paired t-tests between congruent and incongruent trials. These indicated that the gaze cueing effect emerged both at the 200-ms SOA, t(45)\u2009=\u20093.342, p\u2009=\u2009.002, d\u2009=\u2009.493, and at the 700-ms SOA, t(45)\u2009=\u20096.610, p < .001, d\u2009=\u2009.975, but it was greater in the latter case (14 vs. 30\u2005ms). No other results were significant , including the two theoretically relevant spatial congruency\u2009\u00d7\u2009mask, and spatial congruency\u2009\u00d7\u2009SOA\u2009\u00d7\u2009mask interactions  for being preferable over the null model, and very strong evidence (10BF\u2009=\u200940.087) for being preferable over the first best model including the theoretically relevant interaction between spatial congruency and mask .RTs of correct trials were analysed through a repeated measures ANOVA, with congruency (2: congruent vs. incongruent), SOA (2: 200 vs. 700\u2005ms), and mask (2: mask vs. no mask) as within-participants factors. The main effect of spatial congruency was significant, F\u2009=\u200916.955, p < .001, 2p\u03b7\u2009=\u2009.274, with fewer errors at the longer  than at the shorter  SOA. No other results were significant . The Bayesian ANOVA confirmed that the model including only the main effect of SOA was the best one fitting the data. This model received extreme evidence (10BF > 100) for being preferable over the null model, and extreme evidence (10BF > 100) for being preferable over the first best model including the theoretically relevant interaction between spatial congruency and mask .Wrong responses were analysed through an identical ANOVA as that used for RTs analyses. The main effect of SOA was significant, \u03b1\u2009=\u2009.44) and the average index for subjective measures (Cronbach's \u03b1\u2009=\u2009.76) did not show any significant result . This holds true also for the overall index of gaze cueing .Mean scores collected through the questionnaire are reported in Fs > 61.508, ps < .001), as well as the spatial congruency\u2009\u00d7\u2009SOA interaction, F\u2009=\u20097.758, p\u2009=\u2009.007, 2p\u03b7\u2009=\u2009.079, reflecting that the gaze cueing effect, albeit significant at both SOAs , was greater at the longer one. Moreover, the main effect of experiment was not significant, F\u2009=\u2009.461, p\u2009=\u2009.499, 2p\u03b7\u2009=\u2009.005, but the experiment\u2009\u00d7\u2009congruency interaction was significant, F\u2009=\u20099.301, p\u2009=\u2009.003, 2p\u03b7\u2009=\u2009.094, reflecting that the gaze cueing effect, albeit significant in both samples , was greater among Asian than European participants . More importantly, experiment was not involved in any interaction including both spatial congruency and mask , thus indicating a similar gaze cueing effect in response to faces irrespective of whether wearing a mask or not, in both samples. The Bayesian ANOVA, including the same factors as those reported for the frequentist approach, indicated that the best model fitting the data included the main effects of spatial congruency, SOA, and experiment, as well as the spatial congruency\u2009\u00d7\u2009SOA and spatial congruency\u2009\u00d7\u2009experiment interactions. This model received extreme evidence (10BF > 100) for being preferable over the null model, and extreme evidence (10BF > 100) for being preferable over the first best model including the theoretically relevant interaction between spatial congruency and mask .In order to explore whether the gaze cueing effect was different between the two samples, we conducted a mixed design ANOVA, with the same within-participants factors used in previous analyses , and with experiment (2: Experiment 1 vs. Experiment 2) as an additional between-participants factor. Both the main effects of spatial congruency and SOA were significant (d\u2009=\u20090.058) suggests that regardless of the statistical significance, the difference, if any, would reflect a practically-irrelevant effect.As a final note, the effect size of the difference between the magnitude of gaze cueing in the mask and the no mask condition , From a purely methodological point of view, social attention abilities have been widely explored by adopting gaze cueing tasks based on manual responses, such as the one employed here. However, in every day social interactions, we tend to explore the surrounding environment around us through eye movements, which can be considered as a more direct and ecological index of visuo-spatial orienting of attention e.g., , and canOne of the main concerns regarding face mask use is related to the impact that such protective gear can have on interpersonal communication e.g., . Indeed,"}
+{"text": "P < 0.001). Phosphoproteomics analysis showed that 72 phosphosites were differentially regulated between HFpEF and Sham LV. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins and nuclear-localized proteins associated with contractile dysfunction and cardiac hypertrophy. Seven aberrant phosphosites were observed at the z-disk binding region of titin. Additional agarose gel analysis showed that while total titin cardiac expression remained unaltered, its stiffer N2B isoform was significantly increased in HFpEF vs. Sham . In summary, this study demonstrates marked changes in proteins related to mitochondrial metabolism and the cardiac contractile apparatus in HFpEF. We propose that SIRT3 may play a role in perpetuating these changes and may be a target for drug development in HFpEF.Although the prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, evidence-based therapies for HFpEF remain limited, likely due to an incomplete understanding of this disease. This study sought to identify the cardiac-specific features of protein and phosphoprotein changes in a murine model of HFpEF using mass spectrometry. HFpEF mice demonstrated moderate hypertension, left ventricle (LV) hypertrophy, lung congestion and diastolic dysfunction. Proteomics analysis of the LV tissue showed that 897 proteins were differentially expressed between HFpEF and Sham mice. We observed abundant changes in sarcomeric proteins, mitochondrial-related proteins, and NAD-dependent protein deacetylase sirtuin-3 (SIRT3). Upregulated pathways by GSEA analysis were related to immune modulation and muscle contraction, while downregulated pathways were predominantly related to mitochondrial metabolism. Western blot analysis validated SIRT3 downregulated cardiac expression in HFpEF vs. Sham (0.8 \u00b1 0.0 vs. 1.0 \u00b1 0.0;  Heart failure (HF) is a clinical syndrome caused by abnormalities in the heart that limit its ability to fill or eject blood . Heart fProteomic studies are powerful tools that allow for large-scale characterization of the entire protein phenotype in a biological system . AlteratPrevious proteomic studies have identified protein changes in dilated cardiomyopathy, atherosclerosis, and atrial fibrillation \u201323 and tSAUNA model , which recapitulates the human HFpEF phenotype  for 4 weeks via osmotic minipumps  and were maintained on 1% sodium chloride drinking water.As previously described , 35\u201337, in vivo Micro-Imaging System and a Real-Time Micro Visualization 707B Scanhead  as previously described . Transthoracic echocardiography was performed using a Vevo770 High-Resolution escribed . Brieflyescribed . As diasescribed . Pulse wParaffin-embedded sections (5 \u03bcm) of the mid-LV were stained with hematoxylin and eosin  to measure LV cardiac myocyte cross-sectional area. Microscopy images  were analyzed blinded to group identity using ImageJ measuring software .Left ventricle samples from 4 mice/group were processed as previously described , 40\u201342. Peptide concentrations were determined by a colorimetric peptide assay kit  and an aliquot of 100 \u03bcg was placed in 100 \u03bcl of 100 mM triethylammonium bicarbonate. Peptides were labeled with 0.4 mg of TMT label . All samples were labeled in the same TMT-batch, representing reporter tags 126C, 127N, 127C, 128C, 129N, 129C, 130N, and 131N. Labeled samples were pooled, and 95% was set aside for phosphopeptide enrichment. The remaining 5% of labeled peptides and the phosphopeptide enriched samples were analyzed separately by mass spectrometry.2) beads   . Briefly6 ions with a maximum fill time of 60 ms. The AGC for MS/MS scans was 3 \u00d7 104, with 80 ms maximum injection time, 0.1 ms activation time, and 33% normalized collision energy. To avoid repeated selection of peptides for MS/MS a dynamic exclusion list was enabled to exclude all fragmented ions for 60 s.Tryptic peptide mixtures and enriched phosphopeptides were analyzed by nano-scale high-performance liquid chromatography  and online nano electrospray ionization tandem mass spectrometry . Briefly, samples were loaded in aqueous 0.1% (v/v) formic acid via a trap column  and peptides were resolved over an Easy-Spray analytical column  by an increasing mobile phase B. Mobile phase A consisted of 2% acetonitrile and 0.1% formic acid, and organic phase B contained 80% acetonitrile and 0.1% formic acid. Reverse phase separation was performed over 120 min at a flow rate of 300 nl/min. Eluted peptides were ionized directly into the mass spectrometer using a nanospray ion source. The mass spectrometer was operated in positive ion mode with a capillary temperature of 300 C, and with a potential of 2,100 V applied to the frit. Tandem mass spectrometry (MS/MS) was performed using high-energy collision-induced disassociation and 10 MS/MS data-dependent scans  were acquired in profile mode alongside each profile mode full-scan mass spectra  as reported previously . The aut1 under standard settings using the entire Swiss-Prot mouse database2 downloaded January 24, 2019, allowing for two missed trypsin cleavage sites, carbamidomethylation of cysteine (fixed) and variable oxidation of methionine, protein N-terminal acetylation and phosphorylation of STY residues. Precursor ion tolerances were 20 ppm for first search and 4.5 ppm for a second search. The MS/MS peaks were de-isotoped and searched using a 20-ppm mass tolerance. A stringent false discovery rate threshold of 1% was used to filter candidate peptide, protein, and phosphosite identifications. The datasets generated for this study have been deposited and publicly available at the PRIDE Archive, proteomics data repository  with the data set identifier PXD033501.Data files (RAW format) were searched using the standard workflow of MaxQuant (version 1.3.0.5)Omics Notebook  software from the fgsea R package was used to compute gene set enrichment after ranking proteins by differential expression in HFpEF vs. Sham , 47, 48.3 , 50. GSEN = 7) and HFpEF (N = 11) mice were extracted and electrophoresed in 1% agarose gels using a SE600X vertical gel system  as previously described  (t-test (two sided). In those cases when data were not sampled as a normal distribution, non-parametric Mann\u2013Whitney U test was used. P \u2264 0.05 values were considered significant. These statistical tests were performed using GraphPad Prism software .Proteomics and phosphoproteomics differential analysis were based on a moderated Austria) , 55. ForSAUNA) induced hypertension associated HFpEF in mice. Compared to Sham, HFpEF mice demonstrated a moderate increase in systolic blood pressure , lung congestion (4.5 \u00b1 0.1 vs. 4.0 \u00b1 0.1 P < 0.01), and LV hypertrophy, measured by the LV weight-to-total body weight ratio . Additionally, cardiomyocyte size was increased 1.2-fold in HFpEF mice vs. Sham; P < 0.05 . As previously shown (P < 0.05).Echocardiography demonstrated preserved LVEF and increased LV mass  were developed to standardize the clinical diagnosis of human HFpEF. However, a discrepancy exists between these scores , beta (\u03b2)-myosin heavy chain , myosin heavy chain 9 , tropomyosin alpha (\u03b1)-1 chain ; the mitochondria-related proteins mitofusin 1 , mitochondrial dynamin like GTPase  and transcription factor A mitochondrial ; and the NAD-dependent protein deacetylase sirtuin-3 , recently implicated in cardiac function and cardiac stress responsiveness in HFpEF -actin , pyruvate, and ketone bodies. Significant changes are summarized in There was an extensive (I) \u03b2-oxidation related enzymes, implicated in FFA metabolism to acetyl-CoA, such as acyl-CoA dehydrogenase (ACAD) family member 11 , long-chain specific ACAD , short-chain specific ACAD , short-branched chain specific ACAD , 3-ketoacyl-CoA thiolase , enoyl-CoA hydratase , hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex (HADH) beta (\u03b2)-subunit  and HADH alpha (\u03b1)-subunit .pyruvate oxidation enzyme pyruvate dehydrogenase X component , which is part of the pyruvate dehydrogenase complex that catalyzes pyruvate to acetyl-CoA; and(II) the ketone metabolism enzyme succinyl-CoA:3-keto- acid coenzyme A transferase 1 , which catalyzes ketone bodies and produces acetyl-CoA for the tricarboxylic acid (TCA) cycle.(III) the P = 0.00376) in the LV of HFpEF.These cumulative results suggest that the energy substrates for mitochondrial oxidative metabolism may be inefficient in HFpEF. Interestingly, although there were no significant alterations in the protein signature of fatty acid and glucose transporters  in HFpEF, there was an upregulation of the ketone bodies transporter monocarboxylate transporter 1 , succinyl-CoA ligase beta subunit , isocitrate dehydrogenase  and pyruvate carboxylase , are required to catalyze acetyl-CoA and produce essential intermediates for the biosynthesis process, and most importantly, high energy molecules such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) for the electron transport chain (ETC). Subsequent analysis then showed that 27 proteins involved in the ETC  were also differentially expressed between HFpEF and Sham. Of these 27 proteins, 19 proteins were significantly reduced in the HFpEF, suggesting impaired ETC, which was consistent with an additional reduction of the uncoupling protein 3 .Additional analysis revealed that the mitochondrial proteins involved in the TCA cycle were also significantly decreased in the LV of HFpEF mice. These mitochondrial enzymes, namely citrate synthase  and fusion  were similarly decreased in the LV tissue from HFpEF mice.Lastly, additional proteins involved in mitochondrial biogenesis , striated muscle contraction , positive regulation of adaptive immune response , cardiac muscle cell development  and cardiac muscle cell differentiation . In contrast, the downregulated pathways were related to a multitude of GO terms associated with cellular metabolism (P = 0.00000), fatty acid metabolic process , acyl-CoA biosynthesis process , fatty acid oxidation , coenzyme metabolic process  were significantly reduced in HFpEF.Pathway enrichment analyses of the proteomics and phosphoproteomics combined datasets were performed by means of GSEA, which detects biology-driven gene sets of canonical pathways from databases of molecular signatures . These atabolism . This isP < 0.05; We next investigated the phosphoproteomics dataset. Phosphoproteomics analysis profiled 281 mouse reference protein sequences, of which 240 mapped to serine, 37 mapped to threonine and 3 mapped to tyrosine residues, consistent with the expected 90:9:1 cellular distribution ratio . The abuAberrant phosphorylation patterns occurred on proteins linked to disparate subcellular compartments, ranging from sarcomeric proteins , to nuclear-localized proteins  with established links to cardiac contractile function, cardiac hypertrophy and/or cardiomyopathy .P < 0.05, all considered class 1   in the phospho-dataset, and among them, seven phosphosites were 75\u20131.00) . Interes75\u20131.00) , 63.P < 0.05). Neither N2BA expression, N2BA/N2B ratio nor titin degradation were differentially altered between HFpEF and Sham mice , we thus performed additional immunoblot analysis to validate these findings. Indeed, SIRT3 expression was significantly decreased in the LV from HFpEF mice vs. Sham , and pathway alterations in HFpEF. These included but were not limited to: (I) changes in cardiac metabolism, where the predominant components were the mitochondrial metabolic processes and mitochondrial dysfunction; (II) alteration in cardiac contractile function-related proteins; (III) overexpression of pathways related to immune modulation; and (IV) a significant decrease in SIRT3 expression, that was validated by immunoblotting.We found marked changes in signatures of protein expression related to mitochondrial function and oxidative metabolism of energy substrates in HFpEF. There was a significant decrease in targets related to mitochondrial substrate oxidation, suggesting that cardiac mitochondrial metabolic function is impaired in HFpEF. Interestingly, there was an upregulation of the ketone bodies transporter SLC16A1 in the LV of HFpEF, but this was not accompanied by comparable changes in ketone metabolism enzymes. Although not investigated in this study, these findings may contribute to the metabolic impairment seen in HFpEF by increasing the transport of ketone bodies into the mitochondria, but without a compensatory catabolic response. We hypothesize that this mismatch in mitochondrial substrate intake and utilization results in mitochondrial ketone bodies accumulation which may detrimentally affect cardiac function . Ketone Proteomic evaluation of PTMs is essential to understand the function of many proteins in physiological and pathophysiological settings. PTMs are regulators of protein structure and function and, in the heart the predominant PTM is phosphorylation, followed by acetylation , and it SAUNA HFpEF mice most of the significantly hyper-phosphorylated titin residues were located at the Z-disk binding region of the titin protein . Although not investigated in the present study, SIRT3 may also play a role in cardiomyocyte stiffness and impaired diastolic function in HFpEF, possibly by titin acetylation  with the data set identifier PXD033501. Raw unedited gels images are shown in The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Boston University School of Medicine.MV-M and FS contributed to the conception and design of the study. MV-M and ES performed the surgeries and physiological measurements. MV-M, ES, and ZH performed the molecular analysis. RH and BB performed the proteomic sample preparation and carried out the mass spectrometry and bioinformatics analysis. MV-M, ES, and FS wrote the first draft of the manuscript. RH wrote sections of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version."}
+{"text": "Homologous recombination (HR) is a major repair pathway of DNA double-strand breaks and is closely related to carcinogenesis. HR deficiency has been established as a therapeutic target. The aim of this study was to elucidate the functions of a novel HR factor, Mediator complex subunit 1 (MED1), and its association with BRCA1. Formation of the MED1/BRCA1 complex was examined by immunoprecipitation and GST-pull down assays. The transcription cofactor role of BRCA1 was evaluated using luciferase assays. The roles of MED1 on DNA damage response and HR were analyzed by immunofluorescence and HR assays. R-loop accumulation was analyzed using immunofluorescence. R-loop-induced DNA damage was analyzed by comet assays. Immunoprecipitation and GST-pull down assays demonstrated that MED1 is a novel binding partner of BRCA1 and binds to the BRCT domain. Luciferase assays showed that MED1 potentiated the transcription ability of BRCT by two-fold. In MED1-depleted cells, recruitment of HR genes, such as RPA and \u03b3H2AX, to DNA damage sites was severely impaired. HR assays showed that MED1 knockdown significantly decreased HR activity. R-loop nuclear accumulation and R-loop-induced comet tails were observed in MED1-depleted cells. We conclude that the transcription factor MED1 contributes to the regulation of the HR pathway and R-loop processing. The human Mediator complex has a molecular weight of approximately 2\u00a0MDa, with approximately 30 subunits and three modules  and separable kinase modules. It is capable of dynamically changing its conformation to interact with various transcription regulators. During mRNA processing, the Mediator complex forms a bridge between general transcription factors on the enhancer and Pol II, leading to the formation of pre-initiator complexes (PICs), chromosomal loop formation, and transcriptional elongation of mRNA. It also exhibits several functions, including splicing and chromatin reconstitution, and regulates gene expression. Inactivation and hyperactivation of Mediator subunits is associated with the development of various malignancies7.The mechanisms by which genes are transcribed and transcription is dysregulated during carcinogenetic steps have been intensively researched for decades. Initiation, synthesis, and transcription of mRNA is catalyzed by the multi-subunit enzyme RNA polymerase II (Pol II) together with the basal transcription machinery, which comprises the general transcription factors and the Mediator complex9. MED1 overexpression was observed in 40\u201360% of breast cancers and positively correlated with the human epidermal growth factor receptor 2 (HER2) status11. In breast cancer cells, MED1 is phosphorylated in HER2-dependent manner and activates the estrogen receptor target genes12. HER2 activation is a major mechanism of breast cancer endocrine resistance. MED1 simultaneously blocks the estrogen receptor and the HER2 pathway, and thus MED1 is suggested to play a key role in HER2-mediated tamoxifen resistance and MED1\u2019s potential role as a therapeutic target is expected15. Previous large scale next-generation sequence studies have also unveiled the high mutation frequencies of Mediator complex components. MED1 mutations and deep deletions are observed in approximately 5% of both uterine endometrial cancer and cervical squamous cell carcinoma. These data also suggest that the loss of function of the Mediator complex can drive carcinogenesis.Carcinogenesis is a multi-step process involving the alteration of various signaling pathways. The altered gene expression patterns in cancer cells are attributed to gene mutations encoding cellular signaling molecules. Large scale genome rearrangement like fusion genes or sequence-specific variants in DNA binding motifs for protein modulators are also associated with carcinogenesis. To date, large scale cancer genome sequencing studies have revealed that gene alterations also arise in RNA processing machinery components including the Mediator complex. However, the role of these gene alterations and their contribution to carcinogenetic steps remains unclear. The first recognized carcinogenetic process with Mediator complex was the association between MED1 and breast cancer. Initially, in breast cancer tissues and cell lines, MED1 amplification and overexpression were observed16, which included a large number of proteins involved in RNA metabolism. RBMX, an hnRNP associated with spliceosomes that influences alternative splicing, regulates HR in a positive manner, accumulates at sites of DNA damage in a PARP1-dependent manner, and promotes resistance to several DNA damaging agents17. DNA damage-induced changes in the phospho-proteome, acetylome, and proteome of human osteosarcoma cells treated with etoposide also include a significant number of proteins involved in RNA metabolism18.Genomic instability, manifested as alterations in chromosome number and structure and in DNA structure, such as nucleotide substitutions, insertions, and deletions, is characteristic of most solid tumors. To maintain genomic stability and counteract DNA damage, cells have DNA damage response (DDR) pathway. Recently, a novel layer of complexity in the cellular response to DNA damage has emerged with the involvement of RNA metabolism. RNA-binding proteins (RBPs) involved in different steps of mRNA, transcription, splicing, and translation can affect genome stability. For example, proteomic analysis to identify human and mouse proteins phosphorylated in response to DNA damage in ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3 related) consensus sites, revealed approximately 700 targets19. Accordingly, mutations in mRNA processing genes leads to defects in the packaging of nascent mRNAs and, as a result, nascent pre-mRNA hybridizes with the transcribed strand, generating an RNA\u2013DNA duplex known as the R-loop. R-loop, a three-stranded nucleic acid structure, causes genomic instability23. The R-loop impairs replication fork progression, and stalled replication machinery leads to double-strand breaks. The other DNA strand, which does not bind to transcribed RNA, is exposed in a single-strand loop and can be an additional source of genome instability. The R-loop is involved in cancer and neurodegenerative diseases, and many DNA damage repair proteins, RNA binding proteins, and non-coding RNAs participate in the processing of this transcription byproduct. Interestingly, BRCA1 and BRCA2, master regulators of DDR, are also involved in R-loop processing. BRCA1 forms a complex with senataxin and processes aberrant R-loop accumulation at transcriptional pause sites and protects the genome from endogenous DNA damage24. BRCA2 also prevents R-loop accumulation through its interaction with the TREX-2 mRNA export factor PCID225.The mechanism by which mRNA processing factors, including transcription and splicing factors, play a role in genome stability was first reported by Manley et al.27. Compromised Mediator complex function may affect the generation of cancer-driving transcripts and induce aberrant transcription of critical DDR effectors, indirectly altering the cellular response to DNA damage. Although the R-loop may lead to the collapse of replication forks and generation of DSBs, which in turn leads to genome instability in cells with dysregulated mRNA processing, there is still little evidence of the participation of the Mediator complex in the DDR and genome stability. Therefore, in this study, we focused on MED1 to further elucidate the role of the Mediator complex on genome instability and cancer.Pathways related to mRNA biogenesis are unveiled to play an important role in genome stability and are an emerging hallmark of carcinogenesis. Subunits of the Mediator complex are found among candidate genes for homologous recombination pathwaysImmunoprecipitation (IP) of endogenous MED1 and BRCA1 was performed to determine whether MED1 interacts with BRCA1 in cells. The presence of MED1 and BRCA1 was confirmed by anti-MED1 and anti-BRCA1 antibodies in samples treated with anti-BRCA1 and anti-BRCA1 antibodies, respectively, indicating that MED1 and BRCA1 form a specific complex in human cells Fig.\u00a0c.Figure To determine whether MED1 binds to the BRCT region of BRCA1, whole-cell lysates of U2OS cells were incubated with GST-BRCT (wild type) protein or GST protein, and the presence of MED1 was detected by western blotting. As shown in Fig.\u00a0Therefore, we examined whether MED1 overexpression affects BRCT transcriptional activity using a luciferase assay. The results show that MED1 enhanced the transcriptional activity of GAL4-BRCT by approximately 2.4-fold . On the day after U2OS cells were seeded, the cells were knocked down by siRNA followed by DTB and synchronized to the G1 phase. The samples were collected at 0, 3, 6, 9, 12, and 24\u00a0h after tuning and washing thoroughly with PBS and analyzed by FACS. Twelve hours after the end of DTB, the percentage of G2/M cells was significantly increased in MED1-knockdown cells compared to that in the control  and DNA interstrand crosslinks (ICLs). We first performed a colony formation assay in U2OS cells, which were reseeded after MED1 knockdown by siRNA and then irradiated (IR 0\u20134\u00a0Gy) or treated with cisplatin (0\u20134\u00a0\u03bcM) to assess colony formation capacity. Our results show that colony-forming ability was significantly suppressed after exposure to IR and CDDP  repair using DR-GFP assays. HR activity was reduced to approximately 1/7 of the control in BRCA1 knockdown cells and to approximately 1/2 of the control in MED1 knockdown cells Fig.\u00a0a. We alsIn addition, we examined the effect of MED1 on the NHEJ pathway. When c-NHEJ activity was assessed by EJ5-GFP assay, MED1 knockdown significantly reduced GFP-positive cells compared to controls  and the protein was purified. Phosphorylation of ATM occurred after IR for 1\u201312\u00a0h in control cells, whereas it occurred after 3\u201324\u00a0h in MED1 knockdown cells. Phosphorylation of Chk2 also occurred after 1\u20133\u00a0h of IR in the control, whereas it occurred after 6\u201312\u00a0h of MED1 knockdown . Total protein expression of Chk2 and H2AX itself was repressed, whereas ATM was not affected by MED1 knockdown alone. However, after the induction of DSB by hydroxyurea, we observed a reduced expression of ATM in MED1 deficient cells, suggesting that MED1 is involved in the transcription of ATM under replication stress induced by hydroxyurea. When DSBs occur, ATM binds to the MRE11/RAD50/NBS1 complex and is recruited to the DSB site, where it activates and phosphorylates various substrates such as BRCA1, Chk2, NBS1, p53, and TopBP1. Thus, ATM promotes DNA damage repair and cell cycle regulation at the uppermost level and acts as a sensor of the damage response. MED1 knockdown prevented the accumulation of pATM, RPA, and Chk2 at the DSB site, suggesting that MED1 may contribute to DSB repair by directly regulating a wide range of signals downstream of ATM through the transcriptional control of ATM/CHk2/H2AX. MED1 partially contributed to HR activity, but not to BRCA1. The colony formation assay also suggests a partial role of BRCA1 via the MED1\u2013BRCA1 interaction in the DNA damage response. BRCA1 forms diverse complexes in the HR pathway, including the BRCA1\u2013MRN\u2013CtIP, BRCA1\u2013PALB2\u2013BRCA2\u2013RAD51, BRCA1\u2013BACH1\u2013TopBP1, and BRCA1\u2013BARD1\u2013Abraxas complexes, which play different roles in multiple stages of the HR pathway. Briefly, BRCA1 first forms a macromolecular complex that signals the presence of DNA damage and then induces DSB repair by recruiting proteins that process the excisional edges. Immunostaining showed reduced nuclear accumulation of RAD51, \u03b3H2AX, and BRCA1 at the DNA damage site after MED1 knockdown, supporting the aforementioned results. In addition, MED1 knockdown prolonged IR-induced DNA damage, and MED1 knockdown delayed and prolonged phosphorylation of ATM and Chk2. These observations suggest that MED1 regulates the HR pathway, but whether it is dependent on the transcriptional regulation of upstream factors such as CHK2 or on the BRCA1\u2013MED1 complex, or both, cannot yet be determined. Our pulse-chase BrdU labelling assay showed the reduced DNA synthesis by MED1 knockdown and this partially explains the induced replication stress and the reduced HR activity in MED1 deficient cells.We also examined the effects of MED1 on the activity of the NHEJ pathway, which functions as an alternative pathway to the HR pathway. Our results show reduced C-NHEJ activity by approximately 1/2 in MED1 knockdown cells and a significant suppression of DNA-PKcs phosphorylation. MED1 also affected the regulation of the upstream C-NHEJ region after DNA damage. PARP1 expression was preserved after MED1 knockdown, whereas XRCC1 transcription was repressed, suggesting that Alt-NHEJ and BRCA1-independent DNA damage repair pathways were also affected by MED1. We also observed a reduction in NHEJ activity in MED1 deficient cells by the EJ5-GFP assay, also confirmed by reduced 53BP1 accumulation at DNA damage sites induced by IR. As HR and NHEJ are balanced DNA repair pathways for DSBs, this reduced NHEJ activity in MED1 deficient cells partially explains the weaker effect on HR by MED1 knockdown compared to BRCA1. We must also consider the effects of MED1 on the transcriptional regulation and phosphorylation of ATM and H2AX. ATM and H2AX phosphorylation are upstream events of both HR and NHEJ pathways, and our observation suggests that both HR and NHEJ pathways may be repressed by reduced transcription level or phosphorylation of ATM and H2AX upon DNA damage.30. Our data suggest that a novel HR factor, MED1, may be one of the factors involved in both complex HR and R-loop processing.Because MED1 is conjugated to the BRCT region of BRCA1, we also tested the association of MED1 with the R-loop. Our results show that DNA damage caused by MED1 knockdown was cancelled by forced RNaseH1 expression. Furthermore, MED1 knockdown induced R-loop accumulation, suggesting that MED1 may contribute to R-loop processing. Whether R-loop processing is a MED1\u2013BRCA1 complex-dependent phenomenon and the mechanism of MED1 knockdown-induced DNA damage is open to further investigation. Components of the homologous recombination mechanism are involved in R-loop formation and genomic instability. Although the details remain unclear, the breast cancer suppressors BRCA1 and BRCA2 have been suggested to be involved in pathways that prevent R-loop accumulation31. Besides, R-loop formation in cells has recently been shown to be affected by various RNA-degrading enzymes, including RNAseH1 and RNAseH232. R-loops are more robustly detected in cells using DRIP assay and RNAseq assay. However, we have not established these methods in this research, and these are the limitations of our work.In this work, we showed R-loop formation by fluorescent immunostaining using S9.6 antibody, and that the induced DNA damage by MED1 depletion is rescued by overexpression of RNAseH1. We also showed by BrdU assay that replication stress may be induced under knockdown of MED1 through the mechanism of transcription\u2013replication conflict. On the other hand, we have not been able to verify whether the results of our S9.6 antibody-based fluorescent immunostaining assay are affected specifically by RNAseH1, not by RNAse T1 or RNAse III33, and MED1 may also be involved in this mechanism.Although the present study showed that the phosphorylation signals of ATM and H2AX are reduced and both HR and NHEJ pathways, which are regulated by ATM and H2AX phosphorylation, are repressed by MED1 knockdown, the contribution of MED1 to the transcriptional regulation of DNA damage repair factors is not well defined. The decreased protein levels of H2AX at steady state and the decreased protein levels of ATM upon double-strand break induction under MED1 depletion may suggest an aspect of MED1 as a transcription factor that forms a complex with RNA polymerase II and BRCA1. It has been reported that BRCA1 forms an mRNA splicing complex with BCLAF1 to contribute the efficient transcription and splicing of DNA damage repair factors during DNA damage induction35. On the other hand, recent studies have unveiled the mechanisms by which mRNA splicing intermediates and R-loop contribute to ATM phosphorylation37. In these studies non-dividing neuronal cells were used, indicating that replication-dependent conversion of these lesions into DSBs in S phase is not a requirement for ATM activation. The possibility that the R-loops formed by MED1 knockdown may contribute to ATM phosphorylation remains a subject for further investigation.The reduced ATM and H2AX phosphorylation signals under MED1 knockdown may also suggest that the direct contribution of MED1-containing complexes, such as Mediator complex, to ATM phosphorylation. As for phosphorylation of ATM, the intermolecular ATM autophosphorylation and direct phosphorylation by the MRN complex have been elucidatedIn conclusion, our results indicate that MED1, a novel binding partner of BRCA1\u2013BRCT, may contribute to the maintenance of genomic stability through its contribution to the HR pathway and suppression of R-loop accumulation.2. DR-GFP U2OS cells were kindly provided by Prof. Thomas Helleday and Dr. Oliver Mortusewicz and cultured in DMEM with 10% FBS, antibiotics and 1\u00a0\u03bcg/ml puromycin (Gibco). For siRNA-mediated knockdown, cells were transfected with 5\u00a0nM predesigned Silence Select siRNAs or control siRNA (Ambion/Life Technologies) using INTERFERin . For plasmid transfection, jetPEI (Polyplus Transfection) was used. Chemotherapeutic treatment was performed using hydroxyurea  dissolved in H2O, and X-rays were generated using a CellRad X .U2OS osteosarcoma cells, HeLa cells, and 293T cells were purchased from ATCC and cultured in DMEM  supplemented with 10% FBS (Sigma-Aldrich) and 1% antibiotics (penicillin)  at 37\u00a0\u00b0C and 5% COsiRNA-mediated knockdown was achieved using INTERFERin (Polyplus Transfection) following the manufacturer\u2019s instructions. Predesigned Silence Select siRNAs or control siRNA (Ambion/Life Technologies) were used. Each siRNA was used as a pool of three target-specific siRNAs: non-targeting, Mission SIC-001 (Sigma-Aldrich); MED1, #s10889 , #s10890 , and #s10891 ; and BRCA1, #s457 , #s458 , and #s459 . siRNAs were transfected with INTERFERin (Polyplus Transfection) at a final concentration of 5\u00a0nM..38.The pCMV-I SceI plasmid and pcDNA-RNaseH1 vector were kindly provided by Prof. Thomas Helleday and Dr. Oliver Mortusewicz. The pcDNA3, pGEX 4T-1, and pRL Renilla CMV-Luc control reporter vectors were purchased from Invitrogen , GE Healthcare , and Promega , respectively. The pGEX 4T-1 BRCT, GAL4, GAL4-BRCT, and pcDNA-MED1 vectors and reporter constructs (17M8-AdMLP-luc) were described previously by Hiraike et alUsed primary and secondary antibodies are listed in Supplemental Table Total cell extracts were obtained using RIPA buffer  with complete, EDTA-free , and Halt Phosphatase inhibitor cocktail (Thermo Scientific). Sample buffer  was added to the extracts. Equivalent amounts of lysate protein (10\u00a0\u03bcg) were used in Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and electrophoretically transferred onto Trans-Blot Turbo Transfer Packs (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Proteins transferred onto membranes (Bio-Rad) were probed with appropriate primary and secondary antibodies. Target protein expression was detected using ECL Plus WB detection reagents  using Image Quant LAS 4000 mini .The formation of a MED1\u2013BRCA1 complex in U2OS cells was analyzed by IP. Whole-cell extracts of U2OS cells using RIPA buffer were used for IP with anti-BRCA1 and anti-MED1 antibodies or preimmune IgG. The immunoprecipitate was subjected to 30\u00a0ml of protein G Sepharose 4 Fast Flow  and subsequently immunoblotted with anti-BRCA1, anti-MED1, or anti-IgG antibodies. Immunoprecipitation samples were treated with lambda protein phosphatase (Sigma Aldrich), DNase I (Sigma Aldrich), or RNase (Sigma Aldrich) according to the manufacturer\u2019s protocol.2, 10\u00a0mM PIPES, 1\u00a0mM ethylene glycol-bis (\u03b2-aminoethyl ether)-N,N,N0,N0-tetraacetic acid). After fixation, cells were rinsed briefly with 0.05% Tween 20 in PBS twice, and then permeabilized for 10\u00a0min with 0.3% Triton X-100 in PBS. After blocking for 40\u00a0min with PBS\u2009+\u20093% BSA or skim milk in PBS, cells were incubated with primary antibodies diluted in PBS\u2009+\u20093% BSA or skim milk in PBS at 4\u00a0\u00b0C overnight in a wet chamber. Cells were again rinsed twice with 0.05% Tween 20 in PBS and then permeabilized for 10\u00a0min with 0.3% Triton X-100 in PBS. Cells were then incubated with secondary antibodies diluted in PBS\u2009+\u20093% BSA at room temperature in the dark for 1\u00a0h. After washing with 0.05% Tween 20 in PBS, nuclei were stained with 4\u2032,6-diamidino-2-phenylindole (DAPI) for 5\u00a0min, washed with 0.05% Tween 20 in PBS, and permeabilized with 0.3% Triton X-100 in PBS. After rinsing with D2W, slides were mounted with ProLong Gold (Life Technologies) and visualized using a confocal microscope LSM700 . For foci quantification, cells were fixated in 96 well plates  using the same protocol, and images were taken using BZ-X710  and analyzed using Image J software  and BZ-X800 Analyzer . More than 400 cells were counted. Error bars represent SEM from three independent experiments.Cells plated on coverslips were fixed for 10\u00a0min at room temperature with fixative . For RPA staining, pre-extraction was performed for 5\u00a0min with ice-cold 0.3% TritonX-100 in CSK buffer . GST proteins were incubated with whole-cell extracts of U2OS cells. Unbound proteins were removed, and bound proteins were eluted and analyzed by SDS-PAGE.Glutathione-S-transferase fusion proteins or GST alone were expressed in 4/well) in 12-well plates and transfected with the indicated expression vectors using jetPEI . Luciferase assays were performed using the dual luciferase reporter assay system (Promega). Expression vectors\u00a0were co-transfected with reporter plasmid (17M8-AdMLP-Luc). As an internal transfection control, phRL CMV-Renilla vector (Promega) was also transfected in all experiments. After 24\u201348\u00a0h of transfection, cells were harvested, washed with PBS, and fixed following manufacturer\u2019s recommendations. Luminescence was measured using an LB96V luminometer . Individual transfections, each consisting of triplicate wells, were repeated at least three times.293T cells were seeded (6.0\u2009\u00d7\u2009104/well) were seeded in 6-well plates. After 24\u00a0h of incubation, endogenous MED1 was depleted with siRNA-mediated knockdown. Two days after siRNA transfection, cells were collected by trypsinization, treated with different doses of radiation, and reseeded into 6-well plates at 1 000 cells/well. Alternatively, cells were reseeded and cultured in various concentrations of cisplatin. The medium and cisplatin were replaced every 3\u00a0days. After 7\u00a0days of incubation, the cells were fixed with 100% methanol for 2\u00a0h and stained with Giemsa . The dishes were gently washed with water and air-dried. Colonies with more than 50 cells were counted. Results are presented as the mean\u2009\u00b1\u2009SEM from three independent experiments.HeLa cells (8.0\u2009\u00d7\u2009104 cells/well). Twenty-four hours after transfection of siRNA against endogenous MED1 or BRCA1, or non-targeting siRNA, cells were transfected with plasmids expressing I-SceI endonuclease or an empty vector as a control. After 48\u00a0h, cells were harvested, fixed with 4% PFA, and subjected to FACS analysis to determine the efficiency of HR or C-NHEJ repair induced by I-SceI digestion, which reconstituted a functional GFP gene. GFP signals were quantitated on a FACS Navios flow cytometer  and analyzed with Cell Quest Pro software v 3.1 (BD Bioscience). For each experiment, 20,000 cells were scored per treatment group, and the frequency of recombination events was calculated from the number of GFP-positive cells divided by the number of cells analyzed. Data are presented as the mean\u2009\u00b1\u2009SEM from three independent experiments with p-values determined by Student\u2019s t test.HR and NHEJ assays were performed in DR-GFP USOS and EJ5-GFP U2OS cells, respectively. Cells were seeded in 12 well plates (5.0\u2009\u00d7\u2009105 cells/well). Twenty-four hours later, cells were transfected with non-targeting siRNA or MED1 pool siRNAs using INTERFERin (Polyplus Transfection). After another 24\u00a0h, cells were treated with X-rays and incubated for 24\u00a0h before harvesting. After washing with PBS, cells were re-suspended in PBS at a concentration of\u2009~\u20091.0\u2009\u00d7\u2009106 cells/ml. DNA damage was evaluated using the Comet Assay Kit  following manufacturer\u2019s instructions. Electrophoresis was run at 300\u00a0mA, 21\u00a0V for 30\u00a0min in electrophoresis buffer using the Comet Assay Electrophoresis System II (Trevigen). Slides were washed in neutralization buffer (0.4\u00a0M Tris\u2013HCl pH 7.5) and counterstained with SYBR Gold (diluted 1:1000 in PBS) (Invitrogen). Images were acquired using a confocal microscope LSM780 (Carl Zeiss), and comet tail moment was estimated using CometScore software. At least 100 comets per sample were analyzed.U2OS cells were seeded in 6-well plates (1.0\u2009\u00d7\u2009104 cells/well) in 6-well plates and transfected with indicated siRNAs. After 24\u00a0h of incubation, cells were reseeded in 6-well plates (2.0\u2009\u00d7\u2009105 cells/well). For cell cycle synchronization, cells were arrested twice at the G1/S boundary using a double incubation in the presence of 2.5\u00a0mM thymidine for 15\u201318\u00a0h, followed by a 9-h interval of growth without the drug. Cells were released from the cell cycle blocks and harvested using trypsin at the indicated times. Cells were stained in the dark with 50\u00a0\u03bcg/ml propidium iodide (PI) (Sigma-Aldrich) at 4\u00a0\u00b0C for 30\u00a0min. Cell cycle distribution was analyzed by flow cytometry on an Epics XL instrument  using Cell Quest Pro software v 3.1 . The data are presented as the mean\u2009\u00b1\u2009SEM from three independent experiments.U2OS cells were seeded (8.0\u2009\u00d7\u2009104 cells/well) in 6-well plates and transfected with indicated siRNAs. After 48\u00a0h of culture, cells were treated with 10\u00a0\u03bcM BrdU. Cells were collected and fixed at appropriate time points . Following the manufacturer\u2019s protocol, cells were analyzed by flow cytometry on an Epics XL instrument  using the Cell Quest Pro software v 3.1 .FITC BrdU Flow Kit (BD Bioscience) was used. U2OS cells were seeded . Results are shown as mean\u2009\u00b1\u2009standard error of the mean. Data were analyzed using Student\u2019s Supplementary Information."}
+{"text": "Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes. In the manufacture of numerous biological drugs, a large amount of cell-culture-based biomass is required for each batch of the final product, and for some of these cultures, the detachment of cells adherent to glass, plastic or microcarrier surfaces and the type of subcultivation technology is an important process factor to generate that large biomass . For thoTraditional indexes for evaluating digestive performance include cell density, cell viability, and glucose metabolism rate , etc. AlMulti-omics technology refers to the modern biology research system in a series of high-throughput analyses based on testing technology research methods, including metabolomics, proteomics, genomics, and proteomics , etc., wVero is a cell line approved by the World Health Organization for production and is widely used in the production of biological drugs, such as inactivated COVID-19 vaccines , poliomyThe process of cell detachment is affected by many factors, such as temperature, procedure, and time. The most important factor is the type of digestive solution. There are two types of digestive solutions commonly used in the production of biological products: biogenic and abiogenic. Animal-based enzymes are represented by trypsin, which can hydrolyze proteins between cells and disperse cells. Animal-based enzymes can be obtained in large quantities in the pancreas and are easy to purify, so they are widely used in various cell culture processes . HoweverIn recent years, as an alternative to traditional animal-based enzymes, animal-origin-free enzymes have been gradually applied ,24,25; tIn the present study, we established the optimal detachment process for Vero cells, used multi-omics research technology to deeply analyze the physiological state of Vero cells after detachment, and found biomarkers that may be used to evaluate the cell detachment process. Our investigations provide new tools and ideas for the continuous optimization of cell detachment technology, and has a certain guiding significance for the industrial production of biological drugs.2, and passaged when the cells were cultured to 90% density. Trypsin is an example of an animal-based enzyme, while TrypLE is an example of an animal-origin-free enzyme. The 0.25% EDTA-Trypsin was provided by vaccine room six of the Beijing Institute of Biological Products Co., Ltd. . TrypLE was 1\u00d7 and purchased from Gibco , and contains 200 mg/L KCl, 200 mg/L KH2PO4, 8000 mg/L NaCl, 2160 mg/L Na2HPO4-7H2O, 457.6 mg/L EDTA, and 100 mg/L Phenol Red. Non-enzymatic cell lysate was purchased from Sciencell Company , and the product number was 0123. Vero cell growth medium containing 10% bovine serum was provided by vaccine room six of the Beijing Institute of Biological Products Co., Ltd. Newborn bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. .Vero cells were purchased from the UK Health Service . Vero cells were cultured in Minimum Essential Medium (MEM)  supplemented with 10% fetal bovine serum at 37 \u00b0C in a humidified atmosphere with 5% CO5/well, and after 12\u201324 h, we incubated trypsin at 25 \u00b0C, trypsin at 37 \u00b0C, TrypLE detachment solution at 25 \u00b0C, and TrypLE detachment solution at 37 \u00b0C. Detachment took place at 37 \u00b0C with four detachment times of 5 min, 10 min, 15 min, and 20 min. After detachment, we used a growth solution containing 10% serum to terminate the detachment reaction or directly discarded the detachment solution without stopping the detachment reaction. After the cells were treated with the abovementioned detachment processes, the cell viability, cell aggregation rate, and cell metabolism were detected.Vero cells were plated into 24-well plates at a concentration of 2 \u00d7 10An IC1000 Countstar automatic cell counter and cell counting plates were purchased from Shanghai Ruiyu Biotechnology Co., Ltd. . Vero cells were detached with the corresponding detachment conditions: 20 \u00b5L of the cell suspension was aspirated, added to the cell counting plate, and the number of cells, cell viability, and cell aggregation rate were detected using the Countstar automatic cell counter.5 cells per well. After 12\u201324 h, Vero cells were detached with trypsin or TrypLE detachment solution at 25 \u00b0C or 37 \u00b0C, and then the cells were counted and plated into 96-well plates. Five thousand cells were plated in each well, and after culturing in a 96-well plate for 24, 48, 72, and 96 h, the number of cells was detected, and the cell growth curve was drawn.Vero cells were plated into six-well plates, with 5 \u00d7 10Before cell detachment, we used an SBA-40E biosensor analyzer to detect the glucose content in the cell culture medium before detachment. After the cells were detached with the corresponding detachment conditions, we continued to culture them in a petri dish for 1 h; then the cell culture medium was removed, and the glucose content in the culture medium was detected after detachment. The difference in glucose content before and after detachment was calculated, denoting the change in cellular glucose metabolism caused by the detachment process.5 cells. We added 500 \u00b5L of binding buffer to suspend the cells and 5 \u00b5L of Annexin V-FITC, mixed the solution well, then added 5 \u00b5L of PI and mixed well. The mixture was allowed to react in the dark at room temperature for 5\u201315 min. We reserved three groups of Vero cell samples for each experiment, namely, the Vero cell control, FITC single-staining control, and PI single-staining control. During cytometry detection, the boundary between apoptotic cells and normal cells was set, and FITC and PerCP channels were selected for observation and detection using flow cytometry.An annexin V-FITC/PI apoptosis detection kit was purchased from Jiangsu Jikai Biotechnology Co., Ltd. ; the product number was KGA108. We detached the Vero cells, washed the cells twice with PBS (centrifugation at 2000 rpm for 5 min), and collected 1\u20135 \u00d7 10Transcriptomic, metabolomic, and proteomic analyses were undertaken for the cells treated with different detachment processes. The Vero cells were divided into three groups. The first group (Group 1) was detached with trypsin at 37 \u00b0C. The cells collected immediately after detachment were set to 0 h, and the detached cells were placed back into the cell incubator for 8 h. After the cells were detached with a nonenzymatic detachment solution and collected at the 8 h timepoint, the cells after trypsinization at 37 \u00b0C were further cultured for 24 h, and the cells were detached with a nonenzymatic detachment solution and collected at the 24 h timepoint. The second group (Group 2) was detached with TrypLE at 37 \u00b0C, then we set the cells collected immediately after detachment as 0 h, placed the detached cells back into the cell incubator for 8 h, and detached the recovered cells with a nonenzyme detachment solution. After 8 h, we continued to culture the cells detached with TrypLE detachment solution at 37 \u00b0C for 24 h, and then detached the cells with a nonenzyme detachment solution at the 24 h timepoint. For the third group (Group 3), we used a 4 \u00b0C TrypLE detachment solution at 25 \u00b0C detachment; the cells collected immediately after detachment were set to 0 h, and the detached cells were placed back into the cell incubator for 8 h. The cells collected after detachment with the nonenzyme detachment solution were set to 8 h, and detached with TrypLE detachment solution at 4 \u00b0C. After the cells were cultured for 24 h, the collected cells were detached with a nonenzymatic detachment solution and set to 24 h. In addition, Vero cells were detached with an enzyme-free detachment solution at 37 \u00b0C, and the immediately harvested cells were set as the control group. The experimental process and group settings are shown in p-values were adjusted using Benjamini and Hochberg\u2019s approach for controlling the false discovery rate. Genes with an adjusted p-value \u2264 0.05 found by DESeq2 were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented using the clusterProfiler R package, in which gene length bias was corrected. GO terms with a corrected p-value of less than 0.05 were considered significantly enriched by differentially expressed genes. KEGG is a database resource used for understanding the high-level functions and utilities of biological systems, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/ (accessed on 26 May 2022)). We used the clusterProfiler R package (version 3.8.1) to test the statistical enrichment of differential expression genes in KEGG pathways.RNA-seq was performed on three biological replicates for each sample at 0 h, 8 h, and 24 h after treatment with three kinds of detachment solutions . The total RNA of Vero cell samples under different detachment conditions was extracted using Trizol reagent. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in a First-Strand Synthesis Reaction Buffer (5\u00d7). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H\u2212). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3\u2032 ends of DNA fragments, the adaptors with a hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of 370\u2013420 bp in length, the library fragments were purified using an AMPure XP system . Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and the library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS  according to the manufacturer\u2019s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Differential expression analysis of two conditions/groups  was performed using the DESeq2 R package (1.20.0). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting g for 15 min at 4 \u00b0C and the supernatant was reduced with 10 mM DTT for 1 h at 56 \u00b0C and subsequently alkylated with sufficient iodoacetamide for 1 h at room temperature in the dark. Following a BCA, normalized quantities of protein were detached overnight with trypsin. The resulting peptides were fractionated using a C18 column  on a Rigol L3000 HPLC system, with the column oven set to 45 \u00b0C. The eluates were monitored at UV 214 nm, collected, and finally combined into six fractions. All fractions were dried under a vacuum, and then reconstituted in 0.1% (v/v) formic acid (FA) in water. For transition library construction, shotgun proteomics analyses were performed using an EASY-nLCTM 1200 UHPLC system  coupled with a Q ExactiveTM HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode. A half-sample containing 4 \u03bcg of fraction supernatant and 0.8 \u03bcL of iRT reagent was injected into a homemade C18 Nano-Trap column . Peptides were separated in a homemade analytical column . The separated peptides were analyzed using a Q ExactiveTM HF-X mass spectrometer (Thermo Fisher), with a Nanospray Flex\u2122 (ESI) ion source, spray voltage of 2.1 kV, and ion transport capillary temperature of 320 \u00b0C. The full scan range was from m/z 350 to 1500 with a resolution of 120,000 (at m/z 200), the automatic gain control (AGC) target value was 3 \u00d7 106, and the maximum ion injection time was 80 ms. The top 40 precursors of the highest abundance in the full scan were selected and fragmented using higher energy collisional dissociation (HCD) and analyzed in MS/MS, where the resolution was 15,000 (at m/z 200), the automatic gain control (AGC) target value was 5 \u00d7 104, the maximum ion injection time was 45 ms, the normalized collision energy was 27%, the intensity threshold was 1.1 \u00d7 104, and the dynamic exclusion parameter was 20 s.Proteomics was performed on three biological replicates for each sample. Vero cells were lysed with a DB lysis buffer , followed by 5 min of ultrasonication on ice. The lysate was centrifuged at 12,000\u00d7 2O) and B (0.1% FA in 80% ACN) were used to develop a gradient elution. A half-sample containing 4 \u03bcg fraction supernatant and 0.8 \u03bcL iRT reagent was injected into an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q ExactiveTM HF-X mass spectrometer (Thermo Fisher) operating in the data-independent acquisition (DIA) mode with a spray voltage of 2.1 kV, Nanospray Flex\u2122 (ESI), and capillary temperature of 320 \u00b0C. For DIA acquisition, the m/z range was from 350 to 1500. The MS1 resolution was set to 60,000 (at m/z 200), the full scan AGC target value was 5 \u00d7 105, and the maximum ion injection time was 20 ms. Peptides were fragmented by HCD in MS2, in which the resolution was set to 30,000 (at 200 m/z), the AGC target value was 1 \u00d7 106, and the normalized collision energy was 27%. The search and identification results obtained using Proteome Discoverer software  were imported into Spectronaut  software to generate a library. Eligible peptides and product ions were selected from the spectrum by setting peptides and ion pair selection rules [t-test.The raw data of MS detection were used to construct a DDA spectrum library. Mobile phases A (0.1% FA in Hhttps://www.mzcloud.org/ (accessed on 24 May 2022)), mzVault, and MassList database to obtain the accurate qualitative and relative quantitative results. Statistical analyses were performed using the statistical software R , Python , and CentOS ; when data were not normally distributed, normal transformations were attempted using the area normalization method. These metabolites were annotated using the KEGG database (https://www.genome.jp/kegg/pathway.html (accessed on 10 Febrary 2018)), HMDB , and LIPIDMaps databases (http://www.lipidmaps.org/ (accessed on 5 November 2021)). Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were performed in metaX  [t-test) to calculate the statistical significance . Metabolites with VIP > 1, p-value < 0.05, and fold change \u2265 2 or FC \u2264 0.5 were considered to be differential metabolites. Volcano plots were used to filter metabolites of interest based on log2 (FoldChange) and \u2013log10  of metabolites by ggplot2 in R language. For clustering heat maps, the data were normalized using z-scores of the intensity areas of differential metabolites and were plotted using the Pheatmap package in R language . The functions of these metabolites and metabolic pathways were studied using the KEGG database. The metabolic pathway enrichment of differential metabolites was performed; when the ratios were satisfied by x/n > y/N, the metabolic pathways were considered enriched, and when the p-value of the metabolic pathway was <0.05, the metabolic pathway was considered to have experienced statistically significant enrichment.Metabolomics was performed on six biological replicates for each sample. The samples were resuspended with prechilled 80% methanol by well vortex. Then, the samples were melted on ice and whirled for 30 s. After sonification for 6 min, they were centrifuged at 5000 rpm and 4 \u00b0C for 1 min. The supernatant was freeze-dried and dissolved with 10% methanol. Finally, the solution was injected into the LC-MS/MS system for analysis ,32. UHPL, China)   models. Variables in different datasets with different correlations and weights were initially identified by loading plots to filter out important variables affecting other omics .In order to explore the effects of animal-based enzymes and animal-origin-free enzymes on the physiological state of cells, we detached the cells using different detachment processes see , aggregaIn order to further mechanically explore the effects of different detachment processes on the physiological state of cells, we performed transcriptomic detection on cells detached with animal-based enzymes or animal-origin-free enzymes at 37 \u00b0C. The specific experimental process and group settings are shown in Next, we drew Venn diagrams of co-expression by comparing the gene expression 0 h, 8 h, and 24 h after digesting the cells with animal-based enzymes or animal-origin-free enzymes at 37 \u00b0C, as shown in p-value < 0.05, and a total of 1237 differentially expressed genes with significant relationships were obtained, including 531 upregulated genes and 706 downregulated genes, as shown in The differentially expressed genes (DEGs) were analyzed and the total, upregulated, and downregulated DEGs per group were enumerated . A volcaIn order to further compare the effects of animal-based enzymes and animal-origin-free enzymes on gene expression levels, we used the sum of the differential genes of Vero cells (0 h group) and the control group to digest the animal-based enzymes and animal-origin-free enzymes at 37 \u00b0C; as for the gene list, the 0 h group, the 8 h group, and the 24 h group were detached with the two detachment solutions for cluster analysis. The heat map is shown in In order to explore which physiological functions are involved in the genes affected by animal-based enzymes and animal-origin-free enzymes, we performed KEGG pathway enrichment analysis on the differentially expressed genes. The KEGG pathway enrichment bubble map is shown in In order to explore the effects of animal-based enzymes and animal-origin-free enzymes on the protein levels of Vero cells, we treated cells with animal-based enzymes, animal-origin-free enzymes, and nonenzymatic digests at 37 \u00b0C, as shown in p-value \u2264 0.05 and FC \u2265 2 as the thresholds, the main biological functions of the differential proteins could be determined by GO significance analysis. The GO enrichment results of the differential proteins are shown in In order to explore which biological processes are involved in the proteins affected by animal-based enzymes and animal-origin-free enzymes, we performed GO enrichment analysis on the differential proteins of the two. With In order to explore the physiological functions of proteins affected by animal-based enzymes and animal-origin-free enzymes, we performed KEGG pathway enrichment analysis on the differential proteins. Through KEGG pathway enrichment analysis, the most important biochemical metabolic pathways and signal transduction pathways involved in differential proteins can be determined. The KEGG pathway enrichment bubble map is shown in In order to further explore the effects of animal-based enzymes and animal-origin-free enzymes on the metabolic level of Vero cells, cells treated with animal-based enzymes, animal-origin-free enzymes or enzyme-free digestive solution at 37 \u00b0C were subjected to metabolomics detection according to the process shown in p-value < 0.05. Based on the above data, hierarchical clustering analysis was performed on the differential metabolites obtained in each group to determine the differences in metabolic expression patterns between and within the two groups in the same comparison pair, as shown in The results of untargeted metabolomics technology show that a total of 615 positive-ion-mode metabolites and 351 negative-ion-mode metabolites were identified in 60 samples, including the control. Differential metabolites were screened according to the criteria of VIP > 1.0, FC > 1.5 or FC < 0.667, and Volcanic map results show that 210 positive-ion-mode metabolites were significantly different in the G1_0h vs. G2_0h comparison group, among which 196 metabolites were upregulated and 14 metabolites were downregulated d1. ThereThe results show that, in positive ion mode, metabolic pathways, such as pyrimidine metabolism, beta-alanine metabolism, and glutathione metabolism were mainly involved, while in negative ion mode, nicotinate and nicotinamide metabolism, aminoacyl-tRNA biosynthesis, alanine, aspartate, and glutamate metabolism, glutathione metabolism, and other metabolic pathways were mainly involved. We also found that the levels of spermine and spermidine metabolites in glutathione metabolism were significantly reduced under positive ion mode. In conclusion, animal-based enzymes may affect cell glutathione metabolism by downregulating the levels of spermine and spermidine metabolites.The genes with significant differences obtained by transcriptome analysis and metabolites with significant differences obtained by metabolomics analysis were co-enriched to obtain the corresponding metabolic pathway map. In the KEGG pathway diagram, circles represent metabolites; blue circles represent downregulated differential metabolites, and yellow circles represent upregulated differential metabolites. The boxes represent genes or proteins: green boxes represent genes whose expression levels are differentially downregulated, and red boxes represent genes whose expression levels are differentially upregulated.PepN, TryS, TcTryS, and TcTryR. In conclusion, the results of combined metabolome and transcriptome analysis further confirm that animal-based enzymes may affect the metabolism of glutathione by downregulating the levels of spermine and spermidine metabolites, and thus affect the normal physiological state of Vero cells.We found that the 37 \u00b0C animal-based enzymes 0 h group (G1_0h) versus the 37 \u00b0C animal-origin-free enzymes 0 h group (G1_0h) was involved in glutathione metabolism in Vero cells a,b. In pIn recent years, the scale of cell-culture-based production of biologicals in the biopharmaceutical industry has continued to expand. For example, the value of >USD 100 billion in revenue per year generated by antibodies and similar molecules from CHO cells ,38. TherIn our study, we found that different detachment conditions had no significant effect on Vero cell viability, the cluster formation rate, cell growth and proliferation, or glucose metabolism. However, animal-based enzymes are more likely to cause cell apoptosis than animal-origin-free enzymes. Moreover, animal-based enzymes had a greater effect on cell gene expression levels than animal-origin-free enzymes during detachment. Proteomic results show that the differential proteins caused by animal-based enzymes and animal-origin-free enzymes were mainly enriched in steroid biosynthesis, oxidative phosphorylation, programmed necrosis, ribosome, senescence, metabolism, and other pathways. The results of combined metabolome and transcriptome analysis show that the differential metabolites and gene enrichment caused by animal-based enzymes and animal-origin-free enzymes were mainly involved in glutathione metabolism in Vero cells. In positive ion mode, the cell metabolite 5-hydroxyproline was significantly upregulated, and the metabolites spermine and spermidine were significantly downregulated.We found that animal-based enzymes and animal-origin-free enzymes appeared to have little effect on other indicators of cell status, except for the apoptotic indicators, which are commonly used in conventional detachment processes. However, using multi-omics technology, we found that animal-based enzymes had a greater impact on transcription, metabolism, proteins, and other aspects during cell detachment than animal-origin-free enzymes. This indicates that the commonly used indicators of traditional cell detachment technology cannot fully reflect the true state of cells, and some microlevel change indicators need to be identified and discovered.2+.We found that the protein COX17 and the metabolites spermine and spermidine may be biomarkers to evaluate the cell passage process at the microlevel. COX17 is an important protein for cytochrome C oxidase assembly in mitochondria . In the Spermine and spermidine are essential cellular components that play important roles in cell proliferation, transformation, differentiation, apoptosis, protection from oxidative damage, regulation of ion channels, and the stabilization of important cellular components, such as the cell membrane and chromatin structure ,46,47,48Microscopic changes in cellular components, such as the COX17 protein, spermidine, and spermine metabolites may affect the state of the cell, thereby affecting the expansion of the virus, and ultimately the quality of the vaccine product.Our study established the optimal detachment process of Vero cells by deeply characterizing the cell subculture process using multi-omics tools. We found new biomarkers, COX17, spermine, and spermidine, which may help us to evaluate the cell passage process. More refined evaluation tools are of great significance for cell-scale preparation.In future research, animal-based enzymes, animal-origin-free enzymes, and the Vero cell passage process under detachment conditions will be further compared in cell factories or large-capacity fermenters combined with the operation parameters of process equipment, and the state of Vero cells will be actively monitored possibly by using evaluation tools, such as COX17, spermine, and spermidine. By that time, the large-scale production of Vero cells in a minimal-damage state might be used to better guide the industrial production of biomedicines."}
+{"text": "Materials that will publish original and review papers on new scientific and applied research, and the articles it contains will make a contribution to the discovery and understanding of biodegradable and recyclable materials, their functional properties, characterization and applications.\u201cRenewable and Recyclable Polymeric Materials for Food Packaging\u201d is a new open Special Issue of Plastic is a common material used in food packaging. However, as plastics are petroleum-based, they take centuries to degrade. In fact, fewer than 12% of plastics are recycled, and an estimated 95% are discarded after one use. Thus, non-biodegradability and disposal problems are the major challenges associated with synthetic plastic packaging. A vast amount of plastic is disposed of annually by being left in landfills, incinerated, or dumped in the oceans worldwide, as this is the cheapest and fastest way to dispose of generated waste [While researchers have been working on developing biodegradable packaging in recent years, consumers want food packaging materials that are biodegradable, recyclable, environmentally friendly, and capable of extending the shelf life of food. In this context, bioactive packaging has emerged as a way to produce sustainable, efficient, and versatile packaging.An advanced polymer system containing modifying agents is characterized as a combination of materials with improved properties  or functions  that are used for a variety of industrial applications. Blending environmentally friendly biopolymers with renewable natural sources, synthetic polymers, nanofillers, natural anti-browning agents, nutrients, colorants, natural antioxidants, and antimicrobial compounds can result in a unique combination of properties that enhance products\u2019 quality, safety, and shelf life while reducing bacteria growth. Therefore, the development of tailored polymer systems will allow us to meet technological challenges while addressing global sustainability issues .Renewable and Recyclable Polymeric Materials include the development of biodegradable smart/intelligent packaging, characterizations, and the incorporation of natural functional ingredients, antioxidants, antimicrobial compounds, and eco-friendly nanomaterials to enhance the properties of biopolymers.The research interests of the section As Guest Editor, I am delighted to invite contributions on this topic in the form of original research articles or reviews."}
+{"text": "The present article provides a didactic presentation and extension of selected features of Pearl\u2019s DAG-based approach to causal inference for researchers familiar with structural equation modeling. We illustrate key concepts using a cross-lagged panel design. We distinguish between (a) forecasts of the value of an outcome variable after an intervention and (b) predictions of future values of an outcome variable. We consider the mean level and variance of the outcome variable as well as the probability that the outcome will fall within an acceptable range. We extend this basic approach to include additive random effects, allowing us to distinguish between average effects of interventions and person-specific effects of interventions. We derive optimal person-specific treatment levels and show that optimal treatment levels may differ across individuals. We present worked examples using simulated data based on the results of a prior empirical study of the relationship between blood insulin and glucose levels. Psychologists have long distinguished between experimental designs in which participants are randomly assigned to an active treatment or a control treatment and passive observational designs  in which the responses of participants are simply observed e.g., , 1975. GX and Y are each measured at a set of equally spaced measurement waves. Throughout we make a clear distinction between (a) the forecast of the causal effect of a  intervention on an outcome at a future time point and (b) the prediction of the individual\u2019s outcome at a future time point. The machinery for making forecasts of causal effects of interventions is provided by Judea In this paper, we present an approach that shows that under certain conditions longitudinal passive observational designs contain information that allows researchers to forecast the causal effect of a hypothetical treatment, even if that treatment has not yet been carried out. We consider the commonly used cross-lagged panel design in which two variables T = 4 equally spaced time points. We assume that the data are generated in a dynamic system in which the value of X at time t, Xt, influences the value of Y at time t + 1, Yt+1, and likewise Yt influences Xt+1. The system also includes autoregressive effects in which Xt influences Xt+1 and Yt influences Yt+1. In our illustration Xt represents blood insulin levels in non-diabetic adults at each of four equally spaced measurement waves measured in micro international units per milliliter (mcIU/ml). Blood glucose (Yt) is also measured at each measurement wave in milligrams per deciliter (mg/dl). For ease of presentation and without loss of generality, we use variables centered at the individual mean for each person.Using this approach, we illustrate how to forecast the effect of an intervention on the level of an outcome variable at a future time point. We also show how to calculate the variance of the forecasted value and the probability that the outcome variable attains a value within a predefined acceptable range. We initially assume a population of homogeneous individuals and later extend the approach to consider potential individual differences in the mean level of each person\u2019s time series data (unobserved heterogeneity). To facilitate a clear illustration of these ideas, we use as a running example a simple system with two variables measured at directed acyclic graphs (DAG) and represent the researcher\u2019s theory and prior  knowledge about the causal relationships between the variables.1 A key advantage of representing the hypothesized causal process as a DAG is that it allows the researcher to analyze the causal system using mathematical tools from the fields of graph theory and graphical modeling .We initially consider a dynamic system representing a single person from a population of t \u2265 1, for the X-series and the autoregressive coefficients t \u2265 1, for the Y-series are constant over time. Likewise, we will assume that the cross-lagged coefficients from X at time t to Y at time t + 1, denoted by t \u2265 1, and the cross-lagged coefficients from Yt to Xt+1, denoted by t \u2265 1, are constant over time. In addition, we will assume that the variances of the disturbances corresponding to the X-series, denoted by t \u2265 2, and those corresponding to the Y-series, denoted by t \u2265 2, are constant over time.Consider the DAG in error terms covary due to unobserved confounding. For example, we assume an unobserved common cause of X1 and Y1 that implies a covariance between the corresponding error terms X2 \u2192 Y3 relationship. Throughout this article, variances and covariances of disturbances are denoted by \u03c8 and a subscript. For example, Traditionally, error terms are not explicitly drawn in a DAG and the following conventions are used: (a) A bidirected edge drawn between two variables in the DAG indicates that the corresponding 3 Based on these values we obtain a standard deviation of blood insulin of SD(Xt) = 11.48 mcIU/ml and a standard deviation of blood glucose of SD(Yt) = 25.16 mg/dl, t \u2265 1.For our illustration, we simulate data from a known data generating process. Numerical values have been assigned to each parameter in the DAG based on the values from a panel data study by X. Thus, if a dose of insulin were given, the person\u2019s insulin level would be expected to experience a fixed increase to a level that reflects both the dose received and the person\u2019s baseline insulin level prior to the manipulation. In contrast, X to a constant value x. The latter type of manipulation is formalized via the do-operator and has proven to be very useful for theoretically defining and quantifying causal effects. For example, the do-operator applied to variable Xt is denoted by do(Xt = xt) or equivalently abbreviated as do(xt). By definition, the do(xt)-operator has the following properties: It (a) fixes the level of Xt at xt for each unit in the population, where xt is referred to as the interventional level. The interventional level xt is (b) not related to any causally prior variable. Finally, the do(xt)-operator (c) leaves all other relationships in the dynamic system unchanged.Xt is set by an experimenter. By fixing the level of the variable, each unit will have the identical value of Xt following the do(xt)-manipulation and Xt will have 0 variance under the intervention. In other words, the do(xt)-operation reflects a hypothetical experiment in which the treatment Xt = xt is applied uniformly over the whole population. Part (b) of the definition of the do(xt)-operator reproduces the feature of randomized experiments that all prior covariates are independent of the treatment variable. Part (c) of the definition of the do(xt)-operator, an assumption formally known as autonomy or modularity (do(xt) only alters the value of Xt and does not change the relationships between other measured variables not involved in the manipulation.Part (a) reflects the manipulation in a hypothetical experiment in which the level of the treatment variable dularity , impliesdo-operator applied to insulin levels at time t = 2, denoted as do(x2). Here, we imagine that each person\u2019s insulin level is set to the interventional value X2 = 11.48 mcIU/ml, that is, one standard deviation above the person\u2019s mean level (which is 0 for each person after mean-centering in a homogeneous population). The causal links of X2 to its direct predecessors in the causal chain (here: X1 and Y1) have been removed, but all other causal relationships in the model are maintained as before the do(x2)-operation.Up to this point, we have discussed the data generating mechanism but have not yet introduced the statistical model we will use for data analysis. Since we use simulated data the choice of a statistical model is straightforward: We chose a correctly specified statistical model, that is, one that exactly captures the data generating process described above. In our example, we will use a bivariate linear cross-lagged panel model with four measurement waves. This model can be equivalently understood as a linear structural equation model. Thus, it can be represented by a causal path diagram as commonly used to represent linear SEM . These cnon-experimental evidence is combined with our assumptions about the data generating mechanism to forecast the effect of the  intervention -operation depicted in X2 on Y3 and (b) predicting the value of Y3 based on X2 in the absence of interventions.Given the important distinction between the system following the do-operator allows us to represent a specific type of treatment effect by comparing the distribution of an outcome variable under the intervention do(xt) (representing the treatment) to that of a second intervention Y3 given the interventions do(X2 = 11.48)  and do(X2 = 0) .The effect of a treatment is always defined relative to another active or control treatment . The do-Yt+k in the population given the treatment do(xt) as the interventional distribution, denoted by P(Yt+k|do(xt)), where k is the number of waves after the intervention when the outcome is measured.4 In this paper, we restrict our attention to the interventional distribution k = 1 wave after the treatment has been applied. To compare the outcome variable Yt+1 given two distinct interventions do(xt) and Yt+1|do(xt) and Yt+1|do(xt)) and t = 3 given two distinct interventions on blood insulin at t = 2, namely do(X2 = 11.48) and do(X2 = 0). The latter intervention sets the value of blood insulin to the mean value (= 0 in mean-centered metric), the former intervention to one standard deviation above the mean. The upper panel of P(Y3|do(X2 = 0) (dashed line) and P(Y3|do(X2 = 11.48) (solid line).We define the distribution of the outcome value do(xt) relative to treatment Yt+1, abbreviated as the ATE of Xt on Yt+1 (do(X2 = 11.48) relative to treatment do(X2 = 0) on Yt+1, denoted as E(Y3|do(X2 = 11.48)) E(Y3|do(X2 = 0)). The interventional means are depicted in the upper panel of Another common way to compare two distributions is to examine differences in their means. The difference between the interventional means  on Yt+1 . In our do(xt) and widths of the bell-shaped curves in the upper panel of X2 on the level of Y3 is the same for do(X2 = 11.48) and do(X2 = 0).The comparison of two distributions is not restricted to a comparison of their location (means). Another important feature of the interventional distribution is its variance. The variances of the outcome distributions under the distinct treatments do-operation will lead to glucose levels that fall in an acceptable range of values that is specified a priori.Comparisons that go beyond first- and second-order moments (means and variances) may be of importance in a number of research situations involving dynamic systems. For example, Yt+1 given an intervention do(xt), and (b) the prediction of the value of Yt+1 given the passive observation that variable X takes the value x at time t, denoted by Xt = xt. Returning to our example, in case (b) we would like to predict the value of blood glucose at t = 3 based on the passive observation  that the insulin level is one standard deviation above the mean level at t = 2. We will, therefore, use the conditional distribution of Y3, given X2 = 11.48, denoted by P(Y3|X2 = 11.48), to predict the value of blood glucose at t = 3. We use the term predictions  to refer to probabilistic statements that are based on the conditional distribution.Throughout this article, we will make a clear distinction between (a) the forecast of the outcome value X2 = 11.48 yields the conditional distribution P(Y3|X2 = 11.48) represented by the solid line in the lower panel, whereas administering the treatment do(X2 = 11.48) yields the interventional distribution P(Y3|do(X2 = 11.48)) represented by the solid line in the upper panel. The conditional distribution of the predicted values of blood glucose levels is shifted to the right relative to the interventional distribution of the do(x2)-forecast of blood glucose levels.Comparing the two panels of Given this initial impression of the differences between (a) the interventional and (b) the conditional distribution, we now systematically analyze the differences in mean levels and variances, followed by the probabilities of blood glucose levels falling in an acceptable range. As part of this systematic comparison, we present formulae for interventional distributions and the moments thereof see also . Formulacxx instead of X-series, because these coefficients are assumed to be stable over time. Analogously, we will write cyy for the autoregressive coefficients of the Y-series as well as cxy and cyx for the cross-lagged coefficients. Likewise, we will drop the time indices of the variance parameters for t \u2265 2. We will write \u03c8xx instead of \u03c8yy instead of For ease of presentation we will drop the time indices of the structural coefficients in the equations. For example, we will write A complete presentation of all relevant formulae and details regarding the derivation thereof is provided in the t = 3 given (a) a do(x2)-intervention has been applied on insulin levels at t = 2 and (b) prior observations on the levels of X2 are available.In this section we systematically compare the mean level of blood glucose at do-treatment is applied (denoted by do(\u2205)), and (b) no prior observations are available (denoted by the empty set \u2205). In both situations, the best guess of the value of Y3 is zero since the data are mean-centered within each person (see row 1 of Y3|do(\u2205)) and the conditional mean E(Y3|\u2205) are both equal to the unconditional mean, that is, E(Y3|do(\u2205)) = E(Y3|\u2205) = E(Y3) = 0.We start with a comparison of the baseline scenarios in which (a) no do(X2 = 11.48) and in case (b) the value X2 = 11.48 has been observed (see row 2 of We now turn to a comparison of the scenarios where in case (a) the level of blood insulin has been set to do(X2 = 11.48) is performed, which sets the value of blood insulin to one SD above the mean level at time t = 2. Due to the intervention both paths entering into X2 are removed (see Y3 given the intervention do(X2 = 11.48) is \u2212 6.89 mg/dl  relative to the treatment do(X2 = 0). Thus, the resulting ATE is equal to E(Y3|do(X2 = 11.48)) \u2212 E(Y3|do(X2 = 0)) = \u22126.89 \u2212 0 = \u22126.89. In other words, actively changing the level of blood insulin at t = 2 from 0 to 11.48 (= one SD above the mean) results in a difference of \u22126.89 mg/dL in the forecasted blood glucose level at time t = 3.In case (a), the intervention oved see . The fort = 2 leads to a predicted value of 1.71 \u00b7 11.48 = 19.63 mg/dl of blood glucose at time t = 3 , we would obtain a difference of 19.63 mg/dL between the predicted values of Y3 given we observe a change in X2 from 0 to 11.48.In case (b) observing a blood insulin level of one SD above the person\u2019s mean at time \u2192 Y3 see . Observido(x2) treatment level by plus one SD yields a \u22126.89 mg/dL decrease in the forecasted value of blood glucose at time t = 3. In case (b) an observed one SD increase in blood insulin at wave 2 is predicted to produce a 19.63 mg/dL increase in blood glucose at time t = 3. Both findings provide useful information that need to be used for distinct purposes. In case (a), a physician would correctly forecast a negative value (\u22120.6 \u00b7 11.48 = \u22126.89 mg/dl) of blood glucose at time t = 3 after administering the dose do(X2 = 11.48) of insulin at time t = 2. In case (b), a patient obtaining the insulin reading X2 = 11.48 at time t = 2 would correctly predict a positive level (1.71 \u00b7 11.48 = 19.63 mg/dl) of blood glucose at time t = 3.In summary, in case (a) changing the do-operator (case (a)) is used to predict future values of Y in the absence of interventions (case (b)).Incorrect conclusions only arise if the algebraic machinery of conditional expectations (case (b)) is used to forecast the effects of interventions (case (a)) \u2013 or the other way around \u2013 the interventional mean based on the not be identified based on observational data even if the model is correctly specified. X2 on Y3 in the present example, a graphical rule called the backdoor criterion can be used. The backdoor criterion states that observing a set of variables which blocks all backdoor paths from X2 to Y3 provides sufficient information to correctly calculate the ATE based on non-experimental data. In the present example, the minimal sufficient backdoor adjustment set for the ATE of X2 on Y3 is {Y2}.5 Thus, if Y2 is held constant or statistically adjusted for, all backdoor paths are blocked.We have seen that interventional means (forecasts) and conditional expectations (predictions) are different quantities. The latter is a purely statistical quantity that can always be estimated based on observational data in a correctly specified model. The former is a causal quantity that might X2 on Y3 can be estimated using a linear regression of Y3 on X2and Y2 ), and (b) no prior observations are available (denoted by the empty set \u2205), the interventional variance V(Y3|do(\u2205)) and the conditional variance V(Y3|\u2205) are both equal to the unconditional variance V(Y3). All quantities take on the value 632.93, as displayed in row 1 of In the baseline scenarios in which (a) no Y3|do(x2)) can be calculated according to the following formula  (dashed line) and do(X2 = 11.48) (solid line). If a patient is treated at time t = 2 and we change the treatment dose , only the location of the interventional distribution changes and the forecast variance (width of the bell-shaped curves) is not affected by the specific level to which blood insulin is set by the experimenter.In the left part of do(x2) to a formerly untreated (do(\u2205)) person: the forecast variance increases from 632.93 to 951.43. An increase in variance might appear counter-intuitive at first glance. However, our illustration considers the dynamic interplay of blood insulin and blood glucose in a population of healthy (non-diabetic) persons. For such a population the glucose and insulin levels are governed by a self-regulating dynamic system. In our example the external intervention temporarily eliminates the ability of the system to self-regulate. Consequently, the system becomes temporarily destabilized and the variance increases.In contrast, the interventional variance does change as we introduce additional interventions on different variables in the model. This can be verified by comparing the value in row 1 with the value of row 2 in column 3 of Y3|X2 = x2) is calculated according to the following general formula  of shape of the conditional distributions is the same for both observations X2 = 0 (dashed line) and X2 = 11.48 (solid line). The two curves only differ in location but have the same width.In case (b), the conditional variances used for prediction decrease (632.93 > 245.71 > 40.00) as we successively move from row 1 to row 3 in column 6 of  formula :(5)V is identified since the backdoor criterion introduced in the section titled \u201cSo far, we have seen that the interventional variance and the conditional variance are different quantities. The conditional variance is a purely statistical quantity used to represent prediction error, whereas the interventional variance is a causal quantity used to represent forecast error when assessing the effect of a Y3 from observational data. In our example, the question is: Can we compute the numerical value V(Y3|do(x2)) = 951.43  from the numerical values of the statistical quantities in column 6 of Given identification is ensured, the question arises whether there is a simple way to estimate the interventional variance of do-operator can be expressed as a function of the parameters of the statistical model. This idea can be illustrated for the interventional variance as stated in Y3|do(x2)) which is a causal quantity and contains the do-operator. The expression on the right-hand side is a polynomial function of the model parameters cyx, cyy, \u03c8yy and does not contain the do-operator. In our working example all parameters that appear on the right-hand-side of cyx, cyy, \u03c8yy) are identified in the statistical model and can be estimated from observational data. Therefore, the interventional variance V(Y3|do(x2)) on the left-hand side of This problem can be resolved by using the technique for causal identification and estimation in linearly parameterized causal DAG-models suggested by yupper, upper threshold) where she will experience hyperglycemia. Nor should a patient\u2019s glucose level fall below a critical level  where she will experience hypoglycemia  after a treatment. A patient\u2019s glucose level should not exceed a critical level  and lower (60 mg/dl) thresholds were centered around a global mean of 100 mg/dl ) or in brief do(x2 = 11.48).We use the values 00 mg/dl . Thus, ado-treatment is applied (denoted by do(\u2205)), and (b) no prior observations are available (denoted by the empty set \u2205), the probability that blood glucose levels fall in the acceptable range at t = 3 is .94 as displayed in the first row of We now turn to a systematic analysis of interventional probabilities and contrast them with conditional probabilities. In the baseline scenarios in which (a) no do(X2 = 11.48) at time t = 2 yields a probability of blood glucose values within the acceptable range at time t = 3 equal to .86 (see row 2 column 3 in t = 2 in a nonlinear way that exhibits a unique maximum as depicted by the solid line in 6In case (a), administering t = 2, the probability of blood glucose values being within the acceptable range at time t = 3 is equal to .9999 (see row 2 column 6 of t = 2 which is depicted as the dashed line in In case (b), where a value of blood insulin equal to one standard deviation above the person\u2019s mean level is observed at time do(X2 = \u221233.34) to maximize the probability of treatment success . Recall that we use mean-centered variables with SD(X2) = 11.48. Thus, the treatment do(X2 = \u221233.34) corresponds to setting a patient\u2019s insulin level to 2.9 standard deviations below the mean level. In case (b) a patient who is passively measuring blood insulin levels at t = 2 would hope to measure a value of x2 = 11.67, since observing this value maximizes the conditional probability of blood glucose levels within the acceptable range at t = 3 .Both curves displayed in do(X2 = 11.67), this non-optimal intervention would result in a 85.5% probability of treatment success. A physician who correctly used the interventional distribution to choose the optimal treatment would apply do(X2 = \u221233.34), resulting in a 94.8% probability of treatment success. Thus, the consequence of an incorrect decision yields a 9.3% percentage drop in the probability of treatment success.If a physician were to erroneously use the conditional distribution to optimize an intervention and applied the treatment In summary, we have seen that conditional probabilities  and the interventional probabilities  differ. The statistical quantity can always be estimated from observational data in a correctly specified model, whereas the causal quantity needs to be identified. The backdoor criterion ensures causal identification of interventional probabilities in our example model. The exact formula of the interventional probability At the beginning of this article, we assumed that individuals come from a homogeneous population. This assumption will often be unrealistic in practice. A treatment that would be optimal  for an \u201caverage\u201d person is not necessarily optimal for a given patient if the population from which the average was calculated consists of heterogeneous individuals . In thisT = 4, and sample size N as presented in the Introduction. Authors from different disciplines have proposed a variety of approaches for including person-specific differences into this framework  = E(\u03b7y) = 0.Here, we adopt the approach of including the latent variables odel see . The ran7 This approach gives random intercepts the status of latent variables as indicated by the dashed circles around \u03b7x and \u03b7y in the DAG representation of the model displayed in unobserved heterogeneity.Note that these time-invariant variables  are neither explicitly modeled nor measured in the study. Instead, the time-invariant variables are captured in the random intercepts .\u03b7x are assumed to have direct causal effects on the levels of blood insulin as indicated by the directed edges from \u03b7x to X1, X2, X3, and X4 in \u03b7y to Y1, Y2, Y3, and Y4 are included in the DAG. The values of the structural coefficients corresponding to the paths from \u03b7x to Xt, t \u2265 2 are restricted to be equal to 1. These restrictions (a) assign a scale to the latent random intercept \u03b7x and (b) reflect the assumption that the structural coefficients from the random intercepts to blood insulin levels Xt, t \u2265 2 do not change over time. The same reasoning applies to the paths from \u03b7y to Yt, t \u2265 2. The bidirected dashed edge between the random intercepts \u03b7x and \u03b7y indicates that time-invariant variables that determine blood insulin levels might covary with time-invariant variables that determine the level of blood glucose due to unobserved confounding. The random intercepts are assumed to be uncorrelated with the error terms t \u2265 1.The time-invariant variables collected in the random intercept X1 and Y1 since they differ from the subsequent variables X2, X3, and X4 and Y2, Y3, and Y4 in an important way. The levels of Xt and Yt are explained in our model by the values of Xt\u22121 and Yt\u22121 when t \u2265 2. In contrast, the initial variables X1 and Y1 are exogenous in our model, that is, there are no incoming directed edges into X1 and Y1. The dynamics of the insulin\u2013glucose relationship, however, were going on in the individuals prior to the first measurement wave. Thus, the initial variables somehow need to account for the past of the process that is not explicitly modeled  = 5 and V(\u03b7y) = 10, where the time-invariant factors covary with COV = 2.5. All parameters introduced to model person-specific differences in mean levels via random intercepts are collected in A measure of the t \u2265 2, the standard deviation of blood insulin levels increases from SD(Xt) = 11.48 mcIU/ml in the homogeneous model to SD(Xt) = 26.30 mcIU/ml in the model with random intercepts. Similarly, the standard deviation of blood glucose levels increases from SD(Yt) = 25.16 mg/dl to SD(Yt) = 61.83 mg/dl. A complete presentation of all relevant formulae and details regarding the derivation thereof is provided in the A consequence of modeling unobserved heterogeneity via random intercepts is an increase in total variance as compared to the model for the homogeneous population. For example, for T = 4) with random intercepts for data analysis. Since we use simulated data, we know with certainty that the causal model is correctly specified and that our statistical model captures the data generating process. In practical applications, however, one usually does not know the true data generating mechanism and causal assumptions postulated by researchers might be wrong. We discuss statistical tests of model assumptions and methods to analyze the sensitivity of causal conclusions with respect to violations of assumptions in the section on \u201cdo-operator. Since the causal diagram of the model with random intercepts still belongs to the class of DAGs (see do-operator is well-defined and can be directly applied to the model including random intercepts. do(x2) is applied.As in the homogeneous model, we are interested in the effects of an intervention represented by the DAGs see , the do-Y3|do(x2)) describes blood glucose levels in the entire population of individuals, given that the treatment do(x2) has been administered. The inclusion of random intercepts allows us to define the person-specific effects of interventions.The interventional distribution P, \u03b7x = zx, \u03b7y = zy). The person-specific interventional distribution describes blood glucose levels at time t = 3 for an individual who is characterized by the time-invariant characteristics \u03b7x = zx and \u03b7y = zy after the treatment do(x2) has been applied. If multiple individuals have the same time-invariant characteristics the person-specific interventional distribution is also the subgroup-specific interventional distribution of this homogeneous group of individuals.Each individual in the population can be characterized by the values Y3|do(x2)) and the person-specific interventional distribution P(Y3|do(x2), \u03b7x = zx, \u03b7y = zy), the random intercepts \u03b7x and \u03b7y belong to every adjustment set for backdoor identification  has been applied, where 26.30 mcIU/ml is the standard deviation of blood insulin levels in the model with unobserved heterogeneity  In the following sections, we compute the person-specific effects of interventions for Amy, Joe, and Sam and compare these quantities to the average  effect of an intervention in the entire population.In the following, we consider three hypothetical individuals from the population, namely Amy, Joe, and Sam, who are characterized by person-specific characteristics displayed in t = 3 if no further information is available. Since all observable variables are mean-centered and the random intercepts \u03b7x and \u03b7y have zero means, the unconditional mean is equal to zero  has been applied. They are given by the following formulae:Y3|do(x2)) in the presence of unobserved heterogeneity is equal to 5939.03 (see column 5 of t = 3 after the intervention do(x2) has been applied.The interventional mean and variance of t = 3 for an individual who is characterized by the time-invariant features \u03b7x = zx and \u03b7y = zy, after the treatment do(x2) has been applied. The person-specific interventional mean is given by the following formula:x2 and the time-invariant characteristics zx and zy of that person. For example, the person-specific interventional mean for Sam is computed by plugging in Sam\u2019s numeric values do(X2 = 26.30), different forecasts of blood glucose levels at time t = 3 are obtained for different individuals, depending on their respective time-invariant characteristics.The person-specific interventional moments describe the distribution of blood glucose levels at x2 is constant and equal to cyx = \u22120.6 in the equation for the person-specific mean  to change in forecasted values for each individual.Note that the linear coefficient of the treatment level mean see . As a coY3|do(X2 = 26.30)) = \u22120.6 \u00b7 26.30 = \u221215.78 for a new patient under treatment of whom the person-specific characteristics are yet unknown. After learning that a patient is Amy, Joe, or Sam (and their respective person-specific characteristics) the physician updates his or her forecast to \u221258.73, \u221215.78, or 27.17, respectively. Note that by learning the person-specific characteristics of a new patient, the variance of the forecast error drops from 5939.03 to 951.43  interventional moments and the person-specific interventional moments can be illustrated as follows. A physician initially forecasts the value E, an event we refer to as treatment success. Recall that As in the section titled \u201cdo(X2 = 26.30) (see column 1 of do(X2 = 26.30) has been applied to a new patient from the heterogeneous population (a patient whose person-specific characteristics are yet unknown).In the presence of unobserved heterogeneity, the  probability of treatment success is equal to .52 given the treatment Y3 drops from 5939.03 to 951.43 after learning the person-specific characteristics  for Amy, do(X2 = \u221233.33) for Joe, and do(X2 = 38.25) for Sam. In other words, if a given person is administered his or her individually optimal treatment level, the probability of treatment success for that person is 94.8%.new patient from the population for whom the person-specific characteristics are yet unknown, the optimal treatment decision would be do(X2 = \u221233.33). If the physician learns that the new patient is Joe  the initial treatment do(X2 = \u221233.33) coincides with Joe\u2019s person-specific optimal treatment resulting in a 94.8% probability of treatment success. On the other hand, if the new patient turns out to be Amy or Sam, then the treatment do(X2 = \u221233.33) is no longer optimal (given this new person-specific information) and would result in a 71% probability of treatment success in either case. The optimal treatment decision after learning that the new patient is Amy or Sam would be do(X2 = \u2212104.92) or do(X2 = 38.25), respectively, resulting in a 94.8% probability of treatment success in either case. Thus, updating an initial population-based optimal treatment decision to include Amy\u2019s or Sam\u2019s person-specific characteristics, results in an absolute increase of 23.8% in the probability of treatment success.Joe is the average patient, that is, his person-specific characteristics coincide with the respective population means (see row 2 of 11 Throughout we emphasized the important distinction between (a) forecasting the future value of a variable following an intervention at a prior wave and (b) predicting the future value of a variable following measurement of variables at a prior wave. In task (a) a causal effect is computed based on the interventional distribution in a DAG-based framework. We focus on do(xt)-type interventions that set each individual\u2019s value on the variable X at time t to a fixed constant value. In task (b) the future value of a variable is predicted using the conditional distribution . Task (a) requires identification of the interventional distribution and its moments . Causal identification in the case of a homogeneous population can be established using the machinery of In this article, we focused on the example of a four-wave linear cross-lagged panel design to provide a didactic presentation of not available  by including additive random intercepts into the linear cross-lagged panel framework. We showed that \u2013 even in this simple model \u2013 a treatment decision that is optimal on average can be improved substantially if person-specific characteristics can be incorporated into the model. In other words, treating a new, unknown patient with a treatment dose that has proven to be optimal on average  will only be optimal for a specific person as long as additional person-specific information is vailable . As (a) causal misspecification. In linear models with random intercepts additive time-invariant unobserved confounders are being statistically controlled. This, in turn, allows for the identification of causal effects that would not be identified in the most general non-parametric setup.12 In the case of linear models with random intercepts, we can to use the approach developed by do-intervention were forecasted without performing an experiment or otherwise perturbing the dynamic system. For causal conclusions based on observational data to be valid, the underlying assumptions need to hold. The methods discussed in this paper rely on the correct a priori specification of the causal ordering of the variables and the assumption of a linear dynamic process that is both stable over time and that is not altered by the intervention . In the present article, we only allowed for between-person differences in the individual mean levels of the variables. Computing causal forecasts based on models that account for between-person differences in the autoregressive and cross-lagged effects remains a task for future research.The methods presented in this paper permit causal conclusions to be reached based on a combination of observational data and assumptions about the data generating mechanism. The effects of a hypothetical Statistical tests are available that can detect certain types of misspecification. In the present paper the set of possible causal orders is limited in part by the cross-lagged panel design . Further tests of the causal ordering of the variables  can be performed using procedures proposed by In the development of our approach, we did not consider measurement error. Finally, our example was based on simulated data based on empirical findings reported by In conclusion, the proposed method combines the desirable features of cross-lagged panel designs, a sound definition of causal quantities based on DAGs, and the flexibility of SEM for data analysis. We hope that our presentation facilitates the integration of linear SEM and DAG-based causal inference and enables researchers to better distinguish between tools for predictive and causal research questions.Supplemental Material [1]"}
+{"text": "Targeted biologic therapies can elicit an undesirable host immune response characterized by the development of antidrug antibodies (ADA), an important cause of treatment failure. The most widely used biologic across immune-mediated diseases is adalimumab, a tumor necrosis factor inhibitor. This study aimed to identify genetic variants that contribute to the development of ADA against adalimumab, thereby influencing treatment failure. In patients with psoriasis on their first course of adalimumab, in whom serum ADA had been evaluated 6\u201336 months after starting treatment, we observed a genome-wide association with ADA against adalimumab within the major histocompatibility complex (MHC). The association signal mapped to the presence of tryptophan at position 9 and lysine at position 71 of the HLA-DR peptide-binding groove, with both residues conferring protection against ADA. Underscoring their clinical relevance, these residues were also protective against treatment failure. Our findings highlight antigenic peptide presentation via MHC class II as a critical mechanism in the development of ADA against biologic therapies and downstream treatment response.  Monoclonal antibodies comprise the largest family of biologic therapies and have transformed the treatment of immune-mediated inflammatory diseases (IMIDs). However, serial administration of recombinant protein drugs may induce an undesirable immune response characterized by development of antidrug antibodies (ADA) , 2. ThesEstimates of ADA prevalence vary widely depending on the drug, disease, and ADA assay technique, but up to 65% of patients with IMID reportedly develop ADA in response to biologics inhibiting the pro-inflammatory cytokine tumor necrosis factor (TNF) , 4\u20136. HeAlthough several drug-related factors influence drug immunogenicity, not all individuals develop significant immune responses to a given biologic. This implies the existence of host-specific factors that influence variability in the antidrug response in vivo. Major histocompatibility complex (MHC) class II genes have been hypothesized to be centrally involved in the development of ADA, fueling a series of candidate gene studies reporting associations with ADA against TNF inhibitors \u201310. MorePsoriasis provides an optimal clinical scenario to study the genetic basis of ADA development, since it is one of the most common indications for biologic use, with adalimumab a first-line treatment. Critically, biologics for psoriasis are generally used as monotherapy, whereas patients with IBD and rheumatoid arthritis (RA) receiving the same biologic therapies are often coprescribed immunosuppressants, which are known to reduce the development of ADA.The current investigation represents the largest genetic study of ADA against adalimumab and to our knowledge the first genetic investigation of ADA against any biologic in psoriasis. In a real-world data set, we examine the role of genetic variation across the genome and implicate antigenic peptide presentation via MHC class II as a key genetic driver of ADA development. We further demonstrate that the genetic variation associated with ADA development is also associated with clinical outcome, since individuals harboring alleles protective against ADA were also less likely to terminate adalimumab due to ineffectiveness.https://doi.org/10.1172/jci.insight.156643DS1). Consistent with previous investigations, those testing positive for ADA were also more likely to terminate treatment due to ineffectiveness  is a single nucleotide substitution located within the cluster of genes encoding MHC class II antigen-presenting molecules.Genome-wide genotyping was performed in 784 individuals of European ancestry, for whom ADA status had been determined 6\u201336 months after starting adalimumab. Following quality control and genome-wide imputation of common variants with the Haplotype Reference Consortium panel , 16, we P = 3.09 \u00d7 10\u20137), and at position 11, where the presence of either a serine, valine, or aspartic acid residue was associated with risk of developing ADA . These specific allelic combinations at positions 9 and 11 were in perfect LD and anticorrelated. No single classical allele of any of the 5 class II molecules had stronger evidence of association with ADA, although we noted HLA-DRB1*11 to be the classical allele with the strongest evidence of association with ADA , which has previously been reported to be associated with ADA against TNF inhibitors in a population with IBD , inferring the resulting genotypes of variable amino acid residues and classical alleles at 2- and 4-digit resolution. The resulting genotypes of classical alleles and combinations of amino acid residues at specific positions were tested for association with ADA see . The strwith IBD .P = 1.76 \u00d7 10\u201312; position 71 lysine: OR 0.39, 95% CI 0.28\u20130.55, P = 5.77 \u00d7 10\u20138; P = 0.0048). The effects of tryptophan at HLA-DRB1 position 9 and lysine at position 71 were robust to potential confounders, including immunosuppressant cotherapy, threshold of ADA detection, genotype of the psoriasis susceptibility allele HLA-C*06:02, sample timing, comorbidities (including psoriatic arthritis and diabetes), age, age of psoriasis onset, sex, and baseline disease severity . Consistent with these observations, classical HLA-DRB1 alleles harboring either a tryptophan at position 9, or a lysine at position 71, were enriched for protective effects against ADA development  and 11 (P6 pocket) are located on the \u03b2-sheet floor, with their side chains oriented into the groove; position 71 (P4 pocket) is separated by a single turn along the \u03b1 helix, with its side chains spatially close to those of position 11 . AntigenThe specific HLA-DRB1 amino acid positions highlighted by our analysis are well established in numerous disease settings where adaptive immunity plays a critical role. Although there have been no reports of association with psoriasis susceptibility, which is primarily driven by the MHC class I allele HLA-C*06:02 , in RA aThe use of a drug-tolerant assay is a critical aspect of the current study design, due to the predicted impact of variability in sample timing, with respect to treatment administration, on serum drug levels. The proportion of individuals testing positive for ADA against adalimumab was higher than previously reported , 6, 14; We acknowledge the limitations of the study cohort, which includes only individuals of European ancestry with severe psoriasis in the United Kingdom. The inclusion of individuals consenting to different levels of bioresourcing may have introduced selection bias, although baseline demographics are broadly comparable across the individual cohorts. Replication and further investigation in cohorts with larger sample sizes and those encompassing different population ancestries may be informative to disentangle the individual effects of amino acid residues at positions 9 and 11.Adalimumab remains a first-line agent across multiple IMIDs; its recent off-patent status in Europe, its impending patent expiration in the United States, and the growing development of adalimumab biosimilars are converging on high availability of this drug, with a corresponding drop in cost. Even small gains in optimizing use of adalimumab and its emerging biosimilars could translate into clinical and economic benefit. Although further validation is required, pretreatment HLA-DRB1 genotyping to stratify patients according to risk of developing ADA may have clinical utility in guiding treatment selection. In patients lacking ADA-protective HLA alleles, less immunogenic but higher cost drugs may be considered as alternatives to adalimumab  but critThe robust identification of genetic drivers of ADA development and ineffectiveness of treatment should now motivate development of integrated treatment prediction models across genetic and clinical biomarkers, to optimize treatment selection and maximize patient benefit in psoriasis and other IMIDs.The discovery cohort comprised 784 individuals in the BSTOP cohort for whom genotyping data were available and a serum sample was obtained 6\u201336 months after starting their first course of adalimumab see . The repEvaluation of genetic association with treatment failure was evaluated in 716 individuals for whom a serum sample had not been collected for detection of ADA but for whom genotyping data, treatment dates, and clinical outcome data were available see .All patients were on the standard adalimumab dose of 40 mg every 2 weeks at the time of ADA sampling. Adalimumab therapy was considered ongoing where treatment episodes were separated by fewer than 90 days . Immunosg for 10 minutes and stored at \u201380\u00b0C prior to adalimumab ADA detection using a drug-tolerant assay . A threshold of \u226530 arbitrary units/mL .Genomic DNA was extracted from whole blood, and genotyping was performed using the Illumina HumanOmniExpressExome-8 v1.2 and v1.3 and v1.6 BeadChips (Illumina). Genotype calling was performed using Illumina\u2019s GenomeStudio Data Analysis software. Quality control was performed using established protocols \u201341. GenoEvaluation of the association of genetic variants  and ADA status was performed using a logistic regression model in PLINK and R . The firA Cox proportional hazard model was used to test the association between the likelihood of terminating adalimumab due to ineffectiveness and either ADA status or amino acid residues at positions 9 and 71 of HLA-DRB1. Individuals terminating adalimumab for reasons other than ineffectiveness were censored at the adalimumab end date; those without a stop date  were censored at the last visit date. The assumption of proportionality was tested using Schoenfeld residuals. The Kaplan-Meier method was used to estimate the probability of remaining on the drug for 1, 2, or 3 years without terminating the drug due to ineffectiveness.A series of sensitivity analyses were performed to evaluate the impact of immunosuppressant cotherapy, threshold of detection in the ADA assay, HLA-C*06:02 genotype, sample timing, presence of comorbidities (including psoriatic arthritis and diabetes), age, age of psoriasis onset, sex, and baseline psoriasis severity see .n = 87) cohort study, approved by the South East London REC 2 Ethics Committee, London, United Kingdom (11/H0802/7), conducted in the spirit of the 1996 International Conference on Harmonisation in Good Clinical Practice and in accordance with the 2008 Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrollment to the BSTOP bioresource , contributed to study design, and provided scientific input on data analyses and interpretation. JB provided scientific input on study design and data interpretation. TT, JS, TR, FCL, ADV, MHA, IAB, DB, TD, MD, FM, AR, AC, S McBride, KM, GP, HR, RR, KS, AS, ADB, CEMG, NJR, RBW, S Mahil, JB, ND, CS, and MAS provided critical revision of the manuscript for intellectual content."}
+{"text": "Mechanotransduction is the process by which cells convert external forces and physical constraints into biochemical signals that control several aspects of cellular behavior. A number of approaches have been proposed to investigate the mechanisms of mechanotransduction; however, it remains a great challenge to develop a platform for dynamic multivariate mechanical stimulation of single cells and small colonies of cells. In this study, we combined polydimethylsiloxane (PDMS) and PDMS/Mxene nanoplatelets (MNPs) to construct a soft bilayer nanocomposite for extracellular mechanical stimulation. Fast backlash actuation of the bilayer as a result of near-infrared irradiation caused mechanical force stimulation of cells in a controllable manner. The excellent controllability of the light intensity and frequency allowed backlash bending acceleration and frequency to be manipulated. As gastric gland carcinoma cell line MKN-45 was the research subject, mechanical force loading conditions could trigger apoptosis of the cells in a stimulation duration time-dependent manner. Cell apoptotic rates were positively related to the duration time. In the case of 6 min mechanical force loading, apoptotic cell percentage rose to 34.46% from 5.5% of the control. This approach helps apply extracellular mechanical forces, even with predesigned loading cycles, and provides a solution to study cell mechanotransduction in complex force conditions. It is also a promising therapeutic technique for combining physical therapy and biomechanics. Mechanotransduction is the mechanism through which cells sense and transduce mechanical forces into biological signals ,2,3,4. ISome strategies have allowed interactions between isolated cells and realistic and complex biological environments to be studied, such as microfluidic devices ,28,29,30To overcome this issue, we designed an in vitro mechanical loading tool using MNP-based polymer nanocomposites. This bilayer consists of a pure PDMS layer and a PDMS/MNPs composite layer. Specifically, Mxene nanoplatelets (MNPs) are dispersed inside polydimethylsiloxane (PDMS) matrices to produce polymer nanocomposites. This bilayer structure was inspired by the folding phenomenon in which two sheet-like components with different mechanical properties attain a shape that facilitates an equilibrium between their constituent elements. With this tool, as depicted in Nanjing XFNANO Materials Tech Co. Ltd.  and were directly used in their original form. Polydimethylsiloxane  was used as the host matrix with the base agent and curing agent obtained from Dow Corning Corporation . The PDMS/MNPs composite was prepared by weighing the desired amount of MNPs and adding them to the PDMS crosslinker. Then, the PDMS base compound was added at a ratio of 10:1 to the PDMS crosslinker and mixed. The pure PDMS solution was configured using the PDMS base compound and the crosslinker at a ratio of 10:1. The fabrication procedure was facile and scalable. It only involved scrape coating and spin coating for the bottom PDMS/MNPs nanocomposite layer  and the upper pristine PDMS layer , which were measured using an optical microscope .Ti3C2Tx Mxene Nanoplatelets  with 50\u2013150 nm thickness and 2\u201310 \u03bcm diameter were purchased from KEYENCE displacement sensor  Ltd., measurement range \u00b1 10 mm, linear accuracy 0.02% F.S.) was used to record the tip deflection of soft bilayers when the nIR light was incident from the PDMS/MNPs layer. The photomechanical bending process of the soft bilayers was experimentally measured in the case of nIR light intensity ranging from 0 to ~30 mW\u2219mm\u22122. Additionally, a programmable optical shutter was utilized to modulate illumination frequency, and then the displacement sensor recorded the photomechanical bending process. Meanwhile, a handmade polystyrene foam sphere (d = 2 mm) was chosen to demonstrate the mechanical oscillation induced by the two-step photomechanical bending of soft bilayers.The MNP concentrations used in this study ranged from 1 to 5 wt%. All samples were made into strips with dimensions of 12 \u00d7 3 mm (length \u00d7 width). An nIR light source with a wavelength of 808 nm was chosen to actuate the soft bilayer actuator. A 5 cells per well, in which a 12 \u00d7 3 mm soft bilayer structure with 4 wt% MNP concentration was in each well. The cultured cells were allowed to grow and adhere to the thin PDMS layer by incubating them in Dulbecco\u2019s modified Eagle medium  supplemented with 10% Fetal Bovine Serum  and 10% penicillin and streptomycin at 37 \u00b0C for 24 h in an incubator  containing 5% CO2. Afterwards, each soft bilayer was carefully taken out to conduct the actuation process. An nIR light (working frequency with 1 Hz) with a light intensity of 15 mW\u2219mm\u22122 was applied to the left side of the soft bilayer at the actuating time from 0 to 6 min in a 2 min interval. The soft bilayer with cultured cells was then put back into the incubator and cultured from 24 to 72 h.The left side of the fabricated soft bilayers, 3 \u00d7 3 mm (length \u00d7 width), was anchored at the K9 glass bases with PDMS solution as an adhesive layer, in which the width of glass bases is slightly larger than that of soft bilayers. In this case, it was helpful for constructing a cantilever actuator, which was chosen as the matrix for culturing cells onto the PDMS surface. The cell-culture medium of the gastric gland carcinoma cell line MKN-45  was plated in 6-well culture plates at a density of 2 \u00d7 10Soft bilayers without mechanical force loading were set as the control group, while the soft bilayers with mechanical force loading were set as the experimental group. An optical microscope was utilized to record the cell growth states of the control group at 24, 48, and 72 h. The statistics of living cells were conducted based on commercial cell counting boards. Meanwhile, the flow cytometry characterizing apoptosis rates of the experimental group was determined. An Annexin V-binding assay was conducted by using an Annexin V-FITC/PI Apoptosis Detection Kit. A flow cytometer  was used to analyze the expression of Annexin V-FITC+/PI\u2212 (early apoptosis) and AnnexinV- FITC+/PI+ (late apoptosis) cells.The strategy used to produce this soft bilayer structure was based on a straightforward layer-by-layer manufacturing technique. This technique is straightforward and scalable, and it requires only scrape-coating and spin-coating operations for the bottom PDMS/MNPs nanocomposite layer and the top pure PDMS layer, respectively. Mxene acts as a heat source when dispersed in PDMS matrices owing to its remarkable photothermal conversion efficiency in the nIR band. Meanwhile, the relevant properties of nanocomposites containing MNPs are altered as a result of the exceptional thermal conductivity and negative coefficient of thermal expansion of MNPs, which result in photomechanical actuation due to mismatch expansion between the PDMS layer and the PDMS/MNPs layer. As shown in \u22122) is incident from the pure PDMS/MNPs layer, an analogous heat source is exerted at the rear of the PDMS/MNPs nanocomposite layer because the nIR irradiation is regarded as a power source that heats up a restricted region. Heat is transferred from the rear surface of the PDMS/MNPs nanocomposite layer to the top surface of the PDMS layer along the thickness direction through the bilayer actuator. Thus, bending occurs due to differences in thermal expansion between the two thin layers. Using COMSOL Multiphysics software, the two-step analytical bending process is derived by combining the heat transport and thermal expansion processes. When comparing the analytical deflection processes with the experimental results in To illustrate the photomechanical bending mechanism, Timoshenko thermoelastic model, in which the two bonded layers had a mismatch in elongation that caused deflection under heat stimulus. To further explain the photomechanical bending mechanism, a simple method was utilized in our analysis only by calculating the deviation of thermal expansion, i.e., PDMS\u03b5-PDMS/GNPs.\u03b5 As shown in To further explain the two-step photomechanical bending process, the individual thermal expansion process of PDMS and the PDMS/MNPSs layer was calculated with the help of COMSOL Multiphysics, as shown in \u22122) occurred at a particular moment in the rapid bending process. After the cells had adhered to the surface of the bilayer, it was permissible to apply mechanical stimulation (force) to the cells due to the constant and rapid change in velocity and acceleration in accordance with Newton\u2019s second law of motion (F = m \u00d7 a), as shown in Initially, a bilayer structure containing 4 wt% MNPs was chosen as the cell force loading mechanism. Given the two-step bending process in response to nIR light stimulation, it was possible to obtain velocity and acceleration curves as a function of time. As shown in r = 100 \u03bcm), given the objective to develop a force loading tool that is applicable to single cells and small colonies of cells. Due to the light weight of single cells and small cell colonies, it is plausible to equate the acceleration of the bilayer to that of the cell itself. As shown in In addition, mechanical force loading instruments containing varying MNP concentrations were built and evaluated. As shown in \u22122 for a bilayer containing 4 wt% MNPs. All the curves had almost identical bending tendencies, with the exception of the backlash and terminal deflections. In accordance with the analysis shown in \u22122), the amplitude of photomechanical bending increased steadily. Adjusting the operating frequency of nIR light enables programmable modulation of photomechanical actuations in bilayer structures. Thus, it is the benefits of nIR light stimulation that enable bilayer structures to achieve programmable photomechanical deflection, in turn enabling temporal and spatial modulation of force loading on cells.As a result of the controllability and modulation of the nIR light source, it was anticipated that our mechanical cell loading tool would be capable of temporal and spatial modulation, as well as controllable deflection. First, the photomechanical bending mechanisms under different intensities of nIR light were investigated. As shown in To demonstrate the applicability of our bilayer to cell mechanobiology, tests to simulate the circumstances of cell loading were performed. The photomechanical bilayer was used to stimulate a polystyrene sphere, which represented a small colony of cells. 2, was applied to the end of the soft bilayer away from the cell-cultured regions. In this case, the soft bilayer temperature change was lower than its culturing circumstance temperature, i.e., 37 \u00b0C, as shown in Gastric cancer cells (MKN-45) were examined in terms of their growth and differentiation when cultured on the soft bilayer (12 \u00d7 3 mm) with 4 wt% MNPs. This analysis was performed to determine whether the mechanical forces generated in the photomechanical bending process of bilayer were physically transduced to the cells cultured on them. With an actuation time ranging from 2 to 6 min, nIR light at a working frequency of 1 Hz and irradiated from the side of the PDMS/MNPs layer at an intensity of 15 W/cmIn our cell mechanical force loading experiments, soft-bilayer-cultured normal MKN-45 cells that were not mechanically actuated were set as the control group. Soft bilayers that were actuated by 2, 4, and 6 min were set as the experimental group. Commercial cell counting boards recording cell growth states and flow cytometry characterizing apoptosis rates were conducted for the control group and experimental group, respectively. As shown in In conclusion, we produced a novel in vitro mechanical loading tool using a soft and photoresponsive material with a bilayer structure to study mechanotransduction. Due to the photothermal effect of MNPs and the variations in the coefficient of thermal expansion of the two thin layers attributed to MNP incorporation, the soft bilayer demonstrated photomechanical bending under nIR light irradiation with a fast and reversible two-step bending process, which involved fast backlash deflection as the nIR light was switched on and off, and the bilayer bending process. Based on the examination of the velocity and acceleration of the bending process, we determined that the sudden and rapid variations in acceleration made it feasible to load the cells with mechanical forces. In addition, the mechanical force loading tool could qualify temporal and spatial modulations and control and adjust photomechanical deflections due to the controllability and modulation of the nIR source. The movements and oscillations demonstrate the photomechanical bilayer working as an in vitro force loading mechanism. With the gastric gland carcinoma cell line MKN-45 a research subject, it was shown that mechanical force loading conditions could trigger apoptosis of the cells in a stimulation duration time-dependent manner. Cell apoptotic rates were positively related to the duration time, which was attributed to the fact that longer mechanical force loading duration time enabled higher mechanical force accumulation to affect cell growth and viability. Similarly, in the case of 4 and 6 min mechanical force loading, the percentage of apoptotic cells increased up to 28.12% and 34.46% from 4.17% and 5.5% of the control, respectively. In the future, it will be convenient to develop a force-loading tool for use with single cells or small colonies of cells due to the ease of fabrication of the bilayer structure using high-precision microfabrication technology. We anticipate that this will be a significant advance in the development of stimuli-responsive materials that are well-adapted to multivariate mechanical stimulation of specific cells."}
+{"text": "The use of biological systems in manufacturing and medical applications has seen a dramatic rise in recent years as scientists and engineers have gained a greater understanding of both the strengths and limitations of biological systems. Biomanufacturing, or the use of biology for the production of biomolecules, chemical precursors, and others, is one particular area on the rise as enzymatic systems have been shown to be highly advantageous in limiting the need for harsh chemical processes and the formation of toxic products. Unfortunately, biological production of some products can be limited due to their toxic nature or reduced reaction efficiency due to competing metabolic pathways. In nature, microbes often secrete enzymes directly into the environment or encapsulate them within membrane vesicles to allow catalysis to occur outside the cell for the purpose of environmental conditioning, nutrient acquisition, or community interactions. Of particular interest to biotechnology applications, researchers have shown that membrane vesicle encapsulation often confers improved stability, solvent tolerance, and other benefits that are highly conducive to industrial manufacturing practices. While still an emerging field, this review will provide an introduction to biocatalysis and bacterial membrane vesicles, highlight the use of vesicles in catalytic processes in nature, describe successes of engineering vesicle/enzyme systems for biocatalysis, and end with a perspective on future directions, using selected examples to illustrate these systems\u2019 potential as an enabling tool for biotechnology and biomanufacturing. In the most basic terms, biocatalysis is a chemical process in which biomolecules such as enzymes perform reactions between organic components. Biocatalysis encompasses numerous technologies, from small scale to industrial scale, and from in vitro reactions with only enzyme catalysts, substrates, co-factors, and buffer, to fermentations using whole-cell microbial cultures. Backed by advances in synthetic biology that have enabled substantial microbial engineering, biocatalysis is emerging as a competitor to classical chemical routes of synthesis for specialty chemicals, therapeutics, and novel materials.Enzyme-based catalysts can offer numerous advantages over conventional chemical catalysts, including higher efficiency, tremendous specificity , and the potential for customization of protein function using molecular biology techniques . There aEscherichia coli is generally not cost-prohibitive, and the use of whole cells removes the cost in money and time for individual purification of each enzyme required for a desired process. Cells can often be used repeatedly, while the longevity of purified enzymes is more limited and they sometimes cannot be separated in usable form from reaction products once synthetic steps are complete [One of the major strategies for biocatalysis is the use of enzymes in whole-cell microbial cultures. There are advantages in terms of cost, as growth of commonly used chassis organisms such as complete . ContainE. coli, Bacillus subtilis, Saccharomyces cerevisiae, and others using established and well-described protocols and systems for scalable production [Artemisia annua, researchers invested heavily in developing methods of mass producing this high-demand anti-malarial heterologously to meet demand. In a multitude of studies highlighted in a review by Muangphrom et al., recombinant expression of the A. annua biosynthetic pathway was augmented with enzymes from a variety of plant, fungal, and microbial species to create a highly efficient production system in microbes such as E. coli and S. cerevisiae [Advances in our understanding of cellular metabolism, protein function, and gene regulation and the development of corresponding synthetic biology tools to manipulate these systems allow for detailed fine-tuning of a desired metabolic pathway for biocatalysis ,4. Non-noduction ,5. One orevisiae .Biomanufacturing in microbial chassis is not without its limitations, however . Much lMost bacteria studied to date release nanosized proteoliposomes from their outermost membrane, referred to as membrane vesicles (MVs) or outer membrane vesicles (OMVs) . In natuAs alluded to above, OMV biocatalytic systems allow advantages of both cell-based and purified enzyme in vitro biocatalysis . SimilarWithout intervention by engineering, MVs/OMVs produced by certain bacteria have been demonstrated to catalyze a variety of vital reactions through the enzymes they naturally carry. Much of the extant literature on the use of natural MVs/OMVs for enzyme catalysis revolves around the breakdown of lignocellulosic biomass, or dry plant matter, as well as other complex polysaccharides . Since lFibrobacter succinogenes, a cellulose-degrading species found in the cow rumen, produces OMVs that have several different classes of proteins that enable efficient degradation of plant fibers, including fibro-slime proteins, cellulases and hemicellulases that can degrade cellulose, pectin, and hemicelluloses. Importantly, the group reported a significant 2.4-fold increase in sugar equivalents resulting from switchgrass that was pretreated with F. succinogenes OMVs [F. succinogenes to have improved accessibility to the nutrient-rich components of lignocellulose following OMV-led degradation. Interestingly, although F. succinogenes itself only utilizes cellulose as a carbon source, the OMVs it produces contain a cocktail of polysaccharide-degrading enzymes, suggesting a supportive role of its OMVs for other organisms, including the host, in the rumen microbiota [Clostridium thermocellum also produces membrane vesicles that degrade cellulose. However, in the case of C. thermocellum, the group determined via immunoelectron microscopy that the carbohydrate-active enzyme complexes (or cellulosomes) are localized to the outer vesicle surface [Recently, OMVs that degrade lignocellulosic biomass and that are produced by important residents of the rumen microbiota have been discovered . It is tnes OMVs , relativcrobiota . In anot surface .Pseudomonas putida KT2440, they showed that when grown in lignin-rich media, putatively ligninolytic enzymes such as de-colorizing peroxidases are significantly enriched in the exoproteome relative to intracellular proteins. In addition, they identified other enzymes that they hypothesize modify both oligomeric and monomeric aromatic or phenolic compounds, including a 2,3-quercetin dioxygenase, a xenobiotic reductase, and azoreductases, each of which was enriched in the P. putida exoproteome in lignin-rich media. Regarding OMVs, they showed that two distinct populations of OMVs develop in lignin-rich media\u2014as determined by diameter measurements with transmission electron microscopy, suggesting a large corresponding phenotypic shift by P. putida  that inhabit the human gut produce fibrolytic OMVs [Bacteroides preferentially packages a large number of hydrolases in OMVs, many of which were detected exclusively in vesicles as opposed to the bacterial outer membrane. Specifically, they identified xylanase, \u03b2-xylosidase, glycosyl hydrolase, chitinase, among others, exclusive to Bacteroides OMVs, targeting glycans that are not substrates for human hydrolases, but the products of some of which can be utilized by both microbiota members and are beneficial for the host [Bacteroides OMVs may see application outside of the human gut with particular interest in degradation of polymers to D-xylose, where D-xylose works as a feedstock for biosynthetic cascades for other organisms [Similar to the rumen microbiota discussed above, the human gut microbiota has also symbiotically co-evolved systems to convert consumed foodstuffs into a carbon and energy source. Previously, Rakoff-Nahoum et al. demonstrated that OMVs produced by a number of the host . This sttic OMVs . In thisthe host . These Brganisms .Vibrio harveyi [Alteromonas macleodii KS62, were hydrolytic enzymes\u2014approximately 30% [Kappaphycus, a genus of red algae. After 12 h of incubation of Kappaphycus seaweed biomass with A. macleodii OMVs, the group recorded a >10-fold increase in reducing sugars compared to the OMV-free control. Hypothesized to assist Alteromonas to invade and colonize the seaweed biomass, with a potentially large role in this marine environment niche, these OMVs may be of interest for degrading seaweed in biotechnology applications such as bioethanol production and for extracting valuable biomolecules, e.g., carotenoids and vitamins [Trichoderma reesei produces more EVs in the presence of cellulose in comparison to glucose and glycerol, and the vesicles had higher \u03b2-glucosidase activity than supernatant [Shifting to the marine environment, several recent studies have begun to investigate the role of OMVs produced by marine bacteria such as  harveyi ,36. Repotely 30% . Of partvitamins . While nernatant .While the breakdown of lignocellulose, polysaccharides, and other targets of interest can clearly benefit from a biotechnology solution, in practice many processes require a cell-free system if used with cellularly toxic pretreatments. Here, several recent reports have demonstrated the promise of naturally occurring, non-engineered OMVs in the degradation of plant matter, foodstuffs, and other matter, suggesting that, for certain applications, cell-free catalysis systems are readily discoverable in nature. In some cases, these systems may be directly portable into industrial applications; in others, these systems can instead be targets to be engineered into industrially-used model organisms.E. coli [Extensive efforts have been made in the field of OMV engineering to enhance their usage in vaccination, tumor therapy, and antibiotic and small molecule delivery ,38,39. TE. coli ,41. SeveE. coli ,43,44. EE. coli was described by Park et al. [C. cellulolyticum), DocT (from C. thermocellum) and DocF (from R. flavefaciens), and one cellulose-binding module (CBM); the enzymes in turn were fused to corresponding docterin domains. The scaffold was tethered onto the surface of OMVs utilizing a truncated ice nucleation protein anchoring motif (INP). This engineered system led to a 23-fold enhancement in glucose production over the expression of non-complexed enzyme, demonstrating the power of scaffolded multienzyme pathways on OMVs.As described above, one of the natural targets of OMV catalysis is the degradation of complex polymers such as cellulose. A key example of how this has been engineered into the model organism k et al. . The autLoligo vulgaris, paraoxonase (PON1) from liver, organophosphate hydrolase from Agrobacterium radiobacter, and phosphotriesterase (PTE) from Brevundimonas diminuta and Deinococcus radiodurans. OP toxicity has driven the efforts of numerous research groups to develop sensors, therapeutics, prophylactics, and environmental decontamination tools based on these enzymes [Due to their ease of manufacture and range of toxicities, organophosphate (OP) compounds are widely employed as agricultural pesticides and maintained as chemical warfare agents by several nations . Yet, du enzymes ,53. Init enzymes . HoweverE. coli [One of the initial efforts to engineer OMVs towards this goal was made by DeLisa and coworkers in 2008 where they fused organophosphorus hydrolase to the vesicle-associated toxin ClyA of E. coli . This geE. coli . This foE. coli to actively package the phosphotriesterase enzyme PTE from Brevundimonas diminuta into OMVs  and SpyTag (ST) based protein\u2013protein interaction system together with the abundant outer membrane protein OmpA of nto OMVs . PTE is In other work, Su et al. localized OPH and a cellulose-binding domain (CBD) to the surface of an OMV using the ice nucleation protein (INP) . The resE. coli OMVs for bioremediation of antibiotic pollution for the food and agriculture industry by engineering OMVs to encapsulate a class C \u03b2-lactamase (CMY-10) from Enterobacter aerogenes [E. coli-based whole-cell biocatalysts and exhibited greater catalytic stability compared to free enzymes in the aqueous phase [In addition to organophosphate bioremediation, more recently Woo et al. (2022) have engineered erogenes . The engus phase . This isus phase .E. coli outer membrane lipoprotein SlyB  has recently gained importance, as it is an ATP-independent luciferase and displays a relatively long half-life, enhanced thermal and pH stability, and lower background. OMVs have been used as a modular platform for biosensing applications via fusion of NLuc on an ein SlyB . In ordeThe use of NLuc in vivo as a subcutaneous implement can be an extraordinary tool for a variety of biomedical applications and is being investigated with some promising results . BioengiE. coli OMVs to form nano-scale bioreactors for transformation of unsaturated long-chain fatty acids [Stenotrophomonas maltophilia (SmOhyA) and photoactivated fatty acid decarboxylase from Chlorella variabilis NC64 A (CvFAP) into OMVs and transformed oleic acid via a multi-step pathway (E. coli cells. This is the first study on engineering OMVs for biotransformation of unsaturated fatty acids and has opened a new dimension in the field of biotechnology and biomanufacturing.In addition to the above detailed studies, other enzymes have been engineered into bacterial OMVs. Song et al. have engineered ty acids  by packa pathway . While rE. coli; expanding to other organisms could take advantage of their inherent benefits, but this requires the genetic tools for engineering these organisms. To date, researchers have developed methods to anchor recombinant peptides and proteins such as enzymes both within MV/OMVs and on the exterior surface [Given that directed encapsulation of recombinant enzymes and biomolecules within membrane vesicles such as MVs and OMVs is still in its infancy, there remain multiple challenges, and therefore opportunities, to explore. Most engineered systems rely on the model organism  surface ,18,68. T surface ,48,49,50 surface . AdditioDespite these challenges, these methods may provide an ideal alternative to whole-cell biocatalysts and some of the inherent issues associated with those systems. Export or \u201coff-loading\u201d of recombinant enzymes in vesicles could circumvent problems of toxicity due to accumulation of recombinant proteins or the catalysts and formation of products that could impact cellular viability. These biological nanoreactors have been shown to be functional in several chemical reactions including biomass degradation  and as p"}
+{"text": "Outer membrane vesicles (OMVs) are miniature versions of gram-negative bacteria that contain almost the same content as their parent cells, particularly in terms of membrane composition. Using OMVs as biocatalysts is a promising approach due to their potential benefits, including their ability to be handled similarly to bacteria while lacking potentially pathogenic organisms. To employ OMVs as biocatalysts, they must be functionalized with immobilized enzymes to the OMV platform. Various enzyme immobilization techniques are available, including surface display and encapsulation, each with advantages and disadvantages depending on the objectives. This review provides a concise yet comprehensive overview of these immobilization techniques and their applications in utilizing OMVs as biocatalysts. Specifically, we discuss the use of OMVs in catalyzing the conversion of chemical compounds, their role in polymer degradation, and their performance in bioremediation. Outer membrane vesicles (OMVs) are intriguing and versatile extracellular vesicles that have garnered increasing attention in recent years. These nano-sized, spherical proteoliposomes are continuously discharged from the outer membrane of gram-negative bacteria via a vesiculation mechanism that involves the separation of the outer membrane from the underlying peptidoglycan layer . There aOMVs are more than just lipid bilayer structures; they contain outer membrane proteins (OMPs), a thin layer of peptidoglycan, lipopolysaccharides (LPSs), and other periplasmic constituents that give them a complex composition . This coOMVs can transport a diverse range of biomolecules, such as nucleic acids, proteins, and lipids, over extended distances in their lumen. Due to these properties, OMVs play an essential role in intercellular communication and are also involved in forming biofilms ,8,9,10. Numerous studies have employed protein translocation mechanisms from the cytoplasm and periplasm to display enzymes on cell surfaces ,15, whicOMVs were discovered over 50 years ago, and research on them has progressed significantly, particularly in modifying their functionality to expand their application potential. This review focuses on using OMVs as biocatalysts\u2014particularly their functionalization with enzymes and their applications. Functionalizing OMVs for biocatalytic targets involves modified enzymes, with the immobilization of enzymes being the most common and relevant approach. Immobilized enzymes are stronger and more resistant to environmental changes than free enzymes in solution ,21. AddiThere are several methods of enzyme immobilization, including depositing enzymes on solid matrices, adsorption on porous materials, immobilization via covalent bonds, affinity immobilization, and entrapment or encapsulation ,23. EachEscherichia coli K-12 strain, is known to be one of the periplasmic proteins secreted along with the vesicles from the bacterial cell, suggesting its use in a vesicle transport mechanism in bacteria [The recent prevalence of OMV surface decoration may be traced back to one of the earliest investigations of Kim et al. . That stbacteria ,28,29,30Kim et al. conducted experiments in which they fused several heterologous proteins to the ClyA toxin with N-terminus and C-terminus variations. The proteins \u03b2-lactamase (Bla), organophosphorus hydrolase (OPH), and \u03b2-galactosidase (LacZ) were coupled with ClyA to produce synthetic OMVs. Before combining ClyA with the enzymes, they fused it with GFP to assess the effect of fusion on the N- and C-termini. The combination of ClyA with GFP, Bla, and OPH, where ClyA was bound to the N-terminus fusion protein, showed a considerable increase in cell-surface and vesicle activity. On the other hand, Bla bound to the N-terminus of ClyA resulted in a significant decrease in activity. Similarly, ClyA bound to the C-terminus of OPH had no measurable effect on OPH activity. Given that the OPH substrate used in the study, paraoxon, is membrane impermeable, OPH bound outside the cell or vesicle was preferred . In lineStreptococcus pyogenes. SpyTag is a small peptide consisting of 13 amino acids, while SpyCatcher is a larger protein fragment containing 116 residues. This system is based on the covalent peptide interactions between SpyTag and SpyCatcher protein pairs, which result in the formation of an irreversible isopeptide bond between lysine and aspartic acid residues. The isopeptide bond formation is spontaneous and occurs within minutes, without the need for specific pH or reaction temperature conditions. In addition, it is compatible with various buffers [The SpyTag/SpyCatcher system has emerged as a powerful tool for the covalent bioconjugation of proteins. SpyTag and SpyCatcher are derived from the CnaB2 domain of the fibronectin-binding protein FbaB of  buffers ,35,36. T buffers ,38 and r buffers  applicatSpyTag and SpyCatcher can be fused at either the N-terminus or the C-terminus. In addition to directly fusing the functional protein to the anchoring motif, the use of SpyTag or SpyCatcher to fuse with the C-terminus of the outer membrane protein is a way of functionalizing OMVs through surface display. One advantage of using SpyTag or SpyCatcher to fuse with the C-terminus of an outer membrane protein is the ability to display proteins that are challenging to transport out of the cell, such as large proteins or those with limited expression levels in the cytoplasm. To conjugate the fused protein with SpyTag or SpyCatcher, the only requirement is adding the fused protein to the buffer. For instance, the SpyTag/SpyCatcher system was used to display the virulence factor hemoglobin protease (Hbp) on the OMV platform to investigate optimal exposure of multiple antigenic sites to the immune system . The HbpSometimes the functionalization of OMVs for biocatalyst purposes using the Spy system does not require engineering of the OMV surface. Instead, SpyTag is conjugated to the transmembrane porin protein OmpA at the internal, N-terminus, and C-terminus regions to create an entrapment platform for OMVs. For example, the phosphotriesterase (PTE) enzyme is fused with SpyCatcher, and the sequence leader torA is added to localize PTE-SpyCatcher expression in the periplasm. This allows the enzymes to be encapsulated within the OMVs ,17. ThisPseudomonas syringae [Pseudomonas fluorescens [Erwinia herbicola [The ice-nucleation protein (INP) is found in bacteria that actively nucleate ice, such as syringae , Pseudomorescens , and Erwerbicola . It can erbicola ,44. Thiserbicola ,46.E. coli JC8031. This configuration allows the dockerin of each of the three different cellulases to interact with their respective cohesin partners, thereby providing a promising approach for the simultaneous and site-specific expression of multiple enzymes that are useful for cascade reactions [Enzymes can be effectively immobilized on the outer membrane surface using the INP . The INPeactions .E. coli lipidation sequence of major lipoprotein-outer membrane protein A (Lpp-OmpA) to form the pUCBD plasmid, in which Lpp-OmpA was used as an anchoring motif. This approach allows the two engineered proteins to be expressed outside of the cell, with the CBD providing an advantage in terms of OMV recovery and enabling the synthetic OMV-enzymes to be utilized multiple times [Similarly, Su et al. reported using a different design to engineer the surface of OMVs using the INP. In their study, they fused the OPH enzyme directly to the INP in the pVLT33-INPOPH6 construct to immobilize the enzyme on the surface of OMVs. Additionally, they fused cellulose binding domains (CBD) with the le times .Both studies demonstrated that the INP offers construction flexibility for displaying diverse functional proteins on OMV platforms and facilitates recovery and purification. Thus, this anchoring motif represents a promising tool for future biotechnological applications in enzyme immobilization.N-hydroxysuccinimide (EDC/NHS) coupling reaction\u2014as it is not a material encapsulation technique used for functionalized OMVs as biocatalysts. In addition, we examine the genetic-engineering-based functionalization of OMVs specifically for the encapsulation technique.Encapsulation does not provide flexible interaction and high mass transfer between proteins and their environment. However, the encapsulation technique offers enzyme protection and increases enzyme stability by shielding enzymes from harsh environments, since the matrices will carry them in the lumen to travel long distances. The use of OMVs as the encapsulation matrices is more commonly applied in biomedicine for loading small molecules, such as pharmaceutical compounds ,49. HoweIncubation is the simplest way to introduce biomolecules or functional proteins into the lumen of OMVs. However, the loading efficiency is often uncontrolled, requiring large quantities of loading material to be incubated with the growth media. Alternatively, physical functionalization methods, such as electroporation, sonication, and extrusion, have been explored but require greater effort ,51,52,53Most OMVs used as biocatalysts are functionalized using genetic engineering approaches, such as the Spy toolbox mentioned earlier . The ideErwinia carotovora. Fatty acid double bond hydratase from Stenotrophomonas maltophilia (SmOhyA) and photoactivated fatty acid decarboxylase from Chlorella variabilis NC64 A (CvFAP) were fused with PelBSS to allow for periplasmic expression, which was subsequently encased by OMVs [E. coli mutant lacking the tolA and tolR genes [Recent studies have shown the potential of signal peptides for localizing fatty acid-converting enzyme expression in the periplasm. While the Tat signal was previously used for this purpose, the enzyme is now secreted into the periplasm via the Sec pathway, guided by the PelB secretion signal peptide (PelBSS) from pectate lyase B of  by OMVs . SimilarlR genes . The outOverall, using signal peptides for enzyme localization and encapsulation within OMVs represents a promising approach for developing novel biocatalysts and drug delivery systems. While more research is needed to optimize the efficiency and specificity of this approach, it has the potential to revolutionize the field of biotechnology and to open new avenues for developing advanced therapeutics.OMVs have numerous applications in research ,50, drug\u221212\u00a0E. coli cells, indicating that OMVs derived from hyper-vesiculating E. coli can contain extremely concentrated enzymes [Recent studies have explored the potential of engineered OMVs to act as nanobioreactors for biocatalytic reactions. One of the most promising applications is the conversion of fatty acids into high-value compounds. The engineered OMVs containing SmOhyA and CvFAP enzymes catalyzed the conversion of oleic acid to 9-hydroxyheptadecane, a valuable ingredient in the cosmetics and pharmaceutical industries. The reaction involves a cascade reaction, starting with SmOhyA hydrating unsaturated fatty acids into hydroxy fatty acids, which are then converted into final products by CvFAP with the help of light. The specific biotransformation rates of oleic acid were reported to reach 8.0 \u00d7 10 enzymes .E. coli-derived OMVs to convert para-nitrophenoxydodecanoic acid or 12-pNCA substrate to para-nitrophenol, expanding the application of OMVs in the field of biosensors. Immobilization of CYP102A1 with OMVs demonstrates increased the stability and ease of biocatalyst use without the need for expensive enzyme purification [Another example of using OMVs as biocatalysts is the display of CYP102A1 protein, a self-sufficient monooxygenase, on fication . Apparen3 U/g total protein and 11.62 \u00d7 103 U/A600, respectively, indicating that enzyme display on OMVs is preferred in this system [Recent studies have shown that OMVs can be highly effective in bioremediation, where catalysts are often required. OMVs can be decorated with enzymes and proteins, such as Bla and OPH, to enhance their bioremediation potential. The success of displaying Bla and OPH on OMVs surface has been demonstrated by researchers investigating the anchoring ability of ClyA motifs. Synthetic Bla-containing OMVs have been shown to hydrolyze \u03b2-lactam antibiotics, while OMVs with OPH decoration can hydrolyze paraoxon . Interess system .Bacteroides species, was successfully immobilized to degrade cefotaxime [In addition, researchers have immobilized OPH by anchoring it to the INP, which increased the enzyme\u2019s stability and enhanced its degradation rate when tested with paraoxon as a substrate. The addition of CBD further enhanced the system, making these OPH-containing OMVs recoverable and retaining their activity even after 15 cycles . The immfotaxime . RemarkaPTE is an enzyme that has been extensively studied for its ability to detoxify organophosphate compounds, which are used as pesticides and chemical warfare agents (CWA), including paraoxon. Researchers have recently found that OMVs can immobilize PTE and enhance its activity and stability ,17,18. WIn a recent study, researchers have also shown that OMVs can be used to immobilize DFPase, another enzyme with organophosphate hydrolysis activity. The DFPase-OMV complex was found to be stable and effective in hydrolyzing diisopropyl fluorophosphate (DFP) and paraoxon. That study further emphasized the potential of OMVs for enzyme immobilization to preserve enzyme stability in different environments\u2014in that case, by protecting the enzymes from temperatures as high as 80 \u00b0C and preventing catalytic activity loss .Overall, OMVs have emerged as a promising tool for enzyme immobilization and have the potential to be used in a range of applications, including environmental remediation and biocatalysis. The enhanced stability, improved activity, and increased resistance to the environmental stress of enzymes immobilized in OMVs make them an attractive alternative to traditional immobilization techniques, which often involve artificial matrices or support structures. With further research and development, OMVs could offer an innovative solution to the growing problem of environmental pollution caused by various toxic substances, including those that are unsuitable for using living microorganisms.Researchers have developed a novel approach for immobilizing multiple enzymes on the surface of OMVs by constructing Scaf3 on the surface of OMVs. The first study to investigate this approach was aimed at assembling three cellulases for a cascade reaction to increase the efficiency of cellulose hydrolysis. Before incorporating the cohesion\u2013dockerin interaction system, the localization of Scaf3 on the OMVs was confirmed using an immunofluorescence microscopy technique with a fusion tag probe. Subsequently, the researchers conjugated three cellulases  to the surface of the OMVs. These enzymes are exoglucanase, endoglucanase, and \u03b2-glucosidase, respectively. The resulting platform significantly enhanced cellulose hydrolysis, increasing it by 23-fold compared to free enzymes. Moreover, the trivalent enzyme displayed on the OMV surface showed a 9-fold improvement in hydrolysis efficiency, compared to the same trivalent enzyme displayed on the yeast surface . This approach has several advantages over other enzyme immobilization techniques. Using OMVs as a natural membrane-based scaffold for enzyme immobilization ensures stability, high activity, ease of manipulation, and the ability to incorporate multiple enzymes. Moreover, the cohesion\u2013dockerin interaction system in that study enabled the specific binding of enzymes to the scaffold protein, making it easier to manipulate and optimize the enzymatic reaction. This technique could be applied in various fields, such as biocatalysis and biofuel production, where the cascade reaction of multiple enzymes is critical for efficient and cost-effective processes. The scaffold protein and OMVs can also be modified to target specific substrates or reactants, further increasing the specificity and efficiency of the enzymatic reactions.Research on OMVs has rapidly advanced since their discovery. As miniature bacterial cells, OMVs have been found to stimulate immune responses, making them promising for developing vaccines and therapeutic agents. While the use of OMVs has primarily been explored in biomedicine, there has been increasing interest in their potential for other applications, especially for biocatalysis.Although the number of studies of the catalytic potential of OMVs is less than the number of studies regarding its application in biomedicine, several studies have investigated this catalytic potential. One promising approach involves engineering techniques that enable the conjugation of OMVs with enzymes, which can be conveniently facilitated by genetic engineering. The use of OMVs for biocatalytic functions has several advantages, including protection against potential pathogen exposure during the application of the biocatalyst, high loading efficiency for encapsulating functional proteins located in the periplasm and the maintenance of catalytic function and enzyme stability.However, the utility of OMVs still needs to be improved in certain respects, such as the costs and the labor-intensive purification process. Although challenging, the isolation and purification of outer-membrane vesicles are also being developed for industrial use, as documented in several granted patents ,62. GeneDespite the progress that has been made, numerous applications of OMVs for biocatalysis can be explored in future research. For example, OMVs have shown promise in the development of biosensors and the production of fine chemicals. Additionally, there is potential for the use of OMVs in environmental remediation and the degradation of pollutants. Furthermore, OMVs have been shown to play a role in bacterial communication. Future research could focus on understanding the mechanisms behind this communication and how it could be exploited for medical or industrial purposes.In conclusion, OMVs represent a promising platform for biocatalysis, due to their stability, activity, and ease of manipulation. Although there are limitations to their utility, the potential applications for OMVs are vast and future research is likely to focus on overcoming these obstacles and developing new and innovative ways to harness their unique properties for medical, industrial, and environmental purposes."
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