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+{"text": "PLoS Biology, volume 2, issue 10:In Regulation of Muscle Fiber Type and Running Endurance by PPAR\u03b4.Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, et al.10.1371/journal.pbio.0020294The following competing interest should have been indicated in the above research article. R. M. Evans wishes to acknowledge a consulting relationship with Ligand Pharmaceuticals. Under a licensing agreement with GlaxoSmithKline, Ligand receives milestone payments on the development of GW501516, a PPAR\u03b4-specific drug used in this study. For clarification, no materials or support were received from either company, and no agreements were in place concerning the execution or publication of this work.Published January 18, 2005"}
+{"text": "Dendritic cells (DCs) are capable of inducing immunity or tolerance. Previous studies have suggested plasmacytoid DCs (pDCs) are pathogenic in systemic lupus erythematosus (SLE). However, the functional characteristics of directly isolated peripheral circulating blood pDCs in SLE have not been evaluated previously.Peripheral blood pDCs from 62 healthy subjects and 58 SLE patients were treated with apoptotic cells derived from polymorphonuclear cells (PMNs). Antigen loaded or unloaded pDCs were then co-cultured with autologous or allogenous T cells. Changes in T cell proliferation, cell surface CD25 expression, intracellular Foxp3 expression and cytokine production were evaluated. pDCs that had captured apoptotic PMNs -alpha, interleukin (IL)-6, IL-10, IL-18) and toll like receptor (TLR) expression.Circulating pDCs from SLE patients had an increased ability to stimulate T cells when compared with control pDCs. Using allogenous T cells as responder cells, SLE pDCs induced T cell proliferation even in the absence of apoptotic PMNs. In addition, healthy pDCs + apoPMNs induced suppressive T regulatory cell features with increased Foxp3 expression in CD4 + CD25 + cells while SLE pDCs + apoPMNs did not. There were differences in the cytokine profile of pDCs that had captured apoptotic PMNs between healthy subjects and patients with SLE. Healthy pDCs + apoPMNs showed decreased production of IL-6 but no significant changes in IL-10 and IL-18. These pDCs + apoPMNs also showed increased mRNA transcription of TLR9. On the other hand, while SLE pDCs + apoPMNs also had decreased IL-6, there was decreased IL-18 mRNA expression and persistent IL-10 protein synthesis. In addition, SLE pDCs lacked TLR9 recruitment.We have demonstrated that peripheral circulating pDCs in patients with SLE were functionally abnormal. They lacked TLR9 expression, were less capable of inducing regulatory T cell differentiation and had persistent IL-10 mRNA expression following the capture of apoptotic PMNs. We suggest circulating pDCs may be pathogenically relevant in SLE. They are the only cells that can stimulate na\u00efve T cells . Commonlh2 cells  and supph2 cells . IL-10 ih2 cells . IL-18, h2 cells .Considering the role of DCs in the induction of immunity or tolerance in health, changes in DC function in autoimmune diseases such as systemic lupus erythematosus (SLE) which is characterized by loss of tolerance to self antigens have been studied intensively. Actually, DC functional abnormalities in SLE have been reported previously, the main progress of which is on the pathogenic role of plasmacytoid DCs (pDCs) in this condition . Though Apoptotic cells are the primary source of autoantigens in SLE ,12. NuclRecently, an animal study has shown apoptotic cell-pulsed bone marrow-derived DCs (AC-BMDCs) could induce the proliferation of self reactive T-cells resulting in tolerance break down and initiation of autoimmune responses in normal mice . Howevern = 26). Thirty-two patients had inactive disease (SLEDAI < 5). Sixty-two sex- and age-matched healthy volunteers were recruited from the Red Cross Blood Transfusion Section. The study was approved by the Hong Kong West Cluster Institutional Review Board for medical ethics. All subjects provided a written informed consent.Patients who fulfilled the American College of Rheumatology criteria for SLE  were stu+CD25+ Foxp3+ expression and cytokine production were evaluated; and (3) functional changes of pDCs after interaction with apoptotic cells were evaluated by detection of cytokine production and toll-like receptor (TLR) expression.(1) Circulating blood pDCs were isolated and cultured with or without apoptotic cells; (2) Antigen loaded or unloaded pDCs were then co-cultured with autologous or allogenous T cells. The same control T cells were used as responder cells in all allogenic proliferation assays. Changes in T cell proliferation, cell surface marker CD25 expression, CD4A total of 100 mls of sodium citrate anti-coagulated blood were collected between 9:00 and 11:00 AM. Peripheral blood mononuclear cells (PBMCs) were freshly isolated by Ficoll density gradient centrifugation. pDCs and T cells from PBMCs were magnetically sorted with the human BDCA-4 DC and pan T isolation kits respectively according to the manufacturer's description . Briefly, pDCs were positively selected using anti-BDCA-4 conjugated beads . T cells6 cells/ml. PMNs were given 120 mJ/ml ultraviolet (UV) irradiation using the CL-100 Ultraviolet Crosslinker  to induce apoptosis. Sixteen hours after UV irradiation, the rate of apoptosis on PMNs reached around 60% to 90% and was confirmed by microscopic examination of cytocentrifuge stained with May-Giemsa, and flow cytometric detection of annexin V and propidium iodide (PI) staining (double positive cells).After isolating PBMCs from the whole blood, dextran sedimentation was applied to separate polymorphonuclear cells  from red blood cells (RBCs). The remaining RBCs were lyzed with ammonium chloride  and removed by washing with saline. Purified PMNs were then resuspended with complete RPMI 1640 medium at a concentration of 5 \u00d7 10pDCs were incubated with apoptotic PMNs for four hours. pDCs that had captured apoptotic PMNs (pDCs + apoPMNs) were confirmed by flow cytometry detection of surface PE-Cy5 CD123 (for pDC) and 5-(and 6)-carboxytetramethylrhodamine succinimidyl ester (for PMNs) double positive stained cells, the percentage of which was around 15%. pDCs or pDCs + apoPMNs were then treated with mitomycin C, which has the ability to inhibit proliferation without affecting the viability of the feeder cells. The cell cultures were subsequently washed with phosphate buffer solution (0.5% BSA) to remove mytomycin C and apoptotic PMNs, for T cell interaction experiments or for evaluation of cytokine and TLR expression.4) or pDCs that had interacted with apoptotic PMNs for four hours  were used to stimulate autologous or allogenous responder T cells (1.0 \u00d7 105). After five days of culture in a 96-well round-bottom culture plates with 200 \u03bcl of complete RPMI 1640 culture medium, the culture supernatants were collected for the detection of cytokines produced by T cells, and fresh medium was added back. Following that, 0.5 \u03bcCi tritiated thymidine  was added per well for 16 hours. T cell proliferation was measured by detection of the incorporation of (3H)-thymidine. Cells were harvested with a Packard FilterMate\u2122 Universal Harvester  and read with a TopCount\u2122 NXT Microplate Scintillation Counter . After the initial experiments, we found the 1:10 pDC:PMN ratio resulted in maximum T cell stimulation. This ratio was used in later experiments.T cell proliferation and differentiation induced by pDCs were evaluated by MLR. In brief, pDCs (1.0 \u00d7 10+CD123+lin- cells [+ cells and its purification is about 99% (data not shown). Changes in surface CD25 expression of T cells stimulated by pDCs were measured by percentage of CD4+CD25+ cells per CD4+ T cells and compared with that of unstimulated T cells. To further investigate whether the CD4+CD25+ T cells were Treg cells or not, intracellular immunofluorescence staining of Foxp3 was carried out according to the manufacturer's description . Appropriate isotype-matched control Ig was used as negative controls for each analysis. Flow cytometric analysis was performed within 24 hours, on a FACSCalibur flow cytometry using Cellquest software.The purity of pDCs and T cells was confirmed by surface immunofluorescence staining and flow cytometry analysis. pDCs were defined as BDCA-2n- cells  and its n- cells . Comparan- cells . T cellsCytokines produced by T cells after interaction with pDCs or pDCs + apoPMNs were measured using OptEIATM ELISA . Supernatants in the MLR system were collected after five days culture and frozen at -70\u00b0C for subsequent cytokine detection. These cytokines included IFN-\u03b3, tumor necrosis factor (TNF)-\u03b1 and IL-2 (Th1 cytokines); IL-4 and IL-6 (Th2 cytokines); and IL-10 and transfer growth factor (TGF)\u03b2 (Treg cytokines) [Changes in cytokine production by pDCs or pDCs + apoPMNs were evaluated also using ELISA. Supernatants from the pDC culture system were collected after 24 hours' culture and frozen at -70\u00b0C for subsequent detection of IFN-\u03b1, IL-6, IL-10, IL-12, TNF-\u03b1 levels. Human IFN-\u03b1 ELISA kit was obtained from PBL Biomedical Laboratory . ELISA results were read using MQX200 FCC Compliance .-\u0394\u0394Ct, where \u0394\u0394Ct = (Ct.target - Ct.18S)Time.x - (Ct.target - Ct.18S)Time.0h.Semi-quantitative real time PCR was applied to detect changes in cytokine or TLR mRNA gene expression in pDCs after interaction with apoptotic cells for four hours. pDCs without interaction with apoptotic PMNs and PMNs alone were used as controls. Total RNA from the culture system was isolated through PurelinkTM Micro-to-Midi Total RNA Purification System  according to the manufacturer's description. First-strand complementary DNA was subsequently synthesized using the SuperScriptTM First-Stand Synthesis System (Invitrogen). For real-time PCR detection of target and housekeeping gene expression, the fluorescent TaqMan 5'-nuclease assay was performed using 2\u00d7 TaqMan Universal PCR Master Mix and TaqMan Gene Expression assays (two unlabeled primers and one 6-FAM or VIC dye -labeled TaqMan MGB probe) for IL-6 (Hs00174131-ml), IL-10 (Hs00174086-ml), IL-18 (Hs00155517-ml), IFN-\u03b11 (Hs00256882-sl), TLR-4 (Hs00152939-ml), TLR-7 (Hs00152971-ml), TLR-9 (Hs00370913-sl) and 18S (Hs999999901-sl) . The reaction was performed in triplicate on an ABI 7000 Sequence Detector (Applied Biosystems) with a standard run. The expression levels of the target genes were adjusted to that of the housekeeping genes 18S. The fold change in the pDC target gene expression was adjusted to that of 0 hour, represented as the 2P < 0.10), a pair-wise Student's t-test was used to evaluate the explicit P-values. Similarly, the Kruskal-Wallis test was used to compare across different groups of non-normally distributed data. If differences were indicated across groups, a Wilcoxon's rank sum test for two samples was used to evaluate the explicit P-values.As this was the first study on human peripheral circulating pDCs, the data were first tested for normality before statistical analysis to detect group differences was carried out. Normally distributed data are expressed as mean \u00b1 SD while non-normally distributed data as median (interquartile range). For comparison across different groups, a two-factor analysis of variance (ANOVA) was used for normally distributed data. In case the ANOVA indicated differences across groups (n = 36) did not induce autologous T cell proliferation whether they were fed with apoptotic cells or not  pDCs did not stimulate autologous T cell proliferation, irrespective of the overall SLE disease activity  induced allogenous T cell proliferation while both pDCs and pDCs + apoPMNs from SLE patients (n = 30) were able to induce T cell proliferation  while no significant changes were found with either SLE pDCs alone or pDCs + apoPMNs. These results suggest that healthy but not SLE pDCs induced the development of suppressive Treg cells.To evaluate whether the CD25eg cells , intraceeg cells  were cars Figure . With alNeither healthy nor SLE pDCs or pDCs + apoPMNs induced autologous T cells to produce Th1 Figure  or Th2 rBoth autologous and allogenous T cells that were co-cultured with healthy pDCs produced a small amount of Treg cell related TGF\u03b2 Figure  but the Following incubation with apoptotic cells for four hours, both healthy and SLE pDCs showed a slight increase in the expression of IFN\u03b1 mRNA but it did not reach statistical significance Figure . Both heResults of the ELISA of cytokines in supernatants of pDCs + apoPMNs cultures were in accordance with mRNA expression findings above. There were no changes in the level of IFN\u03b1 from healthy and SLE pDCs + apoPMNs Figure . IL-6 prP < 0.05). This was not observed with SLE pDCs + apoPMNs. No significant differences in the changes in TLR7 and TLR4 mRNA expression in between healthy and SLE pDCs + apoPMNs were detected (Figure TLR9 mRNA expression in healthy pDCs + apoPMNs was found to be increased Figure  (P < 0.0d Figure .+ T cell lines [Previous reports have shown pDCs induce immune tolerance after phagocyting apoptotic cells . Howeverll lines . Furtherll lines . Our prell lines .We also investigated the ability of pDCs to stimulate allogenous T cells as an assessment of the antigen-presenting capability of DCs . We foun+ T cells that were co-cultured with either healthy or SLE pDCs + apoPMNs were found to have an increased expression of CD25 (Figure +CD25+ T cells may consist of either specific Th or Treg cell subsets [+ in allogenous CD4+CD25+ T cells induced by pDCs, only healthy pDCs + apoPMNs were found to have increased Foxp3 expression (Figure In accordance with the proliferation results above, allogenous CD45 Figure . CD4+CD2 subsets . As Foxp subsets , we evaln Figure , indicat+ expression in CD4+CD25+T cells (Figure +CD4+CD25+ Treg cells in immune homeostasis is that it controls autoimmunity throughout life. In animal models, Treg cell removal by neonatal thymectomy causes the spontaneous development of various organ-specific autoimmune diseases [+CD25+ T cells than normal persons [+CD25high Treg cells with reduced level of Foxp3 mRNA and protein expression has also been found in active SLE [Contrary to healthy pDCs - apoPMNs, SLE pDCs + apoPMNs did not induce Foxp3s Figure . The impdiseases ,34. Ther persons . In additive SLE . Recentltive SLE .An important feature that differentiates the various subsets of T cells is the characteristic profile of cytokines produced by these cells ,39. Our Functional changes of pDCs + apoPMNs including cytokine production and TLR expression  were stu+CD25+ Treg cells [TLRs play an important role in the presentation of antigens derived from apoptotic cells by DCs -43. In oeg cells . With SLeg cells . We hypoA limitation of this study is that most of the patients were receiving some form of treatment including immunosuppressive agents. It is therefore not possible to confirm whether the pDC changes were a result of the underlying disease or that of the various lupus medications. It should, however, be noted that differential changes were seen in patients with different levels of lupus disease activity. In general, the more active the disease was, the more adverse pDC changes were noted. It is therefore tempting to suggest that our findings reflect the true role of pDCs in lupus disease pathogenesis. Future studies should aim to recruit treatment of na\u00efve or newly diagnosed patients with SLE. However, this will have to involve the collaboration of multiple lupus research units. It has taken the authors over two years to recruit 58 suitable patients from a cohort of over 500 patients for the purpose of this study.Our study has provided further insights into the role of pDCs in SLE. In patients with SLE, the capacity of circulating pDCs to stimulate T cells was increased while their ability to induce Treg cell development was decreased. These may be the results of decreased IL-18 and increased IL-10 transcription which may prime Th2 response, and low expression of TLR9 following pDCs' interaction with apoptotic cells.AC-BMDCs: apoptotic cell-pulsed bone marrow-derived DCs; APCs: antigen presenting cells; DCs: dendritic cells; IFN: interferon; IL, interleukin; ODN: oligodeoxynucleotides; PBMCs: peripheral blood mononuclear cells; PCR: real time polymerase chair reaction; pDCs: plasmacytoid DCs; pDCs: + apoPMNs, pDCs: that had captured apoptotic PMNs; PMNs: polymorphonuclear cells; RBCs: red blood cells; SLE: systemic lupus erythematosus; SLEDAI: SLE disease activity index; TGF: transfer growth factor; TLR: toll like receptor; TNF: tumor necrosis factor; Treg: T regulatory; UV: ultraviolet.The authors declare that they have no competing interests.OJ carried out the whole study, performed the statistical analysis and drafted the manuscript. SK, RF and YN participated in some of the cell isolation and interaction assays. MYM participated in patient recruitment. LS and JG helped to draft the manuscript and performed the statistical analysis. AC carried out the flow cytometry, ELISA and MLR assays. JY participated in real time PCR detection. CSL conceived, participated in the design of and sought funding for the study. He coordinated patient recruitment and helped draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "The exact biologic activity of NHERF1 in mammary glands, however, remains unclear. It was recently proposed that NHERF1 forms a ternary complex with platelet-derived growth factor receptor (PDGFR) and phosphatase and tensin homolog (PTEN), linking NHERF1 suppressor activity to PDGF-initiated phosphoinositide-3 kinase (PI3K)/PTEN signaling.The gene encoding NaNHERF1 background. Levels of active Akt in mammary gland of NHERF1 knockout and wild-type mice were compared. We also examined how NHERF1 expression status affects cell sensitivity to PDGFR inhibitor. A plausible connection between NHERF1 and PTEN pathway was explored at the genetic level.The effect of NHERF1 on the kinetics of PDGF-induced Akt activation was determined in cells with varied NHERF1 knockout mice. In agreement with this, mammary gland tissues from these mice exhibited markedly elevated phospho-Akt. The responses of breast cancer cells to PDGFR inhibition were also altered by changes in NHERF1 expression level. Zr75.1 cells with NHERF1 knockdown were more resistant to STI-571-induced apoptosis than parental cells. Similarly, over-expression of NHERF1 rendered MCF10A cells more sensitive to STI-571. NHERF1-induced apoptotic response relies on an intact PTEN pathway; over-expression of NHERF1 in MCF10A cells with PTEN knockdown did not affect STI-571 sensitivity. It was found that NHERF1 LOH-positive breast cancer cells had reduced NHERF1 expression. Interestingly, these cells more frequently had wild-type PTEN or PI3KCA gene than the LOH-negative lines.We showed that NHERF1, through its PDZ-I domain, interacts directly with the carboxyl-terminal tail of PTEN. Knocking down NHERF1 expression in Zr75.1 cells markedly delayed the turnover of PDGF-induced phospho-Akt. Conversely, NHERF1 over-expression in MCF10A cells led to accelerated phospho-Akt degradation. The slowed decay of phospho-Akt that resulted from NHERF1 loss was evident in mouse embryonic fibroblasts isolated from NHERF1 and PTEN (or PI3KCA) alterations suggest that NHERF1 is an active component of the PTEN pathway. Collectively, our study indicates that the biologic activity of NHERF1 in mammary gland is related to PTEN signaling.Our data indicate that the interaction of NHERF1 with PTEN counterbalances PI3K/Akt oncogenic signaling and may affect how cells respond to PDGFR inhibition in breast cancer. The dependence of NHERF1 responses on PTEN and genetic segregation of  Human NHERF1 cDNA encodes a protein of 358 amino acids in length. NHERF1 and its close homolog NHERF2  share two modular structures: two tandem PDZ domains at the amino-terminus and an ezrin-radixin-moesin (ERM)-interacting domain at the carboxyl-terminus methionine . The translation products were mixed with purified GST-PTEN or GST-NHERF1 immobilized on the beads. Pull-down assays were performed at 4\u00b0C for 1 hour in 1\u00d7 binding buffer . The beads were then washed thoroughly with 1\u00d7 binding buffer. Bound proteins were eluted by boiling in 1\u00d7 SDS sample buffer, separated by SDS-PAGE. Ten per cent of the input TNT lysates was also run on the PAGE to determine relative binding capacity. The PAGE gel was then dried and exposed for autoradiography.Radio-labeled full-length or defined segments of NHERF1 and full-length or truncated PTEN were synthesized by 3VO4 and 1\u00d7 protease inhibitor cocktail ). The cell lysates were then incubated with beads coated with GST fusion proteins. The incubation lasted for 2 hours at 4\u00b0C. The beads were then washed thoroughly with 1\u00d7 binding buffer before being boiled in 1\u00d7 SDS sample buffer. The eluted proteins were then subjected to NHERF1 or PTEN immunoblotting.GST pull-down assay was also used to determine whether endogenous PTEN or NHERF1 binds to recombinant NHERF1 or PTEN, respectively. MDA-MB-468, MCF7, and T47D cells were harvested by adding 1\u00d7 lysis buffer . The soluble proteins (about 600 \u03bcg) were immunoprecipitated with 2 \u03bcg of goat IgG reactive to PTEN  at 4\u00b0C overnight, using normal goat IgG as a control. The immunocomplex was collected by addition of 50 \u03bcl of agarose-conjugated protein G (Roche Applied Science) and detected with anti-NHERF1 antibody.To assess the interaction of PTEN with NHERF1 at the endogenous level, cultured MCF7 or Zr75.1 cells were lysed with 1\u00d7 NETN buffer  and total Akt. Immunoblottings were carried out essentially as described previously . AntibodMTT  assays were used to measure cell viability. Cells were seeded in 96-well cluster dishes at 5,000 cells/well with 100 \u03bcl complete medium. After overnight incubation, medium was replaced to include various concentrations of STI-571 . After 2 days of treatment, cells were fed 100 \u03bcl fresh medium that contained 1 mg/ml MTT (Sigma). The incubation lasted for 2 hours before the medium was removed and cells dissolved in 150 \u03bcl dimethyl sulfoxide. Absorbance was measured using a multiSkan plate reader  at a wavelength of 570 nm. Each sample was processed in triplicate. Experiments were repeated at least three times.in vitro synthesized full-length NHERF1. GST-PTEN but not GST interacted directly with the full-length NHERF1 and the PDZ-I&II domains . p-Akt in NHERF1-/- cells remained at high levels at 90 and 120 minutes, but the p-Akt level in NHERF1+/+ cells deminished markedly , whose NHERF1 genetic background was verified  cells. One day of STI-571 treatment resulted in significant increases in caspase-3 cleavage in Zr75.1 Babe control cells, suggesting that apoptosis is at least partially responsible for STI-571-induced growth inhibition. Interestingly, the same treatment led to markedly lower caspase-3 cleavage in NHERF1-knockdown Zr75.1 cells Figure , which iPTEN-null background. MDA-MB-468 cells are NHERF1 positive but lack PTEN as a result of homozygous deletion. We knocked down NHERF1 expression in MDA-MB-468 cells using siRNA retrovirus, and these cells were tested for STI-571 sensitivity. We found that in the absence of PTEN the NHERF1-knockdown cells responded to STI-571 at a level similar to that in Babe control cells .To explore whether LOH is responsible for a decrease in NHERF1 expression, we correlated the two parameters in 22 breast cancer cell lines that had been tested ,25. AmonNHERF1 and PTEN (or PI3KCA) alterations. We thus evaluated whether NHERF1 LOH is correlated with PTEN/PI3KCA mutational status. A total of 39 breast cancer cell lines with available genetic status were used for the correlative analyses [NHERF1 LOH-positive cell lines, 15 (65%) had an unaltered PTEN or PI3KCA gene. In contrast, out of the 16 LOH-negative cell lines, only 4 (25%) showed wild-type PTEN/PI3KCA status. The genetic alteration in either PTEN or PI3KCA gene was strongly associated with intact NHERF1 alleles .The physical and functional relation between NHERF1 and PTEN indicates they may exist in a common tumor suppressor pathway, which would predict segregation between analyses . Among tNHERF1genetic alterations in human breast cancer prompted us to hypothesize that NHERF1 acts as a tumor suppressor gene in mammary gland [NHERF1 is an estrogen-inducible gene [NHERF1 gene that are responsible for estrogen-stimulated expression [NHERF1. Here we provide additional evidence to support our overall hypothesis.Our earlier results of ry gland . Human Nble gene . Estrogepression . Becausepression , the suppression , which iNHERF1-/- mice . Whether aberrant Akt activation is directly related to hyperplastic morphology in mammary gland warrants further investigation. Also remaining to be established is whether this impaired mammary development is sufficient to increase or accelerate the incidence of mammary tumor.In breast cells, NHERF1 expression accelerated the turnover of p-Akt induced by PDGF stimulation. This finding was obtained in both over-expression and knockdown models, from both immortalized normal mammary epithelial cells (MCF10A) and a breast cancer line (Zr75.1). The effect of NHERF1 to stimulate the decay of p-Akt is probably through PTEN recruitment by NHERF1 to the cytoplasmic membrane compartment, where active phosphorylation and dephosphorylation of Akt occur. The response is probably related to normal mammary biology, as indicated by the markedly increased levels of p-Akt in the mammary gland tissues of e Figure . Elevateeostasis . A deregeostasis ,42. InteNHERF1 LOH and the aggressive features of breast cancer, we hypothesized that NHERF1 tumor suppressor activity was haploinsufficient [NHERF1 gene is obligatory for its haploinsufficient biology. In support of this mechanism, we found that LOH of NHERF1 locus in 22 breast cancer cell lines was strongly correlated with lowered NHERF1 protein level. The haploinsufficient expression of NHERF1 is also supported by in vivo observations. Monoallelic deletion of NHERF1 was shown to decrease NHERF1 expression in kidney epithelial cells [NHERF1 gene resulted in decreased NHERF1 protein expression in mammary gland . The correlation suggested an association between abnormal Akt activation and mammary hyperplasia, highlighting the biochemical and pathological consequences of monoallelic deletion of NHERF1.Because of the strong correlation between fficient . A numbefficient -46. In tfficient ,44. A loal cells . SimilarNHERF1 activity during normal mammary gland development. Whether NHERF1 affects breast cancer susceptibility through the haploinsufficiency mechanism requires further investigation. Haploinsuficient NHERF1 tumor suppressor activity would also explain the relatively low frequency of intragenic mutations, although the possibility that other NHERF1 pathway components are genetically altered cannot be ruled out.Our findings clearly indicated a dosage effect of NHERF1 should be associated with altered PTEN (or PI3KCA) gene in breast cancer. Our data from 39 breast cancer cell lines showed that this was indeed the case. Collectively, our present study indicates that NHERF1 binds to PTEN to downregulate the PI3K-Akt pathway to elicit tumor suppressor activity. Given that PTEN-PI3K-Akt is one of the most prominent pathways relevant to tumorigenesis and targeted therapy of almost all types of carcinoma, studies on NHERF1 should be instrumental to the development of new strategies to overcome chemo-resistance and enhance efficacy.The present study verifies that NHERF1 has tumor suppressor activity, providing evidence to suggest that its function relies on an intact PTEN pathway. First, NHERF1 is associated with accelerated dephosphorylation of p-Akt, presumably through recruitment of PTEN by NHERF1. Second, knockdown of PTEN abolishes NHERF1-induced sensitivity to chemo-agents. If NHERF1 activity is dependent on PTEN, as our functional study had suggested, then intact In this study we also present evidence that NHERF1 expression status significantly affects how cells respond to PDGFR inhibition. PDGF is among the key growth factors and cytokines that breast cancer cells produce via autocrine or paracrine mechanisms that contribute to malignant progression . ActivatNHERF1 gene and wild-type PTEN or PI3KCA in breast cancer cells. Given the critical roles of Akt in cell survival and tumorigenesis, the negative regulatory effect of NHERF1 on Akt activity is highly relevant to NHERF1 mammary tumor suppressor function. Our finding of a higher sensitivity of breast cells to STI-571 in the presence of NHERF1 suggests that future investigations of this important pathway may yield new measures to improve breast cancer treatment.In this report, we show that, by interacting with PTEN, NHERF1 accelerates the turnover of p-Akt and enhances the cell sensitivity to STI-571. That NHERF1 elicits suppressor function through PTEN is also indicated by an inverse correlation between intact +/H+ exchanger regulatory factor; p-Akt = phospho-Akt; PCR = polymerase chain reaction; PDGFR = platelet-derived growth factor receptor; PI3K = phosphoinositide-3 kinase; PTEN = phosphatase and tensin homolog; siRNA = small interfering RNA.ERM = ezrin-radixin-moesin; GST = glutathione S-transferase; LOH = loss of heterozygosity; MEF = mouse embryonic fibroblast; MTT = 3--2,5-diphenyltetrazolium bromide; NHERF = NaThe authors declare that they have no competing interests.YP designed the experiments, carried out technical procedures, and interpreted the data. EJW provided the knockout animals and edited the manuscript. JLD designed the experiments, interpreted the results and wrote the manuscript. All authors approved the final manuscript."}
+{"text": "Clostridium difficile is the main cause of antibiotic-associateddiarrhea, leading to significant morbidity and mortality and puttingconsiderable economic pressure on healthcare systems. Current knowledge of themolecular basis of pathogenesis is limited primarily to the activities andregulation of two major toxins. In contrast, little is known of mechanisms usedin colonization of the enteric system. C. difficile expresses aproteinaceous array on its cell surface known as the S-layer, consistingprimarily of the major S-layer protein SlpA and a family of SlpA homologues, thecell wall protein (CWP) family. CwpV is the largest member of this family and isexpressed in a phase variable manner. Here we show CwpV promotes C.difficile aggregation, mediated by the C-terminal repetitivedomain. This domain varies markedly between strains; five distinct repeat typeswere identified and were shown to be antigenically distinct. Other aspects ofCwpV are, however, conserved. All CwpV types are expressed in a phase variablemanner. Using targeted gene knock-out, we show that a single site-specificrecombinase RecV is required for CwpV phase variation. CwpV ispost-translationally cleaved at a conserved site leading to formation of acomplex of cleavage products. The highly conserved N-terminus anchors the CwpVcomplex to the cell surface. Therefore CwpV function, regulation and processingare highly conserved across C. difficile strains, whilst thefunctional domain exists in at least five antigenically distinct forms. Thishints at a complex evolutionary history for CwpV. Clostridium difficile is a bacterial pathogen that causesantibiotic-associated diarrhea, which can be fatal. Infections often occur inhealthcare facilities, where there is a high population density of susceptiblepatients with many possible routes of transmission. We currently do not know thefunctions of molecules found on the surface of C. difficile,which are likely to facilitate colonization of the host. In this study wecharacterize a protein called CwpV that is found on the surface of C.difficile. We show that bacteria isolated from different patientshave different versions of CwpV and that the immune system's weapons(antibodies) against one version are useless against the others. This suggeststhat CwpV may help C. difficile to escape from our naturaldefenses. We have also found that CwpV promotes aggregation of C.difficile, which may be important for host colonization. To testthese hypotheses in the future we will use animal models to compare geneticallymodified C. difficile expressing different levels of CwpV tosee if they behave differently during infection. We hope that knowing more aboutCwpV will help guide improvements in the prevention and treatment of C.difficile. C. difficile is gram-positive spore forming anaerobe and a majorcause of antibiotic-associated diarrhea C.difficile infection (CDI) often occurs in the nosocomial environmentwhere infection management exerts significant economic pressure on healthcaresystems. The strong association of CDI with antibiotic usage reflects disruption ofthe normal gut flora allowing effective colonization by C.difficile. Strains causing disease produce one or two related toxins,TcdA and TcdB, which modulate the activity of host cell Rho GTPases, destroying theintegrity of the epithelial cell barrier and inducing a variety of effects onintestinal cells C. difficilestrains causing CDI however are remarkably diverse. Despite a high prevalence ofcertain strain types, for example NAP1/027 in North America and Europe around2005\u20136, no particular type dominates and strain prevalences varygeographically and temporally C.difficile produces an S-layer, a paracrystalline proteinaceous arraythat completely coats the bacterial cell wall. The C. difficileS-layer is primarily composed of two proteins, the high molecular weight S-layerprotein (HMW SLP) and the low molecular weight (LMW) SLP. These proteins are derivedfrom the precursor SlpA Factors expressed on the bacterial cell surface are likely to contribute to hostcolonization via interactions with host tissue, the immune system and otherbacterial cells. In common with a large number of bacterial species C. difficile 630 contains 28 paralogs of the HMW SLP in vitroin vivo during infection andaccessible to the host immune system.C. difficile 630 cwpV switch cwpV promoter and open reading frame there is a 195 bpinvertible region flanked by imperfect 21 bp inverted repeats. In the OFForientation this sequence adopts a stem loop terminator, preventing formation offull-length transcripts while in the other orientation (ON), the terminator isabsent so full length cwpV transcription proceeds and CwpV isexpressed.In C. difficile strains. Wedefinitively identify the site-specific recombinase necessary for CwpV phasevariation, and show that the cwpV switch orientation is the primarydeterminant of CwpV expression. We show that the CwpV N-terminal domain isresponsible for cell wall anchoring and is well conserved across strains. Incontrast, we find high sequence variation of the C-terminal repeat sequences acrossC. difficile strains with five antigenically distinct repeattypes identified. Despite this variability, both post-translational processing andfunction of CwpV appear to be conserved across C. difficilestrains. All types of CwpV exhibit an aggregation-promoting function, which isconferred by the variable C-terminal domain. The implications of these findingsrelating to the role of CwpV in C. difficile infection arediscussed.In this study we investigate the regulation, post-translational processing andfunction of CwpV across a diverse panel of C. difficile recV gene as a strongcandidate for the site-specific recombinase responsible for inversion of thecwpV DNA switch recV in inversion of the cwpVswitch, we constructed a recV knock-out in630\u0394erm using ClosTron technology erm(wild-type) and four erythromycin resistant recV mutants,\u0394recV1-4, were confirmed by PCR  was introduced intoC. difficile \u0394recV OFF by conjugation.The orientation of the cwpV switch in four\u0394recV(pRecV+) transconjugants was tested by PCR.In all four strains, cwpV switch inversion was observedindicating successful complementation of the switching defect (Y176F)which was introduced into \u0394recV OFF. All four\u0394recV(pRecVY176F) transconjugants only hadthe cwpV switch in the OFF orientation  was serially subcultured withoutthiamphenicol selection to allow loss of the pRecV+ plasmid.Thiamphenicol-sensitive colonies were tested for cwpV switchorientation and all were found to contain a single orientation, as thepRecV+ plasmid had been lost (data not shown). Four clones, from which onlythe ON orientation of the cwpV switch could be amplified, wereisolated and designated \u0394recV ON .During the plasmid-curing process we noted two distinct colony morphologiesamongst the resulting thiamphenicol-sensitive colonies. Interestingly, thesewere associated with the two orientations of the erm (WT),\u0394recV(pRecV+),\u0394recV(pRecVY176F),\u0394recV OFF and \u0394recV ON wasanalyzed by SDS-PAGE (recV(pRecV+) extracts, the twocleavage products of CwpV, the \u223c42 kDa  and \u223c116kDa (repeat domain) can be seen. However, as CwpV is only expressed by theminority of ON cells, the overall level of expression in these cultures is low.No expression of CwpV is seen in\u0394recV(pRecVY176F) or\u0394recV OFF strains. Compared to WT cultures,\u0394recV ON strains express a high amount of CwpV.Densitometry analysis indicated that in \u0394recV ON strains,CwpV constituted 13.3% of the protein present in the S-layer (data notshown). These findings were confirmed by Western blot using anti-CwpVrptI(detects the \u223c116 kDa CwpV repeat domain) and anti-CwpVNter  antibodies  cultures, phase variable expressionof CwpV can be seen with approximately 15% of cells expressing CwpV. Thehigher proportion of CwpV-positive cells is probably due to the altered level ofRecV expression from the multi-copy plasmid, pRecV+. In\u0394recV OFF(pRecVY176F) no CwpV expression canbe detected, as expected due to a lack of switch inversion from OFF. In\u0394recV ON, CwpV expression can be detected in all cells.This suggests that cwpV switch orientation is the primarydeterminant of CwpV expression.In order to assess CwpV expression at the individual cell level, croscopy . In the C. difficile strain diversitywe sought to determine the level of conservation of cwpV acrossa variety of strains. PCR analysis of a collection of C.difficile strains using primers specific for the 5\u2032 region ofcwpV revealed the presence of a common fragment in allstrains and for type V AY1 . A full multiplesequence alignment of all the repeat sequences is shown in Having characterized these five repeat types, a larger collection of strains wereanalyzed by PCR to determine their repeat types see . Our anaGiven the completely different amino acid sequences for each repeat type wehypothesized that the repeats may be antigenically distinct. CwpV expression wastherefore investigated using antibodies raised against recombinant CwpV domains.The CwpV domains used as antigens are indicated by black lines above the proteincartoons in S-layer extracts were then analyzed using antibodies raised against the specificrepeat types I\u2013V. Antibodies against repeat types I, II, IV and Vrecognized only their cognate strains 630, R20352, M9 and AY1, respectively..Anti-CwcwpV switch has been detected in a wide varietyof strains, but phase variable expression of CwpV has only been reported instrain 630 Inversion of the C.difficile 630\u0394cwpV using a constitutivepromoter. S-layer extracts were prepared from these strains, designated\u0394cwpV(pOE I-V), and analyzed by SDS-PAGE .The five different CwpV proteins from 630, R20352, CDKK167, M9 and AY1 weremodified to include a C-terminal Strep-Tag II and expressed in SDS-PAGE . A high SDS-PAGE . In ordecwpV(pOEI) was purified by affinity chromatographyusing a StrepTactin resin. As shown in cwpV strains over-expressing CwpV types II\u2013V.The complete S-layer extracts (S) and first elutions (E) are visualized bySDS-PAGE . S-layer extracts from wild-type,\u0394cwpV, \u0394cwpV(pOE I) and\u0394cwpV(pOENter) cultures were analysed by SDS-PAGE(cwpV and its identity was confirmed by Western blotwith anti-CwpVNter (cwpV(pOEI) and\u0394cwpV(pOENter) S-layer extracts, showing that thetruncated version of CwpV is equally well expressed on the cell surface as thefull-length CwpV protein. We therefore named the N-terminal fragment of CwpV asthe cell wall anchoring (CWA) fragment. This is the first reported experimentalevidence that PF04122 motifs are responsible for cell wall anchoring.The role of the N-terminal fragment of CwpV containing putative cell bindingmotifs was then investigated. A truncated form of the 630 SDS-PAGE. The \u223c42CwpVNter . This suThe determination of the CwpV cleavage site, evidence of complex formation by thetwo CwpV cleavage products, and role of the N-terminal fragment in cell wallanchoring leads to an overall model for post-translational CwpV processing. Thethree steps involved are depicted in recV ON strains showed a smaller,smoother-edged colony morphology than \u0394recV OFF orwild-type strains  colonieswere seen to have diffuse edges, with directional protrusions of bacterialgrowth common at the colony edge. However, \u0394cwpV(pOEI-V)exhibited denser, straighter colony boundaries, with rare if any directionalprotrusions. The observed macroscopic differences in colony morphology aretherefore due to altered cellular organization within bacterial colonies.As noted previously, \u0394ains see . We thermutants  and8A. C. difficile toaggregate from suspension was assessed across the panel of strains.Over-expression of all CwpV types, except type IV, induced aggregation ofC. difficile from a liquid suspension with a startingOD600 nm of 10  inducedaggregation from an OD600 nm of 6 (data not shown). An OD600nm as high as 40 did not lead to type IV-mediated aggregation, noraggregation of wild-type, \u0394cwpV or\u0394cwpV(pOENter) (data not shown). Therefore it appearsthat the ability to induce aggregation is highest for CwpV from 630, followed byR20352, then CDKK167 and AY1, with no observed propensity of M9 CwpV or the CwpVCWA fragment to induce aggregation of C. difficile fromsuspension.This altered cellular organization may result from CwpV-mediatedauto-aggregation. Therefore the propensity of nm of 10 . AggregaIn vitro colonies are organized populations ofbacteria and differences in colony morphology reflect the cellular organization of apopulation C. difficilecells from suspension strongly suggests that CwpV has a direct aggregation-promotingfunction rather than a direct effect on motility. In fact the motility of strains insoft agar was found to be similar, regardless of whether or not they expressed CwpV. However in infected mice large aggregates of C.difficile cells, described as exaggerated mats, were reported to beassociated with regions of severe inflammation C. difficile during infection.Multispecies biofilms in the human GI tract have been observed C.difficile is likely to require factors that promote appropriate intra-and inter-species interactions to colonize the gut. CwpV may play important roles inthis process.Propensities of different CwpV types to cause aggregation varied, as observed by thedifferent starting densities of suspensions required for aggregation, apart fromtype IV, which did not induce aggregation. This is likely to reflect differentaffinities of the different CwpV proteins for their ligand(s). The lack of anobservable aggregation phenotype due to type IV repeats may be explained by a weakeraffinity between this repeat type and its ligand, however the observed colonymorphology phenotype does suggest that it carries out the same general function asthe other types. Functional interactions between CwpV and motility factors, such astype IV pili and flagella, are worthy of further study. Indeed, the diffusedirectional organization of wild-type colony edges seen in this study is reminiscentof twitching motility mediated by type IV pili in in vivo,and if so whether this is directly due to its aggregation-promoting activity.Understanding the roles of CwpV during in vivo infection is apriority and will be addressed using the isogenic mutants described in this study.Established animal models of infection focus more on the acute stage of C.difficile infection, rather than colonization C. difficilegenes likely to be involved in colonization, such as CwpV, have been described in vivo role; its expression in a sub-set ofcells as a contingency may be beneficial to the survival of the population as awhole.Aside from its aggregation-promoting function CwpV may have other functions. Weinvestigated bacterial adhesion to Caco-2 cells, but no significant difference inadhesion was caused by CwpV expression (data not shown). Further work is required toassess whether CwpV expression affects colonization levels C.difficile chromosome C. difficile and incorporation intothe genome are unclear. Human microbiome sequencing may shed light on the origins ofCwpV sequences. It would seem that CwpV type exchange is occurring at a lowfrequency, as all strains tested from within one ribotype contain the same CwpVtype. Current understanding of the phylogenetics and diversity of C.difficile strains is limited C. difficile are not yet known. As moreC. difficile strains are sequenced and our understanding ofevolutionary relationships between strains improves, it will become clearer as towhen different CwpV types were acquired, and whether single or multiple independentacquisition events for each type have occurred. Host immune pressure is one possibleselection that could promote CwpV variability, as one key difference between CwpVtypes is their antigenicity. Antigenic variability is also seen in the C.difficile surface proteins SlpA and Cwp66 C. difficilestrains or in resistance to bacteriophages.The variation of CwpV repeat sequences uncovered in this study is extensive, withstrains expressing one or two of five antigenically distinct types encoded byunrelated sequences. The lack of sequence homology between repeat types suggestsacquisition by horizontal gene transfer. Any known mechanism of chromosomal sequenceacquisition; homologous recombination, illegitimate recombination or additiveintegration could account for introduction of these sequences to the cwpV switch and the RecV recombinase, is important for optimalfunction of CwpV. Phase variation of bacterial surface proteins has been reported tobe involved in numerous processes including immune evasion and colonization in vivo infection islikely to shed most light on the importance of CwpV phase variability.Apart from the marked diversity in CwpV repeat sequences, other aspects of CwpV areremarkably conserved. Expression of all types of CwpV is phase variable suggestingthat this complex regulatory mechanism, involving the conservedcwp84 mutant recV ON strains, it seems likely thatCwpV interacts positively with other S-layer proteins to maintain the integrity ofS-layer packing.Conserved post-translational processing of CwpV and formation of a CWA-repeat domaincomplex again suggests that these processes are important for optimal CwpVexpression and function. To date we only observe complex formation in cell wallproteins CwpV and SlpA, with all other CWPs studied  beingpresented on the surface without post-translational cleavage between the CWA andfunctional domains. An unidentified protease could mediate cleavage of CwpV, or itcould undergo non-enzymatic auto-processing as reported for a number of proteinfamilies C. difficile S-layer where the presumably mediate a similarrole. They are also found in a large variety of Gram-positive bacteria and someArchaea, where they presumably fulfill a similar function of anchoring cell wallproteins to the underlying structure. This then adds to the diversity of mechanismsused by Gram-positive bacteria to anchor and display cell wall proteins Our finding that the CwpV CWA domain is responsible for anchoring of the CwpVC-terminal domain to the underlying cell wall is the first direct evidence of thefunction of Pf04122 domains. These domains are also present in the HMW SLP of theC.difficile have emerged independently from multiple lineages, indicatingthat virulent strains can emerge from across the diversity of the species, ratherthan being confined to a specific pathogenic lineage. This suggests that certaingenetic elements common to all C. difficile strains underlievirulence. It is likely that the incidences of different lineages causing diseaseare dynamic, and we cannot predict which lineages may cause future outbreaks.Therefore understanding the common features of C. difficile as anancient species is important, as in order for intervention strategies to besuccessful in the long-term they must target the full diversity of C.difficile strains, rather than specific lineages. CwpV appears to be acore component of C. difficile, including its phase variableregulation controlled by RecV, post-translational processing mechanism, andaggregation-promoting function. This highlights the importance of CwpV to C.difficile survival throughout evolution of this diverse species.Despite this conservation CwpV appears to have been under positive selection forantigenic variability. Further investigation into the roles of CwpV in theC. difficile life cycle may teach us a lot about what makesC. difficile a successful pathogen, and provide opportunitiesfor new intervention strategies.Phylogenetic studies C. difficile strains characterised for CwpV type in thisstudy are described in C. difficile laboratory-generated mutant andrecombinant-plasmid containing strains used in this study are described in C. difficile was routinely cultured on blood agar base II(Oxoid) supplemented with 7% horse blood (TCS Biosciences), brain-heartinfusion (BHI) agar (Oxoid) or in BHI broth (Oxoid). Cultures were grown in ananaerobic cabinet (Don Whitley Scientific) at 37\u00b0C in an atmosphere of10% CO2, 10% H2 and 80%N2. C. difficile strains containing recombinantplasmids were grown under thiamphenicol selection. Commercial chemicallycompetent TOP10 E. coli cells (Invitrogen) were used forcloning and recombinant plasmid maintenance. Recombinant expression of CwpVfragments was carried out in Rosetta E. coli cells (Novagen).E. coli strains were routinely grown at 37\u00b0C on LB-agarplates or in LB liquid culture in the presence of selective antibiotics whereappropriate to any transformed plasmid.The C.difficile genomic DNA for use in cloning and PCR analysis wasprepared as described previously DNA manipulations were carried out according to standard techniques. C. difficile 630\u0394ermknock out of recV, a retargeted ClosTron vector was designedusing the intron design tool www.clostron.com. Thetop-scored target site in recV was selected (424/425s) and theintron targeting region required was commercially synthesized and cloned intopMTL007C-E2 (DNA 2.0) to generate a plasmid pLRP028. For complementation of the\u0394recV mutant, the recV gene fromC. difficile 630 was amplified using primers NF1357 andNF1358 and cloned using SacI and BamHI into aC. difficile plasmid expression vector, based on thepMTL960 replicon recV tyrosine176 to phenylalanine, producing pCBR115(pRecVY176F+). To express CwpV fragments for use as antigens(cwpV repeats from strains R20352 (type II), CDKK167 (typeIII), M9 (type IV) and AY1 (type V) were amplified by PCR using KOD polymerase and primers described in NcoI andXhoI into pET28a E. coli expression vectorthen transformed into TOP10 E. coli (Invitrogen) to generateplasmids pCBR069-072 described in cwpVgenes from strains of unknown genomic sequence, genomic DNA was digestedovernight at 37\u00b0C with AseI (NEB) and then ligatedovernight at 16\u00b0C using T4 ligase (NEB). Inverse PCR using primers NF401 andNF654 was carried out and amplification products were cloned into pCR4-TOPO(Invitrogen). pCBR044, containing the full-length cwpV genefrom 630 under the control of the cwp2 promoter within thepMTL960 backbone C. difficile strep-tagged 630 CwpV expression plasmidpCBR080 (pOEI). The cwpV genes from R20352, CDKK167, M9 and AY1were amplified with the primers described in In order to produce a antigenscwpV repE. coli CA434 and then conjugatedinto C. difficile as described previously \u22121) to select for pMTL960-based plasmids and cycloserine(250 mg ml\u22121) to counter-select for E.coli.Plasmids were transformed into E. coli Rosetta  and grownovernight at 37\u00b0C in Overnight Express media .Bacteria were collected, lysed using BugBuster  andthe His-tagged fusion proteins purified by affinity chromatography using Ni-NTAagarose (Qiagen). Following purification, proteins were dialysed into 10 mMHEPES 150 mM NaCl. Antisera to the proteins were generated in rabbits using acommercial service . Anti-CwpVNter and anti-CwpVrptIwere described previously Plasmids pCBR069-072 expressing repeat types II\u2013V were transformed intoC. difficile cultures were grown overnight, harvested bycentrifugation and S-layer extracts were prepared using low pH glycineincubation as described previously C. difficile cells from liquid culture were washed with PBS thenfixed in 8% formaldehyde, which was quenched with 20 mM NH4Clfor 15 min prior to staining. Washed cell suspensions were re-suspended in 40\u00b5l 1% BSA in PBS containing 1/20 dilutions of rabbit primaryantibodies  for 45 min. Bacteria were then washed andincubated with 1/40 anti-rabbit-rhodamine red-X (Jackson ImmunoResearch) in thedark for 45 min. Bacteria were washed with PBS and dH20 then allowedto air dry onto a microscope slide. Coverslips were mounted using ProLong goldanti-fade mounting reagent (Invitrogen) and allowed to set overnight. Bacteriawere visualised using a Nikon Eclipse E600 microscope fitted with a NikonDMX1200 camera.S-layer extracts were separated on 10% SDS-PAGE gels and transferred to aPVDF membrane. The membrane was washed in water for 10 min, stained in0.1% Coomassie Blue R in 50% MeOH for 5 min. After destaining in50% MeOH, 10% acetic acid (3\u00d75 min with rocking) and washingin water (3\u00d75 min with rocking) the membrane was allowed to drythoroughly. N-terminal sequencing by Edman degradation was then carried .StrepTactin resin (Novagen) was used according tomanufacturers' instructions. Briefly, resin was washed withdH2O, equilibrated with wash buffer  and mixed with S-layer extract. The mixture was incubatedovernight at 4\u00b0C with rotation. The next day the resin was washed with washbuffer to remove non-specifically bound proteins. Elution buffer  was then used to elutestrep-tagged proteins. All fractions were collected for analysis using SDS-PAGEand Coomassie staining.C. difficile colony morphologystrains were grown on BHI agar (Oxoid) for 5 days from an appropriate C.difficile inoculum such that isolated colonies grew on the plate.Plates were then scanned using a flat-bed scanner to record images of thecolonies. For microscopic visualization of C. difficile colonymorphology, cultures taken from a 24-hour old colony were inoculated directly onto glass-bottomed dishes (MatTek corporation). Anaerobic BHI 0.7% agar at40\u00b0C was then carefully poured over the glass to cover the dish and allowedto set. C. difficile was grown between the glass and the agarfor 3 days. Dishes were removed and images taken using a Zeiss Axiovert 200widefield microscope at FILM, Imperial College London. Microscope images wereanalysed using Volocity 5.3.2 software (Improvision).For macroscopic visualization of Figure S1C. difficile strains M9 and AY1 encode CwpV withmosaics of type III and type IV or V repeats. A cartoon representations ofthese mosaic CwpV proteins is shown above a ClustalW2 multiple sequencealignment of all type III repeats. (E) The seven type IV repeats in strainM9 CwpV. (F) The five type V repeats in strain of AY1 CwpV. The sequences ofall repeats with a type were aligned using ClustalW2 and are shown in color,corresponding to the blocks in the cartoon, with the exception of type IIrepeats which are shown in black instead of yellow.Sequence alignments of the C-terminal repeats in CwpV types I\u2013V. (A)The nine type I repeats in strain 630 CwpV. (B) The eight type II repeats instrain R20352 CwpV. (C) The six type III repeats in strain CDKK167 CwpV. (D)(PDF)Click here for additional data file.Figure S2C. difficile does not affect flagellarexpression or swimming motility. (A) C. difficile strainswere analyzed for FliC expression based on a published protocol C. difficile FliC expression from the panel of strainswith varying levels of CwpV expression was assessed by SDS-PAGE andCoomassie staining, with 630 FliC running at \u223c33 kDa. Lanes 1: WT, 2:\u0394cwpV, 3: pOE Nter, 4\u20138: pOE I-V, 9:\u0394recVON, 10: \u0394recVOFF. (B) Toassess swimming motility of C. difficile strains, BHIcontaining 0.175% agar was inoculated to a defined depth withC. difficile from overnight liquid cultures. Tubes wereincubated overnight then photographed to document motility. Tubes arenumbered in the same way as the lanes in A. All cultures appeared to beequally motile.CwpV expression in (TIF)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.Table S2Plasmids used in this study.(DOC)Click here for additional data file.Table S3C. difficile strains used in thisstudy.Genetically modified (DOC)Click here for additional data file.Text S1References for (RTF)Click here for additional data file."}
+{"text": "Brevundimonas sp. SGJ. A Plackett\u2013Burman design was used for screening of critical components, while further optimization was carried out using the Box\u2013Behnken design. The optimum conditions observed were pH 5.31, tryptone 1.440\u00a0g\u00a0l\u22121, l-tyrosine 1.872\u00a0g\u00a0l\u22121 and CuSO4 0.0366\u00a0g\u00a0l\u22121. Statistical analysis revealed that the model is significant with model F value 29.03 and R2 value 0.9667. The optimization of process parameters using RSM resulted in a 3.05-fold increase in the yield of melanin. The intermittent addition of l-tyrosine enhanced the melanin yield to 6.811\u00a0g\u00a0l\u22121. The highest tyrosinase activity observed was 2,471 U mg\u22121 at the 18th hour of the incubation period with dry cell weight of 0.711\u00a0g\u00a0l\u22121. The melanin production was confirmed by UV\u2013Visible spectroscopy, FTIR and EPR analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of melanin.Melanins are predominantly indolic polymers which are extensively synthesized in animals, plants and microorganisms. It has wide applications in cosmetics, agriculture and medicine. In the present study, optimization of process parameters influencing melanin production was attempted using the response surface methodology (RSM) from The online version of this article (doi:10.1007/s13205-012-0082-4) contains supplementary material, which is available to authorized users. Escherichia coli W3110  melanin can be used to generate monoclonal antibodies (mAb) for the treatment of human metastatic melanoma. This antibody also binds human melanin since both fungal and human melanins have structural similarities , which provide a great amount of information based on only a small number of experiments  whereas other chemicals were procured from HiMedia (India). The melanin-producing bacterial strain was isolated from garden soil of Shivaji University, Kolhapur, India, using serial dilution technique on a nutrient agar  supplemented with 1\u00a0g\u00a0l\u22121l-tyrosine.Melanin (synthetic) and \u22121 peptone, 1.5\u00a0g\u00a0l\u22121 beef extract, 1.5\u00a0g\u00a0l\u22121 yeast extract, and 0.5\u00a0g\u00a0l\u22121 NaCl, supplemented with 1\u00a0g\u00a0l\u22121l-tyrosine and with a pH of 7. The 6-h grown, 2-ml cell suspension was inoculated in 100\u00a0ml of the same medium for melanin production in 250-ml Erlenmeyer flasks. The flasks were kept in an incubator shaker at 30\u00a0\u00b0C and 120\u00a0rpm; melanin was assayed after 48\u00a0h. Melanin production in the broth was assayed spectrophotometrically at 475\u00a0nm using a calibration curve of standard synthetic melanin   used for the cultivation of the isolated bacterium consisted of 5\u00a0g\u00a0lBrevundimonas sp. SGJ. The factors affecting the yield of melanin were selected by screening various carbon sources, nitrogen sources, and mineral salts and physical conditions such as pH and temperature. In addition, some of these variables were selected from the primary literature review , temperature (X2), tryptone (X3), yeast extract (X4), beef extract (X5), glucose (X6), l-tyrosine (X7), CuSO4 (X8), MgSO4 (X9), K2HPO4 (X10), and NaCl (X11) were added at two levels: low (\u22121) and high (+1). The low and high levels of these factors were taken as pH (5 and 7), temperature (20 and 40\u00a0\u00b0C), while levels of media components were tryptone (0.5 and 2.5\u00a0g\u00a0l\u22121), yeast extract (0.5 and 2.5\u00a0g\u00a0l\u22121), beef extract (0.5 and 2.5\u00a0g\u00a0l\u22121), glucose (0.5 and 2.5\u00a0g\u00a0l\u22121), l-tyrosine (0.5 and 2.5\u00a0g\u00a0l\u22121), CuSO4 (0.01 and 0.05\u00a0g\u00a0l\u22121), MgSO4 (0.001 and 0.005\u00a0g\u00a0l\u22121), K2HPO4 (0.5 and 2.5\u00a0g\u00a0l\u22121) and NaCl (0.1 and 0.5\u00a0g\u00a0l\u22121). This design characterizes a model that identifies the significant variables when no interaction among the factors is expected  was used in the experimental design and data analysis. Response surface graphs were obtained to understand the effect of the variables, individually and in combination, and to determine their optimum levels for maximum melanin production. All trials were performed in triplicate, and the average melanin yield was used as response Y.Once the critical factors were identified via screening, a Box\u2013Behnken design for independent variables was used for further optimization. Four variables at three levels were used to fit a polynomial model (Box and Behnken \u22121l-tyrosine) and after optimization. The biomass trend, tyrosinase activity, and melanin production were observed at 6-h time intervals for up to 48\u00a0h. The tyrosinase activity was determined by the previously described method , the melanin production was observed with the process parameters before optimization . The phylogenic tree was constructed with MEGA4 software  , tryptone (X3), l-tyrosine (X7), and CuSO4 (X8) significantly affected the melanin production, with p values less than the significance level of 0.05. The remaining components were found to be insignificant, with p values above 0.05. The \u2018Pareto chart\u2019  showed that the value of l-tyrosine (X7) was above the \u2018Bonferroni Limit\u2019; this indicates it is certainly significant. Also the values of pH (X1), tryptone (X3) and CuSO4 (X8) were above the t-value limit that implies that these factors are possibly significant whereas the remaining factors were below the t-value limit which indicates their insignificance , tryptone (X3), l-tyrosine (X7), and CuSO4 (X8). Table S3 presents the design matrix and the results of the 29 experiments carried out using the Box\u2013Behnken design. The results obtained were submitted to ANOVA using the Design Expert software , and the regression model was given as:X1 is pH, X3 is tryptone, X7 is l-tyrosine, and X8 is CuSO4. The ANOVA of the quadratic regression model (Table S3) demonstrated that Eq.\u00a0p\u00a0=\u00a0<0.005). The model F value of 29.03 implies that the model is significant. The goodness of fit of the model was checked using the determination coefficient (R2). In this case, the value of the R2 was 0.9667. The value of the adjusted R2 was 0.9334. It was in reasonable agreement with the predicted R2 (0.8212). The lack-of-fit value for regression Eq.\u00a0Optimization of process parameters was carried out using the Box\u2013Behnken design with the parameters found to be significant from the Plackett\u2013Burman design, including pH  response surface curves. The response surface curve in Fig.\u00a0l-tyrosine where the shape of the response surface curve indicates that melanin yield was mainly affected by interaction between these two factors. The slight alteration in concentration of these components leads to higher difference in the levels of produced melanin. Melanin production increased with acidic pH and higher l-tyrosine concentrations. The interaction between pH and l-tyrosine was found to be highly significant because l-tyrosine is the substrate for melanin production and its solubility decreases at neutral and alkaline conditions, while l-tyrosine is soluble at acidic conditions  and after that, remains nearly constant with final weight of 0.540\u00a0g\u00a0l\u22121, while tyrosinase activity increased slowly upto 2,015 U\u00a0mg\u22121 at 18th hour and then decreased suddenly to 1,416 U\u00a0mg\u22121 at 24th hour. The biomass trend during melanin production indicated that melanin production occurs in the stationary growth phase of bacterial cells. The decreased tyrosinase activity after 24\u00a0h might be due to conversion of l-tyrosine to l-DOPA and l-DOPA to dopaquinone, and substrates for the tyrosinase were not available further, during melanin synthesis pathway . The other studies have reported isolation and characterization of melanin includes: Klebsiella sp. GSK which produced 0.540\u00a0g\u00a0l\u22121 within 84\u00a0h of incubation, while melanin synthesis from Frankia strain Cel5 was about 0.180\u00a0g\u00a0l\u22121 . The identical result for bacterial pigment and standard melanin obtained by chemical characterization primarily confirms the melanin nature of the pigment  is 2.004. The EPR analysis of the microbial melanin showed a nearly identical g-value compared to standard synthetic melanin (Sigma), and showed resemblance, which confirmed that the purified pigment was melanin  Phylogenic tree of the Brevundimonas sp. SGJ and related organisms (b) Melanin producing colonies of Brevundimonas sp. SGJ on Nutrient agar plate (c) Optimized medium before inocubation (Colorless) and optimized medium after incubation with melanin production (dark brown colored).Fig S2 Pareto chart showing significant effects of factors above the \u2018Bonferroni Limit\u2019 and \u2018t-value Limit\u2019 and insignificant effect of the factors below the \u2018Bonferroni Limit\u2019 and \u2018t-value Limit\u2019 X1 (pH), X2 (temperature), X3 (tryptone), X4 (yeast extract), X5 (beef extract), X6 (glucose), X7 (l-tyrosine), X8 (CuSO4), X9 (MgSO4), X10 (K2HPO4), and X11 (NaCl). (DOC 529\u00a0kb)Fig. S1 Below is the link to the electronic supplementary material."}
+{"text": "Having crossed the BBB or the BCSFB they reach the castle moat, namely the cerebrospinal fluid (CSF)-drained leptomeningeal and perivascular spaces of the CNS. Next to the CNS parenchyma, the castle moat is bordered by a second wall, the glia limitans, composed of astrocytic foot processes and a parenchymal basement membrane. Inside the castle, that is the CNS parenchyma proper, the royal family of sensitive neurons resides with their servants, the glial cells. Within the CSF-drained castle moat, macrophages serve as guards collecting all the information from within the castle, which they can present to the immune-surveying T cells. If in their communication with the castle moat macrophages, T cells recognize their specific antigen and see that the royal family is in danger, they will become activated and by opening doors in the outer wall of the castle allow the entry of additional immune cells into the castle moat. From there, immune cells may breach the inner castle wall with the aim to defend the castle inhabitants by eliminating the invading enemy. If the immune response by unknown mechanisms turns against self, that is the castle inhabitants, this may allow for continuous entry of immune cells into the castle and lead to the death of the castle inhabitants, and finally members of the royal family, the neurons. This review will summarize the molecular traffic signals known to allow immune cells to breach the outer and inner walls of the CNS castle moat and will highlight the importance of the CSF-drained castle moat in maintaining immune surveillance and in mounting immune responses in the CNS.Neuronal activity within the central nervous system (CNS) strictly depends on homeostasis and therefore does not tolerate uncontrolled entry of blood components. It has been generally believed that under normal conditions, the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) prevent immune cell entry into the CNS. This view has recently changed when it was realized that activated T cells are able to breach the BBB and the BCSFB to perform immune surveillance of the CNS. Here we propose that the immune privilege of the CNS is established by the specific morphological architecture of its borders resembling that of a medieval castle. The BBB and the BCSFB serve as the  Inside the castle, that is the CNS parenchyma proper, the royal family of sensitive neurons resides with their servants, the glial cells. Immune surveillance is restricted to the castle moat, where perivascular and meningeal macrophages serve as guards that continuously collect information from within the castle, and present this information to the messengers, the T cells. If in their communication with the castle moat guards, T cells recognize their antigen they will become activated and open gates in the outer castle wall allowing the entry of more immune cells from outside. Within the castle moat the immune cells then mount an invasion of the castle, breaching the inner castle wall and entering the castle with the aim to eliminate any intruders. If for reasons unknown, T cells turn their attack to the inhabitants of the castle, chronic immune cell invasion of the castle will eventually kill many of the castle inhabitants leading to neuronal deficits as observed in chronic neuroinflammatory diseases of the CNS such as multiple sclerosis [Traditionally, the central nervous system (CNS) was viewed as an immunologically-privileged site, which was interpreted as the complete absence of immune surveillance of this tissue . The theclerosis .Brain barriers are composed of the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB), which protect the CNS from the changing milieu of the periphery. The BBB is formed by highly-specialized endothelial cells that tightly restrict the paracellular and transcellular passage of hydrophilic molecules into the CNS by an elaborate network of complex tight junctions between the endothelial cells, in conjunction with a lack of fenestrations and low pinocytotic activity . To fulfBesides the BBB, epithelial cells of the choroid plexus establish the BCSFB, taking over similar functions as the BBB. The choroid plexus, which secretes the cerebrospinal fluid (CSF), consists of villous structures that extend from the ventricular surfaces into the lumen of the ventricles. It is formed by an extensive network of fenestrated capillaries, enclosed by a monolayer of cuboidal epithelium. In contrast to the CNS parenchyma, where microvessels establish the BBB, the microvessels within the choroid plexus stroma are fenestrated allowing the free diffusion of solutes between the blood and the choroid plexus parenchyma through inter-endothelial gaps (reviewed in ). The acglia limitans perivascularis [glia limitans and the BBB delineate a CSF-filled space, in which host antigen-presenting cells are localized [glia limitans perivascularis [glia limitans superficialis, which resembles the glia limitans perivascularis and surrounds the entire surface of the brain and spinal cord [Proper function of the BBB has been shown to rely critically on the sustained interaction with the adjacent extracellular matrix components and cellular elements . The fulscularis ,12. The ocalized -15. At tocalized . There tscularis . Howevernal cord . Neverthnal cord , althougnal cord ,20, suggIn contrast to the BBB, much less is known about how barrier characteristics of the BCSFB are maintained but it is reasonable to assume that the CSF provides signals for BCSFB maintenance. Cells found in tight association with the apical aspect of the BCSFB have been described as Kolmer cells or epiplexus cells and are most probably antigen-presenting cells (reviewed in ).+ T cell blasts infused into the circulation of Lewis rat that resulted in the development of experimental autoimmune encephalomyelitis (EAE), considered to be the best animal model for multiple sclerosis (MS) .+ T cell subsets [MS and EAE are characterized by a massive infiltration of circulating immune cells into the CNS, resulting in central inflammation, oedema formation and demyelination, the basis for the onset of the clinical manifestations of these diseases. Both diseases show clinical and histopathological similarities, however, whereas MS etiology remains unclear and shows a great variety of neuropathological changes, EAE is a T cell-mediated autoimmune inflammatory disease with a well-described CNS pathology. The model disease of EAE can be generated either upon immunization of susceptible naive recipients with CNS myelin antigens or by intravenous injection of freshly-activated - but not resting - CNS myelin-specific CD4 subsets , support+ T lymphoblasts with the healthy brain barriers represents the critical step in the initiation of the disease. Few studies have, however, directly addressed the molecular mechanisms of T lymphoblast migration across the BBB. We have approached this question by means of intravital microscopy (IVM) of the spinal cord, a technique that allowed us to directly observe the interaction of circulating fluorescently-labeled encephalitogenic T cell blasts with the spinal cord microvasculature [It has therefore been recognized that the interaction of CD4culature . Using tculature . Th1 celculature . Both caculature . Howeverculature  suggesticulature . T cell culature . Thus, Tin vivo homing studies following fluorescently labeled encephalitogenic T cell blasts, showed that two hours following their systemic injection these cells were already found within the leptomeningeal compartment [glia limitans into the CNS parenchyma [Therefore, other routes of T cell entry into the healthy CNS need to be considered. In fact, partment . The molpartment ,28. Thuspartment . Howeverrenchyma ,30.in vivo also detected T cells within the choroid plexus parenchyma two hours following their systemic injection [-/- immune cells were found to have accumulated within the choroid plexus parenchyma, where the chemokine CCL20, the ligand for CCR6, is constitutively produced by the choroid plexus epithelium. CCL20 is not expressed at the BBB and CCR6 was not involved in T cell interaction with the BBB. As CCR6-/- mice developed EAE following the transfer of a subclinical number of fluorescently-labelled myelin-specific Th17 cells expressing CCR6, it was concluded that EAE is initiated by the CCR6/CCL20-dependent migration of Th17 cells across the non-inflamed BCSFB. Additional molecular mechanisms of T cell migration across the BCSFB remain to be investigated. Although the choroid plexus epithelium constitutively expresses ICAM-1 and VCAM-1, these molecules are expressed in a highly polarized fashion at the apical side of the choroid plexus epithelium [+ Th17 cells were exclusively found within the leptomeningeal compartment [Following fluorescently-labeled encephalitogenic T cell blasts njection . Similarnjection , suggestnjection ,32. In fnjection . In thisithelium ,35 and apartment .via the BBB and the BCSFB to the CSF space, where they can interact with the antigen presenting cells. In the absence of antigen recognition these T cell blasts remain confined to the CSF drained spaces and therefore do not impair CNS homeostasis. However, if they recognize their specific antigen, these immune cells will mount an inflammatory response that brings additional immune cells from the periphery into the perivascular spaces and from there they eventually breach the glia limitans, to mount an immune defence within the CNS parenchyma.Taken together, these observations demonstrate that immune surveillance of the CNS is ensured by allowing activated immune cells access Reactivation of the T cells, by their antigen-presenting cells bearing the cognate antigen, within the CSF-filled CNS spaces will lead to inflammatory changes of the BBB and probably also of the BCSFB. This is achieved by the expression of additional trafficking signals on these barriers allowing the recruitment of numerous inflammatory cells from the bloodstream into the CSF-drained perivascular and subarachnoid spaces. The molecular mechanisms involved in T cell migration across the inflamed BBB have been well studied using EAE, the animal model of multiple sclerosis, and will be summarized here.A number of intravital microscopic studies observing the interaction of myelin-specific T cells with inflamed leptomeningeal brain microvessels in EAE have shown that endothelial P-selectin mediated the rolling of T cells via their selectin ligand PSGL-1 in leptomeningeal post-capillary venules -39. Surpper se. These are the lymphoid chemokines CCL19 [+ T cells to inflamed brain microvessels in vitro [Firm arrest of T cells at the inflamed post-capillary venule BBB similarly requires signalling via G-protein-coupled receptors present on the surface of the circulating immune cells, as at the healthy BBB . Thus ches CCL19 -50 and Cin vitro . Endothein vitro . Also, Cin vitro . Taken tG-protein-mediated activation of integrins on the T cells leads to their arrest at the post-capillary venule BBB. Several studies reported the upregulation of the integrin ligands ICAM-1 and VCAM-1 on CNS endothelia ,52-54 asin vitro studies have demonstrated that under static conditions T cells bind via LFA-1 and \u03b14-integrins to their respective endothelial ligands, ICAM-1 and VCAM-1, on inflamed cerebral vessels in a Stamper-Woodruff assay [in vitro and in vivo studies have specified that shear-resistant T cell arrest on the inflamed BBB is mediated by LFA-1/ICAM-1 and \u03b14\u03b21-integrin/VCAM-1 interactions [Numerous ff assay ,60 or onff assay ,61-64. Rractions ,45,65.in vivo proof of concept of this therapy in MS.The development of EAE can be abrogated by blocking \u03b14-integrins or VCAM-1 as shown by many laboratories ,60,66-68Despite the proven contribution of LFA-1 and its endothelial ligands ICAM-1 and ICAM-2 in T cell arrest on the inflamed BBB endothelium, their functional inhibition or absence in a variety of EAE models produced contradictory results, ranging from inhibiting EAE to increasing severity of EAE or having no effect at all -72. Therin vitro and in vivo studies demonstrated that once T cells have arrested on the BBB surface, T cells get polarized and start to crawl on the endothelium against the direction of the blood flow until they find sites permissive for diapedesis [in vitro have doubted this view and support the view that paracellular diapedesis occurs through the BBB tight junctions . Nevertapedesis , passageapedesis -81. Howerized in ). At preeability  demonstreability  or ALCAMeability , which hFollowing their diapedesis across the inflamed BBB, T cells need to make their way across the acellular extracellular matrix components of the endothelial basement membrane. The endothelial basement membrane consists of a number of glycoproteins such as laminins, collagen type IV, nidogens, and heparan sulfate proteoglycans. Recently, it has been observed that T cell extravasation at the BBB occurs preferentially at sites, where the endothelial basement membrane contains laminin \u03b14 rather than laminin \u03b15 ,85. In lIn contrast to the BBB, the molecular mechanisms for T cell diapedesis across the BCSFB during EAE have not been investigated. During EAE, upregulated expression of functional ICAM-1 and VCAM-1 and induction of MAdCAM-1 is observed . As thes+ dendritic cells [in vivo imaging study demonstrated that T cells interact with many leptomeningeal macrophages in search of their specific antigen [Once the endothelial basement membrane has been breached, extravasating encephalitogenic T cells reach the perivascular space, where they have to re-encounter their cognate antigen presented by antigen-presenting cells, in the context of major histocompatibility complex (MHC) class II, in order to pursue their route into the CNS parenchyma. This was demonstrated by the study from Greter and colleagues, in which the clinical development of EAE was shown to be triggered by myelin-reactive T cells following antigen priming by perivascular CD11cic cells . A recen antigen . In the glia limitans and their entry into CNS parenchyma [In EAE it has been demonstrated that the typical perivascular inflammatory cuffs are localized between the outer endothelial and inner parenchymal basement membranes, and therefore remain within the perivascular space. Interestingly, clinical signs of EAE only start following the penetration of inflammatory cells across the renchyma .glia limitans perivascularis and glia limitans superficialis, respectively, surrounding the entire surface of the brain and spinal cord [glia limitans is composed of the parenchymal basement membrane, which is molecularly distinct from the endothelial basement membrane with deposition of laminins \u03b11 and \u03b12 but not the endothelial laminins \u03b14 and \u03b15 . The paglial limitans by inflammatory cells requires the activity of matrix metalloproteinases (MMP-2 and MMP-9), which is probably due to the distinct biochemical composition of the parenchymal basement membrane and the tight seal with astrocyte foot processes [glia limitans and reach the CNS parenchyma where they start their attack on the CNS parenchymal cells. Once immune cells have reached the CNS parenchyma the immune attack starts and CNS parenchymal cells may be sacrificed in order to fight the potential infection. In EAE the autoimmune response leads to chronic attack of oligodendrocytes and neurons eventually leading to demyelination and axonal loss, that is to say ultimately the loss of CNS neurons.In contrast to the penetration of the BBB, penetration of the rocesses . Obviousrocesses  and allomedieval castle that is surrounded by a two-walled moat. Activated memory/effector but not resting T cells, are in possession of the molecular keys to breach the outer castle wall - the BBB or the BCSFB - without impairing its resistance to assault. Behind this outer cellular barrier, T cells enter the CSF drained ventricular, perivascular and subarachnoidal spaces, which are bordered by two biochemically distinct basement membranes. Immune-surveillance of the CNS is confined to this space, which resembles the castle moat bordered by outer and inner walls and patrolled by guards, the perivascular antigen-presenting cells and the epiplexus cells. If T cells encounter their specific antigen within the CSF-drained castle moat, they become activated and will trigger an inflammatory response that leads to the upregulation of novel traffic signals on the BBB and the BCSFB - i.e. opening doors in the outer wall and leading to the recruitment of additional inflammatory cells across this outer barrier. Production of cytokines and proteases allows the degradation of the glia limitans - the inner wall of the castle moat - and in a second step by lowering the draw-bridge across the castle moat, large number of infiltrating inflammatory cells will then enter the CNS parenchyma, leading to the cellular assault of the castle, respectively. Taken together, it has been realized that the CSF-drained fluid spaces play a central role in establishing CNS immune surveillance and therefore in maintaining CNS immune privilege. Whereas our understanding of the traffic signals involved in T cell migration across the BBB has increased enormously, T cell entry across the BCSFB and the following steps of immune cell entry across the basement membranes and the glia limitans remain to be investigated.Originally the CNS was considered an immune-privileged organ in the sense that it is ignored by the immune system. Recent studies on T cell trafficking to the CNS in the context of EAE have identified anatomical routes and molecular mechanisms of T cell entry into the CNS during health and disease and suggest that CNS immune privilege is accomplished by the unique architecture of a The authors declare that they have no competing interests.BE developed the basic concept of the review and CC completed the paragraphs. Final editing of the review was done by BE. Both authors have read and approved the manuscript."}
+{"text": "Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth.Sheath rot disease caused by S. oryzae was 32.78\u00a0Mb with contig N50 18.07 Kb and 10526 protein coding genes. The functional annotation of protein coding genes revealed that S. oryzae genome has evolved with many expanded gene families of major super family, proteinases, zinc finger proteins, sugar transporters, dehydrogenases/reductases, cytochrome P450, WD domain G-beta repeat and FAD-binding proteins. Gene orthology analysis showed that around 79.80\u00a0% of S. oryzae genes were orthologous to other Ascomycetes fungi. The polyketide synthase dehydratase, ATP-binding cassette (ABC) transporters, amine oxidases, and aldehyde dehydrogenase family proteins were duplicated in larger proportion specifying the adaptive gene duplications to varying environmental conditions. Thirty-nine secondary metabolite gene clusters encoded for polyketide synthases, nonribosomal peptide synthase, and terpene cyclases. Protein homology based analysis indicated that nine putative candidate genes were found to be involved in helvolic acid biosynthesis pathway. The genes were arranged in cluster and structural organization of gene cluster was similar to helvolic acid biosynthesis cluster in Metarhizium anisophilae. Around 9.37\u00a0% of S. oryzae genes were identified as pathogenicity genes, which are experimentally proven in other phytopathogenic fungi and enlisted in pathogen-host interaction database. In addition, we also report 13212 simple sequences repeats (SSRs) which can be deployed in pathogen identification and population dynamic studies in near future.The genome size of Sarocladium disease biology. This is the first genome sequencing report globally and the genomic resources developed from this study will have wider impact worldwide to understand Rice-Sarocladium interaction.Large set of pathogenicity determinants and putative genes involved in helvolic acid and cerulenin biosynthesis will have broader implications with respect to The online version of this article (doi:10.1186/s12864-016-2599-0) contains supplementary material, which is available to authorized users. Sarocladium oryzae [(Sawada) W. Gams & D. Hawksw] is an Ascomycetes fungus causing sheath rot disease in rice. It has recently emerged as a major threat for rice production in rice growing ecosystems in the world. In addition to rice, this fungus infects other important cereal food crops such as maize, sorghum, pearl millet, finger millet, and foxtail millet [l millet . The coml millet .S. oryzae produces white, sparsely branched and septate mycelium. Conidiophores are branched once or twice with 3\u20134 phialades in a whorl. The conidium is a aseptate, hyaline, cylindrical in shape and located on tip of phialades [hialades , 6. Desphialades . Also viS. oryzae in public databases (NCBI) are limited to internal transcribed spacer (ITS) region sequences of ribosomal DNA and our previous QTL mapping study [Sarocladium pathosystem has not been studied well at global level. Considering these facts, we sequenced whole genome of highly virulent isolate of S. oryzae (Saro-13) from major rice growing region of South India. This is the first report of de novo genome assembly and annotation of S. oryzae. We carried out detailed analyses of gene families, secondary metabolite gene clusters, pathogenicity related genes, transposable repeat elements, phylogenetic relationship with other fungi and microsatellites. In addition, we analysed putative genes involved in helvolic acid and cerulenin biosynthesis pathways, which are very important in Sarocladium disease biology. The genomic resources generated from this study can be translated into designing better disease management strategies to mitigate sheath rot disease epidemics globally and widen the understanding of rice-Sarocladium pathosystem.The genomic resources for ng study . Due to S. oryzae. The virulence test of S. oryzae was carried out by standard mycelial inoculation [S. oryzae.Diseased flag leaf sheath sampled over 25 locations in major rice growing regions of Karnataka state, India was used for isolation of fungus. Diseased sheath was surface sterilized using 0.05 % mercuric chloride solution followed by three times washing with sterile water. Sterilized diseased sheath pieces were incubated at room temperature for 4\u20135 days and germinating spores were transferred to potato dextrose agar (PDA) medium. Based on morphological features of conidiophores, phialades and conidiospores , the funculation  and detaculation  to confiS. oryzae (Saro-13) was grown on potato dextrose broth (PDB) medium for three days. The mycelium was grinded using liquid nitrogen and genomic DNA was isolated using nucleo-pore gDNA fungal and bacterial mini kit . The DNA quality and quantity was assessed by Nanodrop and Qubit (Applied Biosystems), respectively. The genomic DNA was sheared to generate fragments of approximately 400\u2013600\u00a0bp in Covaris microtube with the E220 system . The fragment size distribution was checked using Agilent Bioanalyzer  with high sensitivity DNA Kit (Agilent Technologies). The fragmented DNA was cleaned up using HighPrep beads . The Illumina paired-end library was prepared as per manufacturers instruction using NEXTflex DNA sequencing Kit . The paired end library was sequenced using Illumina NextSeq 500 in Genotypic Technologies, Bengaluru and the length of read\u00a0sequence was 151 nts from both the ends of the fragment.The virulent strain of http://hannonlab.cshl.edu/fastx_toolkit/index.html).The low quality bases with Phred score less than Q30  and adapter sequence contamination in raw sequence reads of Illumina was discarded using FASTX-Toolkit  [http://www.uniprot.org) using BLASTP with an e-value threshold of 1e-10. The annotation of protein domain structures was performed using InterProScan5 software [ssembler . SPAdes omplete) , 13. Funsoftware . The gensoftware .S. oryzae with other Ascomycetes fungi like Magnaporthe oryzae , Fusarium graminearum , Acremonium chrysogenum , and Fusarium oxysporum . The groups having at least one copy from each genome were considered as core orthologous groups (COGs).Gene families were inferred using orthoMCL   by compaS. oryzae to other Ascomycetes fungi . The consensus tree was drawn using FigTree V1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). The phylogenetic analysis results are deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S19046) and Dryad Digital Repository (doi:10.5061/dryad.674p4).Based on orthoMCL clustering, 100 single copy ortholog gene groups from five fungal species were selected randomly and aligned separately using MUSCLE  version http://www.phi-base.org) and BLASTP was performed against S. oryzae proteome. Protein alignments with more than 40\u00a0% identity and 70\u00a0% query coverage were considered as putative pathogenicity genes in S. oryzae.The fungal pathogenicity genes were retrieved from the Pathogen-Host Interaction (PHI) database   and all proteins with signal peptides were analysed for presence of transmembrane (TM) domains using web servers like Phobius (http://phobius.sbc.su.se) and TMHMM version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Subsequently, mitochondrial and chloroplast targeting signal containing S. oryzae proteins were removed based on prediction by TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/). Finally, proteins containing a potential GPI (glycosyl phosphatidyl inositol)-anchor signal identified by PredGPI (http://gpcr.biocomp.unibo.it/predgpi/) web server were discarded.Signal peptides and cleavage sites of S. oryzae proteins from secretome analysis were subjected for CAZy annotation using CAT [\u221205 and classified as per type of reaction catalyzed like Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs), Carbohydrate Esterases (CEs), Carbohydrate-Binding Modules (CBMs), and Auxillary Activities (AAs) as described in CAZY database (http://www.cazy.org/Welcome-to-the-Carbohydrate-Active.html).The sing CAT  and dbCAsing CAT  servers,sing CAT . The resS. oryzae were analysed for secondary metabolites gene clusters using antiSMASH [The scaffold sequences of ntiSMASH .S. oryzae was done using Fungal Cytochrome P450 Database . The proteins encoding for TFs were classified based on Fungal Transcription Factors Database .The cytochrome P450 gene family classification in Aspergillus genome database  and protein homology search was carried out with S. oryzae genes. The genes with minimum 50\u00a0% identity and 70\u00a0% query coverage were considered as putative candidates in helvolic acid biosynthesis pathway. In addition, a homology search was also performed against NCBI non-redundant protein database to obtain homologous sequences in closely related fungal species. The protein domain based search was performed to identify putative genes involved in Cerulenin biosynthesis.We retrieved amino acid sequences of putative genes involved in helvolic acid biosynthesis from S. oryzae scaffold sequences were subjected for de novo repeat prediction using RepeatMasker [http://www.girinst.org/repbase/). The whole genome of S. oryzae was analyzed to determine the distribution and frequency of various types of SSRs using Microsatellite Identification tool (MISA) [http://pgrc.ipk-gatersleben.de/misa/). The minimum length of SSR motif was set as 10 for mono, 6 for di, 5 for tri, tetra, penta and hexa motifs.The atMasker . Referenl (MISA)   produced sparsely branched mycelium with orange pigmentation on potato dextrose agar (PDA) medium. The conidium was single-celled, cylindrical and hyaline in structure  raw reads were generated and the read length was 151 nts. Discarding low quality reads resulted 17,854,048 reads which corresponds to approximately 82x sequence depth of high quality data and these reads were assembled using SPAdes genome assembler . The assn models , 29. Thenaporthe \u201332. The S. oryzae. Large number of major facilitator superfamily (212 proteins), fungal specific transcription factor (175 proteins), protein kinase (137 proteins), fungal Zn(2)-Cys(6) binuclear cluster (122 proteins), sugar transporters (120 proteins), short chain dehydrogenase (97 proteins), cytochrome P450 (93 proteins), WD domain G-beta repeat (72 proteins), FAD binding (67 proteins), methyltransferase (57 proteins), and pyridine nucleotide-disulphide oxidoreductases (50 proteins) domain containing proteins were enriched in the S. oryzae genome. Majority of these gene families are known to be involved in host-pathogen interactions, indicating S. oryzae emerging as a very important plant pathogen to study arsenal of pathogenicity genes.An InterProScan pfam analysis identified 2,820 protein families containing 7718 proteins in S. oryzae revealed that 12.21\u00a0%, 39.10\u00a0% and 47.33\u00a0% of genes were annotated with biological, cellular and molecular functions, respectively  annotation of 10,526 genes of Ascomycetes fungi , we clustered their proteomes using orthoMCL tool. The clustering of proteomes resulted 13185 ortholog groups  in S. oryzae genome. The largest multigene family was encoding polyketide synthase dehydratase (PF14765), followed by ABC transporter (PF00005), ABC transporter transmembrane region (PF00664), Aldehyde dehydrogenase family (PF00171), Fibronectin type III-like domain (PF14310), PA14 domain (PF07691), Copper amine oxidase (PF02727), WSC domain (PF01822), OPT oligopeptide transporter protein (PF03169). The polyketide synthase dehydratase gene family is known to produce secondary metabolites and essential for fungal virulence [The orthologous genes are resultant of speciation process and clear delineation of orthologous relationship between species helps us to reconstruct evolution of species . Moreoveups Fig.\u00a0, of whicirulence , 36 to iirulence  by exporirulence .Fig. 2ThS. oryzae with other Ascomycetes fungi was inferred based on protein similarity of hundred randomly choosen single copy ortholog genes from orthoMCL analysis. Based on WAG model [S. oryzae was closely related to M. oryzae  followed by A. chrysogenum, F. oxysporum and F. graminearum  gene database, secretary proteins, carbohydrate-active enzymes (CAZymes), secondary metabolites, transporters, and transcription factors that are required to colonize in the host tissue.To our knowledge, pathogenicity genes/factors are not determined so far in S. oryzae were aligned to PHI fungal genes using BLASTP . We identified 953  putative PHI genes in S. oryzae spanning across 59 different fungal species. Highest number of homologs was found in Fusarium graminearum (483 genes), followed by Magnaporthe oryzae (145 genes), Aspergillus fumigatus (66 genes), Candida albicans (36 genes), Botrytis cinerea (20 genes), Cryptococcus neoformans (18 genes), Fusarium oxysporum (18 genes), and other fungal species (167 genes)  [S. oryzae.The PHI database has collection of experimentally verified virulence associated genes from fungi, oomycetes and bacteria . All 10,minearum 83 genes,enicity) . These pS. oryzae proteome revealed 391 proteins harboring signal peptides (SPs) . Another class of secretory proteins like Tyrosinase is known to be involved in melanin production. Other important domain containing secretory proteins like hydrophobic surface binding protein A (HsbA), cupin, fungal hydrophobin, and lipase were enriched in the S. oryzae genome  play a vital role in metabolism of structural components of cell wall and storage glucans in plant pathogens. CAZymes are responsible for breakdown of cell wall components of host to establish successful infection process. Pathogen uses the host as a nutrient source and deploys CAZymes to degrade plant storage compounds. Analysis of CAZymes revealed, 3201 proteins encoding CAZymes, which were distributed across 176 CAZyme protein families. Out of which, 295 proteins had signal peptides and remaining 2906 proteins lacked signal peptides  were majorly distributed followed by C2H2 zinc finger (38 genes), bZIP (14 genes), heteromeric CCAAT type (7 genes), MADS-box (2 genes), Myb (1 gene), and Grainyhead/CP2 (1 gene). Similar level of distribution of Zn2Cys6 TFs in Ascomycota was reported by Todd and co workers [Magnaporthe oryzae [Botrytis cinerea [S. oryzae. The basic leucine zipper (bZIP) domain containing TFs is third largest family in the S. oryzae genome and they are known to regulate cellular growth and differentiation. There were 14 genes encoding for bZIP TFs in S. oryzae. Deletion mutants of this TFs showed defects in mycelial growth, development and reduced pathogenicity in Magnaporthe pathosystem [S. oryzae genome fosters diverse classes of TFs required for activation of most of the fungal pathogenicity genes.Transcription factors (TFs) play a vital role in signal transduction pathways by acting as a linker between signal flow and target gene expression. Mining of specific repertoire of TFs in the genome gives us an overview about active pathways in the genome . Around ae Table\u00a0. Among s workers . These T workers . These Te oryzae  and Botr cinerea  affectedhosystem . The repS. oryzae based on fungal cytochrome P450 database (FCPD)  and 120 genes encoding for sugar and other transporters. As compared to other gene families, MFS membrane transporters were high indicating their role in transporting small solutes in response to chemiosmotic ion gradients during pathogenesis.The cytochrome P450 enzymes in fungi carry out a wide range of bioconversions of complex polyaromatic hydrocarbons (PAHs) and steroid compounds mediated by monooxygenase enzymes . There wS. oryzae genome is enriched with PKSs, TCs followed by NRPSs, NRPSs-PKSs hybrid clusters  cytochrome P450, two (SoG_03552.T1 and SoG_03554.T1) transferase family protein, one each of short chain dehydrogenase (SDR) (SoG_04320.T1), qualene-hopene-cyclase (SoG_05635.T1), and 3-ketosteroid-delta-1-dehydrogenase (SoG_03553.T1) genes  than A. flavus.Secondary metabolites (SMs) are small bioactive molecules and they are essential for fungal growth and development. At the same time SMs provide protection against various environmental stresses. The biosynthesis of SMs is catalyzed by either nonribosomal peptides synthases (NRPSs), polyketide synthases (PKSs), hybrid NRPS-PKS enzymes, prenyltransferases (DMATSs), and terpene cyclases (TCs). The catalytic activity of these enzymes results in production of SMs respectively like nonribosomal peptides, polyketides, NRPS-PKS hybrids, indole alkaloids, and terpenes . Searchiers Fig.\u00a0. Severalenin SMs \u201353. The enin SMs , 54\u201356. s flavus  and Metasophilae . Initialnes Fig.\u00a0. The strS. oryzae is Cerulenin and its biosynthesis is closely related to fattyacid synthesis [S. oryzae genome. These genes are of future interest to understand its biosynthesis since Cerulenin is mainly used as antifungal antibiotic and anticancer agent that inhibits fatty acid and steroid biosynthesis [Another important SM produced by ynthesis . The strynthesis . We lookynthesis , 61. TheS. oryzae. The de novo and reference (by taking Sarocladium repeat library from repbase) based repeat analysis showed that 1.09\u00a0% of genome was repetitive. Interestingly, retro and DNA transposons were absent. However, repetitive DNA was limited to small RNA (0.03\u00a0%), simple repeats (0.70\u00a0%) and low complexity repeats (0.37\u00a0%). Then, we performed reference based repeat analysis by choosing repeat databases of closely related Ascomycetes fungi like Aspergillus, Colletotrichum, Fusarium, Gibberella, Magnaporthe and Sarocladium zeae. However, we did not observe significant increase in content of repeat elements. Many genome drafts of Ascomycetes have been assembled using short read technologies, and have reported repeat percentage in the range of 3-10\u00a0% [N. crassa, F. oxysporum, A. nidulans and A. fumigatus showed lower repeat content coupled with RIP mechanism. Thus, these fungal species and S. oryzae which are closely related, might be sharing similar phenomenon like RIP in their genomes [Repetitive DNA is an integral part of fungal genomes. Repeat sequences play a vital role in generating genetic diversity, genome expansion and might also be detrimental to genome with respect to genome stability . Repetitf 3-10\u00a0% , 62, 63.f 3-10\u00a0% , 65. GenS. oryzae was performed in order to enrich genomic resources for population characterization. The scanning of 32.78\u00a0Mb\u2009S. oryzae genome revealed presence of 13,212 SSRs. Of which, 10650 were simple and remaining 2562 were complex types. The major proportion of simple SSRs was mononucleotide repeats occupying 50\u00a0% of total SSRs, followed by 22.06 % dinucleotides (2349), 18.49 % trinucleotides (1969), and 3.16 % tetranucleotides (337) repeats. The remaining SSRs were complex type, with 0.82 % of penta and 0.57 % of hexa nucleotides.Microsatellites or SSRs markers are highly useful for molecular identification, genetic differentiation among individuals and populations in fungi. The genome-wide identification of SSRs in S. oryzae. The poor distribution (3.16\u00a0%) of tetranucleotides SSRs was observed in the S. oryzae genome. The maximum number of tetra nucleotides repeats was of \u2018TTTC/GAAA\u2019 type followed by \u2018AAGA/TCTT\u2019, \u2018AAAG/CTTT\u2019, and \u2018TCCA/TGGA\u2019. The overall analysis showed that the relative abundance of tetra, penta and hexa SSRs types were low as compared to mono, di and tri SSR types in S. oryzae genome. The similar observation was made in other Ascomycetes fungi like A. nidulans, S. cerevisiae, F. graminearum, M. oryzae, and N. crassa [S. oryzae in the rice growing regions at global level.Among mononucleotide repeats, 55.28 % were poly \u2018A/T\u2019 (3232 microsatellites), and 44.72 % were \u2018G/C\u2019 (2615 microsatellites) types  genes were unique to S. oryzae. Multigene families such as polyketide synthase, ABC transporters and other pathogenicity related genes were distributed across 480 orthologous groups. The expansion of these gene families through natural selection denotes survival advantage of this pathogen for acclimatization to diverse environmental conditions. The overall analysis showed that S. oryzae has large sets of pathogenicity-related genes encoding secreted effectors, proteinases, secondary metabolism enzymes, transporters, carbohydrate-active enzymes, cytochrome P450 enzymes and transcription factors. This diversification and maintenance of more number of arsenal of diverse virulence factors may be required to colonize a wider range of host species by S. oyzae. More interestingly, helvolic acid biosynthesis pathway genes were found in a single cluster encoding for cytochrome P450 monooxygenase, transferase, short chain dehydrogenase (SDR), qualene-hopene-cyclase, and 3-ketosteroid-delta-1-dehydrogenase. Genome-wide identification of microsatellites revealed that around 43.71\u00a0% of SSRs were di, tri and tetra types, which could be explored in pathogen identification and population dynamic studies. Prior to elucidation of this draft genome sequence, very little was known about molecular mechanisms involved in pathogenicity and research in this area was limited to metabolite studies. Indeed, the availability of this genome in the public domain from our sequencing effort will now allow the researchers to carry out accelerated and rational experiments to dissect Rice-Sarocladium interaction that may help to articulate better disease control measures.Rice sheath rot disease caused by The genome assembly/contigs are deposited in NCBI/DDBJ/Genbank genome database under the accession number LOPT01000000. The raw sequence reads are deposited in NCBI SRA database under accession number SRX1639538."}
+{"text": "Escherichia coli (DEC) are important bacterial causes of childhood diarrhea in Brazil, but its impact in adults is unknown. This study aimed at investigating DEC among children and adults living in endemic areas.Diarrheagenic n\u2009=\u2009141) and adults (n\u2009=\u2009186) with diarrhea attending health centers. Diarrheagenic E. coli (DEC) were identified by their virulence genes (multiplex polymerase chain reaction) and HEp-2 cell adherence patterns.A total of 327 stools specimens were collected from children (E. coli (EAEC) (23%) was the most prevalent pathotype, followed by diffusely adherent E. coli (DAEC) (13%), and occurred at similar frequencies in both diarrheal groups. Atypical enteropathogenic E. coli (aEPEC) strains were recovered more frequently from children (6%) than from adults (1%). Twenty-six percent of the EAEC were classified as typical EAEC possessing aggR gene, and carried the aap gene. EAEC strains carrying aggR-aap-aatA genes were significantly more frequent among children than adults (p\u2009<\u20090.05). DAEC strains possessing Afa/Dr. genes were detected from children (10%) and adults (6%). EAEC and DAEC strains harboring genes for the EAST1 (astA), Pet, Pic, and Sat toxins were common in both diarrheal groups. The astA and the porcine AE/associated adhesin (paa) genes were found in most of aEPEC strains. High levels of resistance to antimicrobial drugs were found among DAEC and aEPEC isolates.DEC were detected in 56 (40%) children and 74 (39%) adults; enteroaggregative The results show a high proportion of EAEC and DAEC carrying toxin-encoding genes among adults with diarrhea. Escherichia coli is a major public health concern around the world [Escherichia coli (DEC) strains comprise the most common bacterial pathogens worldwide [Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enterohemorrhagic E. coli (EHEC) [eae) gene causing attachment and effacement on intestinal epithelial cells and the bundle-forming pili (bfp) gene encoded in the EPEC adherence factor (EAF) plasmid. Typical EPEC is characterized by the presence of both eae and bfp genes, while atypical EPEC possess the eae gene alone. EAEC is characterized by the virulence factors that is present in the 60\u00a0MDa plasmid, which includes aggregative adherence factors (AAFs), the transcriptional activator aggR, anti-aggregation protein (aap) gene, and anti-aggregation protein transporter (aatA) gene. DAEC is characterized by the presence of Afa/Dr. adhesin genes. ETEC is characterized by the presence of heat labile (elt) and/or heat-stable (est) toxin genes. EIEC is characterized by the presence of an invasion plasmid, which encodes a number of genes for invasion that includes the ipaH gene. STEC is characterized by the presence of toxin genes (stx1 and stx2) [Diarrheal disease caused by he world . Diarrheorldwide , 3. DEC i (EHEC) . EPEC, EDiarrheal disease remains an important public health problem for children in developing areas of Brazil, including peri urban and rural areas. In these regions, the poor quality or absence of sanitization and of a clean water supply for the population introduce risk factors for the mortality and morbidity of childhood diarrhea. In a previous study conducted in the city of Vit\u00f3ria , DEC strains, especially EAEC, DAEC, and EPEC were found in 45% of cases of diarrhea in children from rural communities [The study was conducted between January 2008 and February 2009 in the city of Vit\u00f3ria, Esp\u00edrito Santo. The study was part of a study with the aim of identifying risk factors for diarrhea in rural and peri urban areas with poor hygiene and sanitation conditions in southeastern Brazil . Thirty-E. coli, Shigella, and Salmonella isolates. After incubation for 24\u00a0h at 37\u00a0\u00b0C, four lactose-fermenting colonies with typical E. coli morphology, and two non-lactose-fermenting colonies were subjected to biochemical tests for identification. All E. coli strains were maintained in nutrient agar  slants at room temperature. Investigation of stool samples for parasites was performed by direct examination of stools after sedimentation in Lugol\u2019s iodine solution [Stool samples were collected and placed in Cary-Blair transport medium, and transported in iced boxes within 4\u00a0h to the laboratory at the Universidade Federal do Esp\u00edrito Santo. Samples were inoculated onto the surface of MacConkey and Hektoen plates  for the selection of solution .E. coli isolates were subjected to two multiplex PCRs, as previously described, with some modifications [E. coli attaching and effacing (eae) gene , EAF plasmid , and the antiaggregation protein transporter gene  (for detection of EAEC strains). Primers specific for the detection of DAEC Afa/Dr. (afaB-C) strains were subsequently included into this multiplex PCR. PCR2 assay contained primers specific for elt and est. (enterotoxins of ETEC), ipaH (invasion plasmid antigen H found in EIEC and Shigella), and stx1 and stx2 . PCR1 assay identified EAEC, DAEC, and tEPEC by the presence of eae and bfpA, and aEPEC by the presence of only eae. PCR2 assay identified ETEC, EIEC, and STEC.All ications . PCR1 as2, 2\u00a0mM of each deoxynucleoside triphosphate), 1.5\u00a0U of AccuPrime Taq DNA polymerase, and 5\u00a0\u03bcM of each set of primers except for the ipaH primers, which used 10\u00a0\u03bcM. The reactions were run in a thermal cycler  with the following cycling conditions: 94\u00a0\u00b0C for 5\u00a0min, 40\u00a0cycles of denaturation at 95\u00a0\u00b0C for 1\u00a0min, annealing at 58\u00a0\u00b0C (assay 1) or 50\u00a0\u00b0C (assay 2) for 1\u00a0min and primer extension at 72\u00a0\u00b0C for 2\u00a0min followed by a final extension at 72\u00a0\u00b0C for 7\u00a0min. PCR products (10\u00a0\u03bcL) were visualized after electrophoresis in 2% agarose gels in Tris-borate-EDTA buffer and ethidium bromide staining. In all assays, a mixture of DNA from the prototype EPEC E2348/69, EAEC 042, DAEC C1845, ETEC H10407, EIEC EDL1284, and STEC EDL931 strains [E. coli K-12 DH5\u03b1 was the negative control [Three to six bacterial colonies from each stool sample were pooled for template DNA preparation immediately prior to PCR testing, suspended in 300\u00a0\u03bcL of sterile water, and boiled for 10\u00a0min. A 5-\u03bcL aliquot of this suspension was added to 50\u00a0\u03bcL of the PCR mixture  against O antigens of EPEC serogroups , and EHEC O157. All E. coli strains where used as controls for detection of target genes: 042  [aggA) [agg3A, irp2) [hdaA, chuA), FBC114 (sat) [, iucA [afaE, daaE) [aida/aah) [paa) [Primers and PCR conditions for detecting sequences encoding 17 putative virulence genes are described in Table\u00a0tA, pic) , 17\u20132 (a) [aggA) , RN785\u20131A, irp2) , EDL933 14 (sat) , iucA [1 [, iucA , C1845 (ida/aah) , and HSPh) [paa) .Table 1PE. coli isolates were subjected to HEp-2 adherence tests by the method originally described by Scaletsky et al. [5 HEp-2 cells were grown in Dulbecco modified Eagle medium containing 10% fetal bovine in 24-well tissue culture plates . Bacterial strains were grown statically in 2\u00a0ml of brain heart infusion for 16\u201318\u00a0h. The monolayers were infected with ~3 X 107 bacteria  and incubated at 37\u00a0\u00b0C for 3\u00a0h. The infected monolayers were washed with sterile PBS, fixed with methanol, stained with Giemsa stain, and examined by light microscopy for adherence pattern.y et al. , with slE. coli isolate taken from a nutrient agar culture was inoculated into 10\u00a0mL of sterile water. The resulting suspension was applied to the surface of a 14-cm plate of Muller Hinton agar (Difco) and spread evenly with a sterile cotton-tipped applicator. The plates were incubated at 37\u00a0\u00b0C for 30\u00a0min before the application of antibiotic discs. The antibiotic discs  were amikacin (30\u00a0\u03bcg), ampicillin (10\u00a0\u03bcg), amoxicillin-clavulanic acid (30 \u03bcg), cefotaxime (30 \u03bcg), choramphenicol (30 \u03bcg), ciprofloxacin (5\u03bcg), gentamicin (10 \u03bcg), imipenem (10 \u03bcg), cotrimoxazole (25 \u03bcg); tetracycline (30 \u03bcg), and trimethoprim (5 \u03bcg). The inhibition zone diameters were measured in millimeters and interpreted in accordance with manufacturers\u00b4 recommendations. E. coli NCTC10418 and E. coli K-12 C600 were used as controls.Antimicrobial susceptibility tests were performed employing the disc diffusion method on Mueller-Hinton agar, following recommendations of the Clinical and Laboratory Standards Institute . One colp value <0.05 was considered statistically significant.The statistical analyses were performed using the SPSS version 17.0 . Statistical differences were evaluated by chi-square or Fisher\u2019s exact tests. A From January 2008 and February 2009, a total of 327 cases of diarrhea were recruited in this study. They were divided into two groups, 141 children (< 18\u00a0years of age) and 186 adults (\u2265 18\u00a0years of age), were recruited in this study. Of the 141 children, 75 (53.2%) were younger than 2\u00a0years, 49 (34.7%) were between 2 and 10\u00a0years, and 17 (12.1%) were younger than 18\u00a0years of age. Among adults, 51 (27.4%) were between 18 and 30\u00a0years, 66 (35.5%) were between 31 and 50, and 69 (37.1%) were older than 50\u00a0years of age.E. coli (n\u2009=\u20091200) strains isolated from 280 of 327 cases were categorized into different pathotypes of DEC based on the results of two multiplex PCRs. Strains negative for DEC markers were further examined for their HEp-2 cell adherence patterns. Tables\u00a0eae) was more frequently detected among diarrheagenic children (5.7%) than diarrheagenic adults (1.1%). LT-ETEC was found in two diarrheagenic adults (0.7%). Mix DEC infections were detect in five patients; two of them harbored EAEC and DAEC, one harbored EAEC and aEPEC, one DAEC and EPEC, and one EAEC and ETEC. No EIEC, EHEC or STEC were detected in this study. Other enteric pathogens isolated were Shigella (1.2%) and Salmonella (0.3%). Parasites  were detected in 7% of stool samples. Mixed infections were presented in 22 (15.6%) cases and 12 (2.9%) controls (P\u2009<\u20090.05).oli n\u2009=\u2009100 strainaatA positive. EAEC aatA-positive strains were isolated significantly more often from diarrheagenic children than diarrheagenic adults (p\u2009<\u20090.05) (Table\u00a0pet was the most frequently detected (55.3%) followed by pic (40.8%), iucA (35.5%), irp2 (28.1%), aap (27.6%), aggR (26.3), and chuA (18.4%). One strain harbored AAF/I (aggA), seven strains harbored AAF/III (agg3A), and five strains harbored AAF/IV (hdaA). EAEC strains carrying the aagR-aap-aatA genes were isolated significantly more often from diarrheagenic children than diarrheagenic adults (p\u2009<\u20090.05)  of EAEC were 5) Table\u00a0. The maj.7% of EAected 55.% followeafaE, daaE and aida , while astA and pic were found in 6 (14.2%) and 3 (7.1%) of strains, respectively.There were a total of 42 DAEC, of which 25 (59.5%) harbored adhesins from the Afa/Dr. family (Table da Table . All DAEeae) was more frequently detected among diarrheagenic children (5.7%) than diarrheagenic adults (1.1%)  gene  Table . All strne Table . StrainsEAEC, DAEC, and aEPEC isolates were tested for their susceptibilities to 12 antimicrobial agents Table\u00a0. The EAEDespite the abundance of reports on diarrheal disease in children under five years of age, this study is one of the few to include the identification of all six DEC pathotypes in all age individuals. Our study has shed light on the little-known issue of DEC infections in adult patients attending health centers. Adults rarely visit a health care when they have diarrhea, unless they perceive the diarrhea as being serious. We demonstrated that DEC pathotypes were commonly found in diarrheagenic adults 40%). EAEC (23%) and DAEC (13%) were the most prevalent DEC pathotypes in both diarrheal groups; whereas aEPEC strains were recovered more frequently from diarrheagenic children (6%) than from diarrheagenic adults (1%). ETEC accounted for 1.5% of DEC, and we did not find EIEC and EHEC strains, indicating their limited role in childhood diarrhea in Brazil. Our findings are in agreement with a previous study conducted in rural communities in the city of S\u00e3o Mateus , showing high prevalence of DEC (45%) in children with diarrhea, EAEC 21%) as the most frequent DEC, followed by DAEC (12%) and EPEC (9%) [0%. EAEC % as the aggR-positive strains were isolated significantly more often from diarrheagenic children than from diarrheagenic adults (p\u2009<\u20090.05). Interesting, AA plasmid-positive EAEC was dominant among children and AA plasmid-negative EAEC was dominant among adults. Two hypotheses would be proposed: one is that there are different routes of infection to adults and children in the study area, another is that AA plasmid-negative strains could survive adaptively in adults, though children and adults are equally infected by both AA plasmid positive and negative EAEC. Twenty percent of our EAEC aatA-aggR positive strains simultaneously harbored the aap gene for dispersin. There appears to be a high conservation of the aatA-aagR-aap locus in the pAA plasmid, as has been shown for the prototype 042 strains [The terms typical EAEC and atypical EAEC have been suggested to refer to EAEC strains harboring or lacking the regulator AggR, respectively. Some studies have demonstrated an association of typical EAEC with diarrhea , 27, 28. strains . Most tE strains , 22, 30.pet and pic, found at similar frequencies in both diarrheal groups.The pathogenic mechanisms of EAEC infection are only partially understood. The varying presence of the different virulent factors indicates heterogeneity of the EAEC isolates . It has + were found in higher frequency in diarrheic patients than asymptomatic controls. However, in some studies, DAEC Afa/Dr.+ strains are isolated from cases of diarrhea and controls in similar frequencies [afaE and daaE (F1845) genes were not found in any DAEC strains. In our study, a significant proportion of DAEC isolates carried a gene encoding for a toxin, such as Pet and Sat. In a recent Brazilian study, DAEC sat-positive strains were found to be associated with childhood diarrhea [The adhesins of Afa/Dr. family have been implicated in DAEC pathogenesis. The prevalence of DAEC possessing Afa/Dr. genes in diarrheagenic children and diarrheagenic adults was similar. Germani et al.  demonstrquencies , 33. Thediarrhea .paa) gene has been found in a higher frequency among aEPEC from children with diarrhea than from controls [astA) has been found in association with diarrheal disease among Brazilian children [astA and 40% of them carried paa genes.The porcine AE/associated adhesin (controls , 35. In children \u201338. The E. coli strains similar to those reported in previous studies [E. coli strains isolated from children compared with strains from adults. Resistance to more than one antibiotic was found in approximately 60% of DAEC and aEPEC strains. The most commonly observed resistance was to ampicilin, cefotaxime and cotrimoxazole.Our data show a high resistance rate in  studies , 40 and Our results show a high proportion of DEC, where EAEC and DAEC predominate among children and adults with diarrhea. In addition, our results suggest that DEC carrying toxin-encoding genes seem to play an important role in causing sporadic diarrheal diseases in Brazil. Moreover, the findings reinforce our previous communications regarding the importance of DEC strains in childhood diarrhea in endemic areas of Brazil."}
+{"text": "Telmatobius marmoratus (marbled water frog), Rhinella spinulosa (Andean toad), and Pleurodema marmoratum (marbled four\u2010eyed frog) have expanded their range vertically within the past century to inhabit newly formed ponds created by ongoing deglaciation. These anuran populations, geographically among the highest  recorded globally, are being impacted by the chytrid fungus Batrachochytrium dendrobatidis (Bd), and the disease it causes, chytridiomycosis. In this study, we report results from over a decade of monitoring these three anuran species, their habitat, and Bd infection status. Our observations reveal dynamic changes in habitat including ongoing rapid deglaciation (18.4\u00a0m/year widening of a corridor between retreating glaciers from 2005 to 2015), new pond formation, changes in vegetation in amphibian habitat, and widespread occurrence of Bd in amphibians in seven sites. Three of these sites have tested positive for Bd over a 9\u2010 to 12\u2010year period. In addition, we observed a widespread reduction in T.\u00a0marmoratus encounters in the Vilcanota in 2008, 2009, and 2012, while encounters increased in 2013 and 2015. Despite the rapid and dynamic changes in habitat under a warming climate, continued presence of Bd in the environment for over a decade, and a reduction in one of three anuran species, we document that these anurans continue to breed and survive in this high Andean environment. High variability in anuran encounters across sites and plasticity in these populations across habitats, sites, and years are all factors that could favor repopulation postdecline. Preserving the connectivity of wetlands in the Cordillera Vilcanota is therefore essential in ensuring that anurans continue to breed and adapt as climate change continues to reshape the environment.The Cordillera Vilcanota in southern Peru is the second largest glacierized range in the tropics and home to one of the largest high\u2010alpine lakes, Sibinacocha . Here,  Five threats have been identified as having an important role in Telmatobius declines: habitat degradation and loss; overharvesting for food consumption; introduced predators including trout; pollution; and infectious diseases, notably chytridiomycosis and ranavirus  and Pleurodema marmoratum (marbled four\u2010eyed frog), exhibiting the clinical symptoms of and/or the disease chytridiomycosis  and Brem, Mendelson, and Lips  network . Each frog was captured in a clean, unused plastic bag and swabbed four to five times each on the underside of the hind feet, thighs, abdomen, and forefeet, using moderate pressure to ensure collection of skin tissue without causing injury to the animal. Tadpoles were swabbed by gently rotating the swab across the mouthparts for five turns. Tadpoles from the same pond, or frogs found sharing a burrow under the same rock, were placed together in the same bag; otherwise, frogs were placed in separate bags. Swab samples were air\u2010dried and stored in airtight plastic containers at ambient temperature. Handlers changed powderless, nitrile gloves between each animal sampled to avoid potential cross\u2010contamination. Footwear, nets, rulers, and instruments were sprayed with ethanol (90% solution) between sites.2.3103001004AC52300 and 1030010049008200, sensor WV2, resolution, 50\u00a0cm, acquisition date, 13 October 2015). The image was rescaled to the base map projection. The brightness gradient of the ice margin was then used to define the isoline. Distances were calculated and map overlays created using GPS coordinates from field observations and Google Earth Pro and Google Maps online software.The generation of the map demonstrating temporal evolution of glacial recession has been previously described  was deployed at 30\u00a0cm water depth at Area D Pond 5 on 14 March 2008 and retrieved on 2 August 2009. A second datalogger, installed and collected on the same dates, was placed in a ventilated and shaded rock cavity on a hill summit 150\u00a0m NE of D5 Pond at 5,250\u00a0m.2.5Servicio Nacional de Meteorologia y Hidrologia (SENAMHI)\u2014at Sicuani , 57\u00a0km to the south; Ccatcca , 54\u00a0km west\u2010northwest; and Macusani , 78\u00a0km east\u2010southeast of Area D, have similar annual precipitation means . Daily temperature maxima and 24\u2010hr precipitation totals from the three sites were averaged and then assessed as seasonal (90\u2010day) moving averages. Unlike daytime maxima, nocturnal minimum temperatures exhibit large variations dependent upon local environmental factors so were not used for the analysis.Meteorological records were used to assess seasonal character leading up to site visits and identify conditions relative to comparable time\u2010of\u2010year averages. Long\u2010term climatological observations are not available from within the Sibinacocha watershed, so to characterize climatic variability and trends, we used 15\u2010year daily precipitation and temperature data series from nearby weather stations to serve as proxies for climatological conditions in the watershed; the data are from 1 July 2000 to 30 June 2015. The stations, operated by the Peruvian 2.6Bd testing were air\u2010dried and frozen until analysis in 2013 and 2015. For Bd analysis, DNA was extracted from the swabs using 150\u00a0\u03bcl of PrepMan  and extracts were diluted 1:10 in RNase/DNase\u2010free water. The samples were analyzed in singlicate by real\u2010time quantitative polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS\u20101) and 5.8S rDNA region using established methods , DNase/RNase\u2010free water, and 5\u00a0\u03bcl of diluted DNA were added to each well of a 48\u2010well plate. The exogenous internal positive control reagents served as inhibition controls in the PCR reactions. PCR amplification was run as previously described  and was diluted to a range of concentrations to generate a standard curve. The copy number was calculated as described previously  during visits between 2003 and 2015. Locations of all 63 individual sites are shown in Figure\u00a0Bd. We also examined local climatological variability and trends in postanalysis using SENAMHI station observations. A variety of these data types are displayed along a common 15\u2010year timeline in Figure\u00a0Three anuran species, 3.1Observations from six field expeditions conducted from 2005 to 2015 demonstrate that deglaciation and associated anuran habitat change, previously reported in Seimon et\u00a0al. , continu3.1.1P.\u00a0marmoratum, first documented in E7 Pond in 2005, were also observed in 2015. The ice margin uphill from Area D receded even more rapidly, averaging 13.1\u00a0m/year  relative to daily means over the period July 2000 to June 2015 /effort  and a mortality event at Area D (T.\u00a0marmoratus) , in Area A in 2003 (dry season), in Area B in 2005 (wet season), in Area C in 2008\u20132012 (wet and dry seasons), and in Area A in 2013  Table . Between013 Fig. . We did T.\u00a0marmoratus encounters from 2008 to 2012  or adjusted encounters  with the amount of survey effort.The relationship of encounters or adjusted encounters to the amount of survey effort\u2014comparing wet versus dry season\u2014is presented as scatter plots in Fig. P.\u00a0marmoratum has been documented at areas E  and F . At Area D, T.\u00a0marmoratus relative abundance was 6\u00a0person\u2010hr\u22121 in 2003 . During Area D surveys in 2008, 2009, and 2012, no T.\u00a0marmoratus were encountered despite similar or higher levels of person effort than in previous years. All three of these survey years were also preceded by below\u2010average precipitation and a water table drop occurring in the dry seasons in 2008 and 2009 , whereas only 44\u00a0m, wheP.\u00a0marmoratum was encountered at Area D during all surveys conducted between 2003 and 2015  during dry seasons, as expected between 2003 and 2015  copies of the ITS1\u20105.8S region per sample, a juvenile R.\u00a0spinulosa with 231,330  copies per sample\u2014both collected under the same rock in Area C in 2012\u2014and a dead adult P.\u00a0marmoratum with 1,713,901  copies per sample, collected in Area D in 2013. The ITS1\u20105.8S region of the Bd genome is multicopy, and copy number varies considerably between strains. If we assume an average of 77 copies per zoospore . The percent positive for Bd for each species and number of individuals tested in each area (A\u2013G) are presented in Figures\u00a0T.\u00a0marmoratus (in 2003) and P.\u00a0marmoratum  have tested positive for Bd, indicating that this pathogen has been in this environment and series of connected ponds for at least 12\u00a0years  emerged from retreating ice\u2014at 5,369\u00a0m in 2005 and at 5,383\u00a0m in 2008\u2014and are examples of several that have formed over the past decade. No anurans have been observed at these ponds to date, but Ecological successions and water table drops from glacial retreat are also changing anuran habitat. Recently developed ponds and habitat colonized by anurans at the highest areas, D\u2013F, continue to undergo successions; this includes progressive die\u2010offs of cushion plants. Our observations indicate that this is tied to the seasonal drop in water table occurring each dry season that may be increasing in amplitude. Negative precipitation anomalies from 2004 to 2009 may partially explain the initial water table decline, although subsequent precipitation increases have failed to reinvigorate desiccating wetland habitat and the water table drop continues to expose the deeper layers and soil root matrix of the underlying peat to an oxygenated and drier environment. This may be hindering growth and causing plant mortality of the vegetation, as has been observed at other sites in the Andes  where it was found only in 2005. However, the repeated observations of P.\u00a0marmoratum at other Area E ponds in the deglaciated zone below E8 since 2005 also indicate that the multidecadal upward range extension of this species continues. These results, paired with the observations of R.\u00a0spinulosa, indicate that expansion of each species to the absolute highest recorded sites is temporary and parallels the highly dynamic metapopulation fluctuations often encountered in these amphibians  due to threats imposed by overharvesting for food consumption, habitat loss, water pollution, and chytridiomycosis , pampas cat (Leopardus pajeros), and avifauna such as mountain caracara  seems unlikely to be drivers of the Telmatobius population reduction as they have coexisted with amphibians in the region for decades (for introduced trout) to millennia, or longer. Human consumption of T.\u00a0marmoratus was reported at Area A, but not at other sites. There is no evidence for the use of agrochemicals at our study sites, which all lie above cultivated terrain. Recently, a high prevalence of co\u2010infection of Bd with ranavirus has been reported in T.\u00a0marmoratus found in the San Pedro Market in Cusco, as well as other species at a nearby field site in Kos\u00f1ipata Valley near Manu National Park  collected in 2014 from areas A, B, D, and E were tested for ranavirus, and all samples were negative . However, Bd has been found at all areas surveyed (A\u2013G), of which three  remained positive when sampled over more than a decade (2003\u20132015). Chytridiomycosis has been documented in T.\u00a0marmoratus from areas A, C, and D, and Bd infection has also been found at Area B. Areas A\u2013D are also where reductions of this species have been documented.Data on natural fluctuations of anuran populations prior to the discovery of Batrachochytrium dendrobatidis has been proposed to have spread across the Andes in three epidemic waves in the late 1970s and early 1980s  where the most recent anuran declines have been occurring (Catenazzi & von May, Telmatobius (Catenazzi et\u00a0al., Telmatobius timens, commonly encountered until 1999, was not observed in surveys from 2007 to 2009, and local herders reported massive die\u2010offs of Telmatobius frogs in 2004 (Catenazzi & von May, Bd over time correlated with the increased percentage of species that disappeared, suggesting that Bd was likely the driving factor of the amphibian declines in the southern Peruvian Andes between 1999 and 2009 (Catenazzi et\u00a0al., Telmatobius mortalities in 2004; and detection of Bd and the disease chytridiomycosis, and amphibian declines during the same decade (Seimon et\u00a0al., Telmatobius mortalities occurred during the dry seasons (Seimon et\u00a0al., Telmatobius occurred in years with reduced precipitation. Dry season conditions in the high Andes can increase Bd prevalence in populations, most likely due to reduced flow of water into ponds (Catenazzi et\u00a0al., Bd, cause stress in amphibians, and make outbreaks of chytridiomycosis or other pathogens and parasites more likely (Catenazzi et\u00a0al., Telmatobius tadpoles can also act as disease reservoirs for other life stages making Telmatobius vulnerable to chytridiomycosis (Catenazzi et\u00a0al., Bd throughout the wet and dry seasons are underway and could identify whether pathogen loads and prevalence are higher as the water table drops and water flow is reduced, and if mortality events correlate with higher Bd prevalence. Critical to understanding the long\u2010term conservation threat of Bd to Telmatobius and other threatened genera in South America is determining whether T.\u00a0marmoratus and sympatric species such as P.\u00a0marmoratum and R.\u00a0spinulosa have the potential to develop, or have already developed, innate tolerance to Bd infection. This has been observed for several anuran species in Africa (Seimon, Ayebare, et\u00a0al., Ongoing studies monitoring Bd infections for over a decade. In the wake of glacial recession, cushion peatlands are continually forming and transforming by ecological successions, presenting resident anurans with rapidly changing habitats. Developing water bodies in the wake of glacial recession evolve into suitable habitat for anurans, which over time expand their range and colonize. However, as the glaciers continue to recede, some perennial ponds lose stable inflow of water from glacial melt. Desiccating perennial ponds promote successions from wetland to dryland vegetation, can promote oxygen depletion in the aquatic habitat, and, as they transition into seasonally dry ponds, may not persist long enough for complete tadpole development. Additional research is needed to understand more about the plasticity and genetic variability of these anuran species; the contributions of inflow from precipitation versus glacial melt in sustaining high Andean amphibian habitats; the degree of infiltration and amplification of the seasonal water table drop over time during the dry and wet seasons; whether the drop in water table correlates with increased Bd prevalence and infection; and how perennial to seasonal pond transitions may affect food resources, thermal regime, chemistry, oxygen content, and tadpole development. Such information will lead to a better understanding of the long\u2010term viability of these ponds as suitable amphibian habitat and the consequences on biodiversity once these glaciers are gone (Quenta et\u00a0al., bofedale expansion (Squeo et\u00a0al., In summary, deglaciation, anuran habitat, and landscape changes all continue to progress at a very rapid rate in the Cordillera Vilcanota. Three monitored anuran species remain extant despite the sustained occurrence of None declared.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "Skor2 . The second SNP rs30415957 resides in the intron of Plce1 (phospholipase C epsilon 1). The remaining two SNPs (rs30768258 and rs31216810) are close to each other on chromosome\u00a019, in the vicinity of Sorbs1 (sorbin and SH3 domain containing 1). Using quantitative RT-PCR, we found that Sorbs1 is highly expressed in the mouse uterus during embryo implantation. Knockdown of Sorbs1 by siRNA attenuates the induction of differentiation marker gene Prl8a2  in an in vitro model of decidualization using mouse endometrial stromal cells, suggesting that Sorbs1 may be a potential candidate gene for female infertility in mice. Our results may represent an opportunity to further understand female infertility in humans.The genetic factors underlying female infertility in humans are only partially understood. Here, we performed a genome-wide association study of female infertility in 25 inbred mouse strains by using publicly available SNP data. As a result, a total of four SNPs were identified after chromosome-wise multiple test correction. The first SNP rs29972765 is located in a gene desert on chromosome\u00a018, about 72\u00a0kb upstream of  P\u00a0<\u00a010\u22126, and with number of children at P\u00a0<\u00a010\u22127 . Female infertility was measured as the percent of matings that were nonproductive (MPD:14934) in 33 inbred mouse strains. The phenotypic data were log transformed. Genotypic data were downloaded from mouse HapMap database at Broad Institute (http://www.broadinstitute.org/mouse/hapmap/). A total of 132,285 SNPs for 25 out of the 33 inbred mouse strains with available female infertility data were retrieved. The 25 inbred mouse strains were: 129P3/J, 129X1/SvJ, A/J, AKR/J, BALB/cByJ, BALB/cJ, C3H/HeJ, C3H/HeOuJ, C3HeB/FeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57L/J, CBA/CaJ, CBA/J, DBA/1J, DBA/1LacJ, DBA/2J, LP/J, NZB/BlNJ, NZW/LacJ, PL/J, SJL/J, SM/J, and SWR/J. The SNPs with >\u00a010% of missing genotype calls were removed.Phenotypic data were obtained from the Mouse Phenome Database at Jackson Laboratory . The weighted F-statistic has the following form:g\u03bc is the mean of phenotypic values in a given inferred haplotype, \u03bcT is the mean of all phenotypic values. The genetic diversity between two strains is defined as the number of disagreed SNPs genome-wide divided by the total number of SNPs under consideration. The g\u03c9 in the weighted F-statistic is the average genetic diversity between all strain pairs contained in the inferred haplotype. We calculated chromosome-wise thresholds for multiple testing using Bonferroni multiple test correction. In this way, the family-wise error rate is 1\u00a0\u2013\u00a0(1\u00a0\u2013\u00a0\u03b1i)n \u2248 \u03b1in where \u03b1i is the individual test rejection level, and n is the number of SNPs on the chromosome under consideration. The weighted F-test algorithm was implemented in the MATLAB computing environment .To determine the association between SNPs and female reproductive traits, we applied a weighted F-test. The use of a single SNP is restrictive in the sense that it allows a representation of the genome only as diallelic. The use of windows of multiple SNPs enables the visualization of more complex genomic relationships between multiple strains. A window of three SNPs was used as described previously (http://www.informatics.jax.org/gotools/MGI_GO_Slim.html). The ontology covers three categories: biological process, cellular component, and molecular function. The Gowinda software (P\u00a0<\u00a00.001) were used as the query set, and all remaining SNPs used in the genome-wide mapping were treated as the background SNP set. The Gowinda software was run with the following parameters:\u2013simulations 100000\u2013gene-definition updownstream10000\u2013mode gene\u2013min-genes\u00a01. In the end, false discovery rate (FDR)\u00a0<\u00a00.05 was used as significance threshold to identify enriched terms.For gene set enrichment analysis, we adopted the classification terms defined by MGI GOslim  and 6\u00a0mg/ml dispase . The remaining tissues were incubated with 0.15\u00a0mg/ml collagenase I (Invitrogen). The supernatants were filtrated through 70\u00a0\u03bcm wire gauze, and centrifuged to collect the stromal cells. The cell pellets were washed twice with HBSS, and resuspended in complete DMEM/F-12 medium (Sigma-Aldrich) with 10% charcoal-treated fetal bovine serum . Cells were seeded onto 24-well culture plates at a concentration of 2\u00a0\u00d7\u00a010Sorbs1 were transfected into isolated mouse endometrial stromal cells using Lipofectamine 2000 (Invitrogen). Nonspecific scramble siRNA was transfected as a negative control. The medium was replaced with complete culture medium containing 2% cFBS 6\u00a0hr after transfection. Following an additional 12\u00a0hr of culture, cells were in vitro decidualized with estrodiol-17\u03b2  and progesterone , respectively. Cells were harvested for further analysis after 48\u00a0hr.The siRNAs targeting Rpl7 was used as a reference gene for normalization. Data were analyzed using the 2\u2013\u0394\u0394Ct method. All of the experiments were repeated independently at least three times. The significance of difference between two groups was assessed by Student\u2019s t-test. Results are expressed as mean\u00a0\u00b1\u00a0SEM. P\u00a0<\u00a00.05 was considered statistically significant.The total RNA from mouse uterus or cultured cells was isolated by using TRIzol reagent (Invitrogen). Potential DNA contamination was eliminated by digestion with RQ1 deoxyribonuclease I . Reverse transcription was performed with the PrimeScript reverse transcriptase reagent kit . For quantitative RT-PCR, cDNA was amplified using a SYBR Premix Ex Taq kit (TaKaRa) on the Rotor-Gene 3000A system . The primer sequences were:Sorbs1-forward: 5\u2032-TTTTCCAGGCAACTATGT-3\u2032;Sorbs1-reverse: 5\u2032-ATGCTTCATCCTCCGATA-3\u2032.Prl8a2-forward: 5\u2032-AGCCAGAAATCACTGCCACT-3\u2032;Prl8a2-reverse: 5\u2032-TGATCCATGCACCCATAAAA-3\u2032.Rpl7-forward: 5\u2032-GCAGATGTACCGCACTGAGATTC-3\u2032;Rpl7-reverse: 5\u2032-ACCTTTGGGCTTACTCCATTGATA-3\u2032.The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.http://phenome.jax.org/). Female infertility was measured as the percent of matings that were nonproductive in 25 inbred mouse strains. The female infertility data along with a phylogenic tree built from the SNP data of all 25 strains are shown in In the present study, we retrieved phenotypic data from the Mouse Phenome Database at Jackson Laboratory . The association results were used to generate a Manhattan plot (Table S1) that reached the statistically significant threshold (P\u00a0<\u00a00.001).Using the female infertility data for 25 inbred mouse strains, we preformed a genome-wide association mapping analysis. For studies using inbred mouse strains, one of the major problems is the lack of randomness in breeding histories. As shown in the phylogenic tree , subgroutan plot . As a reThe location of all significant SNPs relative to annotated genes on the mouse genome was calculated. We found that the vast majority of SNPs were intergenic (67%) or intronic (32%); only one SNP (<\u00a01%) was intragenic, residing on the 3\u2032-UTR, to be exact . To furtP-value was calculated. Finally, a total of four SNPs  were still significant after chromosome-wide Bonferroni multiple test correction (P\u00a0<\u00a00.05) . The second SNP rs30415957 resides on chr19:38799731, in the intron of Plce1 (phospholipase C epsilon 1). The two remaining SNPs (rs30768258 and rs31216810) are close to each other on chromosome\u00a019 in the vicinity of Sorbs1 (sorbin and SH3 domain containing\u00a01)  . The firining\u00a01) . The lin high LD .Sorbs1 is significantly upregulated at implantation sites compared to nonimplantation sites in mouse uterus on d\u00a06.5 of pregnancy . In addition to Skor2, another five genes appeared to locate on the same LD block: Ier3ip1 (immediate early response\u00a03 interacting protein\u00a01), Hdhd2 , Katnal2 (katanin p60 subunit A-like 2), Pias2 (protein inhibitor of activated STAT 2), and St8sia5 . To our knowledge, none of these genes are reported to play a role in female fertility. The second SNP rs30415957 resides on chr19:38799731 in the intron of Plce1 (phospholipase C epsilon 1). Its LD block contained Noc3l (NOC3-like DNA replication regulator), Tbc1d12 (TBC1 domain family member 12), and Hells (helicase lymphoid-specific). Of all genes in this LD block, Hells stands out because its downregulated expression in the endometrium is associated with repeated implantation failure  are close to each other in the same LD block on chromosome\u00a019 in vicinity of Sorbs1 (sorbin and SH3 domain containing 1).In the present study, we identified four significant SNPs after chromosome-wise multiple test correction. The first SNP rs29972765 is located on chr18:77022376 and its nearest gene is Sorbs1. A previous study has shown that Sorbs1 is significantly upregulated at implantation sites compared to nonimplantation sites in mouse uterus on d\u00a06.5 of pregnancy (Sorbs1 is significantly upregulated at implantation sites compared to interimplantation sites on d\u00a08 of pregnancy. The most remarkable event in the uterus on d\u00a08 of pregnancy is decidualization, which is characterized as proliferation and subsequent differentiation of endometrial stromal cells into large epithelioid cells. To test whether Sorbs1 plays a role in this process, we isolated mouse endometrial stromal cells and established a model of in vitro decidualization. We showed that expression of Sorbs1 was induced during in vitro decidualization, mimicking the regulation in uterus on d\u00a08 of pregnancy. Furthermore, we found that knockdown of Sorbs1 by siRNA significantly reduced the expression of decidualization marker gene Prl8a2 on day 2 of in vitro decidualization. Decidualization is required for uterine angiogenesis and hemostasis during trophoblast invasion and placenta formation (Sorbs1 may contribute to female fertility by exerting a role in decidualization. However, genes localized in the vicinity of the SNPs are not necessarily responsible for the trait (Sorbs1 and the female infertility trait.Of all these genes, we focused on ormation . Thus, SSorbs1. Using an in vitro model of decidualization, we provided evidence that Sorbs1 may be a potential candidate gene for female infertility in mice by modulating decidualization of endometrial stromal cells. Our results may represent an opportunity to further understand female infertility in humans.In summary, we performed a genome-wide association study of female fertility in 25 inbred mouse strains by using publicly available SNP data. A total of four SNPs were identified, two of which are in vicinity of"}
+{"text": "Traditionally, human vision research has focused on specific paradigms and proposed models to explain very specific properties of visual perception. However, the complexity and scope of modern psychophysical paradigms undermine the success of this approach. For example, perception of an element strongly deteriorates when neighboring elements are presented in addition . As it was shown recently, the magnitude of deterioration depends not only on the directly neighboring elements but on almost all elements and their specific configuration. Hence, to fully explain human visual perception, one needs to take large parts of the visual field into account and combine all the aspects of vision that become relevant at such scale. These efforts require sophisticated and collaborative modeling. The Neurorobotics Platform (NRP) of the Human Brain Project offers a unique opportunity to connect models of all sorts of visual functions, even those developed by different research groups, into a coherently functioning system. Here, we describe how we used the NRP to connect and simulate a segmentation model, a retina model, and a saliency model to explain complex results about visual perception. The combination of models highlights the versatility of the NRP and provides novel explanations for inward-outward anisotropy in visual crowding. Within the classic framework, vision starts with the analysis of basic features such as oriented edges. These basic features are then pooled along a feed-forward visual hierarchy to form more complex feature detectors until neurons respond to objects. A strength of modeling visual perception as a feed-forward process is that it breaks down the complexity of vision into mathematically treatable sub-problems. Whereas this approach has proven capable of explaining simple paradigms, it often fails when put in broader contexts . To fullEfforts to simulate many models for different functions of perception as a single system can encounter many challenges, including the following.Different models often come with very different computational frameworks. For example, one of the models might be a spiking neural network and another might be an algorithm involving a set of spatial convolutions. The models need a common simulation ground to talk to each other efficiently.Even if models coming from different research groups are simple, producing computer code to efficiently and reliably emulate models can be a daunting task. Few labs have the expertise needed to produce (or reproduce) models that address rather different parts of the visual system.It is necessary, but often complicated, to determine the contribution of each model to the general output of the system. Moreover, competing models and hypotheses might be tested on the same data. To address these challenges, models should be treated as modules that can be easily removed from or added to the system. In the same vein, it is important to have a common visualization interface for the output of all simulated models.It might be difficult to synchronize all the models in a common simulation. For example, one model might be a simple feed-forward input-output transformation, and another model might be a recurrent neural network that evolves through time even for a constant stimulus. It is important to make sure that interactions between those models are consistent with their states at every time-step.For many models, it is not straightforward to simulate the system efficiently and adapt the resource management to the workload of the simulation.It is important for scientists to be able to reproduce and extend simulation results. This means not only access to model code but also the ability to reproduce stimuli. Contextual elements such as lighting, distance to the stimulus, stimulus eccentricity or even the display screen, might matter in a complex model system. The simulated environment should ensure a common set of stimuli for all scientists.The NRP, developed within the Human Brain Project, aims to address these challenges. The NRP provides an interface to study the interactions between an agent  and a virtual environment through the simulation of a brain model . The plahttps://bitbucket.org/albornet/crowding_asymmetry_nrp. In the next section, we define visual crowding and the challenges that is addresses to vision research. Then, we describe the models that are combined in our visual system and their interactions. Next, we present the results of the simulation of the visual system that we built on the NRP. Finally, we discuss the results, followed by a conclusion.Here, we show that the NRP can easily combine different visual modules, even those programmed by different research groups. We show that these combined components can explain complex observations about visual perception, taking visual crowding as an example. We made the code publicly available at In crowding, perception of a target strongly deteriorates when it is presented together with surrounding elements  that share similar features with the target . As for Local models cannot explain these aspects of vision . To fullIn this section, we describe the models that we connected, using the NRP, to explain inward-outward anisotropy in crowding. Then, we describe how the models interact with each other. The visual system is composed of the segmentation model of The Laminart model by The segmentation process is triggered by local selection signals that spread along connected contours . The locPrevious work has integrated a retina model as part of a neurorobotic experiment in the NRP  by usingIn addition, we include space variant Gaussian filters provided by COREM that mimic retinal magnification. Along the retinal layers, visual information is pooled with less spatial precision in the periphery than in foveal locations because the Gaussian integration filters are broader with eccentricity. Finally, the output of the retina, i.e., the activity array of the ON- and OFF-centered ganglion cells, is distorted by a log-polar transform to mimic the magnification that results from the mapping of the retina neurons to the visual cortex. An example of the model\u2019s output is shown in Computational models of saliency aim to identify image regions that attract human eye movements when viewing complex natural scenes. The contribution of stimulus features to the allocation of overt attention can then best be captured in a task-free experimental scenario. As a model of saliency computation, we used a deep convolutional neural network, simulated in TensorFlow , that auThe model is an encoder-decoder network that learned a non-linear mapping from raw images to topographic fixation maps. It constitutes a simplified version of the model introduced by The virtual environment reproduces the conditions of two experiments that measure inward-outward anisotropy in visual crowding see : experimFor each condition of experiment 1b of Those signal and noise arrays are then used to measure the match M between the output of the segmentation model and the target template, according to equation (1).ij and the intensity of pixel  of the noise array by nkl. The weight of interference between those two pixels decreases exponentially with the distance between them. I0 is the strength of interaction and sigma is the rate of exponential decrease. I0 is set to 10-3, a value that was determined to generate sufficient interaction between the target and the flanker, without killing the signal completely. Sigma is set to 30 pixels, a value that was determined to follow approximately the pooling range defined by Bouma\u2019s window  of the signal array is denoted by ss window . Given ti) across the trials and divide this value by the mean thresholds of the unflanked condition, where only the target is presented to the robot. We define this final number as the model measurement of the threshold elevation of the flanking configuration [see equation (2)].Finally, for each condition, we take the mean of the thresholds  is the threshold measurement associated to the segmented output of trial n for condition i, and Tu(n) is the threshold measurement associated to the segmented output of trial n for the unflanked condition.Where EFirst, we reproduced the crowding paradigm of experiment 1b of Next, we reproduced the crowding paradigm of experiment 5 of Using the NRP, we simulated a complex visual system composed of several models coming from different research labs. The platform provides satisfactory answers to many of the challenges described in the Introduction. Here, we summarize these issues and briefly explain how the NRP addresses them.Even if the models that we use have different computational frameworks, the platform allows us to easily integrate them into a common visual system, define their interactions, and simulate them with a minimal amount of code. For example, the segmentation and the saliency models use NEST and TensorFlow, respectively, which the platform supports.The collaborative aspect of the platform made it possible to quickly integrate the retina model to the simulation. The retina-modeling framework was already incorporated to the platform by other users , togetheThe NRP allows researchers to de-activate models, simply by commenting out a single line in the setup file of the virtual experiment. This is a powerful tool to investigate how each model contributes to the general output of the system see , or to tThe platform takes care of the synchronization between the simulated models. In our visual system, the segmentation model is a recurrent network and the saliency model is a feed-forward input-output transform and the NRP ensures that their respective inputs are always consistent. The models are first run in parallel for a short amount of time. Then the platform collects data from the simulation and computes the relevant inputs for the next simulation step.However, some challenges were handled with less success. Simulating the whole visual system with the required input resolution required very long computational times . The platform is currently used online with servers that have rather limited resources. The platform is in development and will soon support high-performance computing.Because of the computational limitations, we could not reach the resolution that was required to identify the high-level features of some stimuli . It would be interesting to check if the \u201cface-ness\u201d of the Mooney faces drastically changes the output of the saliency model and if the model threshold results substantially change.Ultimately, simulating the visual system on the NRP allowed us to enhance understanding about visual crowding. We could show that the segmentation model that explains crowding and uncrowding  is able The full model simulated with the NRP makes the prediction that inward-outward anisotropy can be observed only for a fixed range of eccentricities. If the eccentricity is too small (e.g., 3\u00b0 for the paradigm of Furthermore, it would be interesting to test how inward-outward anisotropy interacts with uncrowding. A new interesting paradigm would be to continue the experiment 1b of Breaking down the complexity of vision into simple mechanisms fails when the simple mechanisms are put in broader contexts. To fully understand human vision, one needs to build complex systems that process large parts of the visual field and combine many aspects of vision that all require sophisticated modeling. Using the NRP, we could start to simulate such a system by connecting a segmentation model, a saliency model, and a retina model, thereby providing explanations for complex results in visual crowding, such as inward-outward anisotropy. Crucially, the explanation is in line with the grouping hypothesis of No datasets were generated or analyzed for this study.AB, JK, AK, and AA substantially contributed to conducting the underlying research. AB, AK, and AA provided the models descriptions to the manuscript writing process. KC provided the description of the Neurorobotics Platform to the manuscript writing process. AB wrote most of the manuscript and put all parts together. GF, MH, EF, JK, and AK gave substantial feedbacks to the writing process.KC was employed by the company Fortiss GmbH. Fortiss GmbH is a public research institute financed by the Bavarian region. It is the principal developer of the NRP. The remaining 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": "Drosophila melanogaster) to linearly polarized light have previously been demonstrated. Using newly designed modular flight arenas consisting entirely of off-the-shelf parts and 3D-printed components we present individual flying flies with a slow and continuous rotational change in the incident angle of linear polarization. Under such open-loop conditions, single flies choose arbitrary headings with respect to the angle of polarized light and show a clear tendency to maintain those chosen headings for several minutes, thereby adjusting their course to the slow rotation of the incident stimulus. Importantly, flies show the tendency to maintain a chosen heading even when two individual test periods under a linearly polarized stimulus are interrupted by an epoch of unpolarized light lasting several minutes. Finally, we show that these behavioral responses are wavelength-specific, existing under polarized UV stimulus while being absent under polarized green light. Taken together, these findings provide further evidence supporting Drosophila\u2019s abilities to use celestial cues for visually guided navigation and course correction.Many navigating insects include the celestial polarization pattern as an additional visual cue to orient their travels. Spontaneous orientation responses of both walking and flying fruit flies ( Amongst these cues, the celestial polarization pattern serves as a robust visual stimulus informing the heading choices of many navigating insects3. Since Karl von Frisch first described the ability of honeybees to orient their waggle dances using merely a small patch of sky that did not include the sun as a landmark, many insects have also been shown to integrate the directional information provided by the skylight polarization pattern into their repertoire of visual cues4. Importantly, this ability is not restricted to central-place foragers like bees or desert ants that rely on visual cues to find their way back to their hive or nest6. For example, both diurnal and nocturnal ball-rolling dung beetles have been shown to use the celestial polarization pattern to set a straight path away from the food source where both predators and competitors may aggregate8. In this case, dung beetles show the tendency to maintain the same heading over repeated trials6. The tendency of other walking insects to set and maintain heading choices under a linearly polarized stimulus remains less well characterized. Although spontaneous behavioral responses to rotating polarization filters (polarotaxis) were demonstrated for crickets and flies when walking on air-suspended balls under laboratory settings11, clear characterizations of angular heading choices are missing for these experiments. Similarly, behavioral data for flying insects (other than honeybees), especially when using virtual flight arenas remains relatively scarce2. Oriented flights of suspended monarch butterflies under a polarized stimulus have been demonstrated, yet its ethological significance remains somewhat controversial, due to conflicting reports14. Probably the most valuable recent progress comes from the fly Drosophila melanogaster: spontaneous responses of flying Drosophila to linearly polarized light using virtual flight arenas have been demonstrated, both under the natural sky, as well as using an artificial stimulus generated in the laboratory using commercially available polarization filters18 (reviewed in19). Most importantly, flies were shown to choose arbitrary angular headings with respect to the orientation of the e-vector of the polarized stimulus and showed the tendency to maintain this navigational decision over several minutes, even when the stimulus presentation was perturbed for several minutes18. Nevertheless, fairly little is known about the navigational capabilities of free-living fruit flies (reviewed in20). Catch-and-release experiments from a fixed point in the desert suggested that Drosophila (melanogaster and pseudoobscura) disperse into all directions equally and are able to keep straight headings over extended periods of time, while flying in environments which provide few visual landmarks22. For a better quantitative understanding of the mechanisms underlying such processes, skylight navigation experiments using virtual flight arenas therefore serve as an attractive platform for the study of the navigation skills of wild type insects, thereby providing the platform for testing transgenic specimens harboring well-defined circuit perturbations24.Like many other animals, insects have developed the ability to efficiently navigate the most complex environments. Over several decades, evidence has accumulated showing that different insect species combine a multitude of visual stimuli in order to take fast and reliable navigational decisions 36, and in some cases green-sensitive Rhodopsins were reported (cock chafers)37. Although these different Rhodopsin choices seem to reflect adaptations to different ecological niches, the exact ethological reason for these differences remain incompletely understood39. Increasing evidence also points towards many insects (including flies) being capable to detect linearly polarized light through a DRA-independent channel (reviewed in40). Experiments from Drosophila have shown that these polarotactic behaviors are not UV-specific, since behavioral responses can be elicited using polarized green light presented to the ventral half of the retina23. Although incompletely understood, these behaviors could be indicative of a so-far poorly understood system in which retinal detectors are used to detect linearly polarized reflections. Such reflections could be used by insects to seek out or avoid water surfaces, evaluate oviposition sites, or even detect prey40.The retinal basis of celestial polarization vision across insects is well understood: in virtually all cases, specialized ommatidia located in the \u2018dorsal rim area\u2019 (DRA) of the adult eye are morphologically and molecularly specialized for this taskWe use virtual flight arenas to test heading decisions of individual flies flying under \u2018open-loop\u2019 conditions under a slowly rotating polarization filter. In agreement with previous studies, we find that flies initially choose a heading with respect to the orientation of the incident polarized light that varies between individuals and shows no preference for certain headings over the entire population tested (arbitrary headings). In this configuration, the rotation of the polarization filter therefore forces the fly to constantly adjust its heading in order to hold its original heading decision constant. By quantifying the fly\u2019s ability to adjust its heading relative to the changing e-vector over time we show that the behavioral performance varies greatly within a population, yet a similar behavior is never observed under unpolarized UV light, or linearly polarized green light. Importantly, flies show the tendency to maintain this heading over several minutes: we show that flies that perform well in following the e-vector within a 5-minute experiment show a high tendency to choose a similar heading in a second experiment, even when interrupted by a 5-minute interval of unpolarized light. These experiments underscore the usefulness of the experimental setups presented here and serve as an \u2018open source\u2019 platform for the development of new assays optimized for different visual behaviors, in flies as well as other species of flying insects.The aim of this study was a quantitative analysis of heading choices recorded from single flies flying under a slowly rotating polarization filter. We reasoned that flies that commit to a specific heading angle with respect to the incident angle of polarization would show a tendency to hold this angle constant and therefore correct for the slow rotational drift of the stimulus.41, the magnetic field created by two magnets kept the steel pins (and therefore the flies) vertical and in the center of the dorsally presented stimulus (see below), while allowing individual flies to rotate around their yaw axis, thereby enabling them to freely choose their headings . On top of the setup, switchable LED light sources (UV or green) were attached to two vertical beams, equipped with a matching set of highly collimated optics , in order to minimize off axis illumination of the polarization filter. The polarization state of the dorsally presented stimulus could be altered by shining light through a switchable filter \u2018sandwich\u2019 consisting of diffuser paper and a linear polarizer with either the polarizer or the diffuser facing the fly, as previously demonstrated23. This allowed for switching between two experimental conditions in which the degree of polarization was either ~0% (unpolarized) and almost 100% linearly polarized while keeping light intensity between trials constant  which was glued to the bottom side of the upper magnet. Due to this placement and size of the upper magnet (for holding the fly in place), the stimulus extended over a 17\u00b0 wide concentric ring in the flies\u2019 dorsal field of view  under a constantly rotating e-vector of linearly polarized light presented dorsally, we used custom built virtual flight arenas assembled from 3D printed and off-the-shelf hardware Fig.\u00a0 and S2. ant Fig.\u00a0. The filant Fig.\u00a0. The uppant Fig.\u00a0 and S2. iew Fig.\u00a0. Keeping17 (see 10.1101/527945), we introduced a new stimulus: flies flying within the virtual flight arena were presented a linearly polarized stimulus rotating slowly with constant angular velocity (~6\u00b0/s). Given that our test subjects were flying, this speed was chosen ~3x faster than what was previously published for walking crickets and houseflies11. In these experiments the 5-minute recording session per trial was split up into 30\u2009\u00d7\u200910\u2009s windows. For each of these 30 windows the mean angular velocity of each fly was then calculated. If the difference between this angular velocity and the filter\u2019s angular velocity was smaller than 3\u00b0/s, the particular time window was categorized as polarotactic behavior . For each of these periods the fly\u2019s chosen heading was then calculated as the mean angular difference between the fly\u2019s body axis and the incident e-vector . A representative fly  we investigated the spread of preferred headings when single flies were flying under a slowly rotating polarization filter. The goal was to investigate whether, in this particular kind of virtual flight arena, certain headings are naturally preferred or avoided, or whether the choice of preferred heading is arbitrary and therefore different between individual flies. The strategy is exemplified by the direct comparison of four representative traces of individual flies in response to a slowly rotating polarized UV stimulus Fig.\u00a0. QualityFinally, we tested whether the amount of time that the flies spent following the e-vector  within the first linearly polarized UV trial (PolUV1) correlates with the tendency of the flies to choose a similar preferred heading in a second consecutive trial (PolUV2) that was separated from the first by an interruption (5\u2009min of UVunpol). We found that the better the flies\u2019 performance within the first trial (more time spent following the e-vector in PolUV1), the higher the likelihood of them choosing a similar heading in the second trial Fig.\u00a0. During 1. In addition, the celestial pattern of linearly polarized light serves as an attractive orientation cue that many insects use43. Spontaneous behavioral responses of both walking and flying Drosophila to linearly polarized light (\u2018polarotaxis\u2019) have been demonstrated in the past, using both population assays, as well as single fly assays45. In all these experiments, much care was given to the control and avoidance of intensity artifacts that can result in behavioral decisions that are in fact independent of the linearly polarized component of the stimulus (reviewed in15). The virtual flight arenas used here have been designed with the dual goal of providing relatively cheap, robust setups that can easily be assembled, while at the same time minimizing intensity/reflection artifacts. The codes, templates and building instructions for the virtual flight arenas are freely available for download to anyone . Due to their modular design, they can be modified for studying behavioral responses to moving stimuli49, shapes50, colors51, or celestial bodies24.Navigating insects rely on the detection and integration of a wide variety of visual cues, like celestial bodies , intensity gradients, and chromatic gradients18. In further agreement with these past studies, we also find that flies show a clear tendency to keep their chosen heading over several minutes, which indicates that any given fly attempts to maintain its chosen heading. Given the considerable gap in knowledge about the ethology of Drosophila, these data provide further support for a potential role of polarization vision in guiding long-range navigation behaviors that have been reported for flies22. In contrast, we show that single flies flying under a linearly polarized green stimulus displayed no comparable polarotaxis, which was to be expected due to the fact that orientation responses to polarized stimuli presented dorsally were shown to be mediated exclusively by the DRA23, whose polarization-sensitive R7 and R8 photoreceptors in the fly both express the UV-sensitive Rhodopsin Rh330. Nevertheless, behavioral responses to linearly polarized green light stimuli have also been reported in the past22, but only when presented ventrally  and the retinal detectors responsible are not well understood40.Using these new virtual flight arenas, we show that individual flies choose arbitrary headings under a linearly polarized stimulus, and when summed over all individuals tested, all chosen headings appear to spread randomly. This finding is in good agreement with recently published studies, although these were using a rather different kind of stimulation system18 and reinforce the idea that a generalist fly like Drosophila melanogaster is indeed capable of using skylight polarization for maintaining a chosen course over longer times, which is crucial for achieving more complex navigational tasks22. Like previous studies, we aimed at quantifying the quality of behavioral responses, since we expected that behavioral performance of individual flies to be greatly variable due to the strong influence of environmental conditions as well as internal states of the animal(s). For this study, we introduced a simple new stimulus, where flies are suspended under a slowly rotating polarization filter (~6\u00b0/s) under \u2018open loop\u2019 conditions. Quantifying the quality of a behavioral response by chopping any given 5-minute experiment into 30\u2009\u00d7\u200910\u2009sec polarotactic periods serves as an attractive new strategy for producing statistically significant data in a reasonable amount of time. Using this method, our experiments indeed revealed that behavioral performance is variable across all individuals tested. Even after tight control of food quality, rearing conditions, temperature, and humidity, the flies\u2019 cooperation in these experiments remains unpredictable. Importantly, the distribution of polarotactic 10\u2009s intervals across any given 5\u2009min experiment did not reveal any obvious pattern, except the trend that probability of polarotaxis tended to improve over time  as a reference to choose a heading (menotaxis)24, a behavior that requires \u2018compass neurons\u2019 in a central brain region known as the central complex54. This function is therefore in good agreement with physiological properties described for these neurons in locusts55. Classic data from larger insects5, as well as more recent studies from Drosophila56 are beginning to elucidate the neural circuitry of the \u2018compass pathway\u2019, along which menotactic and polarotactic information are being integrated by the insect brain, resulting in time-compensated compass information in the central complex57. Despite important similarities, it remains unclear whether flies use their anatomical compass pathway for performing exactly the same computations for skylight navigation62. The experiments presented here therefore serve as an important new platform for the efficient combination of Drosophila molecular genetic tools for the cell-type specific manipulation of neuronal function with quantitative behavior assays for testing skylight navigation.Many insect species use the celestial polarization pattern in conjunction with other visual stimuli like celestial bodies, intensity gradients, chromatic gradients, and landmarksWild type Oregon R flies (isogenized) were reared at 25\u2009\u00b0C and 60% relative humidity on standard cornmeal agarose food under a 12h-light/12h-dark cycle. Care was taken to keep population densities low within fly vials by flipping flies on a daily basis.Experiments were performed at 25\u2009\u00b0C and 50% relative humidity during the flies\u2019 evening activity peaks up until one hour after the light period within the respective rearing incubators would have ended. Flies were glued to 10\u2009mm long, 100\u2009\u00b5m diameter steel pins , so that when positioned vertically, they held the flies at a natural flying angle  and were allowed to recover for at least 20\u2009minutes from the gluing procedure before being tested. To prevent the flies from flying during the recovery phase, small pieces of Kimwipes were transferred to their tarsi. Initial flight behavior was triggered by inducing a little air puff towards the fly from below. This was also quickly done when flies stopped flying during the experiments, but not more than 3 times per experiment without excluding those flies from data analysis.www.flygen.org/skylight-navigation. Furthermore, the experimental setup, including stimulus properties  are described in greater detail in Supplemental Figures\u00a0Detailed building instructions of the virtual flight arenas used in this study including 3D printing and step-by-step assembly instructions and the codes necessary for their operation are freely available  for 5\u2009minutes while synchronously recording the flies\u2019 behavior.Above the fly a switchable cassette holding a 50\u2009mm\u2009\u00d7\u200950\u2009mm sheet linear polarizer  and 13 layers of thin, non-fluorescent diffuser paper  was inserted into a motorized rotatable cassette holder. Light from a collimated UV or green LED  coming from above and passing through the filter cassette was polarized (pol filter at bottom) or depolarized (diffuser paper at bottom) depending on the orientation of the filter cassette and presented to the dorsal part of the flies\u2019 eyes. Using a spectrometer  the intensity of the two LEDs was set approximately isoquantaly at 2\u2009\u00d7\u20091063. In short, it binarizes each video and fits an ellipse around the fly\u2019s body to extract its heading in a range from 0\u00b0 to 180\u00b0, in accordance with the directional ambiguity of the presented e-vector. The Fiji tracking results were analyzed using MatLab. Circular statistics were used. In short, a fly\u2019s heading changes were quantified as polarotactic behavior if the mean difference between the fly\u2019s angular velocity and the e-vector\u2019s angular velocity was smaller than 3\u00b0/s for a given 10\u2009s time window. By calculating the fly\u2019s heading relative to the e-vector during such polarotactic episodes it was possible to calculate a measure of the \u2018preferred e-vector\u2019 for each fly. The custom Fiji- and Matlab-scripts used in this study, as well as setup building instructions and documentation is freely available under www.flygen.org/skylight-navigation.The heading of each fly in each acquired video frame was extracted using a custom-written macro script for the open-source software FijiSupplemental Figures and Legends"}
+{"text": "Systemic sclerosis (SSc) is a progressive fibrotic disease that affects the skin and internal organs. Despite evidence implicating increased interleukin-17 (IL-17) activity in SSc, the role of IL-17 in SSc remains uncertain. The purpose of this study was to investigate whether IL-17 plays a pathophysiological role in SSc in two different murine models of SSc.Bleomycin (BLM)-induced fibrosis and chronic graft-versus-host disease (cGVHD) models were used. Histological analysis was performed using Masson\u2019s trichrome and immunohistochemical staining. Quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunoassays were used to quantify the messenger RNA and protein levels of inflammatory mediators in dermal fibroblasts.In vitro experiments showed that IL-1 and IL-17 exerted synergistic effects on the expression of profibrotic and inflammatory mediators. In the cGVHD model, C57BL/6 mice receiving splenocytes of IL-1Ra-KO BALB/c mice developed more severe cGVHD than did those receiving cells from WT mice. Knockdown of IL-17 in IL-1Ra-KO donor mice significantly attenuated the IL-1-induced acceleration of cGVHD severity.IL-1 receptor antagonist-deficient (IL-1Ra-KO) mice were more severely affected by BLM injection, as shown by dermal and pulmonary fibrosis, compared with wild-type (WT) mice. Increased tissue fibrosis was reversed by knocking down IL-17. Targeting IL-1 and its downstream IL-17 activity may be a novel treatment strategy for inhibiting inflammation and tissue fibrosis in SSc. Systemic sclerosis (SSc) is a progressive fibrotic disease that is characterized by excessive deposition of extracellular matrix (ECM) components such as collagen and glycoprotein . The cli+ T cells, which can produce high levels of inflammatory cytokines such as interleukin 4 (IL-4)  , 3. CD4+ltration . These o+ T cell subset that secrete primarily IL-17 (IL-17A and IL-17F). Th17\u2009cells and the associated signaling pathways have been shown to be involved in the pathogenesis of many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Sj\u00f6gren\u2019s syndrome, and type 1 diabetes -induced model of SSc. We also used a chronic graft-versus-host disease (cGVHD) model, another murine model of SSc, to show that blocking IL-17 activity attenuated the clinical severity of cGVHD. IL-1 and IL-17 had synergistic effects on the expression of profibrotic and inflammatory mediators in dermal fibroblasts from humans and mice. We confirmed the antifibrotic and anti-inflammatory potentials of IL-1 receptor antagonist (IL-1Ra) in vivo. These findings suggest that an IL-17 cytokine-targeting strategy by blocking IL-1 may be a novel therapeutic strategy to inhibit generalized tissue fibrosis in SSc patients.Here, we used two different murine models to examine whether IL-17 activity could augment the fibrotic and inflammatory processes in SSc. ad libitum. All experimental procedures were examined and approved by the Animal Research Ethics Committee of the Catholic University of Korea .B6 (H-2kb) and B/c (H-2kd) female mice, 8\u201310\u2009weeks of age, were purchased from OrientBio . IL-1Ra knockout (KO) and IL-17 KO mice were obtained from Prof. Yoichiro Iwakura . IL-1Ra-KO mice were backcrossed to IL-17 KO mice over 10 generations, and double KO (DKO) mice were selected for use in polymerase chain reaction (PCR) analysis. The mice were given standard mouse chow  and water n\u2009=\u20095 or 6 per group) were killed by CO2 euthanasia on the day after the final treatment, and the back skin and lungs were harvested and fixed in 10% formalin solution for histological analysis.Female mice (aged 6\u20138\u2009weeks) were anesthetized with isoflurane, and their backs were shaved. Mice were treated daily for 4\u2009weeks with subcutaneous injections of phosphate-buffered saline (PBS) or BLM . BLM was given at a dose of 1\u2009mg/ml in PBS and was sterilized by filtration before injection into the shaved back skin. The mice  from wild-type (WT) BALB/c or IL-1Ra-KO or IL-1Ra\u2013IL-17-DKO mice were injected intravenously through the tail vein. Mice were monitored for body weight after BM transplantation (BMT) at least twice weekly. Mice were euthanized on day 60 after BMT, and the histopathology of their cGVHD target tissues  was analyzed by investigators who were blinded to the treatment. Organs were harvested, cryoembedded, and sectioned. The sections were fixed in 10% (v/v) buffered formalin and stained with H&E for histological examination.C57BL/6 recipient mice were intravenously injected with 5\u2009\u00d7\u2009102). To quantify the numbers of infiltrating T cells, B cells, and macrophages, lesional skin sections were stained for CD3, CD22, and F4/80 , respectively. The count of positive cells, identified as a discrete membrane staining in cells, was made in 10 randomly selected HPF .Tissues were fixed in 10% formalin and embedded in paraffin. Sections (6\u2009\u00b5m thick) were stained with H&E and Masson\u2019s trichrome (MT). Dermal thickness was examined as previously described . The lunTo quantify the amount of collagen in mouse skin and lung specimens, hydroxyproline content was measured using a commercial Quickzyme Total Collagen Assay kit (QuickZyme Biosciences). The hydroxyproline assays were performed according to the manufacturer\u2019s protocol.4\u2009cells/well, and the cells were serum starved overnight and then treated with IL-1\u03b2 (10\u2009ng/ml) or IL-17 (5\u2009ng/ml) for 24\u2009h.Mouse skin fibroblasts were isolated by enzymatic digestion of skin from normal BALB/c, as described previously . Human d5\u2009cells per well in a 12-well culture plates in DMEM medium and allowed to adhere overnight at 37\u00b0C in a humidified incubator and 5% CO2 atmosphere. The following day, differentiated Th17\u2009cells (2\u2009\u00d7\u2009106) were added to skin fibroblasts with or without blocking anti-IL-17 antibody (5\u2009\u00b5g/ml). T cells were washed out with fresh media and fibroblasts were then harvested and analyzed for fibrosis-associated gene expression using quantitative real-time PCR.Skin fibroblasts from IL-1Ra-KO mice  were plated at 2\u2009\u00d7\u200910+ T cells, the splenocytes were incubated with CD4-coated magnetic beads and isolated using magnetic-activated cell sorting separation columns (Miltenyi Biotec). Positively selected splenic CD4+ T cells were stimulated with plate-bound anti-CD3 (0.5\u2009\u00b5g/ml), soluble anti-CD28 , anti-interferon-\u03b3 (2\u2009\u00b5g/ml), anti-IL-4 (2\u2009\u00b5g/ml), recombinant transforming growth factor \u03b2 (2\u2009ng/ml), and recombinant IL-6 (20\u2009ng/ml)  for 3\u2009days to achieve polarization of Th17\u2009cells.Spleens that were isolated from IL-1Ra-KO mice were minced and the red blood cells were lysed with 0.83% ammonium chloride. The cells were filtered through a cell strainer and centrifuged at 1,300 revolutions per minute at 4\u00b0C for 5\u2009min. To purify splenic CD4Messenger RNA (mRNA) was extracted using TRI Reagent  according to the manufacturer\u2019s instructions. Complementary DNA was synthesized using a SuperScript Reverse Transcription system . A Light-Cycler 2.0 instrument  was used for PCR amplification. All reactions were performed using the LightCycler FastStart DNA Master SYBR Green I mix (Takara), following the manufacturer\u2019s instructions. The following primers were used to amplify mouse genes: for IL-1\u03b2, 5\u2032-GGA TGA GGA CAT GAG CAC ATT C-3\u2032 (sense) and 5\u2032-GGA AGA CAG GCT TGT GCT CTG A-3\u2032 (antisense); for IL-6, 5\u2032-ATG CTC CCT GAA TGA TCA CC-3\u2032 (sense) and 5\u2032-TTC TTT GCA AAC AGC ACA GC-3\u2032 (antisense); for IL-17, 5\u2032-CCT-CAA-AGC-TCA-GCG-TGT-CC-3\u2032 (sense) and 5\u2032-GAG-CTC-ACT-TTT-GCG-CCA-AG-3\u2032 (antisense); for TNF-\u03b1, 5\u2032-ATG AGC ACA GAA AGC ATG ATC-3\u2032 (sense) and 5\u2032-TAC AGG CTT GTC ACT CGA ATT-3\u2032 (antisense); for MMP-1, 5\u2032-AAC TAC ATT TAG GGG AGA GGT GT-3\u2032 (sense) and 5\u2032-GCA GCG TCA AGT TTA ACT GGA A-3\u2032 (antisense); for MMP-9, 5\u2032-CTG TCC AGA CCA AGG GTA CAG CCT-3\u2032 (sense) and 5\u2032-GAG GTA TAG TGG GAC ACA TAG TGG-3\u2032 (antisense); for Col1A1, 5\u2032-CTC CGG CTC CTG CTC CTC TTA-3\u2032 (sense) and 5\u2032-GCA CAG CAC TCG CCC TCC C-3\u2032 (antisense); and for TGF-\u03b2, 5\u2032-GCC TGA GTG GCT GTC TTT TGA-3\u2032 (sense) and 5\u2032-CAC AAG AGC AGT GAG CGC TGA A-3\u2032 (antisense).The following primers were used to amplify human genes: for IL-6, 5\u2032-AGA CAG CCA CTC ACC TCT TCA G-3\u2032 (sense) and 5\u2032-TTC TGC CAG TGC CTC TTT GCT G-3\u2032 (antisense); for MMP-1, 5\u2032-CTG AAG GTG ATG AAG CAG CC-3\u2032 (sense) and 5\u2032-AGT CCA AGA GAA TGG CCG AG-3\u2032 (antisense); for MMP-9, 5\u2032-CGC AGA CAT CGT CAT CCA GT-3\u2032 (sense) and 5\u2032-GGA TTG GCC TTG GAA GAT GA-3\u2032 (antisense); for Col1A1, 5\u2032-ATG GGA GGA GAG CGT GTG-3\u2032 (sense) and 5\u2032-GAG GTC GGA GAG CAG AGG-3\u2032 (antisense); for TGF-\u03b2, 5\u2032-TGC GGC AGC TGT ACA TTG A-3\u2032 (sense) and 5\u2032-TGG TTG TAC AGG GCC AGG A-3\u2032 (antisense). All mRNA levels were normalized to that of \u03b2-actin.The concentrations of IL-1\u03b2, IL-1Ra, IL-6, IL-17, and TGF-\u03b2 in culture supernatants were measured using a sandwich ELISA . To activate latent TGF-\u03b21 to the immunoreactive form, before being assayed, latent TGF-\u03b21 was activated to the immunoreactive form by acid treatment. Briefly, 125\u2009\u00b5l of cell culture supernatant was incubated with 25\u2009\u00b5l of 1\u2009N HCl for 10\u2009min at room temperature, neutralized by addition of 25\u2009\u00b5l of 1.2\u2009N NaOH/0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid for 10\u2009min. Serum concentrations of IgG antibodies were measured using a commercial ELISA kit .In vitro experiments were independently repeated three or more times and each experiment had at least three samples. One-way analysis of variance followed by Bonferroni\u2019s post hoc test was used to compare differences between \u22653 groups. The Mann\u2013Whitney U test was used to compare numerical data between two groups. To assess the Gaussian distribution and the equality of variance, Shapiro\u2013Wilk test and Levene test were used, respectively. p\u2009<\u20090.05 was considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics 20 for Windows .Data are presented as mean\u2009\u00b1\u2009SDs of at least three independent experiments or at least three independent samples and for 10 mice in each group. Col1A1 and TGF\u03b2 mRNA were not affected by IL-1\u03b2 and IL-17.Many studies have revealed mediators involved in the fibrosis of SSc. IL-6 has been suggested as a treatment target for SSc. IL-6 induces collagen production in skin fibroblasts through JAK2\u2013STAT3-dependent signaling , 20. TheNext, we examined the effects of IL-1\u03b2 and IL-17 on the markers mentioned in human dermal fibroblasts. Although each cytokine increased the mRNA expression of IL-6, MMP-9, Col1A1, and TGF-\u03b2, the changes were marginal. IL-1\u03b2 and IL-17 showed significant synergistic effects on IL-6, MMP-9, Col1A1, and TGF-\u03b2 expression  for binding to the IL-1 receptor. IL-1Ra-KO mice on a BALB/c background were developed by Iwakura et al. for use as an animal model of spontaneous arthritis . Iwakurain vitro differentiated Th17\u2009cells that were generated using splenic CD4+ T cells from IL-1Ra-KO mice. After 3\u2009days of coculture, T cells were washed out and dermal fibroblasts were then harvested and analyzed for fibrosis-associated gene expressions. The results showed that MMP1, MMP9, Col1A1, and TGF\u03b2 mRNA expressions were significantly increased in the dermal fibroblasts cultured with Th17\u2009cells compared to those in single cultured system . The numbers of IL-1\u03b2-, IL-6-, IL-17-, and TNF-\u03b1-expressing cells were significantly greater in BLM-injected IL-1Ra-KO mice than in WT BALB/c mice. By contrast, the number of TGF-\u03b2-expressing cells did not differ between these two groups Figures A,B. ThesTo identify the altered populations of immune cells, the populations of T cells, B cells, and macrophages infiltrating into dermal tissues in each group of mice were evaluated by immunohistochemical staining against CD3 (T cells), CD22 (B cells), and F4/80 (macrophages). Fewer cells of each type were detected in IL-1Ra\u2013IL-17-DKO mice than in IL-1Ra-KO mice  in the BLM-induced fibrosis model in mice with a C57BL/6 background. After 4\u2009weeks of BLM injection, skin and lung tissues were extracted and analyzed. Both BLM-injected C57BL/c mice showed evident fibrosis of the skin and lungs Figure , 29. The7\u2009cells) from one of three different kinds of donor mice with a BALB/c background: WT BALB/c, IL-1Ra-KO, or IL-1Ra\u2013IL-17-DKO mice . Human experiments were approved by the Institutional Review Board (IRB) of human subjects at Bucheon St. Mary\u2019s Hospital , The Catholic University of Korea, and conducted accordance with IRB guidelines and regulations. All patients were informed and gave their written consent and this study was performed in accordance with the Helsinki II Declaration.All the authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. M-LC and S-HP had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. M-JP, S-JM, S-HP, and M-LC contributed to study conception, study design, data acquisition, analysis, and interpretation and drafted the manuscript. E-JL, K-AJ, E-KK, and D-SK contributed to data acquisition, analysis, and interpretation. J-HL, S-KK, and J-KM contributed to analysis and interpretation of data.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": "Rhodococcus fascians exist only as epiphytes on the plant surface whereas others can become endophytic and cause various abnormalities including the release of multiple buds and reduced root growth. The abnormalities reflect the action of cytokinin. The strains that can become endophytic harbour a linear plasmid that carries cytokinin biosynthesis, activation and destruction genes. However, both epiphytic and endophytic forms can release cytokinin into culture, affect cytokinin metabolism within inoculated plants and enhance the expression of sugar and amino acid transporters and cell wall invertases, but only the endophytic form markedly affects the morphology of the plant. A unique methylated cytokinin, dimethylated N6-(\u22062-isopentenyl)adenine (2-MeiP), operating in a high sugar environment, is the likely causative factor of the severe morphological abnormalities observed when plants are inoculated with R. fascians strains carrying the linear plasmid.Some strains of  Rhodococcus fascians. For many decades, cytokinins produced by R. fascians have been implicated as the causative factors inducing shooty galls and reduced root growth, since application of cytokinin can mimic the disease symptoms [fasD), cytokinin activation (fasF) and cytokinin destruction (fasE), and avirulent strains lack such a plasmid [Unique methylated cytokinins account for the morphological abnormalities induced by virulent strains of symptoms . Indeed, plasmid ,3,4,5. C plasmid ,7,8. R. fascians have been shown to extrude multiple different cytokinins into culture, most of which can, however, be derived from tRNA breakdown e.g., [Both avirulent (epiphytic) as well as virulent strains of wn e.g., ,10,11. Lwn e.g., ,13,14,15R. fascians trick the plant into providing a compatible environment for the pathogen. The \u201cTrick-with-the-Cytokinin-Mix\u201d hypothesis is based on the accumulation of several cytokinins in tissue inoculated by a virulent strain. These cytokinins are more-or-less resistant to destruction by cytokinin oxidase/dehydrogenase (CKX) [In a well-cited publication, it was suggested that virulent strains of se (CKX) . HoweverR. fascians can produce the cytokinins implicated in the \u2018Trick-with-the-Cytokinin-Mix\u2019 hypothesis in planta [AAP) and sugar (SWEET and SUT) transporters and cell wall invertases (CWINV) [We have shown that both virulent and avirulent strains of n planta ,14,15. M (CWINV) ,14. A hi (CWINV) , so therN6-(\u22062-isopentenyl)adenine (1-MeiP) and dimethylated N6-(\u22062-isopentenyl)adenine (2-MeiP), in tobacco tissues of plants inoculated with a virulent R. fascians strain [mt1, mt2) and fasD, all from the linear plasmid of a virulent strain, were sufficient to produce 2-MeiP in transgenic E. coli cultures. FasD utilised the dimethylated side chain to produce 2-MeiP, indicating that FasD is a dimethyl transferase rather than an isopentenyl transferase [mt1 and mt2 mutants of R. fascians were non-pathogenic [mt1 and mt2, and fasD, are expressed in peas inoculated with a virulent strain but not in tissues inoculated with an avirulent strain or in controls. 1-MeiP and 2-MeiP were detected in pea tissues inoculated with the virulent strain [In 2015, Sakakibara\u2019s lab identified two previously unknown methylated cytokinins, monomethylated s strain . They shnsferase . 2-MeiP nsferase . Moreovethogenic . We receR. fascians strains  within the generally low level of cytokinins able to be produced by both virulent and avirulent strains, should be the subject of further analysis. As of now, assuming 2-MeiP is responsible for changes in the plant phenotype, it is the simpler explanation for the induction of the morphological abnormalities induced by virulent R. fascians and provides for a more readily testable hypothesis compared to the Trick-with-the-Cytokinin-Mix hypothesis. The interaction of 2-MeiP within a high sugar environment should be sufficient to initiate the substantive morphological differences seen between plants inoculated with virulent compared to avirulent strains of R. fascians, as depicted in We also showed that none of the cytokinins implicated in the \u2018Trick-with-the-Cytokinin-Mix\u2019 hypothesis correlated with virulence . We sugg"}
+{"text": "Approximately 900 children/adolescents are treated with radiotherapy (RT) every year in France. However, among the 80% of survivors, the cumulative incidence of long-term morbidity \u2013 including second malignancies - reach 73.4% thirty years after the cancer diagnosis. Identifying a priori the subjects at risk for RT sequelae is a major challenge of paediatric oncology. Individual radiosensitivity (IRS) of children/adolescents is unknown at this time, probably with large variability depending on the age when considering the changes in metabolic functions throughout growth. We previously retrospectively showed that unrepaired DNA double strand breaks (DSB) as well a delay in the nucleoshuttling of the pATM protein were common features to patients with RT toxicity. We aim to validate a high performance functional assay for IRS prospectively.ARPEGE is a prospective open-label, non-randomized multicentre cohort study. We will prospectively recruit 222 children/adolescents who require RT as part of their routine care and follow them during 15\u00a0years. Prior RT we will collect blood and skin samples to raise a primary dermal fibroblast line to carry out in blind the IRS assay. As a primary objective, we will determine its discriminating ability to predict the occurrence of unusual early skin, mucous or hematological toxicity. The primary endpoint is the measurement of residual double-strand breaks 24\u00a0h after ex vivo radiation assessed with indirect immunofluorescence (\u03b3H2AX marker). Secondary endpoints include the determination of pATM foci at 10\u00a0min and 1\u00a0h (pATM marker) and micronuclei at 24\u00a0h. In parallel toxicity including second malignancies will be reported according to NCI-CTCAE v4.0 reference scale three months of the completion of RT then periodically during 15\u00a0years. Confusion factors such as irradiated volume, skin phototype, previous chemotherapy regimen, smoking, comorbities  will be reported.ARPEGE would be the first study to document the distribution of IRS in the pediatric subpopulation. Screening hypersensitive patients would be a major step forward in the management of cancers, opening a way to personalized pediatric oncology.NCT02827552 Registered 7/6/2016.ID-RCB number: 2015-A00975\u201344, ClinicalTrials.gov Identifier:  AlthHowever, among the 80% of survivors, the cumulative incidence of long-term morbidity \u2013 including second malignancies - reach 73.4% thirty years after the cancer diagnosis, with a cumulative incidence of 42.4% for severe, disabling, or life-threatening toxicities or specific death , 6, the in fine toxicity if not repaired on the one hand, or genomic instability and cancer risk if misrepaired on the other hand [Predictive assays of RT toxicity are subject to a lack of universality, reproducibility and specificity, and are time consuming  so that her hand , 12.max) was found to be the parameter with the best correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions. When taken as a binary predictive assay with the optimal cut-off value of 35 pATM foci, pATMmax foci showed promising predictive performances on a retrospective study, with an AUC of 0.97, a PPV of 99%, a specificity of 92% and a sensitivity of 100% [From 2003, the INSERM UMR1052 radiobiology group has elaborated a collection of primary skin fibroblast lines from patients (the majority of adults) with DNA repair deficiencies or who experienced RT toxicity on various tissues, with various severity and different post RT free intervals. The RT-induced distribution of candidate DSB recognition and repair proteins was measured with an indirect immunofluorescence (IIF) assay , 14 lead of 100% .We designed a prospective open-label, non-randomized multicenter cohort study to address the distribution of IRS and validate the performance of the IRS assay in the pediatric subpopulation. We will prospectively recruit 222 children/adolescents who require RT as part of their care path and follow them during 15\u00a0years to describe the specific morbidity including second malignancies. Prior RT we will collect blood and skin samples to raise a primary dermal fibroblast line to carry out the IRS assay \u2013 the result of which will have no impact on the RT prescription.ARPEGE aims to explore the distribution of IRS in the pediatric population and to determine prospectively the discriminating ability of an IRS assay to predict the occurrence of early cutaneous, mucosal or hematological RT toxicity qualified as unusual in children/adolescents treated with RT for cancer.The short term secondary objectives consist in: 1) identifying thresholds for each biomarker to predict the occurrence of unusual early toxicity in order to define IRS groups, 2) comparing the discriminating ability of IIF biomarkers (pATM and \u03b3H2AX on the one hand and micronuclei on the other hand), 3) developing a multivariate predictive model combining biomarkers.The long-term secondary objectives consist in: 1) identifying the discriminating ability of biomarkers to predict the occurrence of Grade 3\u20134 late toxicities, and thresholds for each biomarker, 2) describing IRS biomarkers in the subset of patients developing secondary malignancies, 3) investigating the correlation between the severity of early toxicity and the occurrence of late toxicity .Grade 2 or higher occurring at low doses (first week of treatment) orGrade 3\u20134 for more than 4\u00a0weeks after completion of RT and / or requiring surgery .The primary endpoint is the skin fibroblast radiosensitivity defined as the residual DSB 24\u00a0h after ex vivo radiation assessed with IIF (\u03b3H2AX marker). Unusual early cutaneous, mucosal or hematological toxicity occurs within 3\u00a0months after RT and is defined by any of the following features appreciated with CTCAE v4.0 morbidity scale:The discriminating ability of this biomarker to predict the occurrence of early toxicity will be assessed by the area under the receiver operating characteristic (ROC) curve.Kinetic data on other DSB repair proteins are collected in order to refine the classification of IRS as secondary endpoints. The other biomarkers studied are therefore: 1) the number of pATM foci 10\u00a0min and 1\u00a0h post irradiation, 2) the average number of micronuclei per cell 24\u00a0h after irradiation (control IRS assay).Late toxicity including second malignancies occurs at least 3\u00a0months after the completion of RT. Severe sequelae are defined by any grade 3\u20134 adverse effect with a progression lasting more than 90\u00a0days .All children and adolescents treated with a curative intent in pediatric oncology and radiotherapy departments of the Grand-Est and Burgundy-Franche-Comt\u00e9 regions, France participating in the GE-HOPE network  as well as Lyon and Toulouse may be included according to the following inclusion criteria: 1) age\u2009<\u200918\u00a0years, 2) indication of RT as part of the local control strategy on the primary tumor, 3) standard fractionation  irrespective of the technique and particle used, 4) patient affiliated to social security insurance, 5) patient and / or holder(s) of parental authority signed a written informed consent.Exclusion criteria are: 1) contra-indication to skin biopsy, 2) contra-indication to RT, 3) RT in a palliative intent, 4) Previous irradiation in the same anatomic site (re-irradiation), 5) hypofractionation, 6) Impossible follow-up, 7) Persons deprived of liberty or under supervision.about 20% of patients could benefit from RT only if the response to neoadjuvant CT in unsatisfactory. This data is known late. The biopsy would therefore be sampled just before the RT 60% of patients could benefit from neoadjuvant CT before RT 20% of patients could benefit from definitive RT not preceded by CT The participation to ARPEGE will be proposed as soon as the indication of RT is collegially validated in the care path of the patient. Three modalities of inclusion are permitted independently of surgery when required Fig.\u00a0:about 20In the latter two cases, the biopsy will be anticipated.All patients providing written informed consent to participate in the study are asked to complete a medical history. Clinical data that will be obtained in the ARPEGE study include patient-related data , cancer-related data , biopsy-related data , treatment-related data ). Comorbities that can affect IRS  will be reported.The following biological data from the radiobiological experiments will be reported in triplicates: \u03b3H2AX foci/nucleus prior irradiation and 24\u00a0h after, pATM foci foci/nucleus prior irradiation, 10\u00a0min and 1\u00a0h after, micronuclei/cell prior irradiation and 24\u00a0h after. 50 nuclei are counted for each condition.Adverse events related to investigations  as well as outcome will be recorded at RT simulation time, weekly during RT, at three months after RT, yearly during 5\u00a0years and every two years during the last 10\u00a0years  of parental authority and/or the child/adolescent, a 2\u20135\u00a0mm skin biopsy will be sampled under general anesthesia  or local anesthesia once the indication of RT is certain. In populations B and C, whenever there is a definite indication of postoperative RT known at the time of surgery, the biopsy may be collected from the operative specimen or at the scar level without modifying the nature or extent of the procedure. In other cases, a 2\u00a0mm (12\u00a0G) punch biopsy will be sampled at the buttock. The biopsy aims to collect the epidermis and superficial dermis without ever coming into contact with the superficial muscular fascia. The use of iodized antiseptics is prohibited. The anonymized specimen will be sent fresh in 10\u00a0mL of appropriate culture medium at ambient temperature and within 48\u00a0h.In parallel, 2.5\u00a0mL of venous blood may be collected to enrich a biological collection that will allow for further radiobiological studies .Patient-specific non-transformed fibroblast primo cultures will be raised. The cell lines will be irradiated (2\u00a0Gy) at early passage in the plateau phase of growth to mimic healthy tissues and to avoid any artifacts resulting from the cell cycle. The irradiated and control cells will be fixed at strategic times .Protocols for immunofluorescence with antibodies against pATM and \u03b3H2AX proteins and with DAPI counterstaining for scoring micronuclei have been described previously , 18. TheRT will be planned according to state of the art recommendations without any individual adaptation. The dose delivered to the organs at risk will be systematically collected on the dose-volume histograms.Early toxicity will be reported once a week during RT and then at 3\u00a0months, and rated on the NCI-CTACE v4.0 scale. Late toxicities, including second cancers, will be collected over 15\u00a0years and rated according to the same scale.0: AUC\u2009\u2264\u20090.7 against H1: AUC\u2009>\u20090.7 will be tested.The discriminating ability of any IIF biomarker to predict the severity of toxicity will be defined by the area under the ROC (AUC) curve as well as its 95% confidence interval. The following hypothesis: HThe discriminating capacity of biomarkers will be compared by the nonparametric Mann-Whitney approach . For eacThe analysis of the main and secondary short-term objectives is planned as soon as all the included patients have 3\u00a0months of follow-up. Intermediate analysis of the long-term secondary objectives will be carried out at 2, 5 and 10\u00a0years.If we hypothesize an area under the ROC curve of 0.85 for the main endpoint, a 15% occurrence of early toxicity  and a riAccording to inter-regional data updated in 2015, the inclusion potential would be 150 patients per year in the territory.From the basic research carried out by INSERM UMR1052 radiobiology group, Neolys Diagnostics proposes a powerful decision-making tool to the radiation oncology community in order to reduce side effects, while optimizing the treatment efficiency. With quasi optimal positive and negative predictive values, it is the only IRS test able to accurately quantify this trait according to a continuous spectrum with a strong biologic rationale when compared with other IRS assays. ARPEGE represents a unique opportunity to validate the skin IRS assay according to an appropriate methodology. To our knowledge, we lead the first study to document the specific distribution of IRS in the pediatric population.The application on the pediatric population is relevant due to the scarcity of cancer prevalence and indications of RT, the specific tissue homeostasis in this population, and the major societal challenges of optimizing the quality of survival of children who will recover.Due to the multiplicity of clinical situations in this heterogeneous population and in particular the protocols of concurrent chemotherapy we had considered to harvest the cells in the presence of drugs before irradiating them - in order to evaluate their radiosensitization potential. We abandoned this idea because of the over-cost, low feasibility and reproducibility, and bias on the constitutional trait pointed out by the assay.With regard to the documents intended for children and their decision-making autonomy, it appeared necessary to cover all the differences in development. Using appropriate language and information materials, we always seek the agreement of the child. For this study, we developed 4 different information materials and consent forms for parents and children aged 13\u201317, 8\u201312 and under 8\u00a0years of age. A Childhood Cancer Parents Association validated the materials.The duration of the study is compatible with an exhaustive collection of late toxicities, including radiation induced malignancies; The French expert centers have set up long-term monitoring structures in order to optimize the quality of survival. A stream wise recording of the dosimetric parameters performed routinely in France will provide new dose-volume-effect data for healthy tissues in pediatrics. Medico-economic data collected in an ancillary study on the same population will provide interesting information on the societal cost of sequelae induced by cancer treatments. Dose adaptation clinical trials integrating IRS a priori will be carried out in a second phase."}
+{"text": "The results showed that the complexation reaction prevailed at pH 6 and 7, whereas protons exchange dominated interactions at pH 3. Stability constant of the complexes increased along with pH (logK increased from ~3.8 to 4.2). Complexation was preferred by less-humidified structures of lower molecular mass containing more oxygen groups. The number of fluorophores available for Zn(II) increased from pH 3 to 7 by ~44%. Depending on the pH, complexation involved a bidentate chelate, monodentate and bidentate bridging mode. Zn(II) binding was insufficiently modeled by the classic Stern\u2013Volmer equation and well described by the double logarithmic equation (R > 0.94) as well as by a modified Stern\u2013Volmer formula assuming the existence of available and unavailable fluorophore populations (R > 0.98). The fluorescence ratio of different fluorophores was proposed as an indicator of the binding affinity of various structures. A positive relationship was found between the fraction of accessible fluorophores and Zn(II) binding at pH 7 determined based on proton release (R = 0.91\u20130.97). The obtained results can find application in controlling the mobility and bioavailability of Zn in different conditions.The aim of the study was defined as a complementary analysis of molecular interactions between zinc (Zn) and fulvic acids (FAs) at a broad pH range (3\u20137), different metal concentrations (0\u201350 mg dm Some exemplary fluorescent data elaborated using the Stern\u2013Volmer equation are shown in The Stern\u2013Volmer equation was used for a quantitative description of fluorescence quenching in the FA-Zn(II) system:SVK from the slope of F0/F vs. [Zn(II)] plots showed that graphs obtained for all the studied systems deviated from the straight-line course, which reveals a rather concave-down response  ions, and Fb0 was related to inaccessible fluorophore moieties. The changes of the fluorescence signal under the influence of metal can be expressed in this case as:aK is the Stern\u2013Volmer constant or conditional stability constant related only to accessible fluorophores. The fraction of fluorophores available for Zn(II) can be introduced as af:An attempt to determine response b. Such an et al.  for the n et al.  reportedn et al.  suggest n et al. ,59,63: is given by:aK and af can be determined from the slope and intercept of F0/(F0 \u2212 F) versus 1/[Zn(II)]. Plots illustrating how the modified equation works at pH 3\u20137 were shown in R > 0.97), which permits to determine the aK and af values for these experiment sets  compounds with a simultaneous increase in the pH may result from higher spatial availability of metal to structures such as carboxyl and phenol groups responsible for the formation of strong complex bonds. In their studies on interactions between humic acids and antibiotics, Wang et al. [ent sets . For theg et al.  reportedg et al.  reportedg et al.  on the ig et al. , bindingHigher values of stability constants for fluorophores from the B area indicated a greater stability of connections of Zn ions with the structures of lower molecular weight, which is typical of FAs. These results correspond to the ones obtained by Wei et al. . They reaf) accessible to Zn(II) increased along with the pH and was slightly higher for the B region and the highest for FA1. An increase in the pH to the neutral or alkaline values results in the dissociation of further functional groups, including also phenolic structures, which can raise af values. Possible participation of phenolic groups of humic substances in metal complexation was previously suggested by Gungor and Bekbolet [af value, was also characterized by the largest number of COOH groups, which confirmed the role of these structures in the B fluorescent region and in Zn(II) binding. For an extra analysis of interactions between FAs and Zn(II), a double logarithmic equation was used:bK) as well as the number of binding sites (n) were determined. The log-log plots (R > 0.94), indicating good fit of the model with experimental data. The obtained results show that the n value was similar for A and B peaks and did not exceed 1, which suggests that more binding sites of FAs were required to form a complex compound with Zn(II). A similar mechanism of interaction was also described in the studies of Fu et al. [bK values generally increased with the pH, however a slight decrease of these parameters was observed for some samples at pH 7, probably due to the formation of hydrolyzed ZnOH+ species [bK values were lower than aK ones, probably due to the macroscopic meaning of bK. However, it should be noted that use of the log-log equation is disputable for the interaction of metals with fulvic acids. The equation was originally proposed to describe protein complexation, and one of its basic assumptions was the slope of the log-log plot greater than 1, which meant \u201cpositive cooperative binding\u201d taking place when the binding at one site enhanced the binding at subsequent sites [Population of the fluorophores  revealed the presence of absorption bands typical of this fraction of humic substances. Slight differences between FAs from different soils concerned mainly the intensity of individual bands, and to a lesser extent\u2014wavenumber shifts. Some exemplary FTIR characteristics are shown in  phenols ,13,16,  n amides  as well n amides , (d) peaormation , (e) vibd ethers  and (f) C\u2013O bond ,13. The \u22121 and at 1220 cm\u22121 indicating the binding of cations by COOH groups  influence. In turn, most COOH groups at pH 7 were already dissociated the revealed a low intensity of these bands and, consequently, not so spectacular absorption weakening after Zn(II) supplementation. Interestingly, fluorescent studies (paragraph 2.4) prove the highest binding of Zn(II) ions at pH 7. It could be explained by the fact that complexation may also take place through dissociated ligands as well as other donor atoms, e.g., nitrogen-containing structures. In the studies on binding Cu, Pb and Cd by FAs obtained from lake sediments, Li et al. [The addition of Zn(II) ions caused a decrease in the absorption bands at ~1710 cmH groups . This meykiewicz  in the sykiewicz  during ti et al.  classifi\u22121 (even at the highest Zn concentration). The absorption band of OH group at ~3435 cm\u22121 changed its location towards longer wavelengths after Zn(II) addition (blue shift), indicating a change in the coordination sphere of the complex [\u22121 for complexes at high pHs indicating a complex formation with a higher covalent nature [Therefore, it should be concluded that the exchange of Zn(II) ions with protons is the only possible reaction mechanism, typical of low pH values. Given the above, an increased number of bound Zn(II) at the higher pH results to a lesser extent from exchanges with protons and more from attraction and complexation of positive cations to negatively charged functional groups, as well as from a more linearly developed structure that facilitates the penetration of zinc cations. These assumptions seem to be confirmed by the studies of Saab et al.  on pH ef complex  and invo complex . The blut nature .\u2212 bands (\u2206COO\u2212) of the FA-Zn(II) compound in relation to a similar distance for the ionic form of FAs [\u2212 parameter  is positively related to the mode of coordination in the sequence: monodentate > uncomplexed carboxylate ion(ionic) = bidentate bridging complexes > bidentate chelate complexes. Changes in this distance presented as a function of Zn(II) concentration are depicted for the studied compounds in \u2212 is observed at pH 3, which suggests the bidentate chelate mode of Zn(II) binding . Such mechanism can be attributed to the spatial shape of particles at acidic conditions. Greater aggregation of the structure means that the functional groups reduce distance to each other and that cations can be bound more easily by two oxygen atoms.The mechanism of interaction between Zn(II) and FAs can be additionally inferred from analyzing the wavenumber difference between asymmetric and symmetric COOm of FAs ,75,76. T\u2212 group) while the bidentate bridging mode begins to be more relevant above 5 mg dm\u22123 of Zn(II) . The observed trends applied to all tested FAs, which suggests the same reaction mechanisms regardless of FAs properties. The factor differentiating the coordination mechanism was the pH  concentrations  complexation. Alloway et al.  as well The results of potentiometric titration prove that one of the mechanisms governing the interaction in FAs-Zn(II) system was the exchange of protons with metal ions. Addition of Zn(II) ions reduced the pH of FAs solutions, indicating that metal binding and proton release into the solution took place. The dynamics of pH decrease was the highest at low Zn(II) concentrations and weakened along with increasing the metal dose until changes became insignificant .+ ions were released to the solution at pH 3 while the lowest number at pH 7. The amount of zinc bound by 1 g of FAs could be calculated assuming the interaction of metal with the active sites of FAs at a ratio of 1:2. These results are shown in The reason for this trend is a gradual decrease in the activity of free functional groups due to proximity to groups occupied with metal ions ,45. The + at pH 5 than at pH 4. Probably, a new population of COOH groups become available and visible at pH 5 due to the reconfiguration and spatial development of the structure [It is not surprising that the highest number of protonated COOH groups was present in pH 3, enabling interaction with a large amount of Zn ions by exchange with protons. However, an interesting fact is the release of a higher number of Htructure . Zn(II) tructure . The proaf, logaK, n, ZnH+/1g FA) and FAs properties , which suggests a complex nature of interaction with the metal\u2014independent of only one feature of FA.The results of the study seem to be important due to fact that Zn is an essential micronutrient necessary for the proper growth and development of plants. So far, numerous studies have revealed that the best digestible forms of Zn are complex compounds with soil organic molecules ,83,84. Taf, increased with Zn(II) binding during the exchange with protons at pH 7. This result, however, should be approached as inconclusive, and it requires further evaluation because a significant part of the functional groups  is dissociated at pH 7, and exchange in the Zn/H+ system plays a less important role. In turn, it should be mentioned that the literature on Zn binding by another fraction of humic substances, namely humic acids  suggest a relationship between the parameters describing Zn binding and some humic acids properties, which could be used for environmental forecasts. For example, negative correlations were revealed for stability constants of humic acids-Zn(II) complexes at pH 5 and 7 and Q4/Q6 and \u2206logK (humification degree) [The only statistically significant relationships were found between some parameters determined for the complexation process (fluorescence studies) and those describing proton exchange of the functional groups (potentiometric titration) . In the  degree) . A relat degree) . In the  degree) ,36,65. SZn(II) also forms compounds of a generally lower stability than that of FAs with other metals . It may \u22123) in 0.05 M NaHCO3, respectively at 465 and 668 nm, as well as at 254 and 436 nm, using an UV-Vis spectrometer . The value of absorbance recorded at 280 nm was used to evaluate structure aromaticity. Three replications were performed for each measurement, and the obtained results were averaged. The content of carboxylic (COOH) and phenolic (OH) groups was determined and described earlier using the method proposed by Dragunowa and Kucharenko [FAs were isolated from A-horizons of five chemically different soils: Haplic Fluvisol, Haplic Chernozem, Mollic Gleysol, Haplic Cambisol and Stagnic Luvisol using the procedure recommended by the International Humic Substances Society (IHSS) . The degcharenko .\u22123. The Zn(II) stock solution of 1000 mg dm\u22123 was prepared by dissolving ZnCl2\u22c52H2O in deionized water. Then, for each FA, five series of solutions were prepared with Zn concentration from 0 to 50 mg dm\u22123 and the final FA concentration of 40 mg dm\u22123. The pH of the series was adjusted to 3, 4, 5, 6 and 7 \u00b1 0.1 using diluted solutions of HCl or NaOH. The solutions were equilibrated under N2 atmosphere for 24 h. Afterwards, the pH was readjusted as needed, and all the series were analyzed for fluorescence signals. The fluorescent spectra were recorded as 3D excitation-emission matrices (EEM) with a scan speed of 12,000 nm min\u22121 using the Hitachi F-7000 FL luminescence spectrometer . The emission wavelength was scanned from 300 to 600 nm, whereas the excitation wavelength was raised sequentially by 5 nm steps in the range of 250\u2013500 nm. The analyses were preceded by fluorescence calibration using quinine sulphate at \u03bbex = 350 nm and \u03bbem = 450 nm. Spectral correction of the instrument was performed using rhodamine B [Stock aqueous solution was prepared for each sample of FA at a concentration of 50 mg dmdamine B ,35. The \u22123) were prepared with selected Zn concentrations  at pH 3, 4, 5, 6 and 7. The solutions were lyophilized. Then, 1 mg FA-Zn preparations were homogenized with 200 mg KBr of spectral purity and analyzed on a FTIR spectrometer  in the range of 400\u20134000 cm\u22121. The characteristics were obtained as an average of three measurements with 256 scans at 2 cm\u22121 resolution each.Five series of solutions for each FA (40 mg dm\u22123) with a fixed pH  were added to FA solutions (40mg dm\u22123) of the corresponding pH  to obtain the final Zn concentration from 0 to 50 mg dm\u22123. The solutions were stirred at a constant stirring speed under N2 atmosphere. The pH value was recorded after each titration step when the signal from the electrode was stable at the 0.001 level. The processing data related to the number of released protons enabled the evaluation of binding on the way of H+-Zn exchange. The measurements were performed in triplicate for each FA and each variant of pH and averaged afterwards.The interaction of Zn(II) ions with FAs by proton exchange was analyzed using the titration method. Portions of Zn solution (1000 mg dmThe relationships between the different mechanisms of Zn(II) binding were investigated using a correlation analysis for parameters describing interactions with Zn ions, i.e., stability constants, the number of binding sites and the fraction of accessible fluorophores (fluorescence spectroscopy method), as well as for parameters obtained from proton binding tests (potentiometric titration methods). The results were presented as a correlation matrix. Statistical significance was analyzed at \u03b1 = 0.05 by using the t-student test. Normal data distribution was verified using the Saphiro\u2013Wilk test.\u22123), as well as the different origin and chemical properties of FAs on the mechanism, stability and efficiency of Zn binding.The studies discussed above involved an effort of employing complementary analyses to molecular interactions between an important micronutrient\u2014zinc, and the most mobile fraction of humic substances\u2014fulvic acids. This article attempts to assess the impact of a wide range of pH (3\u20137), metal concentrations (0\u201350 mg dm\u22123) due to the high number of uncomplexed binding sites. However, ligands complexation was not completed even at excess Zn(II) due to the steric effect of the structures. The higher pH had a positive effect on stability of chemical bonds due to dissociation of subsequent functional groups, which caused greater attraction of Zn(II) cations. Mutual repulsion of charged structures counteracted destabilization of the soluble FA-Zn(II) complex and protected it from precipitation. Simultaneously, conformation of FA structure showed favorable changes from aggregated and coiled shape at low pHs to spatially developed and more linear structure at high pHs where Zn(II) access was easier.The results obtained from the fluorescence, FTIR and potentiometric studies demonstrated that the key factor determining the type and efficiency of interactions was the pH and concentration of Zn(II) ions. The complexation process proved highly relevant at pH 6 and 7, whereas protons exchange prevailed at pH 3. Stability constant of the formed complexes increased along with the pH and exhibited the highest stability at pH7 (logK increase from ~3.8 to 4.2). Complexation was preferred by less-humidified structures of lower molecular mass containing more oxygen groups. The number of fluorophores available for Zn(II) increased from pH 3 to 7 by ~44%, however, the formation of hydrolyzed Zn species decreased the number of the binding sites, thus making the interactions less effective. FTIR spectra revealed that the bidentate chelate mode can be typical for low pHs. On the other hand, the monodentate mode at low Zn concentration and bidentate bridging at higher Zn(II) doses can dominate for higher pHs. The binding of Zn(II) ions revealed the highest dynamics at low metal concentration (below 10 mg dmR > 0.98), as well as by a double logarithmic equation (R > 0.94). The ratio of fluorescence intensities of different fluorophores was proposed as an indicator of binding affinity of various structures.Evaluation of the different models showed that Zn(II) binding was poorly modeled by the classic Stern\u2013Volmer equation, and it was well described by the modified Stern\u2013Volmer formula, assuming the existence of available and unavailable fluorophore populations (R = 0.91\u20130.97). Such a comprehensive analysis enables a better understanding and optimization of the interaction mechanism. We believe that the obtained results can be useful in controlling Zn mobility and bioavailability in different conditions, both in the event of its deficiency and toxic concentration levels.The structural and sorption properties of FAs  did not reveal any statistically significant effect on the intensity and mechanism of interaction with Zn(II). It confirms the complex nature of this process and a probable impact of many FAs properties. A positive relationship was discovered between the fraction of accessible fluorophores and Zn(II) binding at pH7 determined based on proton release ("}
+{"text": "Saccharomyces cerevisiae to remove metal ions from four batch systems, namely Zn(II), Zn(II)-Sr(II)-Cu(II), Zn(II)-Ni(II)-Cu(II), and Zn(II)-Sr(II)-Cu(II)-Ba(II), and one real effluent was evaluated. Yeast biosorption capacity under different pH, temperature, initial zinc concentration, and contact time was investigated. The optimal pH for removal of metal ions present in the analyzed solution  varied from 3.0 to 6.0. The biosorption process for zinc ions in all systems obeys Langmuir adsorption isotherm, and, in some cases, the Freundlich model was applicable as well. The kinetics of metal ions biosorption was described by pseudo-first-order, pseudo-second-order, and Elovich models. Thermodynamic calculations showed that metal biosorption was a spontaneous process. The two-stage sequential scheme of zinc ions removal from real effluent by the addition of different dosages of new sorbent allowed us to achieve a high efficiency of Zn(II) ions removal from the effluent. FTIR revealed that OH, C=C, C=O, C\u2013H, C\u2013N, and NH groups were the main biosorption sites for metal ions.The performance of the brewer\u2019s yeast  Environment pollution with heavy metals is one of the most serious problems, since metals, even in traces, can be toxic and detrimental for living organisms . Zinc isConventional technologies, which include flocculation, precipitation, ion exchange, membrane separation, and solvent extraction, are applied for the treatment of wastewaters containing metal ions. However, these processes are usually expensive and show low efficiency of metal removal from slightly contaminated effluents which contain relatively low metal concentrations ,3,4. BioSaccharomyces cerevisiae (S. cerevisiae) are considered good candidates for wastewater treatment. Application of S. cerevisiae as a biosorbent is attributable to yeast safety, availability in large quantities at a very low cost, and high metal uptake capacity [S. cerevisiae is extensively used in beverage and food production and is obtained in large quantities, as a by-product of the fermentation industry [S. cerevisiae can remove toxic metals and radionuclides from aqueous solutions [S. cerevisiae biomass showed the highest sorption capacity for Pb(II) ions, followed by Ni(II) and Cr(VI). S. cerevisiae crosslinked with formaldehyde was used for Cu2+, Zn2+, and Cd2+ removal from solution. The metal uptake capacities of the biomass for Cu2+, Zn2+, and Cd2+ were found to be 8.0, 7.1, and 14.0 mg/g, respectively [S. cerevisiae toward Cd2+, Cr3+, CrO42\u2212, Cu2+, Pb2+, and Zn2+ were evaluated [Yeast cells of capacity ,4. S. ceindustry . It was olutions ,4,5,6,7.valuated . It shouS. cerevisiae for metal removal from four synthetic effluents with different chemical compositions and one real zinc-containing industrial effluent. For synthetic effluents, the effect of contact time, initial zinc concentration, temperature, and pH on the sorption process was investigated. The kinetic and equilibrium models were applied to describe experimentally obtained values. Thermodynamics parameters were calculated. For real effluent, the effect of pH and sorbent dosage on metal removal was investigated.The present work is focused on the application of yeast 2\u00b76H\u2082O, Cu(NO3)2\u00b72.5H2O, Zn(NO3)2\u00b76H2O, Sr(NO3)2, and Ba(NO3)2\u2014were purchased from Sigma-Aldrich  and were of analytical grade.Chemical used in the present study\u2014Ni(NO\u2083)3)2\u00b76H\u2082O, Cu(NO3)2\u00b72.5H2O, Zn(NO3)2\u00b76H2O, Sr(NO3)2, and Ba(NO3)2. Synthetic effluents were modeled based on the data obtained for real galvanic described in the [Four synthetic effluents, namely Zn(II), Zn(II)-Sr(II)-Cu(II), Zn(II)- Ni(II)-Cu(II), and Zn(II)-Sr(II)-Cu(II)-Ba(II), were prepared from metal salt stock solutions of Ni. The chemical composition of the effluent and the initial pH are given in Saccharomyces cerevisiae used as sorbent was obtained from the company Efes Vitanta Moldova Brewery . The procedure of sorbent preparation for analysis can be found in Reference [The yeast eference .In experiments with synthetic effluents, the effect of time (5\u2013120 min), zinc concentration (10\u2013100 mg/L), pH (2.0\u20136.0), and temperature (20\u201350 \u00b0C) on yeast sorption capacity was investigated. In total, 50 mL of synthetic effluents was added in 100 mL flasks containing 10 g/L of dried biomass. Samples were placed on the shaker and continuously agitated for 60 min. At the end of the experiment, biomass was separated from the solution by filtration, dried at 105 \u00b0C, weighted at analytical balance, and packed in aluminum foil cups for irradiation a the IBR-2 reactor.3 or NaOH solutions. In the experiment with real effluent, the effect of pH and sorbent dosage on metal removal efficiency was studied. To assess the effect of pH on biomass sorption capacity, 10 g/L g of dry biomass was added to 50 mL of effluent in a 100 mL flask and continuously stirred for 60 min. The pH of the effluent varied from 2.0 to 6.0. The pH of the experimental solutions was adjusted to the required pH, using HNOThe experiment on the effect of the dosage of sorbent on metal removal was performed in two stages. On the first stage, sorbent in the dosages ranging from 20 to 40 g/L was added to 100 mL of effluent at initial effluent pH (6.0). The suspension was shaken at 200 rpm for 60 min, and then the sorbent was removed by filtration. The supernatant obtained for each sorbent dosage was divided into three parts. One part was used for ICP-MS analysis, while two others were used for the second stage of the experiment. On the second stage, 1.0 or 10 g/L of new biomass was added to the effluent, obtained after the first stage, and shacked for 60 min. Then, biomass was again separated from the supernatant by filtration. Metal concentration in obtained solutions was determined by using ICP-MS.Experiments were performed in three repetitions, and the average values were used for discussion.q, was calculated by using Equation (1):The metal uptake, E, (%) was calculated from Equation (2):q is the amount of metal ions adsorbed on the biosorbent, mg/g; V is the volume of solution, ml; iC is the initial concentration of metal in mg/L; fC is the final metal concentration in the solution, mg/L; and m is the mass of sorbent, g.The sorption removal efficiency, To assess the effectiveness of zinc, nickel, strontium, and barium sorption from synthetic effluents neutron activation analysis at the pulsed fast reactor IBR-2  was used, while copper concentration in solution was determined by means of atomic absorption spectrometry. The procedure of samples irradiation and measurement is described in detail in Reference .Metal concentration in the real effluent, before and after biosorption experiments, was determined by the mean of Element 2\u2122 High-Resolution ICP-MS systems . The operational parameters and settings are presented in Kuznetsova et al. .Fourier-transform infrared spectroscopy (FTIR) was used to confirm the participation of functional groups in metal ions binding. The spectra were recorded by using a Nicolet 6700 spectrometer .The sorption of Zn(II) ions in the Zn(II)-system increased with the pH increase, and it was completely removed from the solution at pH 6.0 .In Zn(II)-Cu(II)-Sr(II) system, the optimal pH for Zn(II) removal lay in the range 3.0\u20136.0, and in comparison with a system containing only Zn(II) ions, its removal was reduced by 15% and constituted 84\u201386%. The maximum removal of Sr(II) in the same system was attained at pH 6.0 (77%), and of Cu(II) at pH 3.0 (76%). In the Zn(II)-Ni(II)-Cu(II) system, Zn(II) was completely removed from the solution at pH 4.0 and biomass maintained high Zn(II) removal efficiency at pH 5.0\u20136.0. The optimal pH for Ni(II) and Cu(II) removal was 3.0, and their removal was not affected by the pH increase. In the Zn(II)-Sr(II)-Cu(II)-Ba(II) system, the pH 5.0 was optimal for Zn(II), Sr(II), and Ba(II) ions\u2019 removal, while Cu(II) ions were efficiently removed at pH 4.0.S. cerevisiae was a time-dependent process. After 45 min of contact, Zn(II) ions were completely removed in the Zn(II) system  system .Complete Zn(II) removal was also achieved in the Zn(II)-Cu(II)-Sr(II) system after 30 min of contact, and for the other two metal ions present in the solution, Sr(II) and Cu(II), maximum removal was attained in 45 min . In the The adsorption kinetic data were described by using three models: pseudo-first-order model (PFO), pseudo-second-order model (PSO), and the Elovich model (EM) The models are expressed by the following Equations (3)\u2013(5): eq and tq are the amounts of metal (mg/g) adsorbed at equilibrium and at t (min), time, respectively, and k1 (1/min) is the rate constant of pseudo-first-order. The pseudo-first-order model is reflected Equation (3):k2 (g/mg\u00b7min) is the rate constant of second order.The pseudo-second-order model is reflected in Equation (4):\u03b1 (g/mg\u2219min) and \u03b2 (g/mg) are the Elovich equation constants.The Elovich model is reflected in Equation (5):N is the number of data points.In addition to the coefficient of determination, the applicability of kinetic models was assessed by using the sum of error squares , given by Equation (6):The modeling results of the applied models, as well as experimental data, are given in eq values for pseudo-first-order and pseudo-second-order models were in very good agreement. In the dependence of the analyzed system, different models were applicable to describe experimentally obtained data obtained for Zn(II) ions. In the Zn(II) and Zn(II)-Ni(II)-Cu(II) systems, Zn(II) ions\u2019 adsorption followed the pseudo-second-order model. In the Zn(II)-Cu(II)-Sr(II) system pseudo-first-order and pseudo-second-order models were applicable to describe experimentally obtained data, while in the Zn(II)-Cu(II)-Sr(II)-Ba(II) system, it obeys the Elovich model. The Elovich model also better described data obtained for Cu(II) in the same system, in comparison with other applied models. For other elements present in analyzed systems, except for Cu(II) in Zn(II)-Cu(II)-Sr(II), the coefficients of determination obtained for pseudo-first-order and pseudo-second-order models were higher than 0.94, indicating the applicability of both models for the description of experimentally obtained data. The experimentally obtained and calculated Equilibrium sorption studies were performed with different concentrations of Zn(II) ions from 10 to 100 mg/L. The increases in initial Zn(II) ions concentration increased the uptake of Zn(II) ions per unit weight of biosorbent (mg/g) and resulted in the decrease of yeast removal capacity. The maximum Zn(II) ions sorption in all analyzed systems was almost on the same level and lay in the range 5.34\u20136.1 mg/g. The removal of other metal ions present in the analyzed system was not dependent on Zn(II) concentration in solution and remained constant with its increase.eC is metal ions concentration at equilibrium (mg/L), eq is amount of metal adsorbed at equilibrium (mg/g), mq is maximum adsorption capacity of the sorbent (mg/g), and b is the Langmuir adsorption constant (L/mg) [Langmuir, Freundlich, and Temkin isotherm models were used for the equilibrium modeling. The Langmuir model suggests monolayer adsorption and is expressed by Equation (7):t (L/mg) .eq is the amount of metal adsorbed at equilibrium (mg/g), eC is the concentration of metal ions in aqueous solution at equilibrium (mg/L), and FK and n are Freundlich constants that include factors that affect adsorption capacity and adsorption intensity, respectively [The mathematical expression of the Freundlich isotherm model is presented by Equation (8):ectively .Tb indicates the sorption potential of the sorbent, Ta is Temkin constant, R is the universal gas constant (8.314 J K\u22121 mol\u22121), and T is the temperature (K) [The Temkin isotherm model is given below, in Equation (9):ture (K) .The Langmuir, Freundlich, and Temkin constants were calculated by nonlinear regression .The values of the parameters calculated from used models, as well as the coefficient of determination, are shown in R2 = 0.99) indicated that the adsorption of Zn(II) ions in all analyzed systems obey the Langmuir isotherm model. Zn(II) adsorption in Zn(II)-Cu(II)-Sr(II) and Zn(II)-Cu(II)-Sr(II)-Ba(II) systems also follow the Freundlich isotherm model. The Freundlich constant 1/n value less than 1.0 confirms the favorable adsorption [mq values showed that yeast biomass accumulated more Zn(II) ions from Zn(II)-Ni(II)-Cu(II) system, in comparison with other analyzed systems. The coefficients of determination for the Temkin model were lower in comparison with those of the Langmuir and Freundlich models.High values of the coefficient of determination  in Zn(II)-Ni(II)-Cu(II) and Zn(II)-Cu(II)-Sr(II)-Ba(II) systems and Cu(II) in all analyzed system was not dependent on the temperature grow. In Zn(II) and Zn(II)-Cu(II)-Sr(II) systems, an increase of temperature from 20 to 30 \u00b0C resulted in the increase of biomass sorption capacity by 5\u201318%, and then it did not change significantly. Sr(II) uptake by yeast occurred differently in the analyzed system; thus, in Zn(II)-Cu(II)-Sr(II), the temperature rise almost did not affect its removal, while in the Zn(II)-Cu(II)-Sr(II)-Ba(II) system, it decreased by 12%. Ni(II) and Ba(II) ions\u2019 removal declined with the increase of temperature by 7% for Ni(II) and 20% for Ba(II).G\u00b0), enthalpy change (\u0394H\u00b0), and entropy change (\u0394S\u00b0) values were calculated according to Equations (10)\u2013(12), as presented below:dK is the distribution coefficient, and it is calculated according to Equation (12).C0 is the initial concentration of metal, (mg/L); eC is metal concentration in aqueous solution at equilibrium, (mg/L); V is the volume of aqueous solution (L); m is sorbent mass (g); R is the universal gas constant (8.314 J K\u22121 mol\u22121); and T is the temperature (K).The Gibbs free energy change  and Cu(II) in the Zn(II)-Cu(II)-Ni(II) system, and Sr(II) and Ba(II)) in Zn(II)-Cu(II)-Sr(II)-Ba(II), was negative, indicating the exothermic reaction. For the rest of the elements, positive \u0394H\u00b0 values were obtained, thus confirming the endothermic nature of the process. Positive values of entropy, \u0394S\u00b0, were obtained for all elements in the analyzed systems. The negative values of \u0394S. cerevisiae biomass , while the band at about 1510 cm\u22121 is attributed to N\u2013O asymmetric stretching, C=C stretching (in-ring), C\u2013H asymmetric deformation, or vibration of the CH3 group. The peaks at 1225, 1400, and 2800 cm\u22121 are related to the vibration of carboxyl (C=O) and alkyl (\u2013CH3 or CH2) groups, while peaks at 1092 and 1043 cm\u22121 correspond to the phosphodiester groups and vibration of polysaccharide skeleton. The peak at 1730 cm\u22121 is related to the vibration of C=C groups of alkenes and C=O from the acetyl groups. The peaks at 3600\u20133200 cm\u22121 are attributed to hydroxyl (\u2013OH) and amine (\u2013NH) groups and at 2950\u20132800 cm\u22121 to methyl (\u2013CH) groups. The band at 3230 cm\u22121 is relevant to the standard absorption band of the amido group (HN=O). Bands at the 1650\u20131200 cm\u22121 region could be characteristic to the amide I\u2013III bands of polypeptide/proteins [The interaction of metal ions with yeast biomass was also investigated by the FTIR technique. FTIR spectrum of control biomass  biomass  shows abThe analyzed effluent contained Zn(II), Sr(II), Cu(II), Sr(II), and Ba(II) ions in different concentrations, and the dominant element was Zn(II). The concentrations of other metal ions in the effluent were significantly lower and were not considered in further discussion. To study the effect of pH on metal removal from the effluent, the pH of the effluent was changed from 2.0 to 6.0. According to data presented in Lower Zn(II) removal in comparison with the batch system can be explained by its higher concentration in the effluent and presence of other metal ions in solution. The same pattern was noticed for Ba(II) and Sr(II) ions, whose removal grew with the increase of pH of the effluent. Yeast removal efficiency toward Ni(II) ions was very high; at pH \u2265 4.0, it was completely removed from the solution. The pH 3.0 was optimal for Cu(II) removal. To reduce Zn(II) concentration, the effect of sorbent concentration on Zn(II) removal efficiency was investigated in the subsequent scheme with the addition of new sorbent to the effluent. In the first stage, the concentration of sorbent varied from 10 to 40 g/L. The increase of sorbent concentration led to an increase of yeast removal capacity from 44 to 72% a.To increase Zn(II) removal to effluent obtained after its interaction with sorbent in concentrations of 20\u201340 g/L, at the first stage, the new sorbent was added in concentration 1.0 or 10 g/L b. The adS. cerevisiae biomass. In Reference [S. cerevisiae showed higher affinity for Cu(II) ions, while Zn(II) and Ni(II) removal in the ternary system was reduced in comparison with single-element solutions. The effect of different parameters on metal removal from the batch system was investigated. The pH of the solution has a significant impact on metal removal by biological sorbents. The maximum sorption of metal ions present in the studied systems occurred at a pH range of 3.0\u20136.0. The removal of Zn(II) from the analyzed systems lay in the range of 85\u2013100%, and the highest efficiency of adsorption was achieved in the Zn(II) system. The addition of other metal ions in the analyzed systems resulted in the slight decline of Zn(II) removal by S. cerevisiae led to a decrease of potassium and magnesium content in biomass [Sargassum filipendula [The different pH binding profiles for the heavy metal ions present in the analyzed system can be due to the participation of different groups in metal capture, as well as different concentrations of metal ions present in the analyzed systems. For example, high sorption of Cu(II) at pH 3.0 can be connected with the participation of amino groups, besides carboxyl and hydroxyl groups in Cu(II) capture . An impo biomass . Calciumipendula . 82Pb > 28Ni > 24Cr.Differences in metal uptake can also be associated with the metal chemistry and the properties of biosorbent. In Reference , it was Candida rugosa biofilm showed maximum Zn(II) removal of 44%, whereas Cryptococcus laurentii biofilm removed 37% of Zn(II) [2+ and Zn2+ were effectively removed from aqueous solutions by S. cerevisiae crosslinked with formaldehyde at pH 5.3 and 6.0, respectively [At pH 2, for all analyzed systems, metals biosorption was insignificant; this can be explained by a high concentration of protons, which compete with metal cations for binding sites ,5. With ectively . Optimalectively . S. cerevisiae increased sharply in 30 min and then reached the equilibrium [2+, Ag+, Cu2+, Zn2+, Co2+, and Sr2+ by yeast took place in 10 min of contact, and it took 1 h for Cs+. The removal efficiencies of S. cerevisiae biomass toward Pb2+, Ag+, Cu2+, Zn2+, Co2+, Sr2+, and Cs+ was very different and varied from 9 to 80% [Kinetic studies are important in biosorption experiments since they allow us to determine the contact time required for maximum removal of metals from the effluents . The bioilibrium . The sor9 to 80% . SSE   showed t2+-binding sites on the cell surface [2+, Ag+, Cu2+, Zn2+, Co2+, Sr2+, and Cs+ sorption onto waste S. cerevisiae biomass [S. cerevisiae [Metal binding to highly heterogeneous systems can occur concurrently by several mechanism . Since t surface . In diff biomass . The pserevisiae . S. cerevisiae for Zn(II) ions was almost the same in all analyzed systems, while the sorption of other metal ions did not depend on Zn(II) concentration in solution. The metal uptake capacities of the Saccharomyces cerevisiae crosslinked with formaldehyde for Cu2+, Zn2+, and Cd2+ were found to be 8.0, 7.1, and 14.0 mg/g, respectively [S. cerevisiae follows an intermediate behavior between mono- and a multilayer adsorption mechanism [The equilibrium of sorption is of great importance for understanding the sorption process . The incectively . In Zn biosorption was not fully covered [2+, Ag+, Cu2+, Zn2+, Co2+, Sr2+, and Cs+ biosorption by yeast biomass [In all analyzed systems, the  covered . The Lan biomass . Both th biomass .S. cerevisiae showed that an increase of S. cerevisiae uptake capacity at the temperature range of 15\u201325 \u00b0C and its decrease with rise of the temperature up to 40 \u00b0C, probably due to the damage of active binding sites in the biomass. According to the data presented in G\u00b0 between 0 and 20 kJ/mol point out that the adsorption process is physical sorption [H\u00b0 was less than 25 kJ/mol, sorption can be considered as physical [S\u00b0 values obtained for all elements in analyzed systems indicate the increased randomness at the solid/liquid interface during the sorption of metal ions onto the yeast cells, as well as its affinity to analyzed metal ions [-1 of bands corresponding to OH, C=C, C=O, C\u2013H, C\u2013N, and NH groups indicates their involvement in metal ions\u2019 binding. According to data obtained by NAA, sodium content in biomass loaded with metal ions decreased 1.9\u20135.5 times in comparison with the control, of potassium 1.3\u20132.9 times, and of calcium 1.1\u20131.7 times, thus indicating their participation in the ion-exchange process.The increase of the temperature from 20 to \u221250 \u00b0C did not significantly affect metal adsorption in analyzed systems, as its increase or decrease was not more than 20%. Ozer and Ozer  studyingsorption . Positivphysical . Positivtal ions . In the The pH and biomass dosages, as well as their combination, are dominant factors affecting the removal of metal ions from industrial effluents . As in tS. cerevisiae was used for metal removal from four synthetic and one real zinc-containing effluents. The optimal pH for metal removal lay in the range 3.0\u20136.0 and allowed removal of 45\u2013100% of metal from the solution, depending on the analyzed system and metal type. The increase of Zn(II) concentration in solution did not affect the sorption of other metal ions present in analyzed systems. Langmuir and Freundlich isotherm models was suitable to describe Zn(II) biosorption with mq values in the range of 8.99\u201317 mg/g. By evaluating the coefficients of determination of every kinetic model, it is seen that the adsorption of metals ions onto yeast biomass can be described by the pseudo-first, pseudo-second-order, and Elovich models. Surface adsorption, chemisorption, and ion exchange can be considered as the main mechanisms of metal removal from solution. OH, C=C, C=O, C\u2013H, C\u2013N, and NH groups are involved in the biosorption of metal ions. Varying pH at sorbent concentration of 10 g/L it was possible to reduce the concentration of Cu(II), Ba(II), Sr(II), and Ni(II) below maximum admissible level. The highest efficiency of removal of Zn(II) ions from complex real effluent was achieved in a two-stage subsequent system, with the addition of new biosorbent: 20 g/L of biosorbent at the first stage and 10 g/L of sorbent on the second stage, at pH 6.0 and contact time 60 min. Yeast S. cerevisiae showed to be an excellent candidate for the treatment of zinc-containing complex effluents.Biomass of yeast"}
+{"text": "The equilibrium time was reached following 30 min and the maximum adsorption capacities (mg/g), based on Langmuir equation were 10.31 and 8.74 for Pb (II) and Zn (II), respectively. By increasing the initial metal ions concentration, the adsorption efficiencies were decreased and adsorption capacity of RE increased with an increase in the initial pH.This study was carried out to investigate Pb (II) and Zn (II) removal from aqueous solutions by Red Earth (RE) as a new local natural adsorbent in using the batch method. The chemical structure of RE adsorbent was characterized by XRF. Giles, Langmuir, and Freundlich isotherms were used to describe the adsorption data. The effect of metals concentration, initial pH, adsorbent dosage, and agitation time were studied. The results showed that RE contains of SiO Specification TableValue of Protocol2 (58 %), Al2O3 (15.2 %) as major compounds.The adsorbent of this series of tests was a local natural RE from Goud-E-Ahmar region in Kerman province located in the south east part of Iran. It was used for removal of Pb (II) and Zn (II) Ions from aqueous solutions as a new local natural adsorbent. First, RE was crushed using jaw crusher, then sieved and particles less than 50 mesh were selected for tests. Also, the adsorbent was dried for two weeks in the laboratory temperature ,2. Chemi3 with concentration of 1.0 N were used. Batch experiments were applied for Pb(II) and Zn(II) ions adsorption from aqueous solutions, as a single metal system. In this study, the following conditions were constant in all tests; temperature (27 \u00b1 1 \u00b0C), particle size (50 mesh), solution volume (50 mL), and shaking rate (150 rpm). For determining metals ions concentration in all solutions, atomic absorption spectroscopy (AAS) methods were used (Varian AA-975 and AA-1275 models).Solutions and reagent used in this study were all analytical grade reagents which were prepared from E-Merck (with \u226599 % purity). Heavy metal solutions were prepared from nitrate salts at a concentration of 1000 mg/L as a stock solution. Stock solution was stored in bottles with tight lid and other required solutions were prepared from the solution. For measuring and adjusting pH, the YSI portable  and NaOH and HNOThe percentage of metal ions removal by RE was calculated by the following equation Eq. :(1)%RE=CThe adsorption capacity of RE was calculated by applying the mass balance equation Eq. :(2)qe=COe is amount of metal ions absorbed on RE (mg/g); C0 and Ce are the initial and equilibrium concentration (mg/L) of Pb (II) and Zn (II) ions, respectively; V is the volume of meal ions solution (L) and m is mass of adsorbent used (g) [In which, qused (g) .2+ and Pb2+ on RE were fitted by Freundlich , based on Langmuir equation were 10.31 and 8.74 for Pb2+ and Zn2+, respectively  and Zn(II) ions from aqueous solutions by RE is shown in For determining metals concentration, Pb(II) and Zn(II) ions were investigated by adding 0.5 g of RE with 50 ml of meal ions of Pb(II) and Zn(II) solutions at concentrations of 10, 30, 50, 75, and 100 mg/L. The pH of solutions was adjusted in 3.0 \u00b1 0.1 with NaOH and HNOThe dosage of RE adsorbent varied from 4\u221224 g/L with a constant concentration of Pb(II) and Zn(II) ions (50 mg/L). Then solution volume (50 mL) mixed in 300 mL flat bottom Erlenmeyer flasks was moved to rotary shaker and shaken for 300 min with speed of 150 rpm. The suspensions were filtered using Wathman filter paper and then metal ions concentration in filtrate was determined as the amount of adsorbent. The effect of adsorbent dosage is shown in + ions are predominant and compete with metals ions uptake by active sites on the adsorbent. Consequently, the removal of Pb(II) and Zn(II) ions from solutions by RE is less. By increasing pH the amount of H+ ions decreases and then the competition between H+ ions and metal ions for adsorption sites decreases. Thus, removal of Pb (II) and Zn (II) ions by RE increased [For determining the pH effect on adsorption capacity of Pb(II) and Zn(II) ions from solutions by RE, first 50 ml of Pb (II) and Zn (II) solutions (50 ppm) was mixed with 0.5 g of RE adsorbent and then the pH of solutions was adjusted in the range of 1.75\u20135.5 to prevent chemical precipitation phenomena and to guarantee that removal of heavy metal ions are essentially due to the adsorption method, pH was selected below 7.0 level). The effect of initial pH of solutions is shown in ncreased ,9. In bencreased .Fig. 4Ef50 ml of Pb (II) and Zn (II) solutions (50 ppm) were mixed with 0.5 g of RE adsorbent. The pH of solutions was adjusted at 3.0 \u00b1 0.1. Then the suspension was moved in rotary shaker and shaken for 1\u2212300 min at speed of 150 rpm. The suspensions were then filtered using Whatman filter paper and metals concentration in filtrate was determined as the amount of adsorbent."}
+{"text": "Society depends on access to water, electricity, materials, and fuels, and these infrastructure networks are uniquely interlinked. Given this interconnection, it is important that experts in each area, often coming from very different disciplinary backgrounds, consider possible interdependencies. Three assistant professors at New York University's Tandon School of Engineering (NYU Tandon)\u2014environmental engineer Andrea Silverman, electrical engineer Yury Dvorkin, and chemical engineer Miguel Modestino\u2014are combining their distinct expertise to tackle complex societal problems at the intersection of their fields in a way that is often precluded by unique language and conventions used in each of their disciplines. Below, they share their story about building their collaborations, finding a common language, and making strides as early career pioneers of interdisciplinary research.Prof. Yury Dvorkin, Prof. Miguel Modestino, and Prof. Andrea Silverman, all assistant professors at NYU Tandon, have undertaken interdisciplinary research and collaborations early in their careers to engage students, seize funding opportunities, and try to tackle real-world problems facing engineers.Engineering in Service to Society. While our collaborations were meaningful to us personally, they were also supported by our institution.We all started as assistant professors at NYU Tandon within a year of each other. In starting our careers as faculty members, we were individually interested in including interdisciplinary projects in our research programs, given that distinct, yet complementary, skillsets are needed to tackle complex societal challenges. NYU Tandon emphasizes and prioritizes collaborative work and promotes interaction between faculty members across its departments. It was during school-sponsored junior faculty gatherings and small group lunches that we first met and built connections. It was at a multidisciplinary research seminar highlighting new faculty where we learned more deeply about each other's research programs. Additionally, NYU Tandon has a motto that we do A large language barrier exists between chemical or environmental engineers, who describe transformation of matter in the form of chemical reactions, and electrical engineers, who use complex mathematical formulations to describe the interaction of the electricity network with its physical components.This gap meant that significant effort and time was needed to learn each other's language, which often involved writing and editing proposals on a shared document, brainstorming ideas on the board, and going through multiple iterations to establish a common language between the fields. For example, we had to work together to translate the behavior of electrosynthetic reactors into mathematical formulations that could be embedded into power grid optimization models. Ironically, the developed reactor formulations belong to a different class of mathematical optimization models than grid models and, therefore, the team needed to develop a surrogate model that would allow for jointly optimizing the storage reactors and grid. As a result, the surrogate models were trained by experimental data that correlated the performance of the electrochemical devices with the inputs that they receive from the power grids. These models then could be used to identify the optimal scheduling of the operation of the reactors to maximize their profitability when integrated with a real power grid.During the collaboration, although vocabulary was a hurdle to overcome between chemical and electrical engineering, we found it interesting that chemical and environmental engineering use a similar vocabulary to discuss fundamental principles shared by the two fields, such as mass and energy balances, reaction kinetics, and reactor designs. Experimentally, both chemical and environmental engineering can involve material synthesis, degradation, or transformation. However, we found that the target compounds and solutions that both fields work with, and the way they are described, can differ dramatically between the two fields.For example, chemical engineers tend to apply the fundamental principles described above to applications where experimental conditions are controlled and well characterized . Alternatively, environmental engineers apply the same principles to more complex, heterogeneous matrices, such as domestic wastewater, sewage sludge, and environmental water samples, including water and sediment from lakes, rivers, wetlands, and subsurface and marine environments. These uncontrolled solutions can vary greatly between locations and over time, which can present additional challenges in designing reactors and evaluating their performance. Furthermore, environmentally relevant abundances of target compounds can be much lower than what are typically found in chemical processes but can still present risks to environmental quality or public health.Silverman (left), Nicole Schnable (center), and Modestino (right), after Schnable's master's defense on the development of photocatalytic gels for water treatment. Silverman and Modestino were part of her thesis committee, and the work led to their joint publication.convincing research proposals that are attractive to external sponsors. Among the many challenges that such seed grants help overcome is the development of a common language to describe the research that is accessible for a broad audience and is capable of convincing non-domain experts that are solicited to evaluate the intellectual and real-life impacts of the proposed work.For scientists, especially early in their careers, it can be perilous to significantly depart from their core disciplines, as it diverts time and resources from advancing their primary research goals. However, the leadership of NYU Tandon and the National Science Foundation, the primary federal sponsor for foundational multi-disciplinary research, have both recognized the importance of supporting faculty who are willing to jump across disciplinary boundaries. For example, NYU offers seed grants to develop robust, technically meritorious, and In working together, we became keenly aware that different fields have different expectations about publishing, as well as different timelines needed to produce data or findings. For instance, journals in systems engineering and operations research that accept publications on power grid models and algorithms tend to favor theoretical developments with a simulated application motivated by a larger, real-life challenge. These papers rarely report on specifics of real-life applications and rather envision their future development. On the other hand, there are journals that not only support publications that are well grounded in theory but also validate their results via physical experiments rather than simulations. Naturally, these journals vary not only in the scope of research that they cover but also in the way they process submission , which is incredibly important for PhD students, postdocs, and all early career scientists .Reaction Chemistry & Engineering (https://pubs.rsc.org/en/content/articlelanding/2020/RE/C9RE00456D#!divAbstract). In the future, we plan to submit ongoing work targeted at removal of specific contaminants to journals more closely aligned with environmental engineering research.In our experience publishing work at the interface between chemical and environmental engineering, we came to realize that many chemical engineering journals are more focused on material or reactor design, whereas environmental engineering journals are more focused on the use of these materials and reactors to remove or degrade specific environmental contaminants. Given that our collaborative project was a proof-of-concept study for the development of a new material in photocatalytic reactors for water treatment, we decided to publish in the more chemical engineering-focused journal Joule, Nature Energy, Energy & Environmental Science) that are receptive to both system-level power grid simulation studies and laboratory experiments typical for chemical engineering. However, although journals may support interdisciplinary research, it has become evident that reviewers from one specific field are not always familiar with the scope of research across different fields and this complicates the evaluation of the potential impact of the work. We anticipate that this situation will change in the future, as more investigators are embracing interdisciplinary research and as the value that it brings starts to become more evident. In addition to peer-reviewed publications, working at the interface of different fields often leads to inventions and thus patents are very important. As an example, our team has worked on grid-integrated electrosynthetic hydrogen generators , which serve as building blocks of a larger vision to synergistically integrate water-electricity-chemical networks.Similarly, at the interface of electrical and chemical engineering, we have identified broad energy research journals , Dvorkin's PhD student, and Daniel Frey (left), Modestino's PhD student, showcased the results of the collaboration between their labs at NYU Tandon's research expo.Finding solutions to complex societal challenges requires interdisciplinary work. We have seen that students trained in interdisciplinary environments develop a broad skillset, have a better understanding of the context and impact of their work, can effectively communicate with colleagues from different fields, and are well prepared to contribute to teams tackling real-world problems in both industry and academia. Interdisciplinary projects involve exploration of new spaces at the interface between different fields, which can inspire students to pursue entrepreneurship opportunities that they identify at this intersection. From our experiences, achieving these benefits is possible if the deep domain-specific knowledge that comes while doing PhD in a given field is not sacrificed but instead augmented with broad interdisciplinary training."}
+{"text": "Patients in a Minimally Conscious State (MCS) constitute a subgroup of awareness impaired patients who show minimal signs of awareness as opposed to patients in a Vegetative State who do not exhibit any such signs. While the empirical literature is rich in studies investigating either overt or covert signs of awareness in such patients the question of self-awareness has only scarcely been addressed. Even in the occasion where self-awareness is concerned, it is only higher-order or reflective self-awareness that is the target of such investigations. In the first part of this paper, I briefly review the relevant clinical neuroscience literature to demonstrate that the conception of self-awareness at play in such studies is indeed that of reflective self-awareness. In the second part, I present the philosophical notion of pre-reflective  self-awareness. This is shown to primarily refer to the implicit awareness of our embodied subjectivity which essentially permeates all our experiences. As discussed, this minimal self-awareness is not specifically addressed when clinically or experimentally assessing patients in MCS. My suggestion is that neuroimaging studies targeting minimal self-awareness as in First-Person Perspective-taking paradigms could be used with MCS patients to shed light on the question of whether those individuals are minimally self-aware even in the case where they lack self-reflective abilities. Empirical evidence of this kind could have important theoretical implications for the discussion about the notion of self-awareness but also potential medical and social/legal implications for awareness impaired patients\u2019 management. And in an apparent association to the above because of the similar terms involved, in clinical neuroscience, there is a seemingly relevant and thriving discussion about some pathological subjects being in a Minimally Conscious State (MCS) after recovering from severe brain damage.According to a recent discussion in the area of Philosophy of Mind and Phenomenological Philosophy, our psychological states are characterized by an inherent self-awareness considered to be a constitutive part of our experiences. This implicit awareness of the self seems to be necessary for the very existence of subjective states in normal subjects . It is oIn this paper, I intend to query about the following: Should we trace the difference between patients in MCS \u2013 who are minimally capable of awareness \u2013 and patients in Vegetative State (VS) \u2013 who are considered utterly unaware \u2013 back to the former\u2019s exclusively possessing PRSA? My interest is to try to make sense of what is it that clinicians and neuroscientists mean (or should mean) when describing a patient in MCS as being minimally conscious and on the other hand to examine whether the neuroscientific literature on the topic can shed any light on the philosophical debate about self-awareness. To pursue this double interest I will first make a short presentation of the recent literature around the so-called disorders of consciousness (DOC) and then give a brief account of the philosophical notion of minimal self-awareness. I will conclude this paper with a discussion about the respective concepts of minimal awareness in these two fields and their potential interrelatedness.[c]onsciousness is the state of full awareness of the self and one\u2019s relationship to the environment\u201d and it \u201chas two major components: content and arousal\u201d . To better highlight this state as opposed to the case where subjects are indeed capable of awareness these patients are now characterized as not manifesting voluntary behavior. This voluntary behavior is the sign the examiner looks for as evidence of their being aware of themselves and their environment.A few years later, a condition of severely altered consciousness in which minimal but definite behavioral evidence of self or environmental awareness is demonstrated\u201d  or covertly (as indicated by objective studies such as neuroimaging).\u2022A patient is considered to be Minimally Conscious on the other hand if he does show behavioral responses considered to be voluntary and the various clinical assessment tools are designed in such a manner as to pick up these responses.\u2022A totally unresponsive patient, one who shows no voluntary behavior is not to be considered Vegetative unless evidence of covert awareness is ruled out by objective means .voluntary behavior, as this occurs in response to comprehension of aspects of the environment presented by sensory means , that is mostly considered indicative of awareness.But what kind of concept of awareness does clinical neuroscience presuppose when such an awareness is demonstrated by clinicians and laboratory neuroscientists to (overtly or covertly) occur? As we saw, clinical neuroscientists have the notion of two broad kinds of awareness. Self-awareness and awareness of one\u2019s environment. Awareness of one\u2019s environment should be regarded here in quite broad terms involving not only the perceptual comprehension of the space around one, but also understanding of the potential practical use of objects, understanding of the actions of other subjects, mindreading their intentions, language comprehension etc. And according to the more narrow clinical diagnostic setting presented above, it is But what about self-awareness? Does the clinical neuroscience of DOCs as presented in the recent literature on the matter take into consideration this type of awareness? And if it does, what concept of it does it possess? Even if it doesn\u2019t explicitly take into consideration self-awareness what concept of self-awareness does it implicitly presuppose?This is the kind of questions I want to raise in this section in an attempt to force into relief the conception of self-awareness at play in DOC studies. In the next section I will juxtapose this to the notion of minimal self-awareness as presented in the contemporary philosophical literature on self-awareness.I\u2019ll begin by briefly discussing the JFK CRS-R clinical scale because it is widely used among clinicians and because it represents how clinicians expect awareness to manifest in a psychological subject. In the next subsections I will also briefly focus on neuroimaging and neurophysiological studies explicitly designed to present covert awareness.2.This behavioral diagnostic tool is divided into six parts each of which quantifies patient responses to different stimuli\u2022There is the Auditory Function Scale which monitors motor responses to sound. What is considered indicative of  awareness here is whether there is a reproducible motor response to verbal commands. This presupposes that the patient has regained the capacity to understand speech and is at least neurologically able to attempt an appropriate motor response.\u2022The Visual Function Scale monitors responses to visual information and what counts as evidence of awareness is the fixation of gaze or visual pursuit of the subjects\u2019 own-face as seen by him in a mirror. Additionally reaching for seen objects and signs of object recognition is regarded as evidence of environmental awareness.\u2022In the Motor Function Scale, it is the localization to pain and object manipulation.\u2022In the [Oromotor] Verbal Function Scale it is intelligible verbalization as opposed to incoherent vocalizations.\u2022In the Communication Scale is evidence of attempted intentional communication  and the last part of CRS-R detects the level of Arousal by evaluating whether the subject can be awakened and maintain his attention to sensory stimuli.3, when appearing to experience bodily pain (by reacting with an avoidance response), when he tries to communicate and when he shows recognition of his own-face in the mirror.So according to this clinical scale, a patient with impaired consciousness is considered to be minimally conscious when consistently exhibiting appropriate behavioral responses to verbal commands, when demonstrating practical understanding of seen objects (affordances)4.Importantly, self-awareness can be ascribed to this subject by his appropriate response to visual self-referential stimuli, in this case by showing signs of recognition of his own-face. I say this is important because it indicates that the conception of self-awareness at work in the MCS related clinical neuroscience literature is this type of higher-order, thematic self-recognition which is not the same as the philosophical conception of minimal or PRSA that we will tackle laterCurrent cortical functional anatomy models distinguish unimodal, primary information processing areas for each sensory modality from multimodal association areas where higher-order or conceptual information is processed. So the idea was that patients in VS who by definition possess no capacity for awareness would not show significant activation in the association areas . And thaislands of function,\u201d inadequate of supporting actual awareness. That is, the case might be that there is merely a disjoint activation of these brain areas which in the normal case are involved in voluntary imagination tasks in a functionally coherent and non-automatic manner. Responding to this objection the authors of the aforementioned study insisted that since \u201cthe observed activity was not transient, but rather persisted for the full 30s of each imagery task\u201d these \u201ctemporally sustained [fMRI.] activations are impossible to explain in terms of automatic responses\u201d (2013: 118). And indeed it makes good sense to think that a temporally sustained activation that lasts for as long as the prompt to perform an imagery task specifies, and the subsequent change of the pattern of activation at the time the prompt for a new task is given, must be indicative of an alert and comprehending subject capable of having intentions and conscious attention shifting.5One objection raised to interpreting these results as evidence of awareness was that these patterns of brain activation might only be indicative of unconscious processing of auditory stimuli by higher-order regions , functioSo what counts as evidence of awareness in these studies is for the subject to perform a specific imagery act when specifically asked to, a type of voluntary act which resonates with the volitional component which the clinical scale focuses on. The content of the imagery act is a representation of a real-life motor act and the method of detecting this is indirect by neuroimaging.myself acting as such and this presupposes a self embedded in the experience. But the psychological act of imagining that I perform a motor action does not specifically address this embedded self as, for instance, the case is with own-face recognition. The subject in this latter case intends and mentally grasps his own face as his own. Rather, the aforementioned fMRI imagery task addresses neural correlates of such phenomenological data as, say, the very ability of the subject to imagine an action and the action imagined  including the intentional objects involved . It does not specifically address an intended attribute of the subject\u2019s self. In this respect, the currently published neuroimaging studies about DOCs do not seem to directly address self-awareness.Is this a case of addressing self-awareness? In the case where I imagine navigating through the rooms of my house or in imagining playing tennis or any similar action I presumably imagine has been addressed in this occasion. For instance, in various Event-Related Potentials (ERP) studies the so-called P300 wave is detected when attention is grasped on auditory stimuli of interest  and not of one focusing on implicit self-awareness 7.The presence of the P300 wave, in this last setting of self-referential stimulus presentation, is considered by the relevant literature as indicative of own-name recognition in healthy subjects. In DOC patients, though this conclusion is not that straightforward since a number of VS diagnosed patients also seem to present the P300 activation when presented with self-referential stimuliWe now move to a brief presentation of the concept of minimal self-awareness as it manifests in recent discussions in the philosophical literature of self-awareness. I cannot and do not intend to cover here the quite extensive modern literature on the matter of self-awareness from six types of representation about self-awareness\u201d following distinctions previously made by In a recent review article about self-awareness in patients with impaired consciousness by an influential research team, own-face and own-name recognition were presented to be the only types of self-referential stimuli used in functional studies with patients with DOC in agreement with our presentation above . In thei(1)Self-Consciousness as the embarrassment of myself in the presence of others, the colloquial use(2)Self-Consciousness as \u201cself-detection\u201d in the sense of my bodily self-awareness in proprioception, interoception, and awareness of motor agency(3)Self-consciousness as \u201cself-monitoring,\u201d the ability to reflectively grasp my-self as a practical agent in action recall and anticipatory motor planning(4)Self-consciousness as \u201cself-recognition,\u201d as in mirror self-recognition(5)Self-Consciousness as \u201cawareness of awareness,\u201d as the awareness of myself being a subject of beliefs and intentions, a \u201ctheory of mind\u201d consciousness(6)the capacity to relive our past in the form of \u2018mental time-travel\u201d\u2019 (Ibid: 5)Self-Consciousness as awareness of myself as the hero of my personal narrative based in part to \u201cimplicitly aware of my body as the experiential center of my perceptual and practical engagement with the world. Bodily self-awareness is of an altogether different type from the other types mentioned by Zeman in that all of those require me to possess a self-representation. I need to have myself as an intentional object  or to reflectively represent myself as such in order to be able to monitor myself in time, to recognize a face in the mirror as my own or to speak about myself as a diachronic entity in a personal narrative. Contrary to all that, bodily self-awareness as PRSA does not require a representation of myself, rather it is a tacit awareness of myself as subject, the embodied subject of my first personal experience.Let us focus on the 2nd type in Zeman\u2019s taxonomy, that of bodily self-awareness. This type of self-awareness is the awareness of myself as the particular embodied subject of experience that I am (and each one of us is), it refers to the fact that I am at any moment of my conscious life is precisely taken to differ from reflective self-consciousness by being an intrinsic non-objectifying form of self-acquaintance\u201d (2017: 4). Similarly, Gallagher distinguishes minimal selfhood from narrative selfhood. While the first term refers to \u201ca consciousness of oneself as an immediate subject of experience,\u201d the second term refers to the ability to speak of my life story in a thematic grasping of myself as a single individual persisting in long term time  a patient in MCS in considered minimally aware because he shows minimal signs of overt or covert voluntary responses to verbal commands or other sensory stimuli as evidenced in CRS-R and neuroimaging studies. As for self-awareness is concerned, we saw that it is only higher-order self-awareness that this domain focuses on in the few cases that it does. So similarly, such a patient is considered minimally self-conscious when he presents minimal signs of this higher-order self-awareness. But surely, what does the term \u201cminimal awareness\u201d refer to here?minimal awareness is quantitative as it refers to the presence in a subject of a minimum number of voluntary behavior traits. And so is the clinical concept of minimal self-awareness. That is, it does not amount to a qualitative difference in self-awareness  but to a difference in the quantity of self-awareness traits overtly of covertly present in patients with DOC. In other words: According to clinical neuroscience, patients in MCS are minimally aware because they possess a minimal quantity of awareness traits (signs of voluntary behavior etc.) and they are minimally self-aware because they possess a minimal quantity of higher-order self-awareness . Or, a patient in MCS is minimally self-aware because he manifests in less instances higher-order self-awareness traits than a normal subject does  has less functional disability and should be characterized as being in MCS+ .Do patients in MCS, being minimally capable of awareness, differ from patients in VS because the first possess PRSA? If we take this question to be asked from the aspect of clinical neuroscience and its current concept of self-awareness the answer is negative. From that aspect, a patient in MCS is minimally self-conscious because he possesses a quantitatively minimal capacity of higher-order self-grasping. The concept of PRSA is irrelevant (yet) to this research field.In light of the above thoughts, we can now return to our initial question (page 2): but additionally because they become pre-reflectively self-aware when emerging from coma or VS? How should we empirically proceed to explore such a scenario? And what would this potential patient\u2019s possession of PRSA mean for theoretical approaches such as the experiential minimalist one?But if, after clearing our view with our previous analysis, we consider this question anew it is actually a significant question to ask. Indeed, how does the situation stand with PRSA in awareness impaired patients? Is the patient in MCS minimally aware not only because he manifests a minimal number of awareness (and self-awareness) traits For instance, consider the case where an awareness impaired person might be pre-reflectively self-aware without actually exhibiting any explicit  or implicit (neuroimaging) signs of reflective self-awareness. This question is important both from a theoretical and an empirical point of view. It would be theoretically rewarding to gain empirical evidence from studies of MCS about the alleged presence of minimal self-awareness without the additional presence of any higher-order reflective capacities. This might give support to the view of the proponents of experiential minimalism that a reflective grasping of the self is not a necessary condition for awareness and also against the eliminativist view that no self-awareness figures necessarily in our experiences.essentially transform our self-conscious life and whose loss in brain damage renders a person completely unable of being self-aware. If that was the case then, when a patient with impaired awareness lacked the capacity to reflectively grasp oneself then this person would also lack the capacity to be pre-reflectively self-aware.But on the other hand, perhaps PRSA is impossible without at least the potentiality for reflective self-awareness, that is, perhaps in us human beings, self-awareness is only possible when we have already developed our reflective capacities in early childhood. This could be the case even though we might maintain the position that a psychologically normal adult has to be already pre-reflectively self-aware to be able to grasp one-self reflectively. This brings to mind the transformative rather than the additive views of rationality  in the a9.Additionally, it would be empirically useful to demonstrate that a person with impaired awareness can be self-aware even if he lacks higher-order reflective capacities. This information would potentially have important scientific repercussions because it could help embed the notion of minimal selfhood into the neuroscientific models of consciousness influencing any future experimental methodology. And information about the presence of minimal self-awareness in reflectively incapacitated patients will have at least some effect on these patients\u2019 medical management and potentially ethical/legal implications about end-of-life decision makingWith these matters in mind we now turn to the last section of the paper. It is composed of two subsections: A penultimate part where we take up our original title question to discuss the potential importance, the presence of PRSA in patients in MCS might have as empirical evidence, to experiential minimalism and a final part where we propose a way to test PRSA in such patients.10 of mineness Zahavi asks: \u201cIf it is the case that our experiences are accompanied by a sense of self, is it then something that holds with necessity, such that it characterizes all experiences, however primitive or disordered they might be? Is it something that only holds for normal, adult, experiences? Or might it be something that only holds under rather special circumstances, say, when we reflectively scrutinize and appropriate our experiences?\u201d .In his discussion of psychopathology and depersonalization in schizophrenics, in the same article, Zahavi seems to argue that even in those extreme cases where one\u2019s experiences are presented to him as not his own \u201cbecoming aware. Again, the question is whether this primitive awareness that this patient awakens into  involve a pre-reflective awareness of herself. We have seen that the current empirical studies and diagnostic tools are structured in such a manner as to evaluate reflective self-awareness as in own-face and own-name recognition. Being so they do not offer empirical evidence as to whether a PRSA is involved.But let\u2019s ponder a little about the case of the newly diagnosed patient with MCS. This is someone who has previously been completely unresponsive (coma) and who has just acquired some primitive form of awareness. In the typical case, she merely tries to grasp your hand when you pinch her, unsuccessfully attempts to respond to your spoken urges to move a finger, sluggishly fixates on familiar faces or on her own when presented in the mirror or turning toward the sound of her own name. These are individuals who usually cannot communicate, do not show complex semantic understanding of objects of common usage presented to them, and who sometimes grasp but cannot manipulate those objects in a practically rational manner. We can, therefore, imagine the patient in MCS as someone who completely lacked awareness previously (and consequently self-awareness) and who is in the process of a limit case the investigation of which could shed light on the question about what-it-is-like for someone who lacks higher-order conceptual abilities to be nevertheless aware. This would presumably be an occasion of pure self-awareness uncontaminated, so to say, by reflective acts, a chance to scientifically investigate the thin distinction between minimal self-awareness and reflectively laden self-awareness from pathology. Would this limit case of non-reflective self-awareness from pathology be a case of minimal self-awareness as Zahavi or others might suggest?In distinction with schizophrenia where higher-order cognitive abilities are usually preserved, I believe the suggestion can be made that MCS represents So to further narrow our investigation as to its theoretical aspect, I believe the inquiry about PRSA in MCS is vital in clarifying whether either or both of the following two proposals stand:(1)That the self-awareness involved in having experiences is not an occurrence of an actual reflective grasping of those experiences and,(2)That the self-awareness involved in having experiences does not require an overall ability to reflect.actual self-reflection. It refers to actual self-reflection as not being a necessary condition for self-awareness. Actual here meaning a reflective ability presently occurring in contradistinction to a reflective ability dormant at the time but with the potential, the capacity to occur. The second proposal, which to my knowledge is not specifically discussed as a topic in the relevant theoretical or empirical literature, is that this embodied subjectivity can figure in experience even in the limit case where one does not absolutely have the capacity to reflect or conceptualize as maybe the case is with young infants and a subgroup of patients in MCS without reflective abilities. It refers to the possession of PRSA even when there is no potentiality for self-reflection. It refers to the potential for self-reflection as not being a necessary condition for self-awareness12.The first proposal establishes in negative terms that self-awareness is primarily a pre-reflective awareness of the self as an embodied subject of experience and not an occasion of If we now flesh out the above two assumptions with the empirical issue we tackling here, the following two questions will constitute the final incarnation our inquiry takes:\u2022Does the patient in MCS possess PRSA even in the hypothetical case where he is utterly unable to reflect or\u2022Does he possess PRSA only in the case where he is at least able to practice self-reflection?That is, should an awareness impaired patient, who does not exhibit overt or covert responses of reflective self-awareness, considered to be also PRSA possessing? Inversely, should he be considered possessing PRSA only in the case where he exhibits at least some minimal form of reflective capacities?But another important task should also be attended to in consequence: How should the experimental setting be staged so that a pre-reflective aspect be disambiguated from a reflective aspect of self-awareness, in order to be able to test the above distinctive possibilities?I will close this paper with some thoughts about what type of empirical evidence would be instrumental in responding to the above two questions.here\u201d the array of objects that are situated \u201cthere\u201d in my peripersonal space13. And similarly, it is the sense of my embodied \u201chere\u201d when I reach and manipulate objects in motor actions.As we saw, the notion of minimal self-awareness we examine in this paper primarily concerns the implicit and immediate awareness of our embodied subjectivity. A significant characteristic of this is the fact that in our perceptual engagement with our environment, we always have a tacit awareness of our body as the zero perspectival point of orientation in relation to the objects of our perceptual and practical interest. This embodied self of perception is experientially given in a pre-reflective manner whenever we direct our perceptual attention to the spatial objects around us. It is the awareness I have that I am observing from my absolute perspectival \u201c14. These self-reflection tasks, once more, seem to be of limited use to our inquiry about what we might call the neural correlates of PRSA. If we consider the empirical aspect of our investigation here, what we need is to establish an experimental connection between some well-circumscribed aspect(s) of PRSA with a brain area or with an electrophysiological response.Now, if one browses through the currently published neuroscience of self-awareness, there has been empirical interest in recent years in the so-called Cortical Midline Structures (CMS), an extensive area of the medial aspect of the frontal and parietal lobes of cerebral hemispheres. These brain areas show elective and reproducible activity in neuroimaging studies with various self-referential tasks . But aga15. Their assumption that these two mental acts were supported by two different neural processes paid off since different brain regions were consistently activated in each case. More specifically, to cling only on the information presented in that paper that is of interest to us, specific brain areas16 were consistently activated when taking the 1PP. At the same time, other brain areas showed deactivations17.In a 2004 fMRI study, researchers tested the taking of perceptual 1st Person Perspective (1PP) in 11 normal subjects as opposed to them taking the 3rd person perspective, in an attempt to detect the relevant neural correlates during these two psychological stances . To achi18. My suggestion would be: Since some brain regions show consistent activations when normal subjects take the 1PP, then subjects with impaired awareness who show minimal signs of awareness (as patients in MCS do) would show the same activations during similar tasks. Given an experimental paradigm could be developed so that 1PP could be assessed in MCS subjects and given some of those subjects exhibited activation of the relevant brain areas, we would be entitled to infer that those patients are minimally self-aware regardless of the fact that they might exhibit or not exhibit self-reflective abilities on additional testing19. This would be important to see because, on the one hand, it would suggest that these patients in MCS possess PRSA, and on the other, it could help us empirically discern whether this PRSA is present with or without the possession of additional reflective abilities20.Without delving into neuroanatomical details here and whatever the definite relevant brain areas prove to be in future studies, I believe this empirical study constitutes an example of how to evaluate an aspect of minimal self-awareness through neuroimagingexperiential minimalism thesis since it would seem that it is only because self-reflective ability is already available as a potentiality that one is self-aware when one has experiences? That is, would experiential minimalism find experimental support only in the case where 1PP turns out to be detected even in the absence of self-reflective abilities? Or could experiential minimalism rather be compatible with the empirical proof of the presence in a subject of minimal self-awareness only when that subject possesses the capacity for self-reflection?What if these patients exhibited 1PP activations only in the case they also exhibited self-reflective capacities when additionally tested? Wouldn\u2019t that suggest that it is only when someone has the ability to self-reflect that one also possesses PRSA? And would that scenario then announce a blow to the I will leave these important questions open for future discussion. I believe that if this paper has achieved anything was in making an effort to clarify the different notions of minimal awareness as discussed in these distinctive areas of research and practice and to elaborate on a possible way to establish a mutually illuminating link.Minimally Conscious State patients constitute a subgroup of awareness impaired patients who show minimal signs of awareness as opposed to VS patients who do not exhibit any such signs. While the empirical literature is rich in studies investigating either overt or covert signs of awareness in such patients, the question of self-awareness has only been scarcely addressed. Even in the occasion where self-awareness is evaluated, it is only higher-order or reflective self-awareness that is the target of such investigations. In the first part of this paper, I briefly reviewed the relevant clinical neuroscience literature to demonstrate that the conception of self-awareness at play in such studies is that of reflective self-awareness. According to this research area, patients in a MCS are minimally self-aware in the sense that they possess a quantitatively minimal capacity for reflective self-awareness. In the second part, I presented the philosophical notion of pre-reflective  self-awareness. This was shown to primarily refer to the implicit awareness of our embodied subjectivity, which essentially permeates all our experiences. As discussed, this minimal self-awareness is not explicitly addressed when clinically or experimentally assessing patients in a MCS. My suggestion is that neuroimaging studies targeting minimal self-awareness as in First-Person Perspective-taking paradigms might be used with patients in MCS to shed light on the question of whether those individuals are minimally self-aware even in the case where they lack self-reflective abilities.The author confirms being the sole contributor of this work and has approved it for publication.The author declares 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": "Accidents, as the third important cause of mortality, are among main challenges of communities due their costs and irreversible damages. This study aimed to investigate the age distribution of people referred to the hospitals affiliated by Iran\u2019s ministry of health because of accidents.In a cross-sectional study, using a CDC checklist, data related to the injured people who referred to the hospitals affiliated by Iran\u2019s ministry of health in a one year period was gathered from hospital records, as well as interview and observation. The collected data was analyzed by descriptive and analytical statistics in SPSS11 software.The commonest age group involved in accidents during the year 1395 (2016) was middle-aged (37%) followed by youth (25%). The commonest accidents in children were: fall (23%), traffic accidents (19%), and burn (13%). In adolescents, the commonest accidents were: traffic accidents (32%), fall (16%), and violence and poisoning (5%). The commonest accidents in youth were: traffic accidents (35%), violence (9%), and poisoning (6%). In middle-aged group, the commonest accidents were: traffic accidents (30%), fall (12%), and violence and poisoning (7%). The commonest accidents in elderly were: fall and traffic accidents (25%), burn (4%), and violence and poisoning (3%).Making plan and policies to prevent accidents and injuries is a health priority and a key tool for improving the safety of the community. Also, informing the all age groups of the community about preventive actions is necessary.Road Traffic Injury, Trauma, Hospital"}
+{"text": "BAFF\u2212/\u2212 mice displayed an improved response to acute cold challenge, commensurate with the up-regulated expression of thermogenic genes in both brown and subcutaneous adipose tissues. These changes were found to be mediated by both increased M2-like  macrophage activation and enhanced leptin and FGF21 production, which may account for the improving effect of BAFF depletion on insulin resistance. In addition, leptin-deficient mice (ob/ob) showed augmented BAFF signaling concomitant with impaired thermogenic activity, identifying BAFF as a suppressive factor to thermogenesis. Our findings suggest that suppression of BAFF could be a therapeutic approach to attenuate aging-dependent insulin resistance.Impaired glucose tolerance is a common feature associated with human aging, which is caused by defects in insulin secretion, insulin action or both. Recent studies have suggested that B-cell-activating factor (BAFF), a cytokine that modulates proliferation and differentiation of B cells, and its receptors are expressed in mature adipocytes and preadipocytes, proposing BAFF as a potential regulator of energy metabolism. In this study, we show that systemic BAFF depletion improves aging-dependent insulin resistance. In aged (10-month-old) BAFF B-cell-activating factor (BAFF), a member of the tumor necrosis factor (TNF) ligand family, is a cytokine that plays an important role in the proliferation and differentiation of B cells, which has been shown to be a ligand for receptors: BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) . AlthougBrown adipose tissue (BAT) is a thermogenic organ that dissipates energy as heat and is characterized by multilocular lipid droplets, high mitochondrial contents and expression of uncoupling protein 1 (UCP1) . Located\u2212/\u2212) mice. Ten-month-old BAFF\u2212/\u2212 mice showed significantly improved insulin sensitivity despite increased body-weight gain, which was attributed to the elevated thermogenesis and augmented gene expression of M2-like macrophage markers and anti-inflammatory cytokines in subcutaneous and brown adipose tissues. Moreover, BAFF\u2212/\u2212 mice showed enhanced leptin and FGF21 production, which provides another explanation for the improving effect of BAFF depletion on insulin resistance. On the other hand, leptin-deficient (ob/ob) mice exhibited increased BAFF signaling in both SAT and BAT, with significantly impaired expression of UCP1 in BAT. These results demonstrate that BAFF has an attenuating effect on thermogenic capacity and, thereby, BAFF suppression could improve aging-induced insulin resistance by promoting non-shivering thermogenesis.Aging is a degenerative process which is accompanied with functional deterioration in the maintenance of homeostasis . The agi\u2212/\u2212 and WT mice. As shown in \u2212/\u2212 mice showed significantly higher body weight than WT controls, which was associated with commensurate increases in weight of adipose tissues, including SAT, EAT  and BAT (\u2212/\u2212 mice exhibited significantly enhanced glucose tolerance and insulin sensitivity compared to their WT controls (We first examined the consequences of aging on body weight and glucose control in wild-type (WT) mice. Aged (10-month-old) mice showed significantly increased body weight accompanied with impaired glucose and insulin tolerance when compared to young (2-month-old) counterparts, indicating that aging is associated with insulin resistance A\u2013C. Next and BAT E. There  and BAT  and adip and BAT  between controls G,H, indi\u2212/\u2212 and WT mice were exposed to cold (4 \u00b0C) and followed by measurement of rectal temperature with exposure time. In response to the cold challenge, BAFF\u2212/\u2212 mice displayed a strong resistance to acute temperature drop compared to their WT counterparts  exhibit not only obesity-induced insulin resistance but also hypothermia . To examWe next examined signaling molecules connecting BAFF-R to downstream non-canonical NF-\u03baB activation pathway . It was \u2212/\u2212 mice on a high-fat diet, which exhibited improved insulin sensitivity and potentiated adipose tissue function [Several studies have reported positive correlations between plasma BAFF level and metabolic diseases, such as insulin resistance and non-alcoholic fatty liver disease ,4,5,6,7.function . These s\u2212/\u2212 mice compared to WT controls, which was supported by data showing that BAFF depletion caused an attenuated inhibition of PPAR\u03b3 activity and an increased expression of lipogenic genes. In this study, with commensurate increase in the weight of adipose tissues, the weight gain of aged BAFF\u2212/\u2212 mice was higher than that of WT controls, which was not the case for young BAFF\u2212/\u2212 mice . BAFF gene deletion was confirmed by analysis of mRNA and protein expression of BAFF in spleen, epididymal and brown adipose tissue as described previously [BAFFeviously . Mice weMice fasted for 4 h and were sacrificed by cervical dislocation. Tissues of the liver, spleen, inguinal subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT), interscapular brown adipose tissue (iBAT) and quadriceps were harvested, snap-frozen in liquid nitrogen, and stored at \u221270 \u00b0C until processed for RNA and protein analysis. All the experimental protocols were approved by the Committee on the Ethics of Animal Experiments of the Handong Global University .Mice were placed in prechilled cages at 4 \u00b0C with free access to a normal chow diet and water. Core body temperatures were measured at 0, 1, 2, 3, 4, 6 and 8 h during cold exposure using an electronic rectal thermometer Testo 925 .For the glucose tolerance test, mice fasted for 16 h and then received an intraperitoneal injection of glucose (2 g/kg). For the insulin tolerance test, mice fasted for 4 h and were injected with 0.5 U/kg insulin. Blood samples were obtained by tail-bleeding, and glucose levels were measured at 0, 15, 30, 60, 90 and 120 min after glucose or insulin injection by GlucoDr auto AGM-4000 .v/v formalin/PBS, and then embedded in paraffin for staining with hematoxylin and eosin (H&E). Images were obtained under a Carl Zeiss light microscope  at a magnification of \u00d7100. Adipocyte size was quantified using ImageJ software .Adipose tissue samples from each mouse were fixed in 10% TM reverse transcription system . Quantitative PCR of gene transcripts for arginase 1 (Arg1), CD301, cell death-inducing DNA fragmentation factor \u03b1 subunit-like effector A (Cidea), type II iodothyronine deiodinase (Dio2), elongation of very long chain fatty acids protein 3 (Elovl3), epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80), fibroblast growth factor 21 (FGF21), interleukin-4 (IL-4), IL-10, leptin, mitochondrially encoded NADH dehydrogenase subunit 5 (ND5), peroxisome proliferator-activated receptor \u03b3 coactivator 1\u03b1 (PGC1\u03b1), peroxisome proliferator-activated receptor \u03b1 (PPAR\u03b1), sialic acid-binding immunoglobulin-like lectin F (Siglec F), transforming growth factor \u03b2 (TGF\u03b2), tyrosine hydroxylase (TH) and uncoupling protein 1 (UCP1) were performed by using gene-specific primers. Primer sequences are available upon request. Results were presented as mean \u00b1 SD normalized to expression of 36B4 (Arbp) using the \u0394\u0394Ct method.Total RNA was extracted using a TRI reagent  and reverse transcribed with oligo (dT) primer and GoScriptMeasurements of BAFF, FGF21 , insulin  and leptin  were performed with commercial ELISA kits according to the manufacturer\u2019s instructions.Western blotting was performed as described previously . Tissuest-test. p values <0.05 were considered as statistically significant.All data were presented as mean \u00b1 SD Comparisons between two groups were performed by two-tailed Student\u2019s"}
+{"text": "The doping concentrations were measured using gamma-ray spectroscopy and confirmed using SIMS analysis. The data from SIMS analysis also show consistent Ge doping concentration throughout the depth of the GaN wafers. After irradiation, the GaN was annealed in a nitrogen environment at 950\u00a0\u00b0C for 30\u00a0min. The neutron doping process turns out to produce spatially uniform doping throughout the whole volume of the GaN substrate.High quality Ge doping of GaN is demonstrated using primarily thermal neutrons for the first time. In this study, GaN was doped with Ge to concentrations from 10 As these needs increase, the demand for more efficient power conversion in power electronics grows as well. This task is currently met using silicon devices. However, silicon is rapidly reaching its operational limit2. Gallium nitride (GaN) has become the topic of much research in building devices which can replace Si power devices. Gallium nitride is preferred due to its high bandgap energy, a high critical breakdown electric field, and a high thermal conductivity5. Research on many GaN devices such as vertical GaN p-n diodes, p-n diodes with avalanche capability, and vertical transistors has been done8 and shows the need for consistently doped GaN layers to form these devices. All three of these devices rely on a thick (upwards of 180\u00a0\u00b5m) layers of N+ doped GaN. This has been an issue with several other doping methods, especially while maintaining a doping consistent throughout the GaN layer. This research shows the benefits thermal neutron transmutation doping can bring to the doping of GaN.In 2005, approximately 30% of electrical energy in the United States passed through power electronic converters. It is estimated that this number will increase to 80% by 20304 and SiH49. The growth was made by first creating a buffer layer of GaN and then growing the doped layer using two-flow metalorganic chemical vapor deposition (MOCVD). While the surface of the Si doped layers was exceptional the surface of the Ge doped layers contained pits at higher carrier concentrations. Similar results were found by using a hydride vapor phase epitaxy10. Initial experiments used hydrogen as the carrier gas, which led to a pitted surface on the GaN. Using nitrogen as the carrier gas instead of hydrogen removed the pits. However, it led to a hillock surface in the grown crystal. Later experiments used a buffer layer of undoped GaN before the doping process11. Silicon doping of the GaN material significantly affected the surface and degraded the quality. Selenium doping was attempted using low pressure metalorganic chemical vapor deposition (LP-MOCVD)12. This method worked but resulted in surface degradation at higher concentrations.Previous work in GaN doping comes in four forms, during growth, ion implantation, diffusion, and neutron transmutation doping. Doping during growth has been extensively studied with a wide variety of elements to create both n-type and p-type GaN wafers. GaN was doped with both Si and Ge using GeH13. While many elements showed redistribution when annealed at 600\u00a0\u00b0C, Ge did not show redistribution even at 900\u00a0\u00b0C. Experiments studying ion implantation of GaN with Si, Mg, and Ca found that after implantation the sheet resistance of the GaN was on the order of 105 \u2126/sq.14. The GaN was then annealed to electrically activate the dopants. The sheet resistance only dropped for materials that were doped with Si at 1050\u00a0\u00b0C, in the range of 103 \u2126/sq. For the Mg and Ca doped material, the sheet resistance varies\u00a0widely between 104 \u2126/sq.\u00a0and 107 \u2126/sq.\u00a0at various annealing temperatures.Ion implantation with GaN typically involves bombarding the surface of the GaN with the ion needed and then annealing the GaN to redistribute the ions throughout the crystal structure. A wide variety of elements have been tested using ion implantation15. The results of these experiments showed that the diffusion doping was possible, but it relied heavily on the capping layer. In addition to the variation with the capping layer the experiment showed some variability in GaN wafer with the same testing conditions. This implies that the doping is heavily dependent on the structure of the undoped GaN. Attempts to improve this process included first growing undoped GaN around three Mg \u201cspikes\u201d and then annealing the structures at 1060\u00a0\u00b0C for 1.25\u00a0h16. The results of this experiment showed little diffusion of the Mg through the GaN. Another attempt to improve the diffusing process of Mg into GaN was to grow a Mg doped GaN layer on an undoped GaN at temperature between 925 and 1050\u00a0\u00b0C17. Although the results are encouraging, the doping profile is typical of the diffusion doping process and not uniform across the depth of the wafer. Differently from various approaches described above, GaN can be doped using the neutron transmutation doping (NTD) process when it is placed in a high flux neutron field. The necessary irradiation conditions require a nuclear reactor and irradiations are typically done at research reactor user facilities.Diffusion of dopants in GaN has been attempted using a few different approaches. In this method of doping, the dopant element diffuses through the semiconductor at elevated temperatures. The dopant layer may be introduced by sputter coating the dopant onto the semiconductor surface and then heating the semiconductor. When this method was attempted using a sputtered layer of Mg on top of GaN, the layer was then capped with either a metal or silicon oxide and then heated to either 850\u00a0\u00b0C or 900\u00a0\u00b0C for 6 hours18. There, the process was used to transmute silicon in phosphorus. This resulted in n-type doped silicon with an extremely uniform doping profile. This is due to the extremely even flux profile of a nuclear reactor. The process required the annealing of the silicon at 600\u00a0\u00b0C but resulted in a method of bulk doping silicon which is widely used in nuclear reactor facilities today. Several groups have also reported on NTD of GaN. The first results of NTD in GaN came from a study of fast neutron damage in GaN and in n-AlGaN\u2215GaN heterostructures20. The GaN material was irradiated under a Cd cover to filter thermal neutrons and pass epithermal and fast neutrons. The authors reported that neutron damage created a semi-insulating GaN material. In a subsequent experiment the GaN material was irradiated without a Cd filter in a research reactor irradiation position reported to have a 1:1 thermal:fast neutron ratio21. This experiment also produced an insulating GaN substrate. The resistivity of the GaN dropped from the 106 \u2126cm range to the 1 \u2126cm range after annealing at 1000\u00a0\u00b0C. Electron hole traps were present in the material post annealing. Other experiments irradiated GaN in an irradiation position with a thermal:fast neutron flux ratio of nearly 2:122. Significant displacement of the GaN in the <0001> row was observed. The samples were annealed and a photoluminescence measurement showed the presence of lattice damage. Subsequent experiments conducted a test using a 1:1 thermal to fast neutron dose and found this created many electron traps that were not mitigated by annealing at 1000\u00a0\u00b0C23. The trap density was reported to increase with increased Ge levels implying that the creation of the traps is caused by radiation damage. Experiments on the NTD of GaAs showed an increased thermal:fast ratio reduced radiation damage24. This new experiment relies on using a primarily thermal neutron flux in order to avoid the extreme damage encountered in previous experiments. Thermal neutron flux is lower energy than fast neutron flux and is less likely to damage the crystal structure of the GaN.Neutron transmutation doping is a method of semiconductor doping which was first demonstrated in 1961T is the number of target nuclei per unit volume, \u03c3c is the capture cross section with units of cm2 and \u03a6 is the neutron fluence, n/cm2. The cross section represents the probability of interaction between the neutron and the nucleus. Neutron capture reactions occur at higher rates with low energy neutrons because the lower velocity allows greater interaction with the target nuclei. The capture cross section is inversely proportional to the neutron velocity at low energies. Reactor neutrons are parameterized into three groups based on energy; fast neutrons have greater than 100\u00a0keV, epithermal neutrons have between 0.5\u00a0eV and 100\u00a0keV, and thermal neutrons have less than 0.5\u00a0eV with a most probable energy near 0.025\u00a0eV. Especially, NTD of GaN occurs when 69Ga, 71Ga, and 14N undergo neutron capture reactions with thermal and epithermal neutrons to produce unstable 70Ga, 72Ga, and 14C, respectively. The 70Ga and 72Ga have half-lives of 21.1\u00a0min and 14.1\u00a0h and beta decay to 70Ge and 72Ge, respectively. The 14N neutron capture reaction is exothermic and produces 1H+ in the lattice leading to proton knock-on damage. Fast neutrons also cause knock-on damage through elastic scatter.The number of captures per unit volume, N, is described by:Previous research focused on irradiation which had equal or nearly equal thermal to fast neutron fluxes. In this work, NTD of GaN was conducted in the hanging wedge regions of the University of Missouri Research Reactor (MURR) with a thermal to fast neutron flux ratio of 25:1. This irradiation position was expected to reduce radiation damage caused by fast neutron knock-on damage compared to previous studies.72Ga (T1/2\u2009=\u200914.1\u00a0h), 60Co (T1/2\u2009=\u20095.27 y), and 58Co (t1/2\u2009=\u200970.86 d) were measured by gamma ray spectroscopy using a high purity germanium detector (HPGe) running Canberra Genie 2000 software. The dead-time for the measurements was less than 10% and was corrected using a live-time algorithm. The HPGe detector was calibrated using a NIST traceable multi-gamma source purchased from Eckert and Zeigler. The atom density of 72Ge was determined from the measured 72Ga activity.The GaN wafers and neutron flux monitors were placed inside of an Al capsule with two flux wires. The capsule was sealed inside of a welded Al can and leak tested. The thermal neutron flux was monitored using NIST SRM 953 cobalt in aluminum neutron density wire and the fast neutron flux was monitored using nickel wire. During irradiation, the sample can was rotated to minimize variation in the axial neutron flux profile. The wafer thickness was 350\u00a0\u00b5m and changes in the axial flux profile were considered insignificant. Following irradiation, the GaN wafer was decayed for\u2009~\u200910\u00a0days and the activity of 70Ga activity cannot be measured directly using gamma ray spectroscopy because of its short half-life. However, we can have a theoretical calculation using the measured 72Ga activity. The production of 70Ge is calculated based on the neutron flux and the nuclear cross sections of the neutron capture reaction of 69Ga. This gives a theoretical value for the production of 70Ga. The same theoretical calculation is also used for the 72Ga production. The 70Ge concentration can be estimated from the 72Ge assuming that the theoretical ratio of 70Ge and 72Ge concentration is the same with the experiment one. The 70Ge concentration is later confirmed with SIMS measurement.The 18 Ge atoms/cm3, also using 1\u00a0cm\u2009\u00d7\u20091\u00a0cm wafers. Experiment #4 used similar irradiation parameters to Experiment #2 but used sealed containers to protect the surface of the samples from the water in the reactor and resulted in doped 1\u00a0cm\u2009\u00d7\u20091\u00a0cm wafers and one 2\u2033 wafer.Four irradiations were conducted for this research with varying fluxes and irradiation times. These parameters are shown in Table After irradiation the wafers suffer from discoloration, as shown in Fig.\u00a03\u00a0kPa. This pressurized environment is necessary to avoid surface degradation of the GaN as at this temperature the nitrogen in the GaN begins to break off from the crystal structure and the GaN decomposes. The pressurized nitrogen environment prevents this from happening.The annealing process uses a stainless-steel pressure chamber which is filled with ultra-pure nitrogen gas. The chamber is pressurized to 331\u00a0kPa. The chamber is then heated to 950\u00a0\u00b0C. At this point the pressure of the nitrogen gas is 1.38\u2009\u00d7\u200910As detailed in the section III.A above, Fig.\u00a016 Ge atoms/cm3 and the smallest comes from B10 with a 7.98\u2009\u00d7\u20091016 Ge atoms/cm3. This shows a variation of 5.7%. This difference in concentrations may come from a difference in geometry influencing the gamma count. Wafer C is significantly larger than the others and Wafer B10 was broken during the irradiation process.The germanium concentration of A and C samples are very similar to B samples. Wafers A1-A10 and B1-B10 are the 1\u2009\u00d7\u20091\u00a0cm pieces which were arranged to emulate a 2\u2033 wafer while wafer C was a 2\u2033 wafer. The highest concentration comes from the 2\u2033 wafer with 8.47\u2009\u00d7\u20091016\u2009\u00b1\u20092\u2009\u00d7\u20091016 72Ge atoms/cm3 was measured in one of the samples from Experiment #2. The reason for the large error in this measurement is because of how similar the atoms being measuring against are. 72Ge and 71Ga are only one proton different. In the plasma it is possible for the 71Ga to combine with a hydrogen atom to for a gallium hydride. Since ICP-MS measures based on mass size, this means that a 72Ge and a 71Ga with a hydrogen atom bonded will be measured as the same atom. This problem is even worse for the 70Ge measurements. There is an additional polyatomic, 40Ar16O14N\u2009+\u2009, which forms in the plasma and has a mass of 70. Since this test is destructive (the GaN wafer is completely dissolved) and cannot measure with a reasonable amount of certainty, gamma-ray spectroscopy was chosen as the preferred measurement method.In order to confirm the gamma-ray spectroscopy measurements, two other type of measurements were also conducted, ICP-MS and SIMS. Since the laser ablation feature of the ICP-MS was not available, another method to dissolve the GaN had to be used. A process known as borate fusion was employed to break down the GaN into solid borate flux, which could then be easily dissolved in a mineral acid. The dissolved samples are compared against pure gallium standards and pure germanium samples. A concentration of 7\u2009\u00d7\u20091017 Ge atoms/cm3 concentration doped GaN wafers and 1018 Ge atoms/cm3 concentration doped GaN wafers were sent to a partner, EAG Laboratories, for SIMS analysis. Figure\u00a018 Ge atoms/cm3 concentration doped wafer. This SIMS analysis not only helps confirm the estimations used in the gamma-ray spectroscopy, but it also shows how consistent the germanium doping concentration is throughout the wafer. This is to be expected as the neutron flux drop-off through the thickness of the wafer (350\u00a0\u00b5m) is extremely small. Calculation using the thermal cross sections for 71Ga and 69Ga (4.61 barns and 1.68 barns respectively) shows that the flux reduction across the 350\u00a0nm thickness of GaN wafer is 7.7\u2009\u00d7\u200910\u20135%.One information gamma-ray spectroscopy cannot provide, however, is the doping concentration profile. To measure this 10UV\u2013Vis spectroscopy measurements were conducted on the GaN pieces to better quantify the annealing results. This test involved shining light through the samples and recording the transmittance of wavelengths from 200 to 700\u00a0nm. Before irradiation GaN begins as a mostly clear crystal. After irradiation there is damage to the crystal which causes a brown coloration, as shown in Fig.\u00a0The UV\u2013Vis spectrum of a GaN wafer before being treated is the black curve in Fig.\u00a02 for the C-V measurements in Fig.\u00a0Another method employed to characterize the GaN wafers was C-V measurements. To conduct these measurements, a Hg probe was used. The mercury probe uses a vacuum system to create a ring and a dot contact on the surface of the GaN wafer. The back contact of the probe is made of stainless steel. The measurements were done with a voltage sweep from \u2212\u20095 to 0\u00a0V with steps of 0.1\u00a0V. Bellow in Fig.\u00a0\u20134 m2), q is the charge of an electron (1.6\u2009\u00d7\u200910\u201319 C), \u03b50 is the permittivity of space (8.854\u2009\u00d7\u200910\u201312 F/m), \u03b5 is the permittivity of GaN (8.9) and m is the slope of the 1/C2 curve. The main goal was to prove a consistent carrier concentration across a 2\u2033 wafer of GaN. To do so C-V measurements were conducted across 13 points on wafer C. The location of these measurements and the data from them are shown in Fig.\u00a0To calculate the carrier concentration from the C-V measurement results, the following equation is used.17 Ge atoms/cm3 are not shown in this paper as the concentrations at that doping range are near the minimum possible to measure using SIMS. While this is not a problem for the 1018 Ge atoms/cm3 samples, the 1017 Ge atoms/cm3 samples are the ones being heavily produced in this study.When comparing the four methods of measuring doping concentration, ICP-MS, gamma-ray spectroscopy, SIMS, and C-V there are two clear favorites. ICP-MS is by far the least accurate method as the dopant atoms are merely one atomic mass unit larger than the atom they are replacing which makes ICP-MS unreliable. SIMS is effective for finding the depth of the doping concentration; however, it has its limitations. The results for the 1070Ge concentration. This theory has proven to be true after testing with both SIMS and C-V. For C-V measurements there is no way to distinguish between the 70Ge and 72Ge concentrations, but that isn\u2019t necessarily needed when it comes to measuring doping concentration. Either way, the doping concentrations measured using the two methods seem to be close. The gamma-ray spectroscopy measurement for the 2\u2033 wafer was measured to be 8.47\u2009\u00d7\u20091016 Ge atoms/cm3. The C-V measurement had the carrier concentration as 1.806\u2009\u00d7\u20091017\u00a0cm\u22123. The carrier concentration of an untreated GaN wafer is 1\u2009\u00d7\u20091017\u00a0cm\u22123. This would mean that 8.06\u2009\u00d7\u20091016 Ge atoms/cm3 were added which is very close to the Ge concentration results from gamma-ray spectroscopy measurement.Gamma-ray spectroscopy appears to be the best methods available to us to measure the germanium concentration. Gamm-ray spectroscopy relies on the theory behind the neutron transmutation to measure the 16 Ge atoms/cm3 to 1018 Ge atoms/cm3. Before the annealing process, the resistivity of these GaN samples was several orders of magnitude below the results of previous research which used equal doses of fast and thermal neutrons. After annealing the resistivity is even further reduced. During the irradiation, the GaN wafer loses a majority of its transparency. But after annealing, this transparency comes almost entirely back. The neutron doping process creates extremely consistent doping across large areas and throughout the depth of the wafer making it a prime candidate for creating large high-quality Ge-doped GaN substrates.The irradiation of GaN using mostly thermal neutron results in high quality, consistent and uniform doping throughout the GaN wafer. It has been shown that germanium doping of GaN using primarily thermal neutrons can effectively dope GaN in concentrations from 10"}
+{"text": "A diagnosis of mild cognitive impairment (MCI) before dementia is a helpful proxy to explore early diagnosis. This study investigated the association between an early diagnosis of dementia and subsequent hospitalisations and ED attendances.a retrospective cohort study of electronic health care records from 15,836 patients from a large secondary care database in South London, UK. Participants were divided into two groups: those with a diagnosis of MCI before dementia, an early diagnosis, and those without. Cox regression models were used to compare the risk of hospitalisation and ED attendance after dementia diagnosis and negative binomial regression models were used to compare the average length of stay and average number of ED attendances.participants with an early diagnosis were more likely to attend ED after their diagnosis of dementia ; however, there was no difference in the number of ED attendances . There was no difference in the risk of hospitalisation  or length of stay between the groups .the findings of this study do not support the assumption that an early diagnosis reduces the risk of hospitalisation or ED attendance. The patterns of health service use in this paper could reflect help-seeking behaviour before diagnosis or levels of co-morbidity. A previous diagnosis of mild cognitive impairment (MCI) is a useful proxy for an early diagnosis of dementia.An early diagnosis of dementia was associated with an increased risk of A&E attendance after diagnosis.There was no difference in the risk of hospitalisation between those with an early diagnosis and those without.There was no difference in the number of hospital days or A&E attendances between the groups.The frequent use of emergency services and unplanned hospitalisations is reflective of fractured dementia care , 2. It iThere is no fixed definition for early diagnosis in dementia. Early diagnosis could be from the onset of neuropathology, many years before the symptoms become apparent, from the use of reliable predictive biomarkers or the onset of cognitive symptoms . With thIn theory, people with an early diagnosis should receive early treatment, have more contact with primary health services ahead of time and be supported to make advanced plans, which reduce the risk of hospitalisation or ED attendance . HoweverTo address the aims of this study, we conducted a retrospective cohort study using electronic health records from South London and Maudsley NHS Foundation Trust (SLaM). SLaM provides specialist dementia care to people living with dementia in the London boroughs of Lambeth, Lewisham, Southwark and Croydon.Data from SLaM's electronic medical health care records were extracted through SLaM's Biomedical Research Centre Clinical Record Interactive Search (CRIS). Data are stored in both free text and structured fields, the extraction of which has been previously described , 10. AddThe CRIS database has full approval for secondary analysis .Participants were included in the cohort if they received a diagnosis of dementia according to ICD 10 classifications , betweenParticipants with a diagnosis of MCI, as recorded by an ICD-10 code of F06.7, before the index date were classified as having received an \u2018early diagnosis\u2019. This was included as a dichotomous variable.Our primary outcomes of interest were time to first hospitalisation and time to first ED attendance. Our secondary outcomes of interest were the cumulative number of hospital days and number of ED attendances.As co-variates, we extracted whether participants were hospitalised or attended ED in the year before dementia diagnosis, as these are known predictors of ED attendance/hospital admission after diagnosis . DemograT-tests and Chi-squared test were used to compare baseline differences between the early diagnosis and no early diagnosis groups.All analyses were conducted using Stata 15 [We assessed the risk of hospitalisation and ED attendance after dementia diagnosis using cox regression models. Negative binomial regression models were used to compare the length of stay (number of days) and the number of ED attendances by each group. We used negative binomial regression, rather than Poisson regression, as data were over-dispersed. We present an unadjusted model and a multivariable model adjusted for age, gender, ethnicity, physical illness, marital status, prescription of ACHEIs, number of psychiatric symptoms, MMSE scores and previous hospitalisation/ED attendance. Follow-up time was included in both models as an exposure variable.Thirty percent of participants were missing MMSE scores and 13% of participants were missing one or more scores on the HoNOS 65+. Missing data were imputed in STATA using multiple imputation by chained equations . All outn\u2009=\u2009807) were diagnosed with MCI before they were diagnosed with dementia. We identified 15,836 people with dementia; 5.1% of participants  recorded after they were diagnosed with dementia . The medP\u2009=\u20090.4).Over two-thirds of participants attended ED after their dementia diagnosis (75.7%). The median time to first ED attendance in the early diagnosis group was 8.9\u00a0months, compared with 10.6\u00a0months. Adjusted cox regression models showed participants with an early diagnosis were at increased risk of attending ED . There was no significant difference in number of ED attendances between the groups.Negative binomial regressions, adjusted for a range of confounders, showed there was no difference in the count of hospital days between the groups . Similarly, there was no difference in the count of ED attendances .In this study, we investigated whether an early diagnosis was associated with a decreased risk of hospitalisation or ED attendance after a diagnosis of dementia. We found that participants with an early diagnosis were at greater risk of attending ED than participants without an early diagnosis; however, there was no difference in the number of ED attendances between the groups. There was no difference in the risk of hospitalisation or length of stay between participants with an early diagnosis and those without.We found a high level of secondary health service use in people with dementia, 74% of participants were hospitalised and 75% attended ED after their diagnosis. The average time to the first hospitalisation and first ED visit was 11.5 and 10.4\u00a0months, respectively. This is consistent with previous research, which showed that people living with dementia have high rates of admission to hospital within the first year of diagnosis [We found, contrary to popular belief, that the risk of hospitalisation and length of stay did not differ between people with an early diagnosis of dementia compared to those without. Additionally, people with an early diagnosis had a higher risk of attending ED, although there was no difference in the number of times each group attended ED. This group may have had increased contact with health services before their diagnosis of dementia, which increased the likelihood of receiving the early diagnosis of dementia, and this pattern of health service use continued after diagnosis.Many hospital admissions for people living with dementia are necessary and appropriate. However, people living with dementia are at greater risk of negative outcomes arising from hospitalisation than older adults of the same age without dementia. They may be hospitalised for longer , 18, mayThere is a risk that focusing on diagnosing dementia early and investing in treatments for the early stages of the disease diverts resources from meeting other needs in the later stages, including the treatment of co-morbidities . PreviouThe cohort from this study came from a secondary care database, which reflects the high levels of service use. Further research is needed to understand the impact of an early diagnosis or early help-seeking on the use of other types of health services, such as primary care. While we have highlighted the possible role of co-morbidities in driving high levels of health service use, our data are restricted to HoNOS rated levels of co-morbidities without information on individual conditions. This is an interesting avenue for future research. This is a cohort study; therefore, variables used in this study were limited to what is routinely collected, and there may be some residual confounding, which has not been controlled for. While we have previously found a previous diagnosis of MCI to be a useful proxy for early diagnosis , we cannWe have found that early diagnosis alone is not a preventative step for reducing hospitalisations or ED attendances and people with an early diagnosis had an increased risk of attending ED. However, an equal or higher use of health services between people with an early diagnosis and those without is not necessarily a bad thing. People living with dementia should be able to access appropriate health services whenever they are needed. However, people with dementia are at greater risk of negative outcomes following a hospitalisation or ED attendances , 23 and"}
+{"text": "It is hypothesized that cessation of colistin use as a feed additive for pigs will reduce the occurrence and distribution of mcr genes in farms. The aim of this study was to investigate this hypothesis by longitudinal monitoring and characterizing of mcr positive Escherichia coli (MCRPE) isolates after colistin was withdrawn on a central Thailand pig farm that previously had a high frequency of MCRPE. Colistin use ceased at the beginning of 2017, and subsequently 170 samples were collected from farrowing sows and suckling piglets (n = 70), wastewater (n = 50) and farm workers (n = 50) over a 3.5-year period. Bacteria were identified by MALDI-TOF mass spectrometry and minimal inhibitory concentrations were determined by broth microdilution. The antibiogram of mcr positive E. coli isolates was determined using the Vitek2 automated susceptibility machine, and multiplex and simplex PCRs were performed for mcr-1\u20138 genes. MCRPE containing either mcr-1 or mcr-3 were isolated from pigs throughout the investigation period, but with a declining trend, whereas MCRPE isolates were recovered from humans only in 2017. MCRPE were still being recovered from wastewater in 2020. Most MCRPE isolates possessed the virulence genes Stap, Stb, or Stx2e, reflecting pathogenic potential in pigs, and showed high rates of resistance to ampicillin, gentamicin and tetracycline. Pulsed-field gel electrophoresis and multi-locus sequence typing showed that diverse MCRPE clones were distributed on the farm. The study identified a decline of pathogenic MCRPE following withdrawal of colistin, with pigs being the primary source, followed by wastewater. However, short-term therapeutic usage of other antibiotics could enhance the re-occurrence of mcr-carrying bacteria. Factors including the environment, management, and gene adaptations that allow maintenance of colistin resistance require further investigation, and longer-term studies are needed.Colistin-resistant bacteria harboring plasmid-mediated  Enterobacteriaceae infections (mcr gene family has jeopardized the efficacy of colistin. The plasmid mediated colistin resistance gene (mcr-1) was firstly identified in E. coli of porcine origin from China (mcr variants including mcr-2 to mcr-10 were discovered mainly from members of the Enterobacteriaceae family (mcr genes encode phosphoethanolamine transferases enzymes which change the lipid A portion of the lipopolysaccharides (LPS), suppressing colistin binding   and carbapenemase genes (Colistin (polymyxin E) is one of the World Health Organization's highest priority antimicrobials: it is regarded as a last resort antibiotic, and is the treatment of choice for multidrug-resistant fections . Unfortuom China . Subseque family \u20136 from de family , 8. The  binding . The mcr bovine)  and food bovine)  but also bovine) . Since t concern . Moreovese genes , 16. Themcr-1 during nation-wide surveillance in Thailand . Furthermore, some studies have shown that even after drastic reductions of antibiotic use on farms, antibiotic resistant bacteria could be maintained in the farm by various factors  that were obtained are summarized in us study  by usingn = 50). Rectal swab samples from 21-day old suckling piglets belonging to the sows that were sampled were also collected (n = 20). Each farrowing sow with their respective litters were kept in farrowing pens, and at each visit one or two sows were sampled from each zone of the farrowing house. The same pens were visited at each sampling time, although the same sows were not necessarily sampled because of animal movements. In September 2018, only fecal samples from sows were collected since at the time of sampling the newly weaned piglets had been moved to another farm.Approximately 25 g of fecal samples were collected from parity 1\u20136 farrowing sows, aged between 1 to 3 years (n = 50) from the wastewater tanks on the farm were collected, with 10 samples obtained at each visit. The wastewater was composed of pig manure along with the water used to clean the pig housing. Approximately 500 ml volumes were collected from wastewater tanks located before and after-biogas treatment, which were sited close to the sampled pig pens. The biogas process involves anaerobic fermentation by fermentative, acetogenic, and methanogenic bacteria to produce methane, carbon dioxide, hydrogen, and hydrogen sulfide gases. In addition, at the request of the company, at each sample collection time the farm submitted rectal swab samples from the same 10 farm workers for routine diagnostic purposes (n = 50).Wastewater samples . The wastewater sample collection protocol was applied according to HACH water analysis guidelines .E. coli using IMViC biochemical tests and Matrix-Assisted Laser Desorption Ionization combined with time of-flight analysis , according to the manufacturer's recommendations (All samples were enriched in EC broth (Difco) containing 2 \u03bcg/ml colistin sulfate at a 1:9 ratio and incubated at 37\u00b0C overnight. The sample suspensions were grown on eosin-methylene blue (EMB) (Oxoid) agar containing 2 \u03bcg/ml colistin sulfate and were incubated overnight. One to three representative colonies with a characteristic metallic sheen on the EMB plates were randomly chosen and sub-cultured on tryptic soy agar (TSA) (Difco) from the samples from which growth was obtained. The colonies were identified as ndations . For minE. coli isolates was determined using the AST-GN 38 test kit in a Vitek2 compact automated susceptibility level detection apparatus . The antimicrobial groups that were included in Vitek2 were synchronized with veterinary guidelines . Multiplex-PCR was used to detect protocol . E. coliin CUP13  that is cription .Enterobacteriaceae were investigated by a set of multiplex and simplex PCRs. The primers used and the PCR conditions followed previously described methods (The 18 plasmid replicon types of  methods . Brieflymcr positive E. coli were examined for virulence genes that are commonly present in enterotoxigenic E. coli (ETEC) and enterohaemorrhagic E. coli (EHEC) by using previously described PCRs  with the thermocycler conditions being an initial denaturation at 94\u00b0C for 10 min, followed by 30 cycles of denaturation at 94\u00b0C for 30 s, and annealing at 55\u00b0C for 45 s. Extension was at 72\u00b0C for 1.5 min increased by 3 s each cycle, followed by a final extension at 72\u00b0C for 10 min.The bed PCRs . Previoubed PCRs . The PCRmcr genes were located on transmissible plasmids, and their transferability rate, conjugation assays were performed by the broth mating technique  and is sensitive to colistin (MIC <2 \u03bcg/ml). Briefly, an overnight culture of bacterial colonies was diluted in Lysogeny broth (LB) and adjusted to OD600 value 1. A 1:1 ratio of donor and recipient then was mixed to obtain a final volume of 2 ml which was incubated overnight. Ten-fold serial dilutions of the overnight mixture were plated on LB agar (Oxoid) plates containing colistin (2 \u03bcg/ml) and sodium azide (100 \u03bcg/ml). The plates were incubated at 37\u00b0C for 2 days, and the transconjugant colonies were counted. The rate of conjugal transfer frequencies was calculated by dividing the number of transconjugant colonies by the number of donor colonies . Gel electrophoresis was undertaken using a Bio-Rad CHEF-DRIII system, with a 200 V field at an angle of 120\u00b0 run for 17\u201320 h, incorporating Salmonella serovar Braenderup H9812 DNA as a standard. Dendrograms were created using the GeneTool program  and analyzed by the GeneDirectory program .To investigate clonal relatedness, PFGE was performed on all 65 available MCRPE isolates from the 33 positive samples (one to three isolates per sample), following the Centers for Disease Control and Prevention standard protocol . BrieflyE. coli used in the Achtman MLST scheme (icd), ATP/GTP binding motif (recA), adenylate kinase (adk), DNA gyrase (gyrB), malate dehydrogenase (mdh), adenyl succinate dehydrogenase (purA) and fumarate hydratase (fumc). The sequences were obtained using the Sanger sequencing platform. The E. coli MLST database at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli was used to determine allele and sequence types (STs).A representative isolate from each of the 34 PFGE pulsotypes that were identified was randomly selected and included for MLST typing. A simplex PCR was performed for each of the 7 housekeeping genes of T scheme . These gmcr positive rates among the samples and the association between each sample collection time were analyzed using Fischer's Exact Test (p \u2264 0.05).The colistin-resistance rates and virulence gene profiles for the representative isolates were described as percentages compared to different sources in each sample collection. The E. coli, and their MICs to colistin varied from 4 to 8 \u03bcg/ml. These positive samples were isolated from pigs , wastewater  and humans . A comparison of the prevalence of MCRPE isolates for each sample type over the 3.5 years since colistin cessation is shown in A total of 33 of the 170 samples (20.6%) yielded colistin-resistant mcr-1 gene was detected in eight of the pig isolates, while mcr-1 and mcr-3 were detected together in two of these, and mcr-3 alone in one pig isolate. At the same time, mcr-1 was detected in all four of the isolates from workers and in both the isolates from wastewater samples .Of the colistin resistant isolates obtained in 2017, the  samples . In 2018E. coli were identified, and most MCRPE isolates from the first and second samplings were found to demonstrate extreme pan-drug resistance. Interestingly, besides colistin, the isolate from the positive pig sample in 2019 was phenotypically resistant only to ampicillin. On the other hand, the MCRPE isolates from the last sample collection in 2020 were resistant to aminoglycosides, ampicillin, and ceftiofur, and those antibiotics were used for individual treatments on the farm. The antibiogram results comparing isolates between the 4 sampling years are presented in The antimicrobial resistance (AMR) profiles detected in the MCRPE isolates are shown in mcr positive isolates from different sources contained more than one replicon type. The incompatibility group IncFIB and IncI type plasmids were found most commonly. Although a variety of plasmid types were detected in pigs in 2017 and 2018, there was a decrease in varieties of plasmid types in later sample collection years. For the conjugation assay, the donor E. coli transferred mcr-1 and mcr-3 genes (as confirmed by PCR) to recipient J53 strains with a frequency of 1.7\u20132 \u00d7 10\u22124. Phenotypic colistin resistance of transconjugants identified by broth microdilution showed MIC values of >4 \u03bcg/ml .Various plasmid replicon types were detected among the MCRPE isolates . All the>4 \u03bcg/ml . The Incmcr positive E. coli isolates. Most of the isolates from pigs contained genes associated with ETEC strains (enterotoxin genes), with StaP and Stb being the most frequent pathotype found in 2017  occurred rarely and were found mainly in the same set of pig or human samples from the same sampling year. Only the MCRPE strains from piglets and wastewater samples in 2020 showed high clonal relatedness, suggesting that MCRPE strains from the piglets with diarrhea had contaminated the wastewater.Thirty-four diverse PFGE patterns were obtained for the 65 MCRPE isolates from different sources . No domiE. coli clonal complex ST10 were detected in 2 pigs and one human sample on the first sampling, and in one wastewater sample on the last sample collection. Isolates belonging to ST 641 were detected in 2 pigs and one wastewater sample in 2018. In 2020, MCRPE isolates belonging to ST3345  and ST 5218 (n = 2 in pigs) were commonly detected.MLST gave similar results to PFGE, with most isolates belonging to different STs . IsolateE. coli on large-scale pig farms across Thailand has been reported previously , and oxytetracycline .NK contributed to conception and design, critical revision, analysis and interpretation of data, and drafting of manuscript. KL contributed to conception and design, analysis, and interpretation of data. WN performed analysis and interpretation of data. TP organized acquisition of data. DH contributed to critical revision, analysis and interpretation of data, and drafting of manuscript. NP organized study conception and design and contributed to critical revision, analysis, and interpretation of data. All authors contributed to manuscript revision, read, and approved the submitted version.This study was supported financially by Royal Golden Jubilee Ph.D. (RGJPHD) program, from the Agricultural Research Development Agency (ARDA) [CRP6205031110], the CHE-TRF Senior Research Fund (RTA6280013), Thailand Science Research and Innovation, and Pathogen Bank, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "To the Editor,Do the Nike Vaporfly/Alphafly Shoes Provide an Unfair Advantage?\u201d, the author claimed that the performance progression in long-distance, such as the marathon and the half-marathon, has not changed and that \u201cthe shoes\u2019 introduction has not obtained any greater change than the reasons attributed to any former record being broken\u201d.We have read with great interest the manuscript recently published in your journal on the technological unfairness of the use of Nike Vaporfly and Alphafly running footwear models . In the \u201crecent improvements in these events are unlikely physiological but rather technological, if for no other reason that such a step-wise improvement in physiological attributes underpinning performance is unlikely\u201d. A recent meta-analysis [Joyner et al.  evaluateanalysis  confirmsanalysis . Therefor\u2009=\u20090.96).However, recent retrospective studies , 7 analyMoreover, another study from our research group (under review) evaluated the impact of the Vaporfly shoes on the best Marathon times in history, where 23 of the 50 (46%) best times in history (accessed 20 November 2020) have been achieved with Vaporflys, and 21 of them being achieved since 2018 (42%). In addition, from the best performances under 2:03:00, every performance except the one by Dennis Kimetto  has been achieved with Vaporfly shoes.We conclude that the new technological footwear implies a clear impact in long-distance running performance, and probably an unfair advantage due to the greater improvements they provide when compared to the years prior of the technological revolution."}
+{"text": "Does adolescent attachment to parents and peers differ between singletons and twins born with ART or natural conception (NC)?Adolescent attachment anxiety with the father was higher among NC singletons than among ART and NC twins, whereas attachment avoidance with the father was higher in ART singletons than in NC singletons and NC twins. No differences were found in attachment to the mother, best friend or romantic partner.Most studies have not found differences between ART and NC singletons in parent\u2013adolescent relationships, but twin relationships may be more at risk. No previous study has examined all four groups in the same study, or specifically looked at attachment relationships.This was an 18-year, prospective and controlled longitudinal study with families of 496 ART singletons, 101 ART twin pairs, 476 NC singletons and 22 NC twin pairs. Families were recruited during the second trimester of pregnancy; the ART group was recruited from five infertility clinics in Finland and the control group was recruited from a hospital outpatient clinic during a routine visit.Experiences in Close Relationships\u2014Relationship Structures) at 17\u201319\u2009years of age.Mothers and fathers gave background information for this study during pregnancy, and during the child\u2019s first year and early school age (7\u20138\u2009years). For the ART group, infertility characteristics and prenatal medical information was also obtained from the patient registry of the infertility clinics. Children  filled in electronic questionnaires related to their attachment to mother, father, best friend and romantic partner , the Juho Vainio Foundation and the Finnish Cultural Foundation. The authors report no conflict of interest.N/A. WHAT DOES THIS MEAN FOR PATIENTS?This study looks at how singleton and twin adolescents conceived with IVF treatment or naturally differ in their attachment relationships. The attachments refer to the adolescent\u2019s close relationships to their mother, father, best friend and romantic partner. The study followed up on more than 1000 families from pregnancy to the age of 17\u201319\u2009years. Infertility and its treatments, as well as parenting young twins, can be stressful or cause worry for parenting or child development. However, most studies have not found long-term problems in parent\u2013adolescent relationships in families conceived with IVF. Knowledge is still lacking on parent\u2013adolescent relationships in twins, and about adolescent relationships to friends and romantic partners in those conceived with IVF treatment. Our results show that both singletons and twins conceived with IVF had equally good attachment relationships with their mothers, best friends and romantic partners as naturally conceived adolescents. Attachment relationships to the father were, however, somewhat more problematic, but only among singletons. IVF-conceived singletons showed more emotionally distant attachment relationships with their father than naturally-conceived adolescents. Instead, naturally-conceived singletons showed more anxious, over-dependent attachments with their father than naturally or IVF-conceived twins. Our results thus show that both twins and singletons conceived with IVF treatment generally do well in their later close relationships. Considering the more emotionally distant adolescent attachment to fathers, it might be important that men, as well as women, receive enough support in pre- and peri-natal health care during and after experiencing infertility.Although assisted reproductive treatments can make the dreams of parenthood come true for infertile couples, the treatments can also be physically and emotionally demanding .Parent\u2013child relationships may be affected by the stress of sharing resources with two infants. Compared to mothers of singletons, twin mothers have been shown to be less involved, sensitive and communicative with their children , and botFew studies have examined parent\u2013adolescent relationships in twin or even in singleton ART families. For ART singletons, parent\u2013adolescent relationships have been reported as at least equally warm as among NC singletons , althougOne of the most crucial aspects of parent\u2013child relationships is attachment: the close affectional bond children develop toward their caregivers  starting from the first year of life . The evoTo our knowledge, only one previous study has looked at attachment in ART-conceived children, showing no differences in the attachment patterns between ART and NC singletons toward their mother at 1 year of age   and peers (best friend and romantic partner) in adolescence. The current study represents the latest phase of an 18-year longitudinal study, targeting both parents and adolescents.The study sample originally comprised 1095 Finnish families, 972 with singleton pregnancies (513 ART and 473 NC) and 123 families with twins (101 ART and 22 NC) (Mothers and fathers were asked to separately complete questionnaires during the second trimester of pregnancy (T1), child age of 2\u2009months (T2), child age of 12\u2009months (T3) and child age of 7\u20138\u2009years (T4). At 17\u201319\u2009years (T5), both adolescents and their parents separately completed electronic questionnaires. For the ART group, research nurses also collected data from the clinics\u2019 patient registries related to their infertility characteristics and other prenatal medical history at T1.Background variables consisted of T1 maternal and paternal self-reported education level , and ART characteristics (as reported by the research nurse): type of treatment , etiology of infertility  and duration of infertility (in months). The T2 maternal self-reported  child health risk factors: newborn health problems (1\u2009=\u2009yes/0\u2009=\u2009no), gestational week at birth and birth weight. As newborn health problems  were highly intercorrelated, they were combined into a newborn health risk index (0\u20133) for further analyses describing the number of risks. At T5, the adolescents self-reported on whether they were currently dating  and their education level. Education level consisted of upper secondary school , vocational training, working without a professional training and being unemployed without a professional training. Since 75.9% of adolescents were in upper secondary school, while all the other categories were small , the classes were dichotomized into high (i.e. upper secondary school\u2009=\u20091) vs. low education level . Adolescent sex  was based on the questionnaire information from mothers and fathers at T1\u2013T4 . Parental divorce (1\u2009=\u2009no/2\u2009=\u2009yes) and number of siblings were based on the questionnaire information from mothers, fathers and adolescents at T5 , and adolescent age at T5 was calculated based on their birthdate (as reported by mothers at T1) and T5 participation date .Attachment to mother, father, best friend \u00a0and romantic partner was assessed from adolescents with a questionnaire, using the Experiences in Close Relationships\u2014Relationship Structures  and avoidance variables need to be divided by 6 . ECR-RS; . FurtherEthical permissions were obtained from the ethical board of Helsinki University Central Hospital separately at T1\u2013T3, T4 and T5. At T1\u2013T3, it was also obtained from the participating clinics. Participants gave a written, informed consent separately at each time point, regarding the use of their self-report questionnaires in the research. At T1\u2013T4, the consent was obtained from the parents, and at T5 both parents and the adolescent each gave their own consents regarding their own self-reported questionnaire data. According to Finnish law, parental consent is no longer needed for 17\u201319-year-old research participants, so parents were only informed about their adolescents\u2019 invitation to participate. At T1, the ART group also gave their informed consent for the collection of their medical registry data from the clinics\u2019 patient registries.U tests were used to examine the associations of attachment variables with background variables: Adolescent sex, education level, current dating and parental divorce, and Spearman\u2019s correlations to examine their associations with maternal and paternal education level, adolescent age at T5, newborn health risk index  and number of siblings. In the ART group, we further examined the associations of the treatment type and the etiology of infertility with attachment variables with the Kruskal\u2013Wallis test and the association of duration of infertility and attachment variables with Spearman\u2019s correlations. We then examined the associations between group status  and background variables using chi square tests and ANOVAs. The background variables significantly associated with attachment variables were used as covariates in the respective models for the research questions.For descriptive analyses, we used non-parametric tests to address the violation of the normality assumption in parametric tests of the attachment variables. Mann\u2013Whitney To answer our research question about differences between ART and NC twins and singletons in attachment variables, multilevel structural equation models (MSEMs) were built with Mplus version 8  using MaWe used multiple imputation to handle missing data, which are appropriate for nested data structures (in our study: data included both twins and singletons), and for use with a combination of continuous and categorical variables. Multiple imputation is an analysis method utilizing all variables in the data to estimate missing values . MultiplAdolescent attachment anxiety and avoidance variables  were skewed in a way that most values were at the lowest value. To appropriately model this type of data, a censor-inflated regression model was fitted to the data (except for father-avoidance model). Traditional model fit indices cannot be used with partially nested models, so fit indices were not calculated for attachment variables . Dummy c2(3) = 2.09, P\u2009=\u20090.55, health as newborn, \u03c72(1) = 0.83, P\u2009=\u20090.36, gestational week at birth, t(710) = \u20130.37, P\u2009=\u20090.71 or child birth weight, t(970) = \u20131.74, P\u2009=\u20090.082, nor in the ART group, with the etiology , \u03c72(3) = 4.80, P\u2009=\u20090.19, or duration of infertility, t(408) = \u20130.58, P\u2009=\u20090.56, or the type of treatment , \u03c72(3) = 0.45, P\u2009=\u20090.93. However, higher drop-out was associated with lower T1 maternal and paternal education level, \u03c72(3) = 10.54, P\u2009=\u20090.014 and \u03c72(3) = 13.56, P = 0.004 and the child being a boy, \u03c72(1) = 28.67, P\u2009<\u20090.001The participation flow chart is presented in The results in Concerning attachment in peer relationships (best friend and romantic partner), no statistically significant effects were found between any of the four groups. However, two marginally significant trends emerged, suggesting that NC singletons showed more attachment avoidance toward best friend than ART twins, and NC twins showed more attachment anxiety toward romantic partners than NC singletons. None of the covariates were significant for any of the parental or peer models see . To viewThis study examined whether ART and NC singletons and twins differed from each other in adolescent attachment toward parents (mother and father) and peers (best friend and romantic partner). Interestingly, adolescent backgrounds of ART and twin status showed distinct effects on adolescent attachment anxiety and attachment avoidance toward the father, but not with attachment to mother or with peers. Twins in general, and most clearly ART twins, showed lower attachment anxiety toward father compared to NC singletons. Conversely, a background of ART was associated with higher attachment avoidance toward the father compared to NC adolescents (both singletons and twins), but this only applied to ART singletons.Our findings concur with earlier studies showing that ART singletons experience at least as warm and harmonious relationships to their mothers in adolescence as NC singletons . HoweverOur findings on adolescents\u2019 attachment to their father were especially interesting, as ART and twin status showed different dynamics related to attachment anxiety and attachment avoidance. Compared to NC singletons, both ART and NC twin groups displayed lower attachment anxiety, that is, less over-dependent relationships with their fathers, where there are worries of paternal availability. This may be because in the challenging twin context, parents are less likely to overprotect, and have a greater tendency to foster autonomy in their twin children. They simply have to because their energies are distributed across multiple children. While it is not absolutely clear why this is the case for fathers but not the mothers, we believe that the fathers feel this \u2018limited resource\u2019 issue of time and energy even more than the mothers. There may also be something protective in the twin relationship itself: for example, at least adult twins report especially close attachments with each other , which cIn the singleton context, ART singletons did not differ from any of the other groups, suggesting that ART singletons do not display higher attachment anxiety toward their father. These findings are important, given that some early childhood studies have reported early family-level enmeshment in ART singletons . Our self-report-based attachment results should thus ideally be verified with AAI, although it is very time-consuming in larger samples.Our findings differ from Related to adolescent attachment to peers (best friend and romantic partner), no significant differences emerged. Overall, the attachment relationships to best friend and romantic partner showed in average low attachment avoidance and attachment anxiety, further indicating that neither individuals conceived with ART nor twins are likely to show problems in peer attachment. Since this is, to our knowledge, the first study on peer attachment after ART, more research is warranted. However, our results support the findings of Some of our descriptive statistics were also interesting. Especially, there was an association between newborn health risks  and higher adolescent attachment anxiety toward mother and father. A previous study by Our prospective, 18-year longitudinal study was, to our knowledge, the first to compare the four groups of ART and NC twins and singletons on the outcomes of relationship to parents and peers. Additionally, it is the first study to assess attachment beyond infancy as an outcome of ART. Our study was also unique in taking into account the statistical dependence of twins, and making appropriate statistical corrections, which is rarely done in twin studies.The main limitation of the study was the relatively small sample size in twin groups, especially NC twins, which was exacerbated by drop-out over the 18\u2009years of the longitudinal study. There was also more drop-out among boys and families with lower parental education level. However, highly sophisticated methods were used to replace missing data, and based on earlier simulation studies on multi-level partially clustered data , the powA second limitation concerns the generalizability of the results. All participants were native Finnish-speakers, and in Finland, the expenses of infertility treatments are also to a large extent publicly funded, making the treatments available for families with lower socio-economic status (SES) than in many other countries. However, there is evidence that at least early treatment-related outcomes are not associated with family SES .Previous studies e.g. , it was Overall, the results contribute importantly to the accumulating long-term evidence that neither ART treatments nor being a twin place adolescent psychosocial development at a significant risk. Infertility and its treatments, as well as parenting young twins who often have medical problems, are typically stressful experiences. It would be a relief for parents to be informed about the safety of the treatments also in terms of the long-term psychosocial development of the children. However, our findings suggest that fathers conceiving with ART need to be kept in mind by health care providers to ensure they receive the same support in the pre- and post-natal services as the mothers. Although infertility-related counseling services for men have improved after our data collection (20\u2009years ago), these services are still more directed to women . EspeciaHuman Reproduction Open online.The data underlying this article cannot be shared publicly due to the privacy of individuals who participated in the study. The data will be shared on reasonable request to the corresponding author.Participation in study design: A.T., R.-L.P., M.S.F., M.V., J.L., M.P. and P.P. Execution: R.-L.P., M.S.F., M.V., J.L. Analysis: M.P., J.M., M.S.F. Interpretation, manuscript drafting and critical discussion: M.S.F., M.P., M.V., J.L., J.M., A.T., P.P., Z.B., R.-L.P.https://projects.tuni.fi/kehi/project/) and the Finnish Cultural Foundation to Marjo Flykt.Funding for the study was obtained from the Academy of Finland (grant number 2501308988), the Juho Vainio Foundation for the Miracles of Development research project (The authors declare no conflict of interest.hoac012_Supplementary_DataClick here for additional data file."}
+{"text": "Aim: To investigate the compliance and the outcome of Traditional Chinese Medicine (TCM) in patients with coronary heart disease (CHD) after treatment of revascularization.Methods: In this prospective cohort study, the non-exposure group (NEG), low-exposure group (LEG), and high-exposure group (HEG) were divided after 2 years follow-up. The primary outcome was a composite of death from cardiovascular causes, non-lethal myocardial infarction, heart transplantation, or stroke. Time-to-event data were evaluated by using the Cox regression analysis with hazard ratios (HRs) and 95% CIs. Then, the two-sided p-values were calculated by using the Cox models. In order to indicate the therapeutic effects of TCM on the CHD after revascularization, the survival analysis and the nested case\u2013control study were conducted separately.Results: There were 1,003 patients with CHD enrolled, 356 patients (35.49%) did not choose the TCM, 379 patients (37.79%) used the TCM seldom, and only 268 patients (26.72%) used TCM regularly. A total of 653 patients with revascularization participated in the prospective cohort study. Over the duration of the trial, the primary endpoints occurred in 12 (4.35%), 11 (4.80%), and 2 (1.35%) patients in the NEG, LEG, and HEG, while the secondary endpoints occurred in 84 (30.43%), 57 (24.89%), and 15 (10.14%) patients in the NEG, LEG, and HEG, respectively. The occurrence time of secondary endpoint events in HEG was significantly postponed (p < 0.001) compared with the other cohorts. The Cox regression analysis indicated that the HRs in the primary endpoints, the secondary endpoint events, the major adverse cardiac and cerebrovascular events (MACCE), and the composite endpoint events for HEG were all around 0.3 (p < 0.05) and HRs for LEG were all around 0.8. The results of the nested case\u2013control study showed that the TCM exposure was significantly different between the cases and controls in the secondary endpoints (p < 0.05), while no significant difference in the primary endpoints (p > 0.05), but the percentage of HEG in the cases was extremely lower than the controls.Conclusion: The HEG-TCM may improve the outcomes of the patients with CHD after treatment of revascularization.Registration:http://www.chictr.org.cn. Unique identifier: ChiCTR-OOC-17012995. Currently, cardiovascular disease (CVD) is the leading cause of premature morbidity and mortality in the world \u20136, of whCoronary heart disease, as a chronic condition, caused the further cardiac damage and death , 15. PatAccordingly, the TCM treatment accepted by the patients with CHD was investigated and a prospective cohort study was carried out to learn about the compliance and prevalence of endpoint events in the patients with CHD after treatment of revascularization. Then, it presents a valuable individual therapy strategy with implications for disease management and endpoints for the patients with CHD.From October 2017 to October 2019, a prospective cohort study was conducted in three representative hospitals of Tianjin. They were Tianjin Chest Hospital representing Western Medical Hospital, the Second Affiliated Hospital of Tianjin University of TCM, and the TCM Hospital of Beichen District representing TCM hospital to get the situation about the TCM treatment accepted by the patients with CHD and evaluate the therapeutic effects of TCM on the patients with CHD after treatment of revascularization. The medicines used, symptoms, and clinical endpoint events of the participants were collected in the baseline and every 3 months without interfering with the treatment of the patients.This study was registered on the China Clinical Trial Registry with number ChiCTR-OOC-17012995. Additionally, this study was conducted in accordance with the recommendations of Ethics Committees of the Second Affiliated Hospital of Tianjin University of TCM and the protocol was approved by the Ethics Committees with registration number 2017-046-01. All the subjects gave a written informed consent form (ICF) in accordance with the Declaration of Helsinki.The patients who were diagnosed as CHD [the CHD diagnostic criteria were: (1) the lumen diameter of at least one main branch was narrowed by more than 50% which were confirmed by coronary angiography or coronary CT angiography and (2) there was clear evidence for myocardial infarction in the previous or at that time including ST-segment elevation or non-ST-segment elevation] for more than 5 years would be screened. The exclusion criteria were as follows: (1) Patients with severe cardiopulmonary insufficiency and severe primary organ diseases before the recruitment such as tumor; (2) Mental patients; (3) Participants joined in other interventional clinical research; (4) Patients with poor compliance and did not cooperate well; and (5) Those who had no crucial data such as refusing to give feedback to the visit and failing to answer the call for many times. All the excluded cases should be recorded and the case report forms should be kept for future reviewing, but without statistical analysis. The patients who experienced revascularization including percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), or both would be enrolled into the prospective cohort study.Over the duration of the clinical trial, the participants were divided into three cohorts, the non-exposure group (NEG), low-exposure group (LEG), and high-exposure group (HEG), according to the administration amount and duration time of TCM treatment (including proprietary Chinese medicine and decoction). In the NEG, participants did not use any TCM during the visiting periods; in LEG, participants did not follow the direction of the physicians or irregularly used TCM during the visiting periods or they did not use TCM enough, which was defined as the period of continuous using TCM < 2 months or intermittent using TCM (recorded the cumulative time) < 4 months; in HEG, participants continuously followed the prescribed regimens with a long-term using of TCM, which was defined as the period of regular using TCM \u2265 2 months or intermittent using TCM \u2265 4 months.In this study, the primary endpoint was a composite of death from cardiovascular causes, non-lethal myocardial infarction, heart transplantation, or stroke. The secondary endpoint was repeat revascularization. The total of primary and secondary endpoint was identified as the cardiovascular composite endpoint events. The major adverse cardiac and cerebrovascular events (MACCE) included primary endpoint events, secondary endpoint events, and all-cause mortality.All the research-related information was inputted separately by the two researchers who were trained rigorously. The consistency was checked automatically and the queries were sent and responded when the differences were founded during the checking. All the information of the participants would be stored securely in locked file with limited access.p \u2264 0.05 was considered as statistical significant.Continuous variables were expressed as mean \u00b1 SD and categorical variables were expressed as counts and percentages. The SPSS software version 26.0 (USA) was used for all the statistical analysis. A two-sided Baseline characteristics in different cohorts were tested by using the chi-squared test or the Fisher's exact test for categorical variables  and the ANOVA for continuous variables .The incidence of endpoint events and correlation intensity [relative risk/hazard ratio (RR/HR)] was calculated and the statistical inference was carried out by survival analysis. Moreover, the cumulative survival rate and death trend were described by the Kaplan\u2013Meier (KM) curve where long-rank test was used to evaluate the survival rate between cohorts. Then, the multivariate Cox regression models with HRs and 95% CI were used to analyze the multivariate factors in CHD. If HRs \u2248 1 indicating that the factors do not affect CHD; if HRs < 1 indicating a protective factor; if HRs > 1 indicating a dangerous factor.The study flow was shown in A total of 1,003 patients with CHD were investigated for the baseline characteristics . The aveThe baseline characteristics of the cohort participants are shown in p < 0.001).The incidence of endpoint events is shown in p < 0.001) compared with the other cohorts. Additionally, the difference between the NEG and LEG was not significant and the trends were similar in both of the cohorts. The risk of primary endpoint events did not show and significant difference (p = 0.2069) between the three cohorts. In addition, the incidence of primary endpoints was much lower than the secondary endpoints . Then, these significant variables along with the variables , which were reported to impact the CHD significantly , while no significant difference for the primary endpoints (p > 0.05) , which were reported to significantly affect the occurrence of CHD endpoints  by using > 0.05) . The nesIn the prospective cohort study, the participants took TCM in LEG and HEG cohort. It showed the top five used frequency TCM, which were prescribed individually according to the disease syndrome by TCM physicians . There wCoronary heart disease has become a global health problem increasingly . In the The cohort study with long time and large samples can follow the individualized diagnosis and dynamic changes of treatment of TCM . MoreoveInterestingly, the Cox regression models indicated that the simultaneously administration of clopidogrel and aspirin seemed to be a dangerous variable for the CHD endpoints. It is known that dual-antiplatelet therapies (DAPTs) are essential for thromboprophylaxis for the instable patients with CHD and PCI , 31. HowOf all the TCM used in the patients with CHD revascularization, Suxiaojiuxin pill and compound Danshen dropping pill accounted for substantial part. Based on TCM theories, Suxiaojiuxin pills have the function of blood-activating and qi-moving, meanwhile, compound Danshen dropping pill could activate the blood and resolve the thrombosis. The patients with CHD with the blood stasis syndrome (BSS) always manifest symptoms including precordial dull pain or stab pain, dark complexion, cyanotic nails and lips, scaly skin, purple dark tongue with spots or ecchymosis, and rough or knotted intermittent pulse , 33. In There were only 1,003 patients with CHD recruited from Tianjin, which might not very representative. The larger, multicenter cohort study throughout China is needed to reduce the recruitment bias. Second, the baseline characteristics (such as duration of CHD and smoking history) were limited to self-reported data and the recall bias was inevitable. We have checked for the Hospital Information System to remedy some recall bias of the data. Third, the confounding bias might also contribute to the evaluation of the effectiveness of TCM treatment on the CHD. The Cox regression models, the KM curves, and the nested case\u2013control study were employed to minimize the unexpected bias. Fourth, we could not give detail descriptions of doses and formulations of TCM for all the participants because the TCM prescribed individually by physicians was not standardized, which yields to the theory of TCM. Fifth, in this study, the exact time after revascularization was not defined in the inclusion criteria. In fact, 80.27% patients were more than 1 year after revascularization showed that the situation of CHD disease was stable. Some subgroup analysis would be conducted further.The HEG-TCM may improve the outcomes of the patients with CHD after treatment of revascularization.The original contributions presented in the study are included in the article/This study involved human participants and was conducted in accordance with the recommendations of Ethics Committees of the Second Affiliated Hospital of Tianjin University of TCM and the protocol was approved by this ethics committees with number 2017-046-01. All subjects gave written informed consent from (ICF) in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.CL and HY wrote the manuscript. YZ, YL, JZ, SG, and RL designed the research. ZC, HD, YZ, YL, SG, and RL performed the research. CL, ZC, HD, and HY analyzed data. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 81673751) and the National Major Science and Technology Projects of China (No. 2018ZX09734002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Modifying the structure of anti-tumor chemotherapy drug is of significance to enhance the specificity and efficacy of drug-delivery. A novel proteolysis resistant PD-L1-targeted peptide (PPA1) has been reported to bind to PD-L1 and disrupt the PD-1/PD-L1 interaction, thus appearing as an outstanding tumor-targeting modification of synergistic drug conjugate for effective anti-tumor treatment. However, the combination regimen of coupling PD-L1 polypeptide with chemotherapeutic drug in tumoricidal treatment has not been reported thus far.We developed a novel synergistic strategy by conjugating PPA1 to doxorubicin (DOX) with a pH sensitive linker that can trigger the release of DOX near acidic tumor tissues. The binding affinity of PPA1-DOX with PD-L1 and the acid-sensitive cleavage of PPA1-DOX were investigated. A mouse xenograft model of colon cancer was used to evaluate the biodistribution, cytotoxicity and anti-tumor activity of PPA1-DOX.in vitro and specifically enriched within tumor when administered in vivo. PPA1-DOX exhibited a significantly lower toxicity and a remarkably higher antitumor activity in vivo, as compared with free PPA1, random polypeptide-DOX conjugate, DOX, or 5-FU, respectively. Moreover, increased infiltration of both CD4+ and CD8+ T cells was found in tumors from PPA1-DOX treated mice.PPA1-DOX construct showed high binding affinity with PD-L1 We describe here for the first time that the dual-functional conjugate PPA1-DOX, which consist of the PD-L1-targeted polypeptide that renders both the tumor-specific drug delivery and inhibitory PD-1/PD-L1 immune checkpoint inhibition, and a cytotoxic agent that is released and kills tumor cells once reaching tumor tissues, thus representing a promising therapeutic option for colon cancer with improved efficacy and reduced toxicity. Chemotherapy is one of the major categories of the medical discipline specifically devoted to pharmacotherapy for variety of cancers, including colon cancer , 2. Sincin vitro and inhibit the tumor growth in CT26 bearing mice by disrupting the PD-1/PD-L1 interaction. The D-peptide construct of PPA1 may prevent the degradation of proteolytic enzymes in serum  with a pH sensitive linker. The reason that we did not select 5-Fu as the conjugated drug was the lack of suitable synthetic site on 5-Fu. Although the development of resistance to DOX in colon cancer has been shown , DOX wasThe PPA1 (nyskptdrqyhfk) and RNA (rhtndysqfypk) were purchased from Chinapeptides Co.,Ltd., China. The polypeptides were both in D-form.2H4, about 80% in H2O), doxoriubicin, 4A molecular sieves, CuSO4.5H2O, sodium ascorbate, 1-(1-benzyltriazol-4-yl)-N,N-bis[(1-benzyltriazol-4-yl)methyl]- methanamine (TBTA) were purchased from TCI. DOX and 5-Fu were purchased from J&K Scientific Ltd. Those custom peptides of R(2-Azido) were synthesized by ChinaPeptides Co,. Ltd. All of the purchased chemicals were of at least reagent grade and were used without further purification. Reactions were monitored by analytical thin-layer chromatography (TLC) using silica gel 60 F254 pre-coated glass plates (0.25\u00a0mm thickness) and visualized using UV light.Methanol, ethanol, trifluoroethanoic acid (TFA) and N,N-dimethylformamid (DMF) were purchased from Sigma-Aldrich. N,N\u2019-dicyclohexylcarbodimide (DCC), 4-dimethylaminopyrid (DMAP), hydrazine hydrate  spectrometer using tetramethylsilane (TMS) as internal standard at 25\u00b0C. Samples were prepared as solutions in deuterated solvent. Those following abbreviations were used to indicate the observed spin multiplicities on NMR spectra: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet, and br = broad. High resolution mass spectra (HRMS) were recorded on Bruker Autoflex MALDI-TOF mass spectrometer. Purity of all final compounds was 95% or higher as determined by high performance liquid chromatography (HPLC) (SHIMADZU Labsolutions) analysis on the Aglilent C18 column  using gradient elution  at a flow rate of 1.0 mL/min.The 2SO4 and the solvent was removed under reduced pressure. The residue was purified by column chromatography to give 2  as a colorless oil.To a solution of 5-hexynoic acid  in MeOH (30 mL), DCC  and DMAP  were added successively and the mixture was stirred at room temperature for 4\u00a0h, then filtrated and concentrated under reduced pressure. Weak acidic water was added and extracted with EtOAc (4 * 30 mL). The organic layers were dried over Na2  in EtOH (30 mL) was added 80% hydrazine hydrate solution in H2O (1 mL) at room temperature. Then the mixture was stirred at 80\u00b0C for 6\u00a0h and the solution was evaporated in vacuo. The residue was dissolved in EtOAc and washed with aqueous citric acid (*3) and brine (*2). The organic solution was dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by column chromatography to obtain 3  as a yellow solid.To the solution of 3  was dissolved in MeOH (30 mL) and treated with 4A molecular sieves following a drop of TFA (20 \u03bcL). The resulting mixture was stirred at room temperature for 24\u00a0h. Then the solvent was evaporated and the crude product was purified by column chromatography to provide 4  as a reddish-brown solid.Doxorubicin hydrochloride  and 4 , azide end-functionalized peptide (PPA1)  and Tris(benzyltriazolylmethyl)amine (TATB)  were introduced in a Schlenk tube and 4 mL of DMF: H2O (v: v = 3:1) were added. The solution was degassed by bubbling argon for 10\u00a0min. CuSO4.5H2O  and sodium ascorbate  were added to the mixture contained in the Schlenk tube and the mixture was degassed once more by bubbling argon for 10\u00a0min. The Schlenk tube was filled with argon and stirred at room temperature for 4\u00a0h. The solution was filtered and concentrated under vacuum. The resulting crude mixture was purified by HPLC to offer 5  as a reddish-brown solid.6, 7 and 8 were prepared as the synthetic procedure of conjugate 5.The conjugates of 2 and 95% humidity. The cells were cultured in T75 culture flask and the cell density up to 80% was used in experiments. The cell line was negative for mycoplasma.Mouse colorectal cancer cell line CT26 was purchased from Shanghai Cell Bank of Chinese Academy of Sciences. The cells were maintained in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and incubated at 37\u00b0C with 5% COThe three-dimensional models of PD-L1 were downloaded from Protein Data Bank (PDB) (PDB ID: 3BIK). The structure of the peptides was generated by Chimera 1.14. The molecular dynamic for the coarse structures were implemented for energy minimization and optimization in amber force field. Molecular docking was performed to generate the initial complex of PPA1-DOX or PPA1 and PD-L1 by using Cluspro 2.0 web server. The binding free energy was calculated with MM-PBSA algorithm.50 is calculated by fitting the dose-response data to a sigmoidal curve, typically using the Hill equation. The calculation is performed by using ECCpy (https://github.com/teese/eccpy).The interaction between Tag1-PD-L1 and Tag2-PD1 is detected by using anti-Tag1-Europium (HTRF donor) and anti-Tag2-XL665 (HTRF acceptor). When the donor and acceptor antibodies are brought into close proximity due to PD-L1 and PD1 binding, excitation of the donor antibody triggers fluorescent resonance energy transfer (FRET) towards the acceptor antibody, which in turn emits specifically at 665 nm. This specific signal is directly proportional to the extent of PD1/PD-L1 interaction. Thus, compound or antibody blocking PD1/PD-L1 interaction will cause a reduction in HTRF signal. The HTRF PD1/PD-L1 binding assay kit was from Cisbio. The ICTo verify the acid sensitivity of hydrazone bond linking the PPA1 to DOX, a series of PPA1-DOX and DOX solutions with different concentrations were configured in the sodium phosphate buffer (pH= 9.0), respectively. A 5 \u03bcL aliquot of each sample was injected onto an HPLC system with ultraviolet detector wavelength to determine 254 nm absorbance values, using hydrophilic interaction chromatography (HILIC) column separation . Through simulating the linearity fitting from disposed concentrations and detected absorbance values, standard curves of PPA1-DOX and DOX were obtained, respectively.Preparing two partials of PPA1-DOX (1 mg/mol) were dissolved into the 2 mL sodium phosphate buffer (pH = 5.0) and 2 mL mouse serum (pH = 7.4), respectively, and the vials were capped and kept 37\u00b0C under nitrogen with continuously slight oscillaation. Samples (20 \u03bcL) were spiked and analyzed by HPLC under the 254 nm absorbance value after incubating 0.5\u00a0h, 1\u00a0h, 2\u00a0h, 4\u00a0h, 12\u00a0h and 24\u00a0h. For evaluation of hydrazone bond cleavage data was considered the disappearance of the major peak related to the standard curves of PPA1-DOX and DOX.The tumor tissues were fixed in 4% paraformaldehyde, gradually dehydrated, embedded in paraffin, cut into 4um sections, and subjected for hematoxylin/eosin staining. For immuno-histochemical staining of CD4/CD8 positive cells, tissue sections processed through deparaffinage, rehydration and antigen plerosised, and endogenous peroxidase activity blockade, were incubated with mouse anti-human CD4, mouse anti-human CD8 at 4\u00b0C overnight, respectively. The secrions were then washed and incubated with a HRP-labeled secondary antibody at RT for 40\u00a0min. After color development through incubation with diaminobenzidine, the sections were counterstained with hematoxylin. The stained sections were observed and imaged under a light microscope.Animal experiments and maintenance were approved by the Laboratory Animal Ethics Committee of Shenzhen University.6 CT26 cells in the left armpit. The mice were randomly divided into groups with 7 mice in each group after the tumor reached about 100 mm3. The mice were housed in animal house of Shenzhen University. For the survival experiment, the mice were killed when the tumor reached about 1500 mm3. The tumor size and body weight were monitored every day after the injection of the drugs. At the end of the experiment, the tumors and major organs  were collected for immunohistochemistry and/or histological tests. For the PPA1-DOX distribution experiment, the tumors and major organs were subsequently analyzed with an in vivo imaging system .Currently the institute does not provide approval or accreditation number. Six-week-old female Balb/c mice were inoculated subcutaneously with 5 \u00d7 10DOX at 5 mg/kg, 5-Fu at 10 mg/kg, PPA1 and RAN at 15mg/kg, PPA1-DOX and RAN-DOX at 20 mg/kg were injected intraperitoneally to mice twice a week for two weeks. From the fifteen day, no further injections of the drugs were given because mice injected with 5-Fu and DOX became very sick due to toxicity. For the PPA1-DOX distribution experiment, RhB-PPA1-DOX and RhB-RAN-DOX at 20mg/kg were injected into the tail vein of the mice.All the variables were expressed as mean \u00b1 standard deviation. Student\u2019s t-test or one-way analyses of variance (ANOVA) were performed in statistical evaluation. A p-value <0.05 was considered to be significant.A tumor-specific targeted synergistic strategy was designed by coupling anti-PD-L1 polypeptide with chemotherapy for colon cancer, where a proteolysis resistant PD-L1-targeted peptide PPA1 was conjugated to DOX with a pH sensitive linker . The com1 was reacted with methanol under the condensation reagent of DCC and catalysis reagent of DMAP to effectively provide the methyl 5-hexynoate 2. Ester 2 was treated with 80% aqueous hydrazine hydrate in ethanol at 80\u00b0C for 6 hours to smoothly give acyl hydrazide 3. The desired compound 4 with an acid-sensitive hydrazone was afforded by compound 3 coupling to commercially available doxorubicin in methanol. Compound 4 was allowed to undergo cycloaddition reaction with various peptide azides under sharpless click chemistry condition to offer the target compound 5 (PPA1-DOX) in good to excellent yields. Compound 6 (RAN-DOX) was synthesized in the similar routine by using a random polypeptides. Compound 7 and 8 with rhodamine (RhB) were designed and synthesized to verify the distribution of the compounds. The intermediates were characterized by Nuclear Magnetic Resonance (NMR), including 1H-NMR, 13C-NMR, and high resolution mass spectrometry (HRMS), and the conjugates were confirmed by high performance liquid chromatography (HPLC) and HRMS.  and RAN-DOX conjugate exhibited low binding affinity with PD-L1.The HTRF (Homogeneous Time-resolved Fluorescence) PD1/PD-L1 binding assay is designed to measure the interaction between PD1 and PD-L1 proteins. By utilizing HTRF technology, the assay enables simple and rapid characterization of compound and antibody blockers in a high throughput format  showed severe damages in the H&E stained sections of heart, liver and kidney, respectively  and RhB-RAN-DOX (fluorescence of a random polypeptides-DOX) and evaluated the biodistribution of the compound in the CT26-bearing mice after 24h intravenous injection by collecting major organs for\u00a0+ and CD8+ T cells by immunohistochemistry staining. We found that tumors from PPA1-DOX-treated mice showed obviously increased infiltration of both CD4+ and CD8+ T cells, as compared with RNA-DOX-, DOX-, or 5-FU-treated mice, respectively  was conjugated to DOX as a control and the biodistribution of the fluorescence labelled compound was evaluated in the CT26-bearing mice after intravenous injection. As expected, a remarkably higher tumor-specific enrichment and lower distribution in other non-tumor major tissues was achieved by the PPA1-DOX construct, as compared with the RAN-DOX construct. Accordingly, PPA-DOX conjugate demonstrated a significantly increased antitumor activity and remarkably reduced off-target toxicity, in comparison with RAN-DOX construct, which even though showed a weak tumor inhibition effect, probably due to a few of DOX released from RAN-DOX conjugate. Thus, delivery of DOX guided by PD-L1-targeted polypeptide contributes to the improved anti-tumor effect of PPA1-DOX conjugate.etc., could only deliver the drugs to specific target cells or proteins with little activity itself. Compared to the traditional drug delivery system, PPA1 not only acts as a targeting navigator for the drug, but also binds with PD-L1 to improve the antitumor activity of immune cells. It is well known that when PD-L1 is bound to PD-1, these \u2018coinhibitory\u2019 receptors function as breaks for the adaptive immune response to protect the host from being attacked by its own adaptive immune system, serving as immune checkpoints that effector T cells must pass to exert their functions , National Science Foundation of China , Natural Science Foundation of Guangdong Province, China , SZU Medical Young Scientist Program (71201-000001), SZU Top Ranking Project (86000000210) and Medical science foundation of Guangdong province (A2017072).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Hanseniaspora uvarum S-2 and Candida famata WB-1, in pure and sequential inoculation with commercial yeast Saccharomyces cerevisiae QA23 were analyzed in industrial-scale vinification of the grape variety Tamjanika. Their contribution to the quality and aroma profile was investigated by quantifying volatile compounds and wine sensory evaluation. Both yeast isolates were able to complete alcoholic fermentation, to reduce ethanol concentration up to 1.06% v/v (in monoculture) in comparation to S. cerevisiae QA23, and to enhance aroma and sensory profile. Based on calculated odor activity values (OAV), p-cymene, ethyl hexanoate, ethyl octanoate, and ethyl decanoate were the major aroma volatile compounds in all Tamjanika wine samples. Analyzed yeast strains significantly affected relative contribution of volatile compounds and can be considered responsible for the differences and uniqueness of the obtained wine samples. Besides confirmation of good enological and fermentative characteristics, selected isolates can be characterized as high ester-producing strains with potential to enhance the floral and fruity aromas of wine. The present study represents a further step toward the use of indigenous yeast isolates at industrial-scale fermentation in order to ensure the regional signature of Tamjanika wine.The enological potential of two previously characterized indigenous yeast isolates,  Saccharomyces yeasts and their impact on the quality and aroma complexity of the wine. Yeasts are widespread in nature, commonly occurring on sugar-rich substrates such as fruits, but also in soil, plant, and in different natural ecosystems [Saccharomyces yeasts, which play a predominant role as the main fermentation species, several non-Saccharomyces species have attracted considerable attention due to their good enological properties. Recent studies have shown that their winemaking potential relies on the ability to produce wines with lower ethanol content or volatile acidity, release the varietal aroma from non-volatile precursors, enhance wine complexity, and prevent spoilage [Candida and Hanseniaspora are the main yeast genera naturally present in the early stages of alcoholic fermentation (up to 5\u20136% v/v ethanol content), while, subsequently, Saccharomyces cerevisiae becomes predominant and completes the fermentation process. Despite the fact that indigenous non-Saccharomyces yeasts are represented only in the first stage of alcoholic fermentation, they possess the ability to produce high levels of aromatic compounds such as esters, higher alcohols, and fatty acids, sufficient to modify the volatile profile and sensory quality of wines [Saccharomyces yeasts in sequential or simultaneous inoculation with Saccharomyces yeasts could significantly contribute to the wine quality while mitigating the risks of stuck/sluggish or spoiled fermentations [In recent years, many studies have been unravelling the enological potential of non-osystems ,2. Althoosystems . Along wspoilage ,4,5,6,7.of wines . In propntations .Hanseniaspora and Candida genera mainly initiate the fermentation and remain dominant during the initial stage [Saccharomyces starter cultures. Furthermore, the use of indigenous yeast strains as starter cultures could additionally contribute to the unique qualities of wine, enhancing distinctiveness and regional characteristics of the wine. Previous reports indicated that although Hanseniaspora yeast demonstrates low fermentative power, it has a positive contribution on the wine quality related to the high production of primary  and secondary  metabolites [Candida genera are characterized as high glycerol and terpenol producers, and described as lower producers of aldehydes, acetic acid, and acetate esters [Hanseniaspora spp. are known as producers of undesirable compounds , a drastic strain variability was observed so some strains might, therefore, produce these secondary products in an acceptable or lower level [Hanseniaspora spp. and Candida spp. have the ability to excrete a broad range of extracellular enzymes which have an irreplaceable role in releasing aroma compounds from non-volatile precursors [Hanseniaspora or Candida strains in mixed culture with S. cerevisiae could enhance the mouthfeel, flavor, and quality of wines [Knowing that the representatives of al stage ,11,12, tabolites ,14. Yeaser level . In addiecursors ,16, promof wines ,13,15,17Saccharomyces yeasts such as metabolism/enzymatic details or winemaking potential mainly in the laboratory-scale level, lacking information about scale-up procedures and their effect on commercial fermentations. Factors such as geometry and volume capacity, heterogeneity in nutrient or oxygen distribution, or rapid sedimentation of yeast cells may be responsible for differences arising in scale-up procedures [A hunting campaign for non-exploited yeast strains with good enological potential mainly focuses attention on the microbial communities naturally associated with vineyard or cellar. In order to find tailored starter cultures for producing unique aroma profiles and give regional signatures to the wines, it should be extended further to the surrounding ecosystems. Additionally, the majority of the published papers deal with the enological characterization of the non-ocedures ,18.Saccharomyces yeasts belonging to epiphytic microbiota of regionally grown fruit were enologically characterized in order to find non-exploited yeast strains with good enological potential [S. cerevisiae. In this study, the contribution of the selected native Hanseniaspora uvarum S-2 and Candida famata WB-1 strains was assessed by analyzing the kinetics of alcoholic fermentation, standard enological parameters, and volatile and sensorial profile of wines produced from the grape cultivar Tamjanika . Despite the fact that this aromatic white grape variety has been very popular in Serbia for the production of wines with pronounced fruit and floral aroma, there is limited knowledge available about the chemical and volatile profiles of Tamjanika wines produced in Serbia. Hence, the significance of this research is also to provide an insight into the aroma and quality characteristics of the Tamjanika wines.In our previous research, an extensive number of non-otential ,19,20. TH. uvarum S-2 and C. famata WB-1 were previously isolated from the epiphytic microbiota of regionally grown fruit, characterized for their enological properties, and maintained at \u221220 \u00b0C in glycerol [Saccharomyces cerevisiae QA23  was used according to the producer\u2019s instructions.The glycerol ,19. IsolH. uvarum S-2 or C. famata WB-1 with the final cell number of 1 \u00d7 106 CFU/mL. For sequential fermentation protocol, after the initial fermentation stages, when \u00b0Brix decreased by 3 degrees, inoculation with S. cerevisiae QA23 (25 g/hL) was performed. The control wine sample was inoculated only with S. cerevisiae QA23 (25 g/hL). Yeast nutrient Fermaid E  was added in the concentration of 40 g/hL. All wines were fermented to dryness. The kinetic of alcoholic fermentations was monitored by a decrease in sugar concentration . At the end of alcoholic fermentation. Wines were sulphited (25 g/hL). After two months, wines were finned , filtered by Seitz filter plates K 100 , and bottled.Tamjanika grapes were cultivated in the Trstenik wine-growing subregion, Central Serbia  and processed at the \u201cMarco\u201d winery . Must characteristics were 23.6 Brix, 5.95 g/L total acidities, and pH 3.4. Manually harvested grapes were destemmed and crushed. After the addition of potassium-metabisulfite (6 g/hL), pectolytic enzyme , and vitamin C (5 g/hL), before further pressing, the grape juice remained in contact with the grape skin and seeds, for 24 h at a temperature of 4 \u00b0C. The free-run must was clarified (static sedimentation 24 h at 8\u201310 \u00b0C) and then divided into five stainless-steel fermentation tanks (1200 L). Fermentations were performed in duplicate. Inoculation of must for pure and sequential fermentation was performed with prepared culture of native strains 2 were determined according to the methods proposed by the International Organization of Vine and Wine . All measurements were performed in triplicate and results were expressed as the mean value with standard deviation.The alcoholic strength, total dry extract, total and volatile acidity, pH, reducing sugar, and free and total SOThe volatile aroma compounds were determined by solid-phase microextraction coupled with gas chromatography, following the procedure described in detail in our previous papers ,21,22. TThe sensory profile of the Tamjanika wine samples was evaluated  by nine judges officially certified for wine sensory analysis by the Serbian Ministry of Agriculture, Forestry and Water Management . Most significant attributes were defined for Tamjanika wines by the assessors on consensus, while a short training session with reference standards was provided monthly for all the assessors to avoid any bias during the sensory evaluation. A ten-point intensity scale  was used to rate the olfactory  and gustatory  attributes. The samples were randomly numbered and presented to the panelists. Tastings were conducted at room temperature, while unsalted crackers and room-temperature water were provided for mouth rinsing between each sample. The analysis was performed in duplicate and the results are expressed as mean values in a spider chart .\u00ae 26.0  and STATISTICA 7  software. Shapiro\u2013Wilk and Levene\u2019s tests were performed to evaluate the normality of data distribution and homogeneity of variances, respectively. Statistical differences were determined by one-way ANOVA followed by Tukey\u2019s HSD test, while Kruskal\u2013Wallis test with post hoc Dunn\u2019s test were applied for variables that did not meet normality criteria. A significance level of 0.05 was used for all statistical analyses. Principal component analysis (PCA) was performed for the differentiation of samples based on the aroma profile of wines.Statistical analyses of the data were performed using the SPSSSaccharomyces yeasts in pure or sequential fermentation with S. cerevisiae QA23 were used as an alternative solution to overcome the drawback of a single commercial species process and to improve the complexity, sensory properties, and flavor of wines [Non-of wines ,12,23. Aof wines ,27,28,29Saccharomyces yeasts have a positive impact on the quality, volatile, and sensory profile of Prokupac red wines [In our previous studies, we showed that some commercial and native non-ed wines ,19,21. SSaccharomyces strains, fermentation lasted for more than 8 days. About half of the sugar amount in grape juice was depleted after 70 h of fermentation by S. cerevisiae QA23, while more than 80% of sugar remained not consumed by C. famata WB-1 and H. uvarum S-2 strains, regardless of the fermentation protocol, even after 90 h of inoculation. Among both used non-Saccharomyces yeasts, faster reduction of sugar content was observed for H. uvarum S-2, independently on the fermentation protocol, indicating slightly better fermentation ability of this strain. Furthermore, after co-inoculation with commercial yeast S. cerevisiae QA23, the fermentation was accelerated compared to the pure fermentations with C. famata WB-1 and H. uvarum S-2. However, both pure fermentations required an additional 24 h for dryness to be attained, compared to the sequential fermentation inoculated with the same non-Saccharomyces isolates. Additionally, during the monitoring period, all fermentations were performed with no stagnation. Good fermentation ability of C. famata WB-1 and H. uvarum S-2 was consistent with our previously published results for the laboratory fermentation trials [H. uvarum strains isolated from spontaneous fermentation of Negroamaro [Saccharomyces yeasts such as Candida zemplinina [Lachancea thermotolerans [Torulaspora delbrueckii, Kluyveromyces thermotolerans [Kazachstania [Saccharomyces yeasts (C. famata WB-1 and H. uvarum S-2) could be considered as possible starter cultures in industrial-scale vinification, without the risk of stuck or sluggish fermentations. Moreover, the slower alcoholic fermentation observed for tested isolates does not necessarily represent a disadvantage, especially when bearing in mind that slower fermentation rate results in a cooler fermentation, leading to better retention of volatile compounds and lower demand for energy, which prevents overheating during the fermentation [Regardless of the yeast strain or fermentation protocol, a typical fermentation curve was observed for all fermentation trials. In the control sample, fermentation was completed 5 days after inoculation, while for the non-n trials ,19. Simigroamaro  or Montegroamaro  grapes amplinina , Lachanctolerans , Torulastolerans , and sevchstania . Such feentation .v/v to 0.53% v/v, for the H. uvarum S-2 and C. famata WB-1, respectively, while the highest ethanol reduction (1.06% v/v less compared to the control) was found in the wine sample produced with monoculture of C. famata WB-1 strain. Such results indicated that selected strains can be used to reduce the content of ethanol in large-scale wine production, which is in line with the interests of modern winemaking and consumer demands for products with a lower content of ethanol. The ethanol reduction was earlier confirmed for non-Saccharomyces species, and this ability highly depends on the yeast strain or/and fermentation conditions [S. cerevisiae QA23 could be explained by the ability of certain yeast strains to produce different metabolic byproducts  or to accumulate greater yeast biomass [Basic quality parameters for produced wines clearly indicated that yeast strain and fermentation protocol significantly affect the Tamjanika grape wine characteristics . Comparenditions ,36,37. A biomass .C. famata WB-1 and H. uvarum S-2 isolates were not able to ferment sugar to dryness in the sterile must, we highlighted that both isolates can produce dry wines from fresh must in a non-sterile environment that is more similar to the real conditions during wine fermentation. Completed fermentation of fresh Prokupac grape, or Tamjanika grape must in this work, confirmed the presence of grape or winery indigenous microbiota, which is able to prevail and finish the fermentation. It is worth mentioning that in our previous experiment we found that the inoculation with 6 log CFU/mL should be considered sufficient to ensure the growth and proliferation of inoculated isolates and their predominance at least during the early stages of fermentation, which will result in a substantial impact on aroma and sensory quality of produced wines. It has been reported that although non-Saccharomyces yeasts are mainly present in the first stage of fermentation, they significantly affect the characteristics of the wine [Saccharomyces yeast strains in monoculture. These findings are in line with our previous results for C. famata WB-1 strain [C. famata WB-1 and H. uvarum S-2, but still, in all produced wines, this level was far below the limits proposed by the OIV (1.2 g/L of acetic acid). Although different non-Saccharomyces yeasts  are known as the producers of higher amount of acetic acid [C. famata WB-1, H. uvarum S-2) on wine total acidity were different for Prokupac and Chardonnay grape must. Such results emphasize the need to examine the enological potential of different indigenous yeast for each specific grape variety individually in order to find the perfect yeast\u2013grape variety match. Compared to the wines produced with native yeast isolates in both fermentation protocols, the lowest total extract content was present in the control wine sample. Significantly higher content of total dry extract  was present in the wine produced with H. uvarum S-2 compared to the control sample, which is in accordance with the literature data [H. uvarum strains to produce high content of glycerol or other non-volatile compounds [C. famata WB-1 or H. uvarum S-2 could positively contribute to the body and mouth-feel of Tamjanika wines.Although in our previous papers we found that the wine . This isthe wine . The tot1 strain . This ab1 strain ,42. Indetic acid , this chtic acid . Comi antic acid  reportedtic acid . Comparetic acid ,19,20, tompounds . BearingSaccharomyces yeast strains  in pure or sequential fermentation on Tamjanika wine sensory profile, a sensory evaluation was carried out (p < 0.05), except for the taste typicality and astringency (p > 0.05).To evaluate the impact of two native non-ried out . Slight Saccharomyces yeast, but with statistically different scores for the majority of gustatory attributes. The increase in fresh fruit, citrus, and floral odor attributes was observed in wines produced in fermentation with pure C. famata WB-1 and H. uvarum S-2 compared to the control or wine produced in sequential fermentation. The wine produced in sequential fermentation with H. uvarum S-2 was highly rated for all taste attributes, except for the acidity, achieving the highest score for the honey, tropical, or dry fruit olfactory attribute. Additionally, this wine sample had the best scores for complexity (8.3), duration (8), fullness (8), intensity (7.7), and harmony (8). Samples fermented by C. famata WB-1 in sequential fermentation were characterized by the highest score for acidity and spice aroma notes. Compared with the control sample, used non-Saccharomyces strains produced wines with stronger fruity and floral flavor and a weaker herbal flavor.Generally, Tamjanika wines produced in all fermentations resulted in similar taste profile for the wines produced with the same non-A total of fifty-four volatile compounds were identified and quantified in wine samples produced by different yeast strains and fermentation protocols .However, in order to better assess the individual contribution of each quantified volatile compound to the overall wine aroma, odor activity value (OAV) and relative odor contribution (ROC) for compounds present in concentrations higher than their corresponding odor thresholds are shown in H. uvarum S-2 isolate). Such high average content of these compounds in wine samples is in agreement with the literature data, which indicated that higher alcohols commonly represent 80\u201390% of the aromatic constituents of wine [C. famata WB-1 or Metschnikowia pulcherrima B-5 yeast isolates [Issatchenkia terricola SLY-4, and Pichia kudriavzevii F2-24 [C. famata WB-1 isolates [H. uvarum S-2, in both fermentation protocols. Concentrations of phenylethyl alcohol in both samples were higher than its OAV, contributing with fine flower  and honey notes. Previous reports also indicated that H. uvarum is characterized as a good producer of phenylethyl alcohol [The majority of detected compounds are formed during alcoholic fermentation, so it is not surprising that their concentrations were significantly affected by yeast strains. As can be seen, quantitatively, the largest group of the volatile compounds in wine were higher alcohols, which represent about 78% (control wine sample) to more than 85% of all identified compounds  and H. uvarum S-2  yeasts. Furthermore, in our experiments, the commercial yeast strain produces Tamjanika wine with the lowest content of higher alcohol acetates, while sequential inoculation of selected yeast isolates resulted in an increase in the production of these compounds known to contribute to fruity notes (mainly banana and apple odor) of wine. These results indicate that the C. famata WB-1 and H. uvarum S-2 yeasts were able to esterify present higher alcohols better than the used commercial strain, providing about 7- and 2.5-times higher alcohol acetates than in a control sample, respectively. Knowing that the enzymatic formation of ethyl and acetate esters required specific enzymes  [S. cerevisiae QA23 strain. Therefore, the significant differences in the OAV and ROC for each compound confirmed the influence of the yeast strains on the wine\u2019s aromatic profile and justified the need to find the best possible match of the yeast species and grape variety.Esters are one of the most important aroma compounds in wines, typically responsible for the floral and fruity aroma. Their concentration in wine is strongly dependent on the yeast strains, fermentation, and storage conditions, as well as on the concentration of fatty acids, higher alcohols, and their precursors . Referri to wine . The tot species ,47. Indesferase) , such reC. famata WB-1, while only p-cymene and terpinolene were present in all samples. The concentration of individual terpenes quantified in analyzed Tamjanika wine samples was similar to the levels of p-cymene (5\u201335 \u00b5g/L), \u03b3-terpinene (6\u201335 \u00b5g/L), nerol (3\u201343 \u00b5g/L), and linalool (6\u2013375 \u00b5g/L) averagely present in different Muscat wines [H. uvarum S-2, but was not detected in wines fermented by C. famata WB-1 and commercial S. cerevisiae QA23 strain. In addition, compared to the pure fermentations (independently of used strains), sequential fermentation elicited higher concentrations of p-cymene, terpinolene, \u03b2-ionone, and (Z) and (E)-\u03b2-ocimene. Similar results, where different non-Saccharomyces strains positively affected the transformation and release of varietal aromas, were previously reported for some aromatic grape varieties such as Muscat, Gewurztraminer, Sauvignon blanc, and Verdejo [C. famata WB-1 and H. uvarum S-2, respectively) than for S. cerevisiae QA23 fermentation alone (0.253 mg/L), the total ROC of terpenes for the control sample was the highest. This finding can be explained by the lowest number of quantified key compounds with OAV above 1 in the control sample.For the recognizability of the characteristic and unique aroma of the Muscat grape, such as Tamjanika grape variety, different terpenes play a fundamental role. Linalool and geraniol are recognized as predominant terpenic aroma compounds present in Muscat grapes, along with citral, citronellol, nerol, and \u03b1-terpineol ,58. In aat wines . Accordi Verdejo ,60,61. AFurthermore, the release and transformation of varietal compounds, which are specific for certain grape varieties and are mainly present in odorless form as aroma precursors ,61, is pThe PCA was used to visualize and reveal the diversity or common characteristics among the wine samples produced by different yeast strains .H. uvarum S-2 from the other samples. However, samples fermented by H. uvarum S-2 were negatively correlated with p-cymene and \u03b2-ocimene. On the other hand, Tamjanika wines produced in both inoculation protocols by C. famata WB-1 were located in the lower right quadrant . The wine produced in pure fermentation with C. famata WB-1 was characterized by a higher relative contribution of acetate esters, isoamyl octanoate, and ethyl hexanoate. These compounds were related to fruit, herb, and pleasant aroma notes. The control wine sample was clearly different from the wine samples fermented by both native yeast strains in both inoculation protocols, with p-cymene as the compound that was considered most responsible for wine aroma.According to the PCA, four factors  had eigenvalues higher than 1, cumulatively explaining 99% of total variance of the initial data set. PC1 and PC2 comprised 74.52% of the variability. A clear differentiation among the Tamjanika wine samples can be observed, while it was found that samples fermented by the same yeast isolates were relatively similar because they were loaded closer to each other in the PCA plot. Methyl decanoate, methyl octanoate, phenylethyl alcohol, and linalool showed high negative loading scores in PC1 that distinguished both wine samples fermented by C. famata WB-1 and H. uvarum S-2 strains can be considered as alternative starter cultures, since they are able to positively modulate wine quality parameters, along with the aroma and sensory profile of the wine, in both pure and sequential fermentation on the industrial level. We have shown that native non-Saccharomyces strains can serve as a tool for ensuring the unique aroma profiles, while the sensory analysis demonstrated that all Tamjanika wines produced in different fermentations resulted in a similar taste profile, but with statistically different scores for the majority of gustatory attributes.This study indicates that both The fact that native yeast strains proved good fermentation capability, and that a variety of aroma compounds were produced in different Tamjanika wine samples, proves the uniqueness of the sensory and aroma profile of yeast isolates\u2013grape combination, indicating its potential in the winemaking industry."}
+{"text": "High-quality wines in industrial winemaking frequently require a professional winemaker to make adjustments according to the wine of single-batch fermentation. Blending can improve the chemical composition and certain organoleptic properties of wine, promote copigmentation, and increase the complexity of the wine body and aroma. In this study, high-performance liquid chromatography (HPLC) and headspace solid-phase microextraction with gas chromatography coupled to tandem mass spectrometry (HS-SPME-GC-MS/MS) were used to study the effects of adding 20% of Merlot, Marselan, Syrah and Pinot Noir and different blending methods on the nutritional, taste, color and aroma components of Cabernet Sauvignon wine. The results showed that the highest total phenols and flavonoids, the greatest content of antioxidant characteristics, the optimal color according to the parameter of T, red% and blue% and the most abundant aroma were observed both in CGM (grape blending Cabernet Sauvignon and Merlot) and CGS (grape blending Cabernet Sauvignon and Marselan), thus indicating the higher quality and complexity of these wines. In addition, the co-grapes treatment afforded more color and hue value than co-wines, which indicates co-grapes had more stable and more varied colors than co-wines. Our findings provide theoretical support for improving wine quality and craftsmanship. Wine is a very complex mixture of compounds such as carbohydrates, polyphenols, organic acids, proteins, minerals and more . It can What is memorable about wine is its consummate quality, such as taste, nutrition, color and aroma, etc. . OrganicTherefore, it is very important to study the technology and application of wine brewing to improve the complexity and quality of wine. Polyphenols and volatile compounds, along with their interactions, are responsible for color and aroma, respectively . The conSome grape varieties may be rich in certain cofactors, while others may be deficient. Furthermore, not all grapes contain the same number of anthocyanins and polyphenols, so some varieties may benefit from the presence of other varieties that may contain an excess of these compounds. In addition, Escudero Gilete et al. demonstrate that blended wines from different grapes provide a more equilibrated ratio of anthocyanins/flavanols , which mThe Cabernet Sauvignon grape is in a dominant position in the market because of its easy planting, high tannin, deep color, abundant style and a strong sense of structure . HoweverThe wine grapes  used in this study were harvested from the Manas production area of Xinjiang in China on 3 September 2021 at optimum maturity. The physicochemical indexes of different grape varieties are shown in 2 and 50 mg/L of pectinase were added to the musts [2 [All wine samples were separately fermented at the same condition (as follows). CS, ML, MS, SY, PN, CGM, CGS, CGY and CGP musts were crushed and collected into 50 L stainless-steel containers. Then, 60 mg/L of SOhe musts . After bmusts [2 . The win2 in wine samples were tested according to the methods of the International Organization of Vine and Wine [The soluble solids content was measured by a digital refractometer . The pH of the wines was monitored by a digital pH meter . Titratable acidity, alcohol and free SOand Wine , Residuaand Wine .v/v) formic acid at a flow rate of 1 mL/min.The organic acids in wine samples were based on a previous method with some modifications . Each saTotal phenolic content of wine samples was determined by a spectrophotometer according to the Folin-Ciocalteu colorimetric method . The totHPLC analysis was performed based on the methods of Alejandro, C\u00e1ceres-Mella et al. with some revisions . Wines w2 = 0.9982), catechin , epicatechin , rutin , coumaric acid , procyanidin B1 , chlorogenic acid , vanillic acid , ferulic acid , quercetin  and kaempferol  standards, and phenolic compounds were quantified using a standard calibration curve. All qualitative and quantitative analyses of the phenolic composition were performed in triplicate.Phenolic compounds were identified by comparing retention times and chromatographic spectra of samples with gallic acid  according to previous reports with some modifications to the temperature program .The GC-MS system was an Agilent 7890B GC incorporated with an Agilent 5977 mass spectrometer . The column used was a 30 m \u00d7 0.25 mm HP-INNOWAX capillary with 0.25 \u03bcm film thickness . The carrier gas was helium, and the flow rate was 1 mL/min. The temperature program was as follows: 40 \u00b0C for 5 min, 50 to 86 \u00b0C at 3 \u00b0C/min, 86 to 90 \u00b0C at 0.8 \u00b0C/min, 90 to 180 \u00b0C at 2.5 \u00b0C/min, 180 \u00b0C for 3 min, then at 25 \u00b0C/min, up to 230 \u00b0C, with a final holding time of 5 min.The mass spectra of the sample volatile compounds were compared with those in the NIST library, and the volatile aromatic compounds were identified by comparing the retention time and mass spectra of the sample volatile compounds with the standard display . UtiliziWine sensory analysis was conducted according to Peng et al. . The welThe one-way analysis of variance (ANOVA) with a Duncan test of the SPSS 19.0 software was used to examine the variation between samples . The effects of different samples of the aroma profiles were evaluated by principal component analysis (PCA) using SIMCA 14.1 . Origin (2021) was used to generate histograms, radar charts and overlays. For each sample group, three parallel samples were analyzed.2 in wine (13\u201329 mg/L) was sufficient to protect wine from further oxidation and microbial contamination [2 (<100 mg/L) content were under the legal limit established for wines [mination . Of noteor wines ,18.The organic acids have a great influence on the flavor balance of the wine, as well as on the chemical stability and pH, thus affecting the quality of wine . In addiRegarding the varietal factor, it can be summarized that the MS and PN wines showed higher tartaric and malic acids than the other wine samples. Citric acid slows down yeast growth and participates in biochemical and metabolic processes , up to 1p < 0.05). Before fermentation, MS wine had the highest total phenol concentration of 1272 mg/L . The total alcohol content of CS and ML was significantly higher in all wines than the others, and the co-grape treatment had a higher total alcohol content compared to the co-wines . The upsp < 0.05). The content of 1-hexanol in CY wine is the highest, reaching 85.73 mg/L, followed by CS wine, 51.05 mg/L. The blending treatment reduces the n-hexanol content to two to three times the original, which suggests that blending results in a significant reduction in the green character of the wine [Alcohol may be derived from yeast fermentation. C-6 alcohol is a kind of common plant volatile with a typical \u201cgreen\u201d odor . They arthe wine ,15. In athe wine . These cthe wine .n-octanol compared with other wines, with pleasant rosy, citrus aromas. Additionally, the highest levels of citronellol, 6-Nonen-1-ol and 3-Methylthiopropanol were found in blends of Cabernet Sauvignon and Syrah varieties. In the blended wine, it was observed that the content of higher alcohol was significantly affected by the blending method (p < 0.05). Compared with the co-wine treatment, the content of 1-heptanol, phenylethyl alcohol, isoamylol and ethyl alcohol was significantly higher in the co-grape treatment, which may be related to the maceration effect in the early stage of the co-grape treatment, which is consistent with the research of S\u00e1nchez-Palomo, who found that the concentration of higher alcohol is significantly correlated with immersion time [Wines blended with Pinot Noir and Cabernet Sauvignon grapes had significantly the highest concentrations of benzyl alcohol and ion time .Esters have long been considered an important contributor to wine aroma as they are the main source of fruity aromas . Most esThe production of fatty acids is related to the initial composition of the must and fermentation conditions. These compounds have been described as fruity, fatty cheese, and rancid notes . VolatilPrincipal Component Analysis (PCA) was used to assess the effect of parameters (different blended varieties and blending methods) on the aroma profile of the analyzed wines . The firn-propyl benzoate and 3-hexenyl isobutyrate. They were negatively related to PC1 and were specific for PN and ML.The projection of the wine samples on PC1 showed a clear separation according to the grape variety A. TherefRegarding PC2, further differences in wine samples were related to the method of blending. Therefore, as shown in Of the 53 compounds analyzed and quantified, only 9 had OAVs greater than or equal to 1.00 in wine . The intThe floral series is the most intense main aroma series of all treatments. The main contributors to this series are leaf salicylate, citronellolformate, phenethyl acetate, ethyl laurate, 1-octanol and 1-decanol. The effect of different varietal blends on this aroma family is markedly different. Leaf salicylate, citronellolformate and phenethyl acetate reached the highest levels in CGW wines. It is worth noting that the concentration of floral series in the blended wine samples is distinguished higher than the blended wine.The fruit series is another main aroma series in this study, dominated by ethyl octanoate, ethyl hexanoate, ethyl laurate, ethyl 9-decenoate, isoamyl acetate and phenethyl acetate. The highest concentration of fruit aromas was found in CGP wine samples, followed by CGW wines. Similarly, the concentration of fruit series in the blended wine samples is about twice that of the blended wine.Fats and nuts were the other two main aroma families in this study. The fats series was dominated by five compounds , and the nuts series was dominated by four compounds . The highest fat and nut concentration was found in CGY wines, followed by CWY. Interestingly, the fat series by the co-wines treatment is significantly higher than co-grapes.The results of the sensorial analysis are shown in The bitterness of CS and SY was higher than other wines B, but thThis work provides a better knowledge of the nutritional, taste, color and volatile composition of Cabernet Sauvignon wines elaborated by the blending technique. The results showed that the wines brewed by the blending technique present a more complex chemical characteristic and aroma complexity than monovarietal wines. High total phenols, total flavonoids and antioxidant activity were found in CGM, CGP and CWP wines. In terms of color parameters, CGM and CGS have a higher red tint, and CGM has a higher blue tint. These wines show better colors. In addition, the wines made by the CGP combination performed best in the fruity characteristic due to its high ester concentrations. The CGY and CWY wines had a high content of ester, alcohol and aldehyde compounds, showing the characteristics of nutty and fatty aromas. Notably, the co-grape treatment had higher antioxidant properties and aroma complexity compared to the co-wine treatment. Our research provides a feasible alternative to traditional methods to improve the chemical and organoleptic characteristics of Cabernet Sauvignon wines."}
+{"text": "Saccharomyces species, can contribute to increasing the sensory profile of the wine thanks to the release of volatile terpenes or other molecules. Therefore, this review will highlight the main aroma compounds produced by Saccharomyces\u00a0cerevisiae and other yeasts of oenological interest in relation to process conditions, new non-thermal technologies, and microbial interactions.The aromatic complexity of a wine is mainly influenced by the interaction between grapes and fermentation agents. This interaction is very complex and affected by numerous factors, such as cultivars, degree of grape ripeness, climate, mashing techniques, must chemical\u2013physical characteristics, yeasts used in the fermentation process and their interactions with the grape endogenous microbiota, process parameters , malolactic fermentation (when desired), and phenomena occurring during aging. However, the role of yeasts in the formation of aroma compounds has been universally recognized. In fact, yeasts  can contribute both with the formation of compounds deriving from the primary metabolism, with the synthesis of specific metabolites, and with the modification of molecules present in the must. Among secondary metabolites, key roles are recognized for esters, higher alcohols, volatile phenols, sulfur molecules, and carbonyl compounds. Moreover, some specific enzymatic activities of yeasts, linked above all to non- Of course, the balance and interaction of all the present compounds (volatile or not) determine the wine aromatic quality [Wine aroma is a very complex concept since it is provided by a hundred different compounds with concentrations varying between 10 quality ,3. HowevAmong all the compounds present in wine, volatile organic compounds (VOCs), with different polarities and volatilities, produced in widespread concentrations ranging from ng to hundreds of mg/L ,5, have Vitis vinifera are essentially: terpenes, norisoprenoids, and methoxypyrazine [Primary or varietal aromas are defined as compounds that derive directly from the variety of grapes and, for this reason, directly contribute to wine typicity. While the qualitative varietal aroma depends on the grapevine cultivar, the amounts present in the grape are strictly related to the environmental and pedo-climatic conditions. The compounds involved in the varietal aroma of the cultivars of pyrazine ,8. Seconpyrazine .The secondary aroma, called fermentation aroma, represents the main part of the transformation of grape must into wine and is operated by yeasts. The fermentation of grape must into wine involves the conversion of grape sugars to ethanol and carbon dioxide through the metabolism of yeasts, which, during alcoholic fermentation, produces other compounds besides ethyl alcohol and carbon dioxide. In particular, the so-called \u201csecondary aromas\u201d are linked to the activity of yeasts as they can only occur during the transformation of grape must into wine ,11. FermSaccharomyces cerevisiae is able to overcome all fermentation stresses, it, despite being in very low populations in vineyards or grapes, becomes prevalent in fermentation and dominates over the other yeast species abundant in natural must [S. cerevisiae has been designated with the title of \u201cwine yeast\u201d, being the main workhorse of the world wine industry [Natural grape must is hostile to microbial growth due to its low pH and high sugar concentrations. Furthermore, high concentrations of the antimicrobial sulfur dioxide are also added in most industrial fermentations, thus increasing the difficult conditions. Consequently, as fermentation proceeds, stress multiplies for additional reasons, including anaerobic conditions, depletion of nutrient reserves , increased acid concentrations, ethanol toxicity, and temperature changes. As ral must ,14. ThisAs far as aroma is concerned, higher alcohols, esters, aldehydes, and terpenes are among the compounds that have the greatest impact on the \u201cfermentation bouquet\u201d . The maiAlthough the secondary metabolism is of fundamental importance in the generation of the sensory profile of a wine, the aromatic bouquet of a wine is the result of a complex interaction between numerous volatile chemical compounds and the macromolecules of the system. From a quantitative point of view, the metabolites that originate as products or by-products of the glycolytic process and alcoholic fermentation are undoubtedly the most abundant and mainly include ethanol, glycerol, acetic acid, and succinic acid .Although they have different, but still very high, odorosity thresholds, due to their presence at high levels, they are able to directly or indirectly affect the final aroma of the wine, influencing the effect of secondary metabolites on the sensory profile of the wine. This is the case for succinic acid, a compound absent in the raw material and produced by yeast during fermentation, which is able to modify the interactions between macromolecules of the system  and aromatic compounds modifying the vapor pressure and, consequently, also the perception of the latter. Some studies have shown, for example, that, in a wine model system, the reduction in the concentration of ethanol from 10 to 7% determined a marked increase in the odorous perception of fruity and floral . AnotherS. cerevisiae or, preferably, with the strategy of co-inoculations or scalar fermentations with non-Saccharomyces yeasts that will be subsequently described [High levels in ethyl alcohol determine a different perception of the final bouquet since they increase the herbaceous hints compared to the fruity ones, also causing a greater astringency on the part of tannins and, therefore, bitter notes. In addition, the high presence of ethyl alcohol can activate a stress response in the yeast responsible for fermentation, influencing both the expression of specific genes and the modulation of the composition and fluidity of the cell membrane. However, market trends, inevitably linked to consumer preferences, are those preferring products with reduced alcohol content . This puescribed . The amoescribed .As previously described, some of these compounds originate from the primary metabolism of yeasts; others are instead generated by the regulation of the secondary metabolism of the latter and follow peculiar metabolic pathways, as reported in Associated with floral scents, esters are the group of secondary aroma compounds with the highest importance in the wine. Formed by the combination of alcohols and organic acids with the elimination of water, esters are produced both during fermentation and aging processes. During alcoholic fermentation, esters produced by enzymes are divided into two main categories: acetate esters and ethyl esters ,18. AcetRegarding higher alcohols , together with acetic and ethyl esters, they are the most important metabolites for defining wine aromas. The effect of fusel alcohols on wine aroma is dose-dependent and, for these reasons, an excess could negatively affect the wine aroma profiles.Their presence and concentration in wine is influenced by various factors, including grapevine cultivation methods, as well as the yeast strain used in fermentation. Other parameters, such as degree of grape ripeness, grape must pH, the presence of nitrogenous substances (including amino acids), the quantity of suspended solids, must aeration conditions, and the fermentation temperature, can also play a fundamental role for the qualitative and quantitative accumulation of higher alcohols in the final products . The bioThe fusel acid formations occur predominantly in aerobic condition , while fusel alcohols formation is favored by anaerobic conditions ,22. SincThis could be explained by the physiological function of the Ehrlich pathway. Apparently, this metabolic pathway appears as a catabolic process for the degradation of amino acids, but the decarboxylation and reduction that lead to the formation of higher alcohols are related to the amino acid biosynthetic processes. One of the most accredited hypotheses is that Ehrlich\u2019s pathway is a part of a scavenger system for the intracellular detoxification of excess of \u03b1-ketoacid resulting from sugars metabolism, especially in the fermentation process of musts with a high sugar concentration, as well as a metabolism/catabolism regulation system of amino acids . In thisS. cerevisiae present in spontaneous fermentation is characterized by a high genetic polymorphism that is made up of genetically different strains and, therefore, potentially capable of influencing the organoleptic and sensorial characteristics of the finished product. Therefore, it should be emphasized that different strains of S. cerevisiae exhibit a high degree of variability in all technological characteristics [S. cerevisiae accompanies different wild strains with the same characteristics (same genes that code for the various characters) but at a considerably different level and, as such, they produce, from the point of view of flavor and taste, wines with very different characteristics, even though they come from the transformation of the same grape must [S. cerevisiae, the production of higher alcohols involves a vast number of genes and Ehrlich pathway proteins. The initial transamination step involves at least four genes. Twt1p  and Twt2p (Bat2p or Eca40p) encode, respectively, for two branched-chain amino acid aminotransferases. The first (Twt1p) has a mitochondrial localization, while the second (Twt2p) is a cytosolic isozyme [S. cerevisiae cultures express the mitochondrial enzyme during the exponential growth phase, while it is repressed during the stationary growth phase. By contrast, the Twt2p cytosolic form has the opposite regulatory pattern [S. cerevisiae wine strains from spontaneous fermentations has demonstrated the existence of such strong polymorphism within this species [The population of eristics ,25 and eeristics ,27,28. Tape must . In S. c isozyme . Batch S pattern . Collect species . Therefo species .S. cerevisiae produces during fermentation through the Ehrlich pathway [From a quantitative point of view, higher alcohols are the most important group of compounds that  pathway . Typical pathway . Table 1 pathway . The cor pathway ,35. Neve pathway .S. cerevisiae synthesizes two major groups of esters during fermentation, namely the acetate esters of higher alcohols and the ethyl esters of medium-chain fatty acids. Such esters include ethyl acetate, isoamyl acetate, isobutyl acetate and phenylethyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate [S. cerevisiae, six genes and their expression products have been identified to be involved in the ester biosynthesis, as well as hydrolysis ester pathways. Specifically, these genes are ATF1, Lg-ATF1,  ATF2,  EEB1,  EHT1, and IAH1 [ATF1, Lg-ATF1, and ATF2 genes encode for alcohol acetyltransferase (AATases) proteins, which are directly involved in the volatile acetyl ester biosynthesis. EEB1 and EHT1 are both involved in the synthesis and hydrolysis pathways of medium-chain fatty acids ethyl esters [EEB1) and medium-chain fatty acid ethyl ester synthase/esterase II (EHT1). The first enzyme participates in the ethanol acyltransferase and ethyl hydrolase mechanism, the second in the ethanol hexanol-transferase mechanism [IAH1 gene encodes for esterase enzyme, which contributes to the hydrolysis of acetate esters. Although the precise physiological function of acetyl and ethyl esters is still unknown, the intracellular turnover of these compounds is strictly regulated. Ester production could be a part of a detoxification process for the removal of excess metabolites produced from sugar fermentations [Esters are the most desirable group of compounds contributing fruity and floral aromas to the wine bouquet . S. cereecanoate . The proand IAH1 ,40. ATF1l esters ,41. Thesechanism ,41. The ntations . The odontations . Taking ntations .S. cerevisiae during wine fermentation is acetaldehyde, constituting over 90% of the total aldehyde content of wine [2, the basic antimicrobial agent, which forms a complex compound, consequently limiting the protection to the produced wine [S. cerevisiae produce considerably different amounts of acetaldehyde, underlining the fundamental importance of choosing a suitable strain according to the type of wine produced [The major aldehyde synthesized by  of wine ,43. Acet of wine ,43. Of sproduced ,45.In addition, other precursors that do not possess odoriferous characteristics are involved in the development of other aroma substances  .S. cerevisiae strains, were generally characterized by different qualitatively volatile molecule profiles in relation to the must employed but also in the function of the inoculated strain [The variability in the formation of volatile compounds during grape must fermentation depends on several factors, including, in particular, the inoculated yeasts, which, transforming the precursors initially present in the must, determine the final organoleptic characteristics of the wine . Recentld strain .S. cerevisiae is found also in the example reported in The high strain variability in S. cerevisiae strains, able to impart specific sensorial and aromatic features, could be a useful tool to increase the gamma of products present on the market. Moreover, in addition to the ability to produce specific volatile profile fingerprinting [S. cerevisiae strains in order to increase the molecule volatile profiles. In fact, mannoproteins, constituted by mannose (85\u201390%) and proteins (10\u201315%), in addition to some positive effects on the technological properties of wines , can also affect the release of volatile compounds, affecting the final perception of the wine [In this perspective, the selection of new printing , some otthe wine . In winethe wine .The production of aromatic compounds that develop during fermentation processes is strongly influenced both by intrinsic parameters of the raw material, such as the presence of varietal aroma compounds, the qualitative and quantitative composition of the carbonaceous (sugars) and nitrogen  fraction, amino acids and inorganic nitrogen, both from the metabolic characteristics of the yeast strain used and from the fermentation parameters , as well as from the refining and aging practices. At the industrial level, the tendency is to optimize the fermentation process in order to enhance the intrinsic characteristics of the raw material, choosing the most appropriate winemaking conditions and fermentation agent, as well as in relation to the type of product to produce .S. cerevisiae by the metabolic regulation protein complex TOR (target of rapamycin), a state of catabolic inhibition by nitrogen, known as nitrogen catabolite repression (NCR). Nitrogen inhibition determines both the inactivation of the genes coding for permeases and enzymes responsible for the absorption of nitrogen sources present in the extracellular space and the modulation of energy catabolism, stimulating glycolysis and tricarboxylic acids cycle [S. cerevisiae strain used in the fermentation process, the amino acids will be consumed by the medium in an order of preference. Typically, glutamic acid, aspartic acid, asparagine, glutamine, serine, threonine, and arginine are elective nitrogenous sources for S. cerevisiae [S. cerevisiae during fermentation processes are known, whereas the exact relationship between the balance of carbon sources and the dynamics that occur in the formation of the compounds has not yet been fully clarified [In addition to carbon, nitrogen is of fundamental importance in the yeast cell for growth, cell division, and the production of secondary metabolites with a high aromatic impact. Yeasts are able to assimilate nitrogen both in amino acid form and in ammonia form. High concentrations of intracellular and extracellular nitrogen induce, in ds cycle . When nids cycle . Dependirevisiae . The prorevisiae . Thereforevisiae ,55. Althlarified .S. cerevisiae EC1118, considered an aromatic reference strain [Saccharomyces eubayanus to ferment Chardonnay must at different temperatures  over two vintages (2013 and 2014), highlighting also the volatile molecule profile in relation to the used temperature. The effect of added nitrogen was also evaluated. S. eubayanus showed its best fermentation performance at low temperatures (10 \u00b0C and 12 \u00b0C), with optimal kinetic parameters and high sugar consumption. The performance of S. eubayanus (EU) was compared with that of a commercial strain of S. cereviaise (CE). In particular, in the EU wine, 2-phenethyl alcohol (rose aroma) reached the highest OAV  compared to other aromatics, regardless of the temperature. Among esters, the ethyl nonanoate  was detected in the EU wine only, with a higher OAV in the wine fermented at 16 \u00b0C. The ethyl decanoate  displayed an opposite trend in the EU and CE wines at both temperatures. Ethyl myristate  was detected only in the EU wine produced at 10 \u00b0C, whereas methyl myristate  was produced at the highest concentrations by both yeasts at 16 \u00b0C. As expected from manufacturer information, CE wines reached high concentrations of ethyl hexanoate  and octanoic acid (fruit), with OAVs in the interval 6237\u201310471. Isoamyl octanoate (fruit) was found only in EU and CE wines at 10 \u00b0C. At 10 \u00b0C, the EU strain produced less ethyl acetate (which has negative aroma characteristics) than the CE strain. Interestingly, the EU wines were also characterized by a higher concentration of phenethyl acetate (rose) than CE wines at both temperatures; this aromatic compound is considered a marker in fermentation performed with S. eubayanus [The fermentation temperature is one of the technological process parameters capable of influencing the metabolism of the yeast and the aromas deriving from it. White wines are generally produced at lower fermentation temperatures than red wines to preserve the fruity and fresh notes typical of these wines. In fact, it has been shown that higher concentrations of esters are obtained for fermentation temperatures between 15 and 18 \u00b0C. This is due to the greater stability of the esters at these temperatures, the reduced loss due to evaporation, and the different metabolism of the yeast due to the modulation of membrane fatty acids. However, although the fermentation temperature significantly affects the growth of yeast and its central metabolism, the impact of this process parameter and the biosynthetic pathways associated with the increase in aroma are not fully known. In fact, although it is proven that high concentrations of esters are obtained at low fermentation temperatures, the effects on the production of higher alcohols are still poorly elucidated and more controversial. For example, some authors have shown that only the concentration of 2-phenylethanol, a higher alcohol attributable to the rose aroma, increases as the fermentation temperature increases. Other authors have shown, for all higher alcohols, a tendency to increase as the fermentation temperature increases. Some authors, using synthetic musts and fermentation temperatures of 15 and 28 \u00b0C, which, respectively, simulate the vinification temperatures of white and red wines, have analyzed the profiles in volatile molecules of the commercial yeast e strain . The rese strain . On the e strain . Therefoe strain  investigubayanus . IsoamylDuring the last decade, the application of non-thermal technologies to produce, age, and preserve wine has become an area of great interest. In particular, several authors have underlined the potential of high-pressure homogenization (HPH), pulsed electric fields (PEF), high-pressure processing (HPP), ultrasound (US), low electric current (LEC), pulsed light (PL), ultraviolet irradiation, and filtration applied to the must or wine as stabilization methods ,60.In particular, PEF and HPH are considered as promising technologies for the beverage industry as they are continuous processes, requiring only microseconds or milliseconds, respectively, of processing time for the inactivation of undesirable microorganisms in wines, with commercial-scale and higher throughput production potential. However, some authors have underlined the role of some non-thermal technologies, applied at sublethal level to bacteria or yeast cells used as culture starters, in order to modify their metabolism and, consequently, the production of aroma compounds, as reported by Saccharomyces bayanus L951 and S. cerevisiae ML692 and commercial strains S. bayanus Lalvin CH14, S. bayanus IOC 18-2007, S. bayanus Lalvin EC1118, and S. bayanus IT 1818), prior to use or prior to their use in the preparation of tirage solutions for sparkling wines, induced an acceleration of yeast autolysis with a consequent release of mannoproteins and different patterns in volatile profiles. In fact, the SPME-GC-MS and electronic nose analyses indicated significant changes in volatile patterns due to HPH treatment. In fact, the sparkling wines produced with HPH-treated cells, with the exception of those by strain L951, were significantly different on the basis of PLS analysis from the control wines. The wines obtained from treated cells were characterized by high acid contents and, particularly, short-chain fatty acids, associated with low levels of sulfur compounds. The wines obtained from HPH-treated strains had significantly modified ester profiles, with lower concentrations of those compounds with medium- and long-chain fatty acids with respect to the control wines. Moreover, the autolytic patterns of the HPH-treated cells can contribute to the different volatile profile recorded. The volatile profile of sparkling wine is reported to be dependent on the kinetics of volatile compound retention and release by yeast less during the aging process [For example, some authors have hypothesized how HPH, when applied to bacteria or yeast cells at levels ranging between 50 and 100 MPa, induces changes in gene expression that regulate specific properties (such as autolysis or production of aroma compounds) without affecting the cell viability or the fermentation kinetics ,62,63,64 process . The abi process . However process . Accordi process . While mS. cerevisiae ALEAFERM AROM grown in synthetic must induced a gene expression response of HPH-treated cells with a massive rearrangement of gene expression due to the identification of 1220 differentially expressed genes (DEGs). Most of the genes related with energetic metabolic pathways and ribosome structure were down-regulated, while genes involved in ribosome maturation, transcription, DNA repair, response to stimuli, and stress were up-regulated. Moreover, the samples produced by HPH-treated cells, compared to the control samples (cell not HPH-treated), after 48 h from the inoculation, were characterized by higher production of 2-phenylethanol, together with a significant increase in benzaldehyde , ethanol, isoamyl alcohol , acetic acid, and ethyl octanoate . Moreover, Comuzzo et al. [S. bayanus powder after HPH treatment, confirming the changes in the volatile molecule patterns. Moreover, according to the authors, the presence of higher amounts of alcohols and ethyl esters also represented evidence regarding the ability of HPH to induce autolysis in wine yeasts. These findings suggest that HPH induces or promotes an autolytic-like behavior that can be exploitable in winemaking also for the differentiation of the gamma of products. Some authors also reported, the use of some non-thermal technologies, such as high-pressure homogenization (HPH), for the treatment of yeast cells also in scalar fermentation.The accelerated autolysis and the increased exposure of the hydrophobic region of protein due to HPH treatment  can accoo et al.  showed aCandida zemplinina (CZ) and a commercial strain of S. cerevisiae (SC) after 21 days of fermentation and in relation to the initial HPH treatment.In the S. cerevisiae certainly represents the main yeast of alcoholic fermentation in relation to its technological performances, such as the ability to ferment the sugars of the grape must quickly and the ability to survive at low pH values and high alcohol levels. Due to these characteristics, it is the prevalent yeast present at the end of the vinification process. On the other hand, the presence of S. cerevisiae strains on grapes is very low in comparison to non-Saccharomyces yeasts, which can start the fermentation process but, due to their low alcohol tolerance, are usually replaced by S. cerevisiae [Saccharomyces yeasts have been associated with a negative role in winemaking because many strains were linked to phenomena of alteration, mainly due to their ability to increase the volatile acidity of wines. Nowadays, it is widely recognized that non-Saccharomyces yeasts contribute to the fermentative process and improve the aroma profile of the final wine despite their low fermentative power. In the past, they were considered as spoilage agents for the high production of undesirable metabolites, such as acetic acid, ethyl acetate, and off-flavors, resulting in anomalous analytical and sensorial profiles of the wine [Saccharomyces yeasts were positively reevaluated in winemaking due to their ability to produce aromatic compounds during the first stages of fermentation that contribute to enhancing the wine complexity. They are considered as a potential biotechnological tool, in mixed fermentations with S. cerevisiae, for their metabolic and enzymatic activities affecting the formation of the final bouquet [Saccharomyces yeasts hydrolyze the glycosidic bonds of the odorless, non-volatile glycoside, determining the release of the aromatic component as terpenols, norisoprenoids, and alcohol benzoics due to the \u03b2-glucosidase activity. The use of non-Saccharomyces yeasts could satisfy both the growing need of winemakers to innovate and diversify wine production in order to better meet the request of the globalized market and the interest of the consumers oriented towards increasingly aromatic products but with lower alcohol levels than in the past [Saccharomyces yeasts belonging to Torulaspora delbrueckii, Lachancea thermotolerans,  Metschnikowia pulcherrima,  Schizosaccharomyces pombe,  Starmerella bacillaris (synonym Candida zemplinina), and Pichia kluyveri are marked by the important manufacturers [Saccharomyces yeasts is fundamental in the early stages of alcoholic fermentation, before S. cerevisiae becomes dominant. In fact, yeasts contain genes that code for specific enzymes responsible for maintaining their survival in the ecosystem in which they are present. Some of these enzymes catalyze reactions involved in the transformation of sugars into ethanol; others are involved in the formation of the primary or secondary aromas described in the previous paragraphs. Due to the high biodiversity of the indigenous non-Saccharomyces population in the initial grape must, the range of extracellular enzymes produced during a spontaneous fermentation is much wider than that obtained by the inoculation of a S. cerevisiae starter. The most modern genetic sequencing techniques have shown that the non-Saccharomyces population is able to produce a greater variety of extracellular enzymes than S. cerevisiae. Consequently, isolation and characterization of these yeasts have become areas of research of great interest in the oenological field for the development of starters and co-starters, with the aim of innovating and improving wine production, even the most traditional [The species revisiae ,74. For the wine . However bouquet ,77. In fthe past . Nowadaythe past . Becauseacturers ,81 and uacturers . In bothditional .Saccharomyces yeasts in terms of flavor production are reported below and summarized in The characteristics of the most employed non-Torulaspora delbrueckii is one of the most studied non-Saccharomyces species in winemaking, being a yeast with interesting metabolic properties, and it is already marked as active dry yeast [T. delbrueckii allows to increase the glycerol content from 0.2 to 0.9 g/L [S. cerevisiae alone [T. delbrueckii and S. cerevisiae), a high content of acetate esters, medium-chain fatty acids, thiols, \u03b1-terpineol, and linalool, which is derived from monoterpene alcohols, were found. T. delbrueckii can produce higher levels of fruity esters and terpenes and lower amounts of higher alcohols when used in sequential fermentations with S. cerevisiae, respecting the varietal character of the grape [T. delbrueckii is characterized by high levels of extracellular enzymes, such as \u03b2-glucosidase, with an impact on the wine sensory profile, by modulating the levels of norisoprenoids, terpenols, and lactones in consequence of the hydrolysis from their respective precursors [T. delbrueckii strains can decrease the final ethanol concentration in wines by about 1% (v/v) [T. delbrueckii is easy compared to other non-Saccharomyces yeasts for its relatively high fermentative power and the ability to tolerate ethanol concentration up to 9\u201310% (v/v). Due to the high ethanol resistance, this species influences the characteristics of final wine during almost all the alcoholic fermentation, although S. cerevisiae is necessary to complete the fermentative process.ry yeast . It contry yeast , one of  0.9 g/L ,85,86 anae alone . Moreovehe grape ,89,90. Secursors ,92. The 1% (v/v) ,93 more 1% (v/v) . Other m1% (v/v) . The manLachancea thermotolerans is actually marked by important manufacturers and used to acidify grape juices [L. thermotolerans has other important oenological characteristics, such as the ability to reduce the volatile acidity in wine [S. cerevisiae [Saccharomyces species, L. thermotolerans shows moderate ethanol resistance (10% (v/v)) [S. cerevisiae strain [L. thermotolerans and S. cerevisiae reduce ethanol concentrations, ranging from 0.2% to 0.4% (v/v) [L. thermotolerans enhances floral and tropical fruit aromas during mixed fermentations of grape musts, producing wines with high concentrations of glycerol and 2-phenylethanol [e juices  because e juices . The rede juices . L. ther in wine  by produrevisiae ,100. Add% (v/v)) ,101. In e strain . Some st4% (v/v) ,93. Furtlethanol .Metschnikowia pulcherrima is actually available on the market and commonly associated with grapes and wine [Saccharomyces species are characterized by an extra-cellular \u03b1-arabinofuranosidase, influencing the content of desirable varietal aromas, such as terpenes and volatile thiols. In particular, M. pulcherrima releases varietal thiols, such as 4-methyl-4-sulfanylpentan-2-one, in concentrations higher than those produced by S. cerevisiae due to the cystathionine-\u03b2-lyase activity [M. pulcherrima is also able to improve the levels of methyl butyl-, methyl propyl-, and phenethyl esters [S. cerevisiae, with delays in fermentation. This effect was correlated to the killer character as a consequence of the production of pulcherrimin pigment by M. pulcherrima [and wine . In receand wine  due to sand wine , and glyactivity . Studiesl esters ,103. In cherrima . This spcherrima ,104,106.Schizosaccharomyces pombe is usually used for the deacidification of musts, mainly from cool areas, such as those from the north of Europe, as it can metabolize malic acid into ethanol and CO2, reducing the total wine acidity [Sch. pombe releases higher amounts of polysaccharides  [Sch. pombe can be used as a new tool to assure wine safety because it could be used to reduce the urea content  by the urease activity [Sch. pombe is the risk of production of high levels of acetic acid [S. cerevisiae or T. delbrueckii or the use of cells immobilized in alginate. Another undesirable effect of the use of Sch. pombe is an increase in the ethanol concentration as the degradation of malic acid produces additional ethanol [ acidity . It is p acidity . This ch acidity . Sch. poglucans) ,107,108,activity . However ethanol ,103.Pichia kluyveri is studied by wine researchers because it has been shown to release flavor precursors from grape juice, with a potential enhancement in the wine aroma and flavor. Mixed fermentation with P. kluyveri has been reported to lead to higher levels of varietal thiols, especially 3-mercaptohexyl acetate (3MHA), 2-phenylethyl acetate, and ethyl octanoate. Moreover, an increase in the total terpene concentration has been reported, improving the grape variety typicity [typicity ,103.Hanseniaspora. The yeasts belonging to this genus show a typical apiculate shape, and, among these, four species are associated with winemaking: H. guilliermondii, H. osmophila, H. vinae, and H. uvarum. They compose the common microbiota of grape berries and are found in the highest numbers in grape must [Hanseniaspora strains cannot survive due to their low tolerance to ethanol; however, they contribute significantly to the character and quality of the final wine [Hanseniaspora species, in particular H. guilliermondii, H. uvarum, and H. vinae, are good producers of interesting enzymes in winemaking, such as \u03b2-glucosidase, \u03b2-xylosidase, and protease, in particular for their application at an industrial scale. These enzymatic activities are correlated with the higher production of 2-phenylethyl acetate, often associated with rose, honey, and flowery sensory descriptors [Hanseniaspora generally exhibit low fermentative power, but the use in mixed fermentation with S. cerevisiae allows the production of some interesting volatile compounds. For example, co-fermentation of must with H. osmophila and S. cerevisiae has produced wines exhibiting an increase in fruit sensory compared to wines produced with S. cerevisiae monoculture, while wines produced with H. uvarum/S. cerevisiae showed increased concentrations of higher alcohols, acetate, and medium-chain fatty acids [ape must . Therefonal wine . The Hancriptors , acetatecriptors ,103, impty acids .Starmerella bacillaris (synonym Candida zemplinina) positively contributes to the production of secondary metabolites, and, in particular, it is known as a high glycerol producer, with concentrations in wine up to 14 g/L during alcoholic fermentation [S. cerevisiae, which preferably utilizes glucose [St. bacillaris persists up to the middle-end phase of the fermentation process due to its ability to tolerate high concentrations of ethanol [S. cerevisiae, it is not able to consume all the sugar in the must, reaching ethanol levels of about 5.8% (v/v) [St. bacillaris/S.cerevisiae wines are characterized by high concentrations of linalool, citronellol, nerolidol, geraniol, and terpenes, with a consequent increase in aroma complexity [entation . These centation . Anotherentation , which ientation ,108, unl glucose . St. bac ethanol . In mixe8% (v/v) . On the 8% (v/v) . Generalmplexity .Wickerhamomyces anomalus, also known as Pichia anomala, is another yeast frequently isolated from grapes and must. Some studies have reported that W. anomalus produces a very high level of extracellular enzymes, such as \u03b2-glucosidases and \u03b2-D-xylosidases, a high level of monoterpenes, and a high level of fruity acetate esters, such as 2-phenylethylacetate [S. cerevisiae [lacetate . It is qrevisiae .foods-1Saccharomyces yeasts is the \u03b2-glucosidase activity, carried out by these yeasts in the early stages of alcoholic fermentation, hydrolyzing the glycosidic bonds of the odorless non-volatile glycoside and determining the release of the aromatic component, which consists of terpenols, norisoprenoids, and benzoic alcohols. Generally, monoterpenic glycosides in grapes are molecules linked to a sugar and, therefore, are non-volatile and odorless, but they can be used as potential indicators of quality. Grapes may naturally contain \u03b2-glycosidase, whose activity is, however, minimal due to the fermentation temperature, low pH, and high sugar concentration of grape must. For this reason, in recent years, the selection of wine yeasts with positive glycosidase activity has been extensively applied, with the aim of enhancing the biotechnological potential of the grape microbiota or of inoculated yeasts in order to increase the varietal aroma without resorting to the addition of exogenous enzymes. In this regard, many non-Saccharomyces yeasts, albeit producing low levels of ethanol, produce \u03b2-glucosidase, whose activity, although influenced by the chemical\u2013physical characteristics of the must and the technological parameters of winemaking, is able to improve the wine sensory profile in a decisive way. Obviously, even the species considered and especially the characteristics of the strain used can significantly affect the persistence and activity of these enzymes in must and wine. Otherwise, this character is poorly expressed between S. cerevisiae and S. bayanus strains, and few strains of the genus Saccharomyces are able to carry out both the primary oenological function  and the production of enzymes capable of enhancing the varietal aroma. Wide interest is being shown by many researchers and operators in the oenological field on the use of sequential inoculation, whose goal is to imitate spontaneous fermentation by inoculating non-Saccharomyces species at the early stage and successively S. cerevisiae, with the aim to guarantee the expression of the metabolism by non-Saccharomyces at the initial phase of the fermentation process, increasing the overall aroma of the wine. In any case, the selection and choice of the non-Saccharomyces starter to be inoculated are crucial for the quality of the final product, also according to the sensory characteristics that are to be conferred to the wine. Furthermore, it should be emphasized that even the inoculum levels between non-Saccharomyces and Saccharomyces in relation to the winemaking conditions can strongly influence the profile of the final aroma, as reported in Of particular importance for non-S. cerevisiae strains and non-Saccharomyces ones, or the presence of mannoproteins affecting the release of aromatic components. However, in order to enlarge wine production and its taste, one of the most ambitious challenges of the modern wine industry is to innovate and diversify wine production processes in order to better meet the needs of the globalized market and consumers oriented towards increasingly aromatic products but with low alcohol content compared to the past. However, the sensory appeal of a wine always remains the first driver of consumer choice and, consequently, the agronomic practices adopted in the field for the cultivation of the vine, along with the choice of strains to be used in fermentation, as well as the parameters of the winemaking process and aging of the wine need to be aimed at achieving this goal. According to the findings of this review, one of the most important tools to increase the aroma compounds in wine is the suitable use of microbial biodiversity. In fact, as previously shown in S. cerevisiae species is a useful example to modulate the final wine aroma compounds. Moreover, other important factors are the proper use of non-Saccharomyces yeasts in the generation of wine aroma compounds and the enhancement of the biodiversity of the grape microbiota to select strains with specific attributes that, if used wisely, can contribute in a positive way to the improvement and characterization of the aromatic profile of the wine. In particular, this can be due, as underlined before, to the \u03b2-glucosidase activity carried out by these non-Saccharomyces yeasts in the early stages of alcoholic fermentation, determining the release of the aromatic component, which consists of terpenols, norisoprenoids, and benzoic alcohols. Another interesting aspect underlined in this review is the set-up of protocols based on non-thermal technologies (such as high-pressure homogenization) useful to treat the initial starters, modifying their metabolism and the release of secondary metabolites.The literature data have long established that taste is the primary choice and driver factor for wine consumption among other factors, such as emotion, social/peer pressure, and human physiological factors. In turn, the final wine taste can be related to several aspects, among which the aromatic wine complexity depends on countless factors and their complex interactions . One of However, to achieve innovation in a mature sector, such as the oenological one, it will be necessary to strengthen the collaboration between the oenological world and scientific research so as to ensure an effective industrial implementation of the results obtained on a laboratory scale."}
+{"text": "Although early diagnosis has been recognized as a key strategy to improve outcomes for those with dementia, receiving a diagnosis may negatively influence social relationships. Absent from the literature are considerations of how race/ethnicity may alter these associations. This study examines racial/ethnic variation in the effects of receiving a diagnosis of dementia on social relationships of older adults. Data from the three waves of the Health and Retirement Study were utilized as part of this study. This study examined whether receiving a new diagnosis of dementia changed subsequent social relationships . Regression analyses with inverse probability weighting were performed to estimate the impact of receiving a dementia diagnosis on changes in social relationships. Receiving a new diagnosis of dementia reduced both informal  and formal  social engagements. The negative impact of receiving a diagnosis on formal social engagement was stronger among non-Hispanic Blacks . We found no statistically significant impacts of receiving a diagnosis of dementia on social networks and social support. Results suggest that receiving a new diagnosis of dementia may have unintended negative consequences on social engagement and may be more salient for racial/ethnic minorities, such as non-Hispanic Blacks. Practitioners and policymakers should be aware of these consequences and should identify strategies to alleviate the negative implications of receiving a diagnosis."}
+{"text": "It is found that \u00b5 reduces as InAlN barrier (TB) and gate length (LG) scale down but increases with the scaled source\u2013drain distance (LSD). Polarization Coulomb Field (PCF) scattering is believed to account for the scaling-dependent electron mobility characteristic. The polarization charge distribution is modulated with the vertical and lateral scaling, resulting in the changes in \u00b5 limited by PCF scattering. The mobility characteristic shows that PCF scattering should be considered when devices scale down, which is significant for the device design and performance improvement for RF applications.We have experimentally investigated the impact of vertical and lateral scaling on low-field electron mobility ( Jeong-G\u03a9\u22121\u00b7cm\u22122 . Xiaoyu \u03a9\u22121\u00b7cm\u22122 . Maddaka\u03a9\u22121\u00b7cm\u22122 . Kedhare\u03a9\u22121\u00b7cm\u22122 .To further improve device performance, device scaling in GaN HEMTs is necessary ,11,12. T0.17Al0.83N/GaN HEMT structure is grown by metal\u2013organic chemical vapor deposition on a Si substrate, as shown in 0.12Ga0.88N back-barrier and a 2 \u03bcm undoped GaN buffer. Here, two different InAlN layers with the thicknesses of 8 nm and 5 nm are grown. The device process started with mesa isolation with Cl2-based inductively coupled plasma (ICP) etching. Then, Ohmic contact was formed with Ti/Al/Ni/Au metal deposition and annealed at 850 \u00b0C for 40 s. Ni/Au gate Schottky contact was deposited in the center of the source\u2013drain region to complete the process. For the large devices, the gate length (LG), gate\u2013source distance (LGS), and gate\u2013drain distance (LGD) of the devices are all 2 \u00b5m. For the RF devices, two types of devices are fabricated. For type I, LG of the devices is fixed at 50 nm and LSD is 2, 1, and 0.6 \u00b5m, respectively. For type II, LSD of the devices is fixed at 1 \u00b5m and LG is 50, 100, and 150 nm, respectively. Here, the gate of all the devices is located between the source and drain regions, and the gate width is 2 \u00d7 20 \u00b5m. The current\u2013voltage (I\u2013V) and capacitance\u2013voltage (C\u2013V) measurements were carried out by using an Agilent B1500A semiconductor parameter analyzer .The lattice-matched InC) of the InAlN/GaN circle diodes with both InAlN barrier thicknesses (TB). Here, six devices are measured and a good consistency is presented. An improved C and a subthreshold voltage (VT) shift are observed due to the reduced InAlN barrier thickness . Through integrating C-V curves, electron density (n2D) is extracted as shown in LG, LGS, and LGD of the devices are all 2 \u00b5m. To extract low-field mobility, the drain current (ID) at VDS = 0.1 V in the output characteristics are used. At VGS = 0 V, the total source\u2013drain resistance (RSD) can be written asRC is the ohmic contact resistance, q is the electron charge, and \u00b50 and n2D0 are the electron mobility and electron density under the gate region with VGS = 0 V. Here, only \u00b50 and RC are unknown. Electron mobility in GaN HEMTs is limited by polar optical phonon (\u00b5POP), polarization Coulomb field (\u00b5PCF), acoustic phonon (\u00b5AP), interface roughness (\u00b5IFR), and dislocation (\u00b5DIS) scatterings [VGS = 0 V, the polarization charge distribution is uniform, and the PCF can be neglected. Based on the two-dimensional (2D) scattering theory and the obtained n2D0 [\u00b50 can be calculated with 1/\u00b50 = 1/\u00b5POP + 1/\u00b5AP + 1/\u00b5IFR + 1/\u00b5DIS, and then RC can be determined with (1). Based on the obtained n2D0 and \u00b50, the electron mobility \u00b5 under the gate region under different VGS can be extracted fromtterings ,27,28. Ptterings ,22. At V\u00b5 versus VGS for both samples. At VGS = 0 V, \u00b5 of the devices with 8 nm InAlN and 5 nm InAlN is 1221 and 1651 cm2/V\u2219s, respectively. The improved electron mobility with a thinner barrier is also confirmed with the Hall measurement (1242 cm2/V\u2219s for 8 nm InAlN and 1663 cm2/V\u2219s for 5 nm InAlN) and the electron mobility of Fat-FETs  [m InAlN) .\u00b5 presents a different trend versus VGS for the devices with different InAlN thickness. As VGS increases, \u00b5 of the device with 8 nm InAlN deceases, but that of the device with 5 nm InAlN increases. \u00b5 limited by different scatterings for both devices [\u00b5  by using 2D scattering theory shows good agreement with the extracted \u00b5 (scatters in the figures), which proves the accuracy of the calculation. As VGS increases, \u00b5POP and \u00b5IFR decrease, \u00b5DIS and \u00b5PCF increase, and \u00b5AP presents a slight change. \u00b5POP and \u00b5PCF play more significant roles among all the scatterings. \u00b5POP and \u00b5PCF for both devices. When the InAlN barrier decreases from 8 nm to 5 nm, \u00b5POP increases while \u00b5PCF decreases. The reduced n2D with a 5 nm InAlN barrier decreases the collision probability between channel electrons and polar optical phonons (POPs), resulting in the improved \u00b5POP [0) in the InAlN barrier near the InAlN/GaN interface. When VGS is applied on the gate terminal, the polarization charges (\u03c1G) under the gate region are changed due to the inverse piezoelectric effect [0 \u2212 \u03c1G) and is written as [\u03b5 is the dielectric constant of GaN and WG is the gate width. Based on inverse piezoelectric effect, \u03c3 can be calculated by using \u03c3 = \u03c10 \u2212 \u03c1G = \u2212ne332VGS/(C33d) [n is the fitting parameter, and e33 and C33 are the piezoelectric coefficient and the elastic stiffness tensor of InAlN, respectively. d is the gate-to-channel distance, which is the sum of the thicknesses of the GaN cap layer (2 nm), InAlN barrier (8 or 5 nm), and AlN interlayer (1 nm). VGS with an 8 and 5 nm InAlN barrier. \u03c3 increases with the decreased TB and VGS, resulting in the enhanced PCF scattering as the InAlN barrier thickness and VGS decrease. Therefore, \u00b5PCF increases with VGS. Because the device with a 5 nm InAlN barrier shows an enhanced PCF scattering, \u00b5 increases with VGS. This fact is more pronounced, especially in the more negative VGS region. For the device with an 8 nm InAlN barrier, the PCF scattering became weaker and the POP scattering dominates \u00b5, leading to a slight decrease in \u00b5 when VGS increases.As shown in  devices ,30,31. Tved \u00b5POP ,28. Due c effect , as showitten as ,22(3)V(\u00b5. The lateral scaling is also experimentally investigated on the devices by varying LSD and LG using the same electron mobility extraction methodology. As the device laterally scales, n2D is not changed, so POP, AP, IFR, and DIS scatterings are not affected. Only PCF scattering can be changed due to the modulation of the polarization change distribution. \u00b5 versus VGS at VDS = 0.1 V for the devices with LG of 50 nm and LSD of 2, 1, 0.6 \u00b5m with 8 nm and 5 nm InAlN. \u00b5 presents an increase with the decrease in LSD. The corresponding \u00b5PCF is also calculated and plotted in LSD scales down, the number of \u03c3 is reduced and the effect of \u03c3 on the electron under the gate region is weakened, resulting in the increased \u00b5PCF and \u00b5. Because PCF scattering in the device with 8 nm InAlN is weaker, the increase in \u00b5 due to the downscaling of LSD is more significant. Here, \u00b5 of the devices with LG of 2 \u00b5m is also plotted for comparison, and a significant decrease in \u00b5 in the device with an LG of 50 nm is observed. Although the number of \u03c3 is the same under the same VGS, the effect of \u03c3 on the 50 nm gate is stronger and thus PCF scattering is enhanced, leading to a decreased \u00b5.From the above discussions, the vertical scale will increase \u03c3 and thus enhance PCF scattering, leading to a reduced \u00b5 versus VGS for the devices with LSD of 1 \u00b5m and LG of 50, 100, 150 nm with 8 nm and 5 nm InAlN. The electron mobility of all devices presents an increase with VGS. This means PCF scattering plays a dominant role in the electron mobility. As VGS increases from a negative value to 0 V, the electric field under the gate region decreases, resulting in the increase in \u00b5PCF and \u00b5. For the devices with different gate lengths, \u00b5 presents an increase as LG increases. This means the increase in gate length can increase the electron mobility. To explain this phenomenon, the corresponding \u00b5PCF is calculated and plotted in LG can weaken PCF scattering and increase \u00b5PCF. Because LSD is fixed, as shown in LG means the increased LGS and LGD, resulting in the enhanced effect of \u03c3 on the electrons under the gate region. Thus, PCF scattering becomes stronger and \u00b5 reduces with the downscaled LG.LG results in a decrease in \u00b5, but downscaled LSD leads to an increase in \u00b5. This is because the polarization charge distribution is modulated with the vertical and lateral scale, resulting in a change in PCF scattering. When GaN HEMTs scale down, the effect of PCF scattering should be considered, providing an insightful guidance for the device geometry design and performance improvement for RF application.In summary, the effect of down-scaling on electron mobility is experimentally demonstrated. It shows that the downscaling of barrier thickness and"}
+{"text": "In this paper, we derive the Cram\u00e9r\u2013Rao lower bounds (CRLB) for direction of arrival (DoA) estimation by using sparse Bayesian learning (SBL) and the Laplace prior. CRLB is a lower bound on the variance of the estimator, the change of CRLB can indicate the effect of the specific factor to the DoA estimator, and in this paper a Laplace prior and the three-stage framework are used for the DoA estimation. We derive the CRLBs under different scenarios: (i) if the unknown parameters consist of deterministic and random variables, a hybrid CRLB is derived; (ii) if all the unknown parameters are random, a Bayesian CRLB is derived, and the marginalized Bayesian CRLB is obtained by marginalizing out the nuisance parameter. We also derive the CRLBs of the hyperparameters involved in the three-stage model and explore the effect of multiple snapshots to the CRLBs. We compare the derived CRLBs of SBL, finding that the marginalized Bayesian CRLB is tighter than other CRLBs when SNR is low and the differences between CRLBs become smaller when SNR is high. We also study the relationship between the mean squared error of the source magnitudes and the CRLBs, including numerical simulation results with a variety of antenna configurations such as different numbers of receivers and different noise conditions. In signal processing, the direction of arrival (DoA) is a popular topic, and there exists a rich literature of estimation of the DoA of incoming sources from measurements of an antenna array. These approaches include the multiple signal classification (MUSIC) algorithm ,2, estimSparse Bayesian learning (SBL)  can be cThis paper is organized as follows. In M identical elements with the ith element located at distance In this paper, a nonuniform linear array, shown in ith element, k is the wave number. In matrix form logP\u03bc\u00af=\u03c3In summary, we first update the unknown parameters used in the hierarchical model iteratively until the marginal likelihood converges, and then we use  and (13)n is the dimension of the vector In this section, we will derive the CRLBs for the DoA estimator described in the previous section. There exist several types of CRLBs, and the appropriate CRLB should be used depending on the configuration of unknown parameters of the estimator ,27,28. Trix I(\u03d5) ,(16)\u03c3\u03d5\u00afIn the ideal case, we assume the noise variance nt y\u00af in  can be wt y\u00af in  in , 19) ca caPI an an24) anI(\u03d5)=A\u00afTAI(\u03d5)=A\u00afTAs matrix . TherefoWe now consider the second case where fined in , so whenfined in , 24), , and eaEquation , we can The Fisher information matrix is written asIn this case, the Fisher information matrix can be computed without knowing the realization of By using ( and 5)))5)) and Now, the unknown parameters become The joint distribution \u03bb). With  and ignoWe continue to obtain the second derivative of From , we can Comparing  with ourIn order to obtain the complete Fisher information matrix, the derivatives of Thus, the Fisher information for By using the source distribution from , we can K.Plugging (48) into (46), we have Equation  will resWe now derive the noise lower bound; we use rived in  and 49)\u03d5=M is the number of antenna elements. It is clear that The second derivative of the corresponding log likelihood can be written asTherefore we haveIn this subsection, we assume that multiple measurements are available. We are interested in multiple measurements because they can improve the accuracy of the estimated results according to experiments, the improvement can be explained by noting that the effect of noises is mitigated and multiple measurements can reduce the correlation between the columns of the measurement matrix if the measurement matrix changes with time which is common under several scenarios such as distributed ground communication where the locations of receivers could change in each measurement. Thus the difficulty in recovering the source is decreased. For example, the Gram matrices 58), we cSimilarly to , we can L compared with the single measurement.For the DoA estimation problem, L measurements obtained from an array of M elements and a set of N incoming sources, the results are obtained by using MATLAB R2021a on a laptop with 8 GB RAM and 1.6 GHz Intel Core i5. The position of the M array elements are drawn i.i.d. from the distribution mth element, a and b, r denotes the feasible range for the position of one element. All the results in this section are generated by setting To test the sharpness of the CRLBs obtained in this paper, we generate numerical results from a simulation involving L. We chose L and the SNR increase which explains the effect of multiple snapshots to this DoA estimation method in the perspective of lower bounds. With the increase of SNR, the difference of the MSE and CRLBs decreases for M. This CRLB and MSE decrease as the number of receiving elements increases which verifies the effect of increasing the number of receiving elements to this DoA estimation method. We also notice that as the SNR increases, the difference between MSE and the CRLB becomes larger for the case with M under various SNRs. We see that the CRLB decreases as the number of receiving elements increases, which means increasing the number of receivers can also improve the estimation accuracy for the noise, and the difference between the MSE and the CRLB also decreases as SNR increases in terms of magnitude.Finally, In this paper, we derived the CRLBs of estimated source, hyperparameters and noise in terms of MSE by using the Laplace prior in sparse Bayesian learning and we explored the several factors that can affect the lower bounds. The numerical simulation result is presented for demonstrating the derived lower bounds under different conditions. Among the derived CRLBs, the marginalized Bayesian CRLB is the tightest one. The relationship between the CRLB of the source to be estimated and the number of receiving elements and the number of snapshots is provided in the DoA problem, as we expect, the CRLB decreases as the number of receiving elements and the number of snapshots increase. In the future, we can extend the lower bounds to planar arrays and exploring the lower bounds of off-grid DoA estimation method can be another topic of a future paper. The effect of the convergence threshold is also worth further investigation."}
+{"text": "Introduction: Low-flow anesthesia (LFA) has gained more interest worldwide owing to its economic and ecological advantages compared to normal-flow anesthesia (NFA). Desflurane is one of the commonly used anesthetic agents for LFA, but it may prolong myocardial repolarization. Frontal QRS-T angle (f[QRS-T]a)\u00a0is a novel marker of myocardial repolarization. To our knowledge, no study has compared the effect of LFA and NFA on f(QRS-T)a. In this study, we aimed to compare the effect of the LFA and NFA with desflurane on f(QRS-T)a in patients undergoing rhinoplasty operation.Methods: A total of 80 patients undergoing rhinoplasty operations were included in this prospective study. The patients were randomized into two groups as follows: LFA (n = 40) and NFA (n = 40). The frontal QRS-T angle was calculated from the automatic report of the electrocardiography device . It was recorded at the following time points: T1: preoperative , T2: immediately after anesthesia induction, T3: immediately after endotracheal intubation, T4: 5 min\u00a0after endotracheal intubation, T5: 15 min\u00a0after endotracheal intubation, T6: 30 min\u00a0after endotracheal intubation, T7: 60 min after endotracheal intubation, T8: end of the operation, T9: 15 min\u00a0after the end of the operation.Results: Baseline clinical characteristics and laboratory parameters were similar between the two groups. In the LFA group, f(QRS-T)a was significantly increased at only the T3 time point when compared to T1 (P = 0.003). However, in the NFA group, f(QRS-T)a was significantly increased at T3, T4, T5, T6, T7, T8, and T9 time points when compared to the T1 value . On the other hand, fQRS-Ta was significantly higher in the NFA group than in the LFA group at T4, T5, and T6 time points.\u00a0Conclusion: In our study, we have shown for the first time that NFA significantly increased the f(QRS-T)a, whereas LFA did not significantly increase the f(QRS-T)a except for immediately after the endotracheal intubation. It was also detected that f(QRS-T)a was significantly higher in the NFA group compared to that in the LFA group. Therefore, it can be concluded that LFA has more protective effects on myocardial repolarization than NFA. Low-flow anesthesia (LFA) is an anesthesia technique and has recently gained more interest worldwide. Compared to normal-flow anesthesia (NFA) in which the flow rate of fresh gas to the breathing system is generally at 2 L/min, the gas flow rate is \u22641 L/min, and the re-inhaled fresh oxygen flow rate is at least 50% with a sufficient amount of volatile agent in LFA . TherefoVolatile anesthetic agents have been reported to prolong myocardial depolarization and repolarization with a direct effect -7. ProloAnesthesiologists tend to use LFA with desflurane and sevoflurane owing to their cost . DesflurA total of 80 patients undergoing rhinoplasty operation with the American Society of Anesthesiologists (ASA) status I or II were included in this randomized prospective study. Patients under the age of 18 and over the age of 65, those with hypertension, diabetes mellitus, heart failure, coronary artery disease, moderate/severe valvular disease, chronic renal or hepatic disease, complete/incomplete bundle branch block, anemia, electrolyte imbalance, and those taking drugs known to prolong myocardial repolarization were excluded from the study. Harran University Clinical Research Ethic\u00a0Committee approved the study design (number: 03/10) and informed consent was obtained from all patients. Our study was also registered at the Australian New Zealand Clinical Trials Registry (number: ACTRN12621000408886).2). Anesthesia induction was performed with propofol (2 mg/kg) and fentanyl (1 \u00b5g/kg). Endotracheal intubation was performed after muscle relaxation with rocuronium 0.5 mg/kg. Patients were then connected to the mechanical ventilator. Anesthesia was maintained with desflurane . Patients were randomly allocated according to sealed opaque envelopes into two groups as LFA and NFA. LFA (n=40) was performed as the fresh gas flow rate was at 4-6 L/min for the first 6-8 minutes. After achieving MAC\u00a0+1, the rate of fresh gas flow was reduced to 0.5 L/min. NFA (n=40) was performed as the fresh gas flow rate was at 4-6 L/min for the first 6-8 minutes. After achieving MAC +1, the rate of fresh gas flow was reduced to 2 L/min.\u00a0During anesthesia maintenance, the target end-tidal CO2 value was 35-45 mmHg. Fifteen minutes before the end of the surgical operation, the vaporizer was turned off. In addition, 100% oxygen was performed at a 5-6 L/min\u00a0rate for 3-5 min\u00a0before extubating. The following time points were defined for the evaluation: T1: preoperative , T2: immediately after anesthesia induction, T3: immediately after endotracheal intubation, T4: 5 min\u00a0after intubation, T5: 15 min\u00a0after intubation, T6: 30 min\u00a0after intubation, T7: 60 min after intubation, T8: end of the operation, T9: 15 min\u00a0after the end of the operation. Baseline characteristics, ASA status, duration of the procedure, laboratory parameters, HR, MAP, and electrocardiographic variables were recorded for all patients.\u00a0Patients were taken to the operation room equipped with ASA monitoring, i.e ECG, heart rate (HR), mean arterial pressure (MAP), and oxygen saturation . The automatic report of the ECG was also printed. The frontal QRS axis and T axis were available in this automatic report of the ECG device. The frontal QRS-T angle was calculated from these axes as follows: the absolute difference between the QRS axis and T axis . If this angle exceeded 180rom 360o ,16. An eStatistical analyses were performed with SPSS 21.0 . Kolmogorov-Smirnov test was used to evaluate the distribution of variables. Continuous variables were expressed as mean \u00b1 standard deviation and compared with an independent-sample t-test. Categorical variables were expressed as numbers (%) and compared with the chi-square test. Repeated variables were compared with a one-way analysis of variance. Post hoc comparisons among the repeated variables in each group were performed by Bonferroni, if appropriate. A P-value of <0.05 was considered statistically significant.Forty patients in the low-flow desflurane anesthesia group and 40 patients in the normal-flow desflurane anesthesia group were evaluated in this study. The baseline characteristics and laboratory parameters of the study groups are demonstrated in Table MAP and HR values of the patients at each time point during the procedure are shown in Figure Frontal QRS-T angles of the patients at each time point during the procedure are shown in Table In this study, we aimed to compare the effect of LFA and NFA with desflurane on f(QRS-T)a. The main findings of our study were as follows: (I) although normal-flow desflurane anesthesia significantly increased the f(QRS-T)a during the procedure and this increase continued at 15 min\u00a0after the postoperative period, low-flow desflurane anesthesia did not significantly increase the f(QRS-T)a except for immediately after endotracheal intubation, (II) f(QRS-T)a was significantly higher in the normal-flow desflurane anesthesia group compared to the low-flow desflurane anesthesia group at T4, T5, and T6 time points. To our knowledge, this is the first study reporting the protective effect of low-flow desflurane anesthesia on f(QRS-T)a.Myocardial repolarization has a crucial role in the cardiac cycle, which plays a principal role in the development of arrhythmias. It has been traditionally calculated with QT and Tp-e interval measurements . HoweverEndotracheal intubation, anesthetic agents, and the type of anesthesia may affect myocardial repolarization. Previous studies demonstrated that endotracheal intubation itself prolonged myocardial repolarization ,21. SimiThe effect of volatile anesthetic agents on myocardial repolarization parameters has been investigated in previous studies . It was The effect of LFA on hemodynamic parameters was evaluated in many studies ,24-26. IOur study had some limitations. First, the sample size was relatively small. Second, anesthesia maintenance was provided according to the MAC, and it might be more suitable to maintain the anesthesia according to the MACage. However, we excluded the patients under the age of 18 and over the age of 65 in this study. Therefore, we do not\u00a0think that using MAC instead of MACage affects our results.\u00a0Third, 24-h Holter ECG recording was not performed. It would provide additional contribution to perform 24-h Holter ECG recording and evaluating the effect of different gas flow rates on arrhythmias. However, we did not observe any clinical arrhythmia during the hospital stay. Fourth, we evaluated the low-risk patients (ASA I and II) without any cardiac disease in this study. Therefore, our results cannot be generalized to all patient\u00a0groups.Frontal QRS-T angle is a novel marker of myocardial depolarization and repolarization heterogeneity. In this study, we found that NFA significantly increased f(QRS-T)a, whereas LFA did not significantly increase the f(QRS-T)a except for immediately after endotracheal intubation. It was also detected that f(QRS-T)a was significantly higher in the NFA group compared to the LFA group. Therefore, it can be concluded that LFA has more protective effects on myocardial repolarization than NFA."}
+{"text": "Antrodia camphorata is an endemic mushroom in Taiwan. This study was designed to screen anti-inflammatory compounds from the methanolic extract of the mycelium of A. camphorata on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA) known to be present in viral infection. A combination of bioactivity-guided isolation with an NMR-based identification led to the isolation of 4-acetylantroquinonol B (1), along with seven compounds. The structure of new compounds (4 and 5) was elucidated by spectroscopic experiments, including MS, IR, and NMR analysis. The anti-inflammatory activity of all isolated compounds was assessed at non-cytotoxic concentrations. 4-Acetylantroquinonol B (1) was the most potent compound against poly I:C-induced NO production in RAW 264.7 cells with an IC50 value of 0.57 \u00b1 0.06 \u03bcM. Inflammation is part of the innate, or nonspecific, immune responses against invading pathogens. It is generally initiated when pathogen-associated molecular patterns (PAMPs), such as pathogen-specific carbohydrates, lipoproteins, and nucleic acids, are recognized by a large family of pattern-recognition receptors (PRRs), expressed by macrophages, monocytes, dendritic cells, and neutrophils . Among tNotable outbreaks in the last two decades mostly involved respiratory viruses\u2014such as the 2003 severe acute respiratory syndrome (SARS) epidemic, 2009 A(H1N1) pandemic, Middle Eastern respiratory syndrome (MERS) epidemic, and ongoing coronavirus disease 2019 (COVID-19) pandemic\u2014that represent a considerable challenge to worldwide public health . Novel rAntrodia camphorata is an edible and precious mushroom endemic to Taiwan, which has been used as traditional medicine. A. camphorata has been found to exhibit a broad range of pharmacological effects, which include anti-microbial, anti-oxidative, anti-inflammatory, anti-diabetic, anti-aging, anti-carcinogenic, neuroprotective, hepatoprotective, cardioprotective, and immunomodulatory effects +. The IR spectrum showed peaks at 3437 (OH), 1670 , and 1593 and 1493 (aromatic) cm\u22121. The 1H and 13C NMR data revealed the presence of a benzene ring with five substituents\u2014an aldehyde , an aryl proton , a hydroxyl proton , two methoxy groups , and a deshielded methyl group , together with six aromatic carbon signals . The methyl signal (\u03b4H 2.51) resonated at a higher frequency, then indicated that the methyl group is located at the ortho-position to the aldehyde group. The relative positions of aryl substituents were confirmed by HMBC correlations (\u03b4H 2.51) to C-1 (\u03b4C 129.7), C-2 (\u03b4C 121.4), and C-3 (\u03b4C 148.0), from the aldehyde proton  to C-1 (\u03b4C 129.7) and C-6 (\u03b4C 105.0), and from the aryl proton  to C-2 (\u03b4C 121.4), C-4 (\u03b4C 140.1), and C-1\u2032 (\u03b4C 191.2). Thus, compound 4 was identified as 3-hydroxy-4,5-dimethoxy-2-methylbenzaldehyde.Compound elations  from the5 was determined to have the molecular formula of C15H17NO4 with eight degrees of unsaturation according to its HRESIMS peak at m/z 298.1051 [M + Na]+. The IR spectrum showed peaks at 3312 (N-OH), 1776 and 1709 (imide), and 1607 and 1514 (aromatic) cm\u22121. The 1H NMR data showed the presence of a para-substituted aromatic ring  and 6.92 ), a hydroxy group , a methoxy group , and an isobutyl group , 2.05 , and 0.90 ). The COSY correlations between H-2\u2032 and H-1\u2032 and two methyl groups further confirmed the presence of the isobutyl group. The 13C NMR data revealed the presence of two carbonyl carbons (\u03b4C 167.8 and 167.1) and two quaternary olefinic carbons (\u03b4C 136.3 and 135.9), which was characteristic of a maleimide moiety. The relative connectivity was further deduced from the HMBC correlations from H-1\u2032 (\u03b4H 2.51) to C-2 (\u03b4C 167.8) and C-3 (\u03b4C 136.3), and the HMBC correlations (\u03b4H 7.47) to C-4 (\u03b4C 135.9) and C-4\u2032\u2032 (\u03b4C 157.4). The overall spectroscopic data of compound 5 were similar to those of antrocinnamomin B +.3-Hydroxy-4,5-dimethoxy-2-methylbenzaldehyde (4): Amorphous colorless powder; IR (KBr) \u03bdmax 3312, 2955, 2924, 2866, 1776, 1709, 1607, 1580, and 1514 cm\u22121; UV \u03bbmax  232 (3.63), 284 (3.00), 370 (2.87); 1H and 13C NMR data: Shown in m/z 298.1051 [M + Na]+.3-Isobutyl-4-(4-methoxyphenyl)-1H-pyrrol-1-ol-2,5-dione (5): Amorphous colorless powder; IR (KBr) 2 at 37 \u00b0C. After being seeded into 96-well plates (5 \u00d7 104 cells/well), the cells were incubated overnight and then treated with filtered, tested samples with different concentrations, followed by poly I:C (50 \u03bcg/mL). After incubation for 24 h, NO concentration in the culture medium was measured with the Griess reaction and calculated using a standard curve. Each supernatant (100 \u03bcL) was reacted with the same volume (100 \u03bcL) of Griess reagent (containing 0.05% N-(1-naphthyl)ethylenediamine dihydrochloride, 0.5% sulphanilamide, and 2.5% phosphoric acid) for 5 min at room temperature. The absorbance at 540 nm was detected using a microplate reader. The CCK-8 assay was applied for cell viability assessment according to standard protocols. The absorbance at 450 nm was detected using a microplate reader. The experiments were performed in triplicates.RAW 264.7 macrophages were cultured in DMEM supplemented with 10% FBS and 1% antibiotics in an incubator with a humidified atmosphere of 5% COt test. p values of less than 0.05 were considered statistically significant.The results were expressed as mean \u00b1 SD of triplicates. Significant differences were examined using a two-tailed unpaired Student\u2019s A. camphorata were assessed on viral infection-associated inflammation using poly I:C-stimulated NO production in a RAW 264.7 mouse macrophage model. Our study provides a precise strategy for screening anti-inflammatory compounds from the mycelium of A. camphorata by a combination of bioactivity-guided isolation with an NMR-based identification approach, which led to isolation and identification of eight compounds, including two new compounds 4 and 5. Among them, 4-acetylantroquinonol B (1) was not only the most abundant compound, but the most potent compound in the bioactive Fraction 5 from the mycelium of A. camphorata. Thus, it could be a chemical marker for the potential anti-inflammatory agent A. camphorata mycelium during viral infection.In this study, the inhibitory effects of the methanolic extract of the mycelium of"}
+{"text": "Based on proteomic data, olfactomedin 4 (OLFM4) related to immunity and inflammation was selected as a potential target. Western blot analysis further confirmed the moderating effect of QBH downregulation on OLFM4 in the lung tissue. Our findings demonstrated that QBH alleviated lung tissue damage and inflammatory reaction via inhibiting OLFM4 expression in LPS-challenged immature rats. Our research indicates that QBH may have therapeutic potential for treating ARIs-related ALI in pediatric patients, which also serves as a candidate target for drug therapy of ALI by intervening OLFM-related signaling pathways.Acute respiratory infections (ARIs) are a common public safety threat with high morbidity and mortality in pediatric patients worldwide. Qinbaohong Zhike oral liquid (QBH), a marketed traditional Chinese medicine product, has been widely used to cure respiratory diseases. QBH is reported to have antitussive, expectorant, and antiasthmatic properties. However, its treatment effect against ARIs is not elucidated. This study aimed to explore the therapeutic efficacy of QBH in the treatment of ARIs-induced pneumonia. Network pharmacology was used to predict the possible targets of QBH against ARIs. Next, the tracheal lipopolysaccharide (LPS-)-induced acute lung injury (ALI) immature rat model was constructed to evaluate the therapeutic effect of QBH. Tandem mass tag (TMT-)-based quantitative proteomics was then used to screen the in-depth disease targets of QBH. QBH exerted a protective effect against LPS-induced ALI by inhibiting pulmonary pathological damage. QBH also reduced the levels of interleukin (IL)-6, tumor necrosis factor (TNF)- Acute respiratory infections (ARIs) such as bronchiolitis and pneumonia in pediatric patients remain a major public health problem worldwide . ARIs-inCurrent clinical treatments are available to relieve symptoms and shorten illness duration, albeit these therapies have limited efficacy as well as side effects. For example, although vaccination helps prevent infections, they are insufficient during outbreaks of new infectious with pandemic potential . FurtherRhododendron dauricum L. (Man-Shan-Hong), Syringa reticulata (Blume) H. Hara (Bao-Ma-Zi-Pi), and Scutellaria baicalensis Georgi (Huang-Qin). QBH has been clinically used to treat lung phlegm heat syndrome in patients with acute or chronic bronchitis. QBH has antitussive, expectorant, and antiasthmatic properties [Traditional Chinese medicine (TCM-)-based herbal therapies are being used to treat infectious diseases for almost two thousand years in China . These toperties , but theoperties . PresentIn the current study, we used network pharmacology to predict the possible pharmacological mechanism of QBH in the treatment of ARIs, which could provide the theoretical foundation . Then, whttps://tcmspw.com/tcmsp.php). Documentary records were also used to supplement the TCMSP database result [https://hpo.jax.org/app/) and DisGeNET databases (http://www.disgenet.org), due to ARIs not on the list of search terms in these two databases.The information about the chemical components of QBH was collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (e result \u201322. The http://string-db.org) to acquire the core protein-protein interaction (PPI) network. The PPI network was built by the Cytoscape software . To determine the potential biological functions of QBH, we used the Metascape database (http://metascape.org/gp/index) to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The GO enrichment analysis included three terms: biological process (BP), cellular component (CC), and molecular function (MF).We merged drug targets of QBH and disease targets of respiratory tract infections. These targets were loaded into the STRING database  with the license number SCXK (Jing) 2016-0004. The rats were raised in the SPF animal room of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China. All animal procedures were performed according to the local ethical guidelines for animal experiments of the Animal Research Committee of the China Academy of Chinese Medical Sciences .QBH consists of three TCMs: Huang-Qin, Man-Shan-Hong, and Bao-Ma-Zi-Pi (weight ratio10: 21: 21). The herbal extract was prepared by Heilongjiang Bifu Jinbeiyao Biopharmaceutical Co. Ltd., China. The extract was dissolved in distilled water. The clinical oral dose of QBH was 0.5\u2009mL/kg/day , which was equivalent to the gavage dose of 3.125\u2009mL/kg/day for the rats (200\u2009g) . Thus, w\u03bcL LPS  through an endotracheal tube . All rats were randomly assigned to six groups (n = 12 each): control group (control), ALI model group (ALI), QBH low-dose group (ALI\u2009+\u2009QBH 1.5\u2009mL/kg), QBH medium-dose group (ALI\u2009+\u2009QBH 3.0\u2009mL/kg), QBH high-dose group (ALI\u2009+\u2009QBH 6.0\u2009mL/kg), and dexamethasone-treated group (ALI\u2009+\u2009DXMS 0.42\u2009mg/kg) [\u03bcL sterile saline through the trachea. At 6\u2009h after anesthesia, QBH or DXMS group rats were administered intragastrically with QBH or DXMS for 3 consecutive days. Control and ALI group rats were given distilled water by gavage.The immature rat model of ALI was established as described . Briefly2\u2009mg/kg) , 25. ConOn the 4th day, the immature rats were killed, and the lung tissue of each rat was removed. The lobe of the left lung was isolated and weighed quickly to prevent fluid loss (wet weight). Next, it was dried in an oven  at 80\u00b0C for 24\u2009h and then measured (dry weight). The calculated W/D weight ratio reflected the extent of pulmonary edema.\u03bcm-thick sections, and stained with hematoxylin and eosin (HE). The tissue slides were observed under a light microscope  for conventional morphological evaluation. The histological score was calculated as follows: (i) alveolar congestion; (ii) alveolar hemorrhage; (iii) interstitial edema; (iv) neutrophil infiltration; and (v) the thickness of the alveolar wall. Each category was scored as 0 for no injury, 1 for slight injury, 2 for moderate injury, and 3 for severe injury [The middle lobe of the right lung was immediately removed, fixed in 4% paraformaldehyde solution , embedded in the paraffin wax, sliced in 5-e injury .g for 10\u2009min, and the serum in the upper layer was gained. The tissue sample was lysed using ice-cold RIPA lysis buffer and protease inhibitors . Subsequently, the sample was crushed by the ultrasonic wave  and then centrifuged at 13,000g for 15\u2009min (4\u00b0C). The concentration of each tissue sample was determined by a BCA protein assay kit .Peripheral blood from the retro-orbital venous plexus and lung tissue from the middle lobe of the right lung was collected for detecting the inflammatory cytokines. The blood sample was centrifuged at 1,500\u03b1, IL-1\u03b2, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (GRO/KC), IL-10, IL-12 (p70), IL-13, IL-17A, IL-18, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1\u03b1, MIP-3\u03b1, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), vascular endothelial growth factor (VEGF), interferon (IFN)-\u03b3 and tumor necrosis factor (TNF)-\u03b1.Luminex technology-based multiplex system  combined with a multiplex immunoassay kit  was used to determine the levels of the serum and tissue samples . A totaln = 5/group) were examined from the control group, ALI group, and QBH 6.0\u2009mL/kg treated group. A protein sample from the middle lobe of the right lung tissue was extracted by lysis solution (2% SDS and 7\u2009M urea) mixed with a protease inhibitors cocktail  and centrifuged at 13,000g for 15\u2009min at 4\u00b0C. The supernatant was collected, and the concentration of protein sample was measured by the BCA protein assay kit . In brief, the sample was precipitated with acetone-TCA and digested by trypsin to generate proteolytic peptides. The peptides were labeled with 16-plex TMT reagents  according to the manufacturer's instructions. The TMT-labeled samples were separated by the Ultimate 3000 HPLC system . The peptide samples were isolated, centrifuged and dried in a vacuum. The dried samples were dissolved in 0.1% formic acid solution, and 2\u2009\u03bcg of the prepared sample was analyzed by an Easy-nLC1000 system . The DAVID database (https://david.ncifcrf.gov/) was used for functional enrichment analysis including GO and KEGG, and the differentially expressed proteins (DEPs) between the groups were analyzed. A p value <0.05 with fold change \u22651.2 (upregulated) or \u22640.83 (downregulated) was considered statistically significant.15 biological replicates (\u03bcg) was separated by 10% SDS-PAGE and transferred to PVDF membranes . After blocking with 5% non-fat milk at 4\u00b0C overnight, the membrane was incubated with primary antibody olfactomedin 4  overnight at 4\u00b0C. \u03b2-actin  was used as an internal control. The membrane was then incubated with a secondary antibody for 4\u2009h at room temperature, and the intensity was measured by an enhanced chemiluminescence agent .The middle lobe of the right lung tissue was lysed with RIPA lysis buffer and protease inhibitors . Protein concentrations were measured by the BCA protein assay kit . The protein sample  were used for comparisons. A p value\u2009<\u20090.05 was regarded as statistically significant.Statistical analysis was performed by SPSS 20.0 software. The data were expressed as mean\u2009\u00b1\u2009standard deviation (SD). Student's \u03b2, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17\u03b1, and TNF-\u03b1 were centrally located at the core position of the PPI network. Moreover, the horizontal stack plot displayed the above 8 inflammatory cytokines were also enriched in the top 20 of the PPI network (In all, 324 ingredients of QBH were identified after removing the duplicated data, including 154 in Man-Shan-Hong, 41 in Bao-Ma-Zi-Pi, and 143 in Huang-Qin, and then, 1,245 potential targets of QBH were collected. Meanwhile, we obtained 358 target genes related to ARIs. There were 65 overlapping genes between QBH targets and ARIs targets . To dete network . These rTo further figure out the possible therapeutic targets, a drug-component-target interaction network was then developed. The network included 178 nodes and 700 edges, indicating the characteristics of multiple target effects correlated with QBH in the treatment of ARIs . FunctioIntratracheal administration of LPS in immature rats led to expected pathological changes including pulmonary hyperemia, edema, thickened alveolar walls, and inflammatory cell infiltration. Moreover, the severity of lung tissue injury was alleviated in the QBH 3.0 and 6.0 groups . These h\u03b1, IFN-\u03b3, and GM-CSF in the blood serum, and IL-1\u03b2, IL-6, IL-8, TNF-\u03b1, IFN-\u03b3, and GM-CSF in the lung tissue (Figures \u03b1, and GM-CSF. However, the serum level of IL-8 and IFN-\u03b3 was downregulated only in QBH 3.0 and 6.0\u2009mL/kg groups. Additionally, the levels of IL-1\u03b2, IL-6, IL-8, TNF-\u03b1, IFN-\u03b3, and GM-CSF in the lung tissue were significantly decreased in all QBH-treated groups compared with the ALI group.To validate the network pharmacology result, we determined the levels of 23 inflammatory cytokines in the blood serum and lung tissue. Compared with the control group, the ALI group had increased levels of IL-6, IL-8, TNF- Figures . CompareTo elucidate the probable therapeutic targets of QBH, we quantified 3,385 proteins from the 15 samples. Data showed that 79 and 8 proteins were significantly upregulated and downregulated, respectively, in the ALI group compared with the control group. We identified 91 DEPs including 52 upregulated and 39 downregulated DEPs in the QBH 6.0\u2009mL/kg group compared with the ALI group. There were 10 overlapping DEPs both changed in these two comparisons.GO-enriched categories in BP, CC, and MF were achieved between the ALI group and QBH 6.0\u2009mL/kg group . The BP Two PPI networks were constructed to further explore the potential therapeutic targets underlying the therapeutic mechanism of QBH against ALI . One sigTo validate the differential expression data of proteomic analysis, western blot was performed to detect the expression of OLFM4 in the lung tissue . OLFM4 eARIs are one of the primary infections in the pediatric population especially in developing countries . Most chLipopolysaccharide is the most common toxin used to stimulate pulmonary inflammation in rodent models of bacterial infection-induced ALI. The resultant innate immune response has a profound effect on the pathogenesis of ALI . Neutrop\u03baB, PI3K/Akt, JAK/STAT, and AMPK, are involved in mediating pulmonary inflammation [\u03baB signaling pathway and promote the transcription of downstream cytokines, such as IL-6, TNF-\u03b1, IL-1, and chemokines [Past reports suggest that multiple signal transduction pathways, including TLR4/NF-ammation . For exaemokines . The PI3emokines . The abnemokines . The sigemokines . Increasemokines . These remokines .\u03b2, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17\u03b1, and TNF-\u03b1. Moreover, the signaling pathways such as a cytokine-cytokine receptor, T cell receptor, Toll-like receptor, JAK/STAT, PI3K/Akt, and AMPK may be involved and play an important role in the therapeutic mechanism of QBH against ARIs. Animal experiments confirmed that QBH downregulated the levels of IL-6, TNF-\u03b1, IFN-\u03b3, and GM-CSF in the blood serum, and IL-1\u03b2, IL-6, IL-8, TNF-\u03b1, IFN-\u03b3, and GM-CSF in the lung tissue, and thus protected the lung tissue from LPS-induced injury. The data of IL-1\u03b2, IL-6, and TNF-\u03b1 were consistent with the network pharmacology result.In our study, network pharmacology results showed that QBH may probably regulate inflammatory reaction and immune response through the modulation of the critical inflammatory cytokines including IL-1To further elucidate the potential therapeutic targets of QBH in the treatment of ALI, we used quantitative proteomics to discover the candidate biomarkers. OLFM4 was selected and then validated by western blot. Western blot result was similar to the result of quantitative proteomic analysis, which indicated high reliability and consistency. The human OLFMs family includes OLFM1, OLFM2, OLFM3, OLFM4, myocilin, gliomedin, latrophilin1, latrophilin2, and latrophilin3 . They ar\u03baB signaling pathway via feedback control [2O2-induced NADPH oxidase activation and cellular apoptosis in mouse neutrophils [OLFM4 regulated the host defense mechanism against H. pylori infection and negatively regulated the NOD-induced NF- control . OLFM4 d control . OLFM4 e control . OLFM4 e control . OLFM4 g control . OLFM4 ctrophils . Converstrophils .Briefly, current evidence demonstrates that OLFM4 is an important regulator of inflammatory and immune responses. In our study, we found that OLFM4 expression was upregulated in the lung tissue of the immature rats with ALI, and QBH significantly inhibited the expression of OLFM4. These results meant that QBH exerted considerable influence on inflammatory and immune responses by altering OLFM4 expression. OLFM4 may be a potential target marker correlated to the pathogenesis of ALI and the therapeutic mechanism of QBH.In summary, we used network pharmacology to search for the potential molecular targets of QBH in the treatment of ARIs. An experimental study on immature rats suggested that QBH inhibited lung injury and inflammatory reaction, resulting in ameliorating the severity of ALI induced by intratracheal LPS. Quantitative proteomics and western blot further provided evidence that OLFM4 might be the critical candidate biomarker involved in the therapeutic mechanism of QBH against ALI . Our res"}
+{"text": "Molecular cloning is a crucial technique in genetic engineering that enables the precise design of synthetic transcriptional units (TUs) and the manipulation of genomes. GreenGate and several other modular molecular cloning systems were developed about ten years ago and are widely used in plant research. All these systems define grammars for assembling transcriptional units from building blocks, cloned as Level 0 modules flanked by four-base pair overhangs and recognition sites for a particular Type IIs endonuclease. Modules are efficiently assembled into Level 1 TUs in a hierarchical assembly process, and Level 2 multigene constructs are assembled by stacking Level 1 TUs. GreenGate is highly popular but has three main limitations. First, using ad-hoc overhangs added by PCR and classical restriction/ligation prevents the efficient use of a one-pot, one-step reaction to generate entry clones and domesticate internal sites; second, a Level 1 TU is assembled from a maximum of six modules, which may be limiting for applications such as multiplex genome editing; third, the generation of Level 2 assemblies is sequential and inefficient. GreenGate 2.0 (GG2.0) expands GreenGate features. It introduces additional overhangs, allowing for the combination of up to 12 Level 0 modules in a Level 1 TU. It includes a Universal Entry Generator plasmid (pUEG) to streamline the generation of Level 0 modules. GG2.0 introduces GreenBraid, a convenient method for stacking transcriptional units iteratively for multigene assemblies. Importantly, GG2.0 is backwards compatible with most existing GreenGate modules. Additionally, GG2.0 includes Level 0 modules for multiplex expression of guide RNAs for CRISPR/Cas9 genome editing and pre-assembled Level 1 vectors for dexamethasone-inducible gene expression and ubiquitous expression of plasma membrane and nuclear fluorescent markers. GG2.0 streamlines and increases the versatility of assembling complex transcriptional units and their combination. Molecular cloning is an integral part of genetic engineering. It allows for the precise design of synthetic transcriptional units (TUs), the manipulation of genomes and the alteration of gene expression. Several methods allow the modular combination of pre-generated modules into destination vectors and were developed in the last 20 years . The GatGolden Gate cloning provides the highest level of modularity and reusability of modules . It is bOver the years, multiple variants of Golden Gate-based cloning toolkits have been developed for plant systems: GreenGate , GoldenBThe different systems have their specificities. While GreenGate relies on a single Type IIs enzyme  for theHere, we introduce GreenGate 2.0 (GG2.0), a backwards-compatible add-on to the GreenGate system that expands the current GreenGate features. GG2.0 presents an expanded repertoire of overhangs that allow the combination of up to 12 Level 0 modules in a Level 1 TU. GG2.0 introduces a Universal Entry Generator plasmid (pUEG) that streamlines the generation of Level 0 modules. GG2.0 introduces GreenBraid, a convenient method to iteratively stack TUs for multigene assemblies. GG2.0 is compatible with most readily existing GreenGate Level 0 modules. GG2.0 also features a set of Level 0 modules for multiplex expression of guide RNAs for CRISPR/Cas9 genome editing and pre-assembled Level 1 vectors for ubiquitous expression of plasma membrane and nuclear fluorescent markers as well as dexamethasone-inducible gene expression. The GG2.0 plasmids are available as a kit (Addgene 83189).Restriction enzymes were obtained from New England Biolabs (NEB) and Thermo Scientific/Fermentas. T4 Ligase and T4 polynucleotide kinase were obtained from NEB. Plasmids were isolated using the alkaline lysis method . PCR reaE.coli (DH5\u03b1), and desired clones were identified through plasmid extraction, digestion and sequencing. The chromogenic bacterial selection markers were added to each GreenBraid vector . The chromogenic marker genes  was obtained by gene synthesis (Thermo Scientific). For the new GreenBraid vectors, we replaced the cloning sites of the pCAMBIA-based GoldenBraid vectors , 13, 14), 1413, 1isPink, (\u201313)) werisPink,  can be found in the E.coli, tested by colony PCR and verified by sequencing. For pU3-containing entry vectors, pU3 and the scaffold+term were independently amplified by PCR from pRU42  containing 1% agar (Duchefa). Following stratification , plants were grown under fluorescent illumination (90 \u03bcE.m-2.s-1) in long-day conditions (16 h light / 8 h dark) at 22\u00b0C.For transformations, A. tumefaciens-based transformation was performed using the floral dip method [For plant transformation by the Level 2 GreenBraid proof-of-principle construct in Col-0, p method .Five days after germination, transgenic first-generation seedlings containing the below-mentioned GreenBraid proof-of-principle construct were mounted on a slice of 1/2 MS containing 1% agar on top of a microscope slide. Confocal Laser-Scanning Microscopy (CLSM) was performed on a Leica SP5 confocal microscope with a 63x, NA = 1.2 water immersion objective. mTurquoise2 fluorescence was detected using the 458 nm excitation laser line and a detection range of 470\u2013510 nm. mVenus fluorescence was detected using the 514 nm excitation laser line and a detection range of 520\u2013540 nm. mScarlet fluorescence was detected using the 561 nm excitation laser line and a detection range of 570\u2013650 nm.The modular cloning system GreenGate uses Eco31I (BsaI) to combine six individual Level 0 entry vectors into a destination vector (Level 1) in a single reaction .These Level 1 constructs can subsequently be used for the expression in plants. Over the years, hundreds of individual entry vectors have been created by many laboratories which can easily be exchanged and combined. Two TUs can be combined into a single destination vector (Level 2) through an adaptor module, and more than two TUs can be iteratively assembled. In its first iteration, GreenGate suffers from three main limitations. First, to generate a Level 0 part, the ad-hoc overhangs are added by PCR and the part is cloned in a dedicated entry clone for the specific part by classical restriction/ligation using Eco31I. This process prevents using the efficient Golden Gate one-pot, one-step reaction for generating the Level 0 clone and the easy domestication of internal sites . Third, GG2.0 addresses these limitations. It introduces a Universal Entry Generator plasmid that relies on a second Type IIs enzyme (PaqCI / AarI) to enable a one-pot, one-step assembly of several elements to domesticate internal sites and create Level 0 modules easily; GG2.0 expands the repertoire of available overhangs in its grammar to enable the assembly of Level 1 vectors from up to 12 Level 0 modules; Finally, GG2.0 features a new set of destination vectors to use as Level 1 expression vectors in plants . These nGG2.0 features a Universal Entry Generator plasmid (pUEG) designed to serve as a polyvalent entry vector for all the GreenGate modules regardless of their category .E. coli strains that do not contain the lacZ\u0394M15 deletion mutation necessary for X-Gal screening.The desired part is PCR-amplified as a single fragment or, if restriction sites need to be domesticated, as multiple fragments . pUEG coTo provide higher granularity and modularity during the assembly, we added six new overhangs to the GreenGate repertoire, which allowed the subdivision of existing GreenGate A, B, C, D and E modules into 14 submodules .We created 21 Level 0 entry vectors that use the same backbone as GreenGate entry clones and contain two convergent Eco31I sites that flank a unique 20 bp sequence and can thus be used, if necessary, as dummy modules during a Level 1 assembly. As several new entry clones share the same new overhangs, not all GG2.0 Level 0 modules can be freely combined in a Level 1 assembly. The GreenGate2.0 modules A1-A3, B1-B2, C1-C4 (ABC set) can be combined with the GreenGate modules D, E and F; alternatively, the GreenGate modules A, B and F can be assembled with the GG2.0 modules C1-C4, D1-D3 and E1-E2 (CDE set). This new set allows up to 12 Level 0 modules to be assembled in a Level 1 TU, dramatically increasing the modularity of GreenGate assemblies. Entry clones spanning multiple overhangs are also available to reduce the complexity of the assembly when a particular module is not required. As up to 12 Level 0 modules and one destination are used in Level 1 TU assemblies, we optimised the protocol and used a higher concentration of T4 ligase. The efficacy of Level 1 assembly remained high when eight or more Level 0 clones were assembled . AlthougTaking advantage of the extended set of Level 0 modules, we designed nine Level 0 entry plasmids to express gRNAs from Pol III promoters. These plasmids are in the CDE set and contain the U3 or U6 Pol III promoter, two PaqCI/AarI sites, a Cas9 scaffold sequence and a Pol III terminator . One or The PaqCI/AarI overhangs are common to all entry clones for maximum modularity, so the user can insert the gRNAs in any entry clone. Once loaded with a gRNA, these entry clones can be combined in a single GreenGate assembly to generate a Level 1 T-DNA vector containing up to 18 gRNAs and a promoter-Cas9-terminator cassette for multiplex CRISPR/Cas9 genome editing.The original GreenGate cloning offers two methods for stacking TUs on a single T-DNA. The first involved assembling individual TUs in intermediate vectors combined in a second assembly reaction in a regular destination Level 2 vector. The necessity to maintain the Eco31I sites in the intermediate vectors led to reduced assembly efficiency for single TUs compared to a regular GreenGate assembly. Additionally, the intermediate vectors were unsuitable for Agrobacterium-mediated plant transformation. The second method enabled the assembly of more than two TUs on a single T-DNA thanks to a particular F adapter module that exploited the sensitivity of Eco31I sites to cytosine methylation to sequentially add one TU to an existing Level 1 vector. This sequential method was increasingly time-consuming as the number of desired TUs increased and was associated with fewer transformants or a higher number of false-positive colonies than a regular GreenGate assembly .To circumvent these limitations, GG2.0 introduces a set of plant-compatible Level 1 and Level 2 destination vectors that enable the parallel and iterative two-by-two stacking of TUs, a process called GreenBraiding .GreenBraiding is a two-step process that can be looped indefinitely. The assembly of single TUs takes place in Level 1 vectors that can be combined to form a Level 2 vector with two TUs. Two Level 2 vectors can be assembled to combine four TUs and generate back a Level 1 destination vector. By alternating between Level 1 and Level 2 vectors , GreenBrFour Level 1 vectors pGB-Zx, , S1 Fig Four Level 2 vectors pGB-Yx, , S1 Fig All Level 1 and Level 2 destination vectors are built on the pCAMBIA backbone  and can In addition to the above-described core set of Level 1 and Level 2 destination vectors, we generated several auxiliary plasmids  containing a dummy 162nt-long insert. These auxiliary plasmids, dubbed \u201cJokers\u201d, can substitute for TUs during GreenBraiding and enable the assembly of an uneven number of TUs .Arabidopsis thaliana. Four individual TUs were assembled from Level 0 entry clones . In plants, ~5% of primary transformants expressed all four reporters at high levels, where expected  and mTurquoise2 expressed from the RPS5A promoter active in dividing cells . TU2 is  vectors , and theexpected : the plaexpected . This exLive imaging and targeted manipulation of gene expression frequently rely on fluorescent plasma membrane (PM) and nuclei markers, along with inducible expression systems for transgenes. We have developed a series of pre-constructed, ubiquitously expressed PM markers, nucleus markers, and an LhG4 driver in GreenBraid Level 1 Z vectors see . These vGG2.0 overcomes the limitations of the original GreenGate approach. GG2.0 introduces a Universal Entry Generator plasmid (pUEG) that allows one-pot, one-step assembly of Level 0 modules, facilitating easy domestication of internal sites and creating entry clones efficiently. By expanding the repertoire of available overhangs, GG2.0 enables the assembly of Level 1 vectors from up to 12 Level 0 modules, greatly increasing the modularity of GreenGate assemblies. GG2.0 also offers a set of Level 0 entry plasmids for multiplex CRISPR/Cas9 genome editing. This feature allows the generation of T-DNA vectors containing up to 18 gRNAs and a promoter-Cas9-terminator cassette for efficient multiplex genome editing. Additionally, GG2.0 introduces GreenBraiding, a novel method that enables iterative stacking of transcriptional units (TUs) using plant-compatible Level 1 and Level 2 destination vectors. This process facilitates the parallel and efficient two-by-two combination of TUs, offering limitless potential for TU stacking. We also present a set of pre-assembled Level 1 vectors featuring fluorescent plasma membrane (PM) and nuclei markers, and for LhG4-driven inducible gene expression. These vectors are readily usable for Level 2 GreenBraid assembly with any desired construct, providing a comprehensive toolkit for researchers to study various cellular processes. GreenGate 2.0 is significantly improved over the original GreenGate, while being backwards compatible. It significantly broadens the capabilities and applications of the GreenGate system, enabling precise and efficient genetic engineering in plant research.S1 FigKey components and cloning sites of the GreenBraid destination plasmids (pGB). The PaqCI/AarI and Eco31I recognition sites are depicted as arrows, and the corresponding overhangs are filled boxes with the overhang in lower letters.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S2 Protocol(PDF)Click here for additional data file.S3 Protocol(PDF)Click here for additional data file."}
+{"text": "Probability of atypical ANCA in diseases other than vasculitis should also be considered in case of ambiguous results.Anti-neutrophil cytoplasmic antibodies (ANCA) are mainly associated with medium and small vessel vasculitis. Two main methodologies currently available for detection of these antibodies are indirect immunofluorescence (IIF) and monospecific proteinase 3 (PR3) and myeloperoxidase (MPO) based immunoassays. However, well-defined guidelines regarding mode of testing for ANCA in laboratories still don\u2019t exist, leading to problems in diagnosis and further patient management. Anti-neutrophil cytoplasmic antibodies testing by IIF and enzyme linked immunosorbent assay (ELISA) often pose a significant challenge in diseases other than vasculitis and in overlapping autoimmune conditions. Anti-neutrophil cytoplasmic antibodies reporting by IIF can be challenging in certain circumstances. This case series aims to discuss four cases with probable interference of anti-nuclear antibodies (ANA) during ANCA testing by IIF resulting in ANCA false positivity. All four cases on subsequent reflex testing by line immunoassay (LIA) for PR3, MPO and glomerular basement membrane (GBM) antigens proved otherwise. While analysing for the presence of ANCA by IIF, the possible interference of ANA leading to a false positive ANCA result should be kept in mind and alternative methods of testing like ELISA, extended granulocyte based IIF assays with MPO and PR3 coated beads,  They are recommended as primary screening methods for detection of PR3-ANCA and MPO-ANCA as i.v. antibiotics and anti-fungals namely cefoperazone, sulbactam, azithromycin, voriconazole, colistin were administered in view of raised procalcitonin. A dermatology opinion was sought in view of persistent hairfall, butterfly rash, oral ulcers and photosensitivity for the last five months. A provisional diagnosis of lupus was made and confirmed on laboratory investigations which revealed ANA screening positivity for speckled pattern (3+) suggestive of anti-Ku antibodies and antimitochondrial antibody (AMA). Her ANA profile by LIA was positive for dsDNA (+++), SmD1(+++), histone (++), nucleosome (++), Ku (+++), SSA/Ro60(+), SSB/La, AMA-M2, Scl-70(++), Jo1. Perinuclear-ANCA (pANCA) positivity was also noted by IIF. However, vasculitis profile by LIA comprising PR3, MPO and GBM antibody testing proved negative. Ultrasound guided renal biopsy revealed type I, type II and type V mixed lupus nephritis. Patient was administered methyl-prednisolone, mycophenolate, hydroxychloroquine and angiotensin-converting enzyme (ACE)-inhibitors. Her condition improved but she developed an episode of severe hypoxic respiratory failure and was intubated and mechanically ventilated. One episode of seizure also occurred post intubation. She was managed by intravenous phenytoin. Her condition gradually improved and finally she was extubated after 5 days. In view of her deranged kidney function tests (KFT) as revealed by raised urea and creatinine at 64.08 mmol/L and 198.06 \u00b5mol/L respectively, she was referred to a nephrology health care center. Indirect immunofluorescence images are shown in A 24-year-old female presented with complaints of pain in the joints for last five months, fever for 2 months, abdominal pain for 15 days and occasional dry cough for 10 days. She had severe anaemia, mild ascites, and bilateral pleural effusion on clinical examination. Four units of packed red blood cells were transfused and An 18-year-old female presented with chief complaints of generalised body swelling since last 3 months, generalised body pain since 10 days and fever since 2 days. The swelling followed a blister in left lower limb and was associated with breathing difficulty and decreased urine output. There was history of prolonged fever of one month duration in the past in 2021. She has been treated with oral L-thyroxine (25 \u00b5g) for hypothyroidism. During examination she had significant pallor and oedema of bilateral feet and face. Arterial blood gas analysis (ABG) revealed metabolic acidosis with high anion gap. X-ray chest showed cardiomegaly, pulmonary oedema and pleural effusion. Ultrasound abdomen revealed gross ascites and renal disease. In view of microscopic and strip-based tests positive for haematuria and proteinuria (3+) and 24-hour urine sample positive for proteinuria (984 mg/day) with raised blood urea and creatinine, urine output monitoring and judicious fluid management were started. Direct Coombs\u2019 test was positive. An echocardiogram (ECHO) revealed moderate mitral regurgitation (MR), mild tricuspid regurgitation (TR), mild pulmonary arterial hypertension (PAH), left ventricular (LV) systolic dysfunction, mild to moderate pericardial effusion and LV ejection fraction 55-60%. One episode of generalised tonic-clonic seizure (GTCS) was noted. Anti-nuclear antibody screening revealed homogenous pattern (grade 3) and ANA profile was strongly positive for dsDNA, nucleosome, histones, smD1, ribosomal- P-protein Po (+++). U1snRNP (++), PCNA (+), Ku (+), DFS 70(+), SSA/R0 60(++) and SSB/La  were also reported by LIA. Anti-neutrophil cytoplasmic antibodies screening was strongly positive for pANCA but vasculitis profile by LIA was negative for antibodies to PR3, MPO and GBM. Renal biopsy revealed an immune complex mediated diffuse endocapillary proliferative glomerulonephritis. The pattern of DIF studies (paraffin retrieved tissues) suggested activation/involvement of classical complement pathway in disease pathogenesis. Indirect immunofluorescence images are shown in 9/L. Her KFT was deranged with serum urea at 57.69 mmol/L and serum creatinine at 137.94 \u00b5mol/L. Renal biopsy revealed co-existing lesions of diffuse lupus nephritis (ISN/RPS Class IV) and membranous lupus nephritis (class V). Her ANA screening was positive with homogenous pattern and reflex ANA profile testing was also reported positive for dsDNA (+++). Her ANCA screening revealed pANCA pattern on IIF but vasculitis profile was negative by LIA for antibodies to PR3, MPO and GBM. She was administered pulse methylprednisolone therapy for three days followed by wysolone 40 mg per oral once daily. Indirect immunofluorescence images are shown in A 16-year-old female presented to the hospital with generalised body swelling, fever with reduced appetite for last one month and abdominal pain for 10 days. There was no associated history of shortness of breath, weight loss and rash. There was history of cough with mucoid sputum production since past two days. On investigation, nephrotic range proteinuria was reported (5055 mg/day). Her haemoglobin was low (70 g/L) with decreased total leukocyte counts at 1.63 x10A 16-year-old female presented to the hospital with complaints of nasal bleeding off and on for past 8 years. Multiple episodes of nose bleed were noted in past which started spontaneously and occurred more in summer season but of late it had been occurring almost round the year. Each episode lasted from thirty minutes to six hours. There was no history of nasal trauma. She also complained of a feeling of mass in the nose. There was history of easy fatigability with no history of fever and weight loss. She also reported of cough for last seven days. It was acute in onset and present uniformly throughout the day. There was no history of sputum production. There was history of breathlessness since 6 months which was aggravated with activity. An otorhinolaryngology consultation revealed a deviated nasal septum towards right side. Her ANA screening was positive for mitotic spindle apparatus 1 grade 2 in intensity. Her ANCA screening by IIF reported pANCA pattern. However, her vasculitis profile by LIA was negative for antibodies against PR3, MPO and GBM. In view of severe anaemia, haemoglobin electrophoresis was done which was reported as normal. She had normal bone marrow studies except for low iron as confirmed by Perl\u2019s stain grade 0 and low serum iron concentration at 6.2 \u00b5mol/L (6.6-25.9 \u00b5mol/L). She was administered injection methyl prednisolone 500 mg for 3 days followed by oral wysolone in view of ANA and ANCA positivity. Indirect immunofluorescence image are shown in The detailed laboratory findings for case series based on IIF and LIA have been shown in The IIF kits (granulocyte mosaic 13) were manufactured by Euroimmun, Lubeck, Germany. Each slide is capable of processing 3 samples. Each kit comes with a positive and negative control. Each reaction well , has three biochips: an ethanol fixed granulocyte substrate, a formalin-based granulocyte substrate and a granulocyte-Hep-2 mixed substrate. Upon addition of patient samples in recommended dilution of 1:10 and subsequent incubation, specific antibodies of classes IgG, IgA and IgM attach to the antigens in case of a positive result. A second step where the bound antibodies are stained with fluorescein isothiocyanate labelled anti-human antibodies are visualised under fluorescent microscope. Line immunoassay for vasculitis profile is an indirect membrane-based enzyme immunoassay for qualitative measurement of IgG class of antibodies directed against PR3, MPO and GBM in human serum. Dilution used is 1:100. Anti-nuclear antibodies and vasculitis profile were performed on fully automated line-immunoassay analyser .Ethical approval is not required for case reports/case series from our institute. However, written informed consent has been taken from the concerned patients/patient\u2019s relatives for possible publication in a medical journal.In all the four cases discussed above, the patients tested positive for ANA and ANCA screening (by IIF) and ANA profile (LIA). Vasculitis profile (LIA) was negative in all four patients. The first three cases were diagnosed as lupus on the basis of clinical presentation and laboratory investigations. Final diagnosis was not made in Case 4 due to varied clinical presentation not conforming to the diagnosis of systemic lupus erythematosus (SLE). Due to discrepancy in results between ANCA screening by IIF and vasculitis profile by LIA and based on clinical presentation, AAV was ruled out in all four cases. A probable interference due to ANA positivity resulting in false positive ANCA was suspected.We reported four cases of false positive ANCA testing by IIF which on subsequent testing by LIA turned out to be negative. This discrepancy needs to be introspected as ANCA screening by IIF is often performed as a first line test for evaluation of suspected cases of AAV. A false positive or false negative result can have grave implications in further diagnosis and patient management. Most often ANCA testing is an emergency and reflex testing for confirmation after primary screening may result in unnecessary delay in clinical interventions. The aim of this case series is to discuss reasons responsible for the discrepancy of results in ANCA reporting by IIF and LIA and what can be done to ensure rapid and accurate results in such cases.per 1999 International Consensus on ANCA testing, IIF should be used to screen for ANCAs and samples with ANCA positivity should be tested by immunoassays for PR3 and MPO  and atypical ANCA. These patterns reflect the fixation and staining of the antigenic material but do not indicate antigen specificity. While cANCA pattern is usually due to a specific serine protease 3, a pANCA pattern can result mainly from MPO apart from a number of not so common target antigens such as elastase, cathepsin G, lactoferrin, etc. (Although the clinical relevance of ANCA detection in non-AAV conditions is limited, several approaches have been adopted to explore these \u201catypical ANCA\u201d by use of IIF fixatives like methanol and formalin and development of ELISA comprising the ANA profile capable of detection of autoantibodies against antigens such as lactoferrin, elastase, BPI, cathepsin G, \u03b1-enolase, \u03b2-glucuronidase, lysozyme, azurocidine, Indirect immunofluorescence is still considered as a primary screening technique for detection of ANCA in many clinical laboratories using ethanol fixed neutrophils as substrate. A big disadvantage would be the failure to differentiate pANCA patterns of AAV and non-AAV (However, IIF for ANCA detection has few drawbacks. Most important in these four cases was the probable interference of a positive ANA, resulting in a false positive ANCA report as reported in previous studies (etc. Cathepsin G, elastase, BPI, MPO, PR3, lactoferrin, comprises \u201cANCA Profile ELISA\u201d. It is capable of detecting aforementioned antigens in a single run. Each strip of ELISA plate consists of \u201cblank\u201d and \u201ccalibrator\u201d apart from the six antigens coated in respective wells. It holds promise for quick and simultaneous detection of antibodies in both AAV and non-AAV. Novikov et al. abandoned IIF for ANCA screening more than 15 years ago (To overcome difficulties faced while using IIF as the primary screening method of ANCA reporting, ELISA has been recently suggested as the primary screening modality (Indirect immunofluorescence methods are often considered labour intensive, cumbersome and time consuming apart from high variability of results due to subjective differences in observation. ELISA methods may be more specific but may miss out non-AAV antigens due to wide use of only PR3 and MPO based monospecific assays. Finally, in order to assess the clinical and laboratory performance of an ANCA assay for use in clinical practice, adequate samples with relevant clinical information should be available. ANCA associated vasculitis is a rare disease posing a big challenge in the form of limited samples leading to questions regarding sufficient expertise in interpretation of the test. This also calls for the need to address issues like number of tests required in a defined time span to maintain the sufficient expertise in laboratory reporting.etc.In conclusion, ANCA is a very sensitive and specific biomarker for diagnosing AAV. However, the correct methodology of their detection plays a very important role to ensure correct diagnosis. A positive ANCA needs to be correlated with clinical presentation and histological findings for diagnosing AAV. While reporting a positive ANCA by IIF, probable interference of a positive ANA result should be always kept in mind. On the other hand, a negative ANCA does not rule out AAV, since AAV without positive ANCA does exist. Atypical ANCA may be associated with other autoimmune diseases, inflammatory bowel diseases, autoimmune hepatitis ANCA testing by IIF in case of a sample significantly positive for ANA, namely homogenous and some cytoplasmic patterns, needs to be cautiously interpreted keeping chances of false pANCA positivity in mind.ANCA testing primarily by ELISA may be preferred if the sample is simultaneously positive for ANA.Non-AAV antibodies may be missed by IIF and ELISA for monospecific antigens PR3 and MPO, and ANCA profile ELISA may be considered in such cases.As ANCA testing by IIF is subject to observer subjectivity apart from false positivity in cases of a simultaneous ANA positivity, variations in interpretations are bound to occur. This may result in delayed reports by laboratories. In such cases where ANCA reporting is also an emergency for patient management, ANCA reporting by IIF using granulocyte mosaic 32 may be opted for."}
+{"text": "Background: Artificial intelligence (AI) is the process by which it is possible to program computers to mimic human thoughts. AI and its subsets machine learning and deep learning have been developed to analyze complicated data gathered from many sources using algorithms built into decision support systems. It has been widely used in the field of dentistry.Aim: The study aimed to evaluate the knowledge, attitude, and practice (KAP) of AI among dental students and dentists.Methodology: The present study is a descriptive cross-sectional online survey that was carried out among dentists and dental students in South India. A self-structured, close-ended questionnaire that was administered that consisted of 25 questions was included. The questions were circulated through Google Forms , and it was circulated among the study participants through online mode. The data were collected systematically, and SPSS Statistics version 22.0  was used for data analysis.Results: One thousand  participated in the study through Google Forms. Among these, 700 (70%) were females and 300 (30%) were males. In the study group, 635 (63.5%) were aware of AI, and 365 (36.5%) were not aware . Among the 21 questions used to assess the KAP, 14 questions were significant with a p-value less than 0.05. More than 60% agreed that the dental curriculum has to be updated with AI. About 269 (26.9%) agreed that AI will replace the role of dentists in the future. There were no significant results in comparing dental surgeons and dental students.Conclusion: The present study showed that the KAP among dental surgeons and dental students was the same. They believe that the dental curriculum has to be updated with AI. This study shows that there is a lack of knowledge about deep learning models and websites used for AI among dentists. Thus, it is necessary to include evidence-based teaching and training about the application of AI in dental practice to improve the future of dentistry. John McCarthy in 1956 first proposed the applied computer science known as artificial intelligence (AI). It is the reproduction of human intelligence in devices that have been designed to reason and acquire knowledge like humans. Robotics, computer vision, machine learning, and natural language processing are all sub-fields in this branch . AI has A core curriculum of dental education needs to be updated in this fast-changing environment since AI in healthcare is fundamentally changing the methods utilized in diagnosis, treatment plans, and prognosis . The varIn research writing, AI tools have been broadly classified into two categories. One is that supports writers throughout the writing process and the other one is those that review and assess the quality and validity of written material . AI imprMethodologyTo assess the KAP among dental students and dental surgeons in various districts of Tamil Nadu, a cross-sectional questionnaire survey was conducted through Google Forms .\u00a0The Institutional Ethical Committee of Priyadarshini Dental College and Hospital issued approval IEC-PDCH 4/2 2023. After obtaining ethical clearance, a self-structured questionnaire consisting of 25 questions was framed. Among these first four questions were about demographic details including age, gender, and qualifications. The remaining 21 questions were used to assess the knowledge about AI. One thousand randomly selected dental surgeons from registered dental councils and dental students from various parts of Tamil Nadu participated in this survey through Google Forms. About 595 dental surgeons and 405 dental students were included in the study. Among the 595 dental surgeons, 433 were Bachelor of Dental Surgery (BDS), 96 were Master of Dental Surgery (MDS), and 66 were post-graduate students. All the participants were explained the purpose of the study and informed consent was obtained through Google Forms.Statistical evaluationRandom sampling techniques and cross-sectional design were used to analyze the KAP among 1,000 individuals. Results were evaluated using SPSS Statistics version 22.0 . Descriptive statistics were used. To compare between dental surgeons and dental students, a chi-square test was used. A p-value less than 0.05 was considered significant.A total of 1,000 responses from dental students and dental surgeons were collected. The study group falls within the age group of 18-60 years. About 825 (82.5%) were within the age group of 18-25 years. Among the study groups, 700 (70%) were females and 300 (30%) were males. About 595 dental surgeons and 405 dental students were included in the study. Among the 595 dental surgeons, 433 (43.3%) were BDS, 96 (9.6%) were MDS, and 66 (6.6%) were post-graduate students. Among the 21 questions used to analyze the KAP, 14 questions were significant.Six hundred thirty-five (63.5%) were aware of AI and the remaining 365 (36.5%) were not aware of AI with a significant p-value of 0.000. Among the websites used for AI, only 372 (37.2%) were aware of the AI apps and the remaining 628 (62.8%) were not aware of what app to use specifically . About 534 (53.4%) used AI to specifically improve knowledge about a particular topic, 394 (39.4%) said no, and 72(7.2%) answered don\u2019t know . The various large language models (LLM) app usage among dental surgeons and dental students was significant . The awareness of the various subsets of AI such as deep learning, recurrent neural networks, convolutional neural networks, and machine learning between the groups was significant  (Table Two hundred sixty-nine 26.9%) agreed that AI in the future will replace the role of dentists. Four hundred twelve (41.2%) disagreed that AI will not replace the role of dentists, and 319 (31.9%) said they don\u2019t know . About 597 (59.7%) agreed that the dental curriculum has to be updated with AI, 100 (10%) disagreed, and 303 (30.3%) answered as don\u2019t know . Four hundred fifty (45%) accepted that the advancement of AI in the field of dentistry can affect creativity. About 222 (22.2%) said that AI will not affect creativity, and 328 (32.8%) answered don\u2019t know . Four hundred five (40.5%) answered that AI in the field of dentistry can violate ethical principles, and 188 (18.8%) answered that it won\u2019t violate ethical principles  . Questions regarding the ethics violations by using AI in dentistry were significant  Table .AI enables the creation of intelligent machines. AI has begun to have an impact on diagnostic and treatment modalities in the health sector . AI has Our findings show that 635 (63.5%) were aware of AI and 380 (38%) were aware of AI apps. A study conducted by Shiva Thulasi et al. [More than 60% were about LLM, similar to studies done by Shiva Thulasi et al.  showed tCancer is not easily detected in its early stages. Thus, a computer-operated system that can distinguish between benign and malignant cells is used in diagnosis . In the In the present study, 590 (59%) believed that the dental curriculum had to be updated with AI. More than 75% agreed that AI be included in undergraduate and postgraduate dental education in a study conducted by Emir Y\u00fczba\u015f\u0131o\u011flu . The widIn this study, there is not much difference between dental surgeons and dental students regarding the KAP of AI. This is similar to the study conducted by Shiva Thulasi et al. . Some deStill, the use of AI is not integrated into dentistry. The biggest limitations were inadequate information and a lack of awareness regarding integrating AI. Additionally, the participants were professionals with clinical expertise as well as dental students who may have contributed distinct AI conceptualizations to the overall study output ,17. ThisThe limitation of this study includes the years of experience among students and dentists, which have varied results in various levels of awareness about AI. The study has to be conducted on a large population of dentists to know the KAP of AI among dentists in Tamil Nadu.The present study shows that awareness of AI is not satisfactory. Therefore, awareness about AI has to be achieved through dental associations, research institutions, and technology companies by promoting discussions and educational resources related to AI. Research papers, publications, and presentations on AI in dentistry could contribute to spreading awareness among dental students. The field of AI is rapidly evolving, and new applications and tools are being developed regularly. Thus, staying updated on advancements in AI is essential for dentists interested in incorporating AI into their work. To improve the future of dentistry in this revolutionary AI period, the curriculum has to be updated."}
+{"text": "Introduction: The utilization of artificial intelligence (AI) and machine learning (ML) models has brought about a significant transformation in the manner in which periodontists gather information, evaluate associated risks, develop diverse treatment alternatives, anticipate and diagnose dental conditions that compromise periodontal health. The principal objective of this prospective study was to examine periodontists\u2019 understanding and acceptance of the application of AI in the realm of periodontology.Materials and methods: This observational study was conducted on 275 participants based on questionnaire using Google Forms. These forms were pre-validated and subsequently circulated among periodontists in Maharashtra via various social media platforms. The study, in its entirety, comprised four open-ended questions on participants\u2019 demographics and 14 closed-ended questions, all of which were presented to the participants in English. These questions aimed to elicit participants' awareness, knowledge, attitudes, and perspectives regarding emerging applications of AI in the field of periodontology. To analyze the collected data, researchers employed the widely utilized Statistical Package for Social Sciences (SPSS) version 22.0.Result: A 75% response rate was achieved and 68% of the respondents were female. 62% periodontists were aware of AI; however, only 24% were aware of its working principles. Most respondents agreed with the use of AI in periodontal diagnosis; however, they disagreed with the use of AI in predicting clinical attachment loss (69%). 80-82% respondents felt that AI should be a part of postgraduate training and should be implemented in clinical practice. However, most periodontists do not use AI for diagnostic or research purposes. 49% periodontists felt that AI does not have better diagnostic accuracy than periodontists, and therefore cannot replace them in the future.Conclusion: Most periodontists possessed a reasonable level of understanding regarding the utilization of AI in the domain of periodontology and expressed a desire to incorporate it into their diagnostic and treatment planning processes for periodontal conditions. Additional endeavors must be undertaken to enhance periodontists\u2019 awareness concerning the effective implementation of AI within their professional practice, with the aim of facilitating personalized treatment planning for their respective patients. It is postulated that the integration of AI will augment the likelihood of achieving favorable outcomes within the realm of periodontology. Artificial intelligence (AI) refers to the imitation or improvisation of human intelligence in machines specifically designed and programmed for numerous cognitive functions, such as problem solving. An exemplary transformation occurs in almost every industry and sector owing to advancements in machine learning (ML) and deep learning (DL) .In 1955, the term \u2018artificial intelligence\u2019 was introduced by John McCarthy, a mathematician widely known as the father of AI. This term was chosen to elucidate the machine\u2019s capability to perform tasks that could be interpreted as \u201cintelligent\u201d activities . AI has AI programs can significantly benefit novice periodontists. A convolutional neural network (CNN), used as an unsupervised diagnostic tool, allows periodontists to view computed tomography dental images to facilitate the accurate diagnosis of periodontal diseases and the detection of plaque, gingivitis, and implant design systems .Despite the numerous advantages of AI, its application in periodontics remains markedly restricted. This can be attributed to a myriad of factors, such as the lack of comprehensive familiarity among periodontists with the underlying principles of AI as well as their limited awareness regarding its true potential domains. Furthermore, the general population is reluctant to place trust in the outcomes offered by AI in the context of robot-assisted surgery. Consequently, a multitude of challenges persist and necessitate a confrontational resolution.Many studies and reviews have reported on the use of AI in periodontics, but no study has been conducted to date to assess the current knowledge and perception of periodontists on the use of AI for periodontal diagnosis and treatment planning -9. ThereStudy designA cross-sectional questionnaire study was conducted with periodontists in Maharashtra to assess their knowledge, attitudes, and perceptions of the use of AI in the field of periodontology. The data were collected over a period of one month from March 2023 to April 2023. This study was approved by the Institutional Ethical Committee of the Jawahar Medical Foundation ACPM Dental College, Dhule (EC/NEW/INST/2022/2959/Y23/212). This study was conducted in accordance with the ethical standards of the Declaration of Helsinki. Participants were granted the opportunity to complete the form on a single occasion. Subsequently, participants' responses were collected after obtaining their informed consent and willingness to participate in the study as well as to elucidate the objective and ensure the preservation of anonymity.Study area and populationThe study was conducted in the Department of Periodontics, Jawahar Medical Foundation ACPM Dental College, Dhule, on the faculty members who were working as periodontists in the dental colleges of Maharashtra state.\u00a0Inclusion and exclusion criteriaPeriodontists employed as faculty members who agreed to participate in the study were included. Undergraduates, postgraduates pursuing dental education, private practitioners, and participants who did not give their consent were excluded from the study.Sample size estimation2 \u00d7 standard deviation (SD) x (1-SD) / (margin of error)2. The confidence level was 95% and the margin of error was 5%. The standard deviation of the unknown population was 50%. The expected sample size was 207. The study was conducted with 275 periodontists, considering a 30% non-response rate of emails.The sample size for the online survey was calculated using GPOWER (version 3.1) software developed by Franz Faul at the University of Kiel in Germany. The formula used for analysis was (Z-score)Sampling and data collection procedureThe selection of respondents for the study was accomplished using convenience and non-probability sampling. A link to an online survey was created using Google Forms and distributed among periodontists in Maharashtra via 42 WhatsApp groups (one WhatsApp group per college)\u00a0between March and April 2023. The examiner explained the study objectives to the participants and they were given a brief introduction to the AI. Participants were asked to select one option from the answers provided to each question. Interested participants entered their names and contact information on Google Forms. Responses were made on a single webpage with a \u201csubmit\u201d button that allowed only one submission through the link. This study aimed to examine respondents\u2019 approach to AI and its possible future in periodontology. The repeated reminders were sent every five days for one month to complete the forms.Testing the validity and reliability of questionnaireThe questionnaire was formed in consultation with six experts: three periodontists, two AI experts, and one researcher with more than 10 years of experience who were not involved in the study. Following the assessment conducted by these six specialists, Aiken's V statistic was derived, exhibiting a value of 0.85, signifying favorable content validity. A meticulously crafted survey was developed to accomplish the research objectives, drawing on the content validity. To evaluate the dependability of the inquiries, a preliminary examination or pretesting of the questionnaire was carried out on 40 individuals who were not involved in the study. The reliability of the questionnaire was tested using Cronbach\u2019s alpha, which was 0.88. The questionnaire was retested after a period of two weeks using the same cohort to assess the level of agreement among the questions. Inter-observer agreement was assessed using kappa coefficient, which was 0.96.Tools and techniqueThe survey served as the primary instrument for the data collection. The questionnaire was divided into four sections. The first section, known as Part A, focused on four open-ended questions on sociodemographic characteristics, where participants entered their name, age, gender, academic affiliation, and institution in which they are currently working. Part B consisted of seven closed-ended questions, identifying the basic knowledge of the periodontists participating in AI using a Likert three-point scale  . Part C Statistical analysisData obtained from the questionnaires were entered into an Excel spreadsheet to serve as a database. The acquired data were subjected to statistical analysis using the SPSS software version 23 . The Shapiro-Wilk test was used to assess the normal distribution of the data. Frequency distributions and tables were used to summarize and present the sociodemographic variables and participants' responses. To determine the significance between variables, non-parametric tests, such as the Chi-square test, were employed. Spearman\u2019s correlation coefficient test was also used to assess the relationship between gender, and designation with knowledge, attitude, and perception. The level of significance was set at P \u22640.05.Demographic details of the respondentsThe study involved 275 participants, of whom 207 individuals filed the online Google Form, generating a response rate of 75%. Descriptive analysis showed that 68% of females and 32 % of males responded to the questionnaire. Of the responded periodontists, 28% were professors, 33 % were readers, and 39% were senior lecturers. 73% of the respondents were in the age group of 25-40 years, as shown in Table Response assessing knowledge of periodontist about AI and its applicationsA total of 129 periodontists (62%) were aware of the term 'artificial intelligence' with a statistically significant difference (p<0.05); however, only 55 (24%) of them were aware of the working principle of AI with no statistically significant difference (p>0.05). Non-significant differences were noted when participants were asked about the use of AI for diagnosing periodontal bone, aggressive and chronic periodontitis, predicting the prognosis of periodontally compromised teeth (PCT), predicting the prognosis of treatment, and clinical attachment levels (p>0.05). However, most of the periodontists agreed with the use of AI in diagnosing periodontal problems, except for the prediction of clinical attachment level, where 69% of respondents disagreed, and assessment of prognosis of PCT, where 51% respondents gave a neutral response . 60% periodontists did not use AI for research purposes, and this difference was statistically significant (p<0.05). 95% periodontists did not use AI to diagnose periodontal problems (p>0.05), as shown in Table Response assessing perception of periodontists towards AI48% respondents disagreed with the better diagnostic ability of AI than periodontists, with a non-significant difference (p>0.05), and 49% of respondents disagreed that AI could replace periodontists in the future, with a statistically significant difference (p<0.05). 81% periodontists felt that AI can be a beneficial tool in pandemic situations such as Covid-19 with a non-significant difference (p>0.05), as shown in Table Correlation between knowledge, attitude, and perception with gender and designation of the participantsThe knowledge, attitude, and perception of the participants demonstrated a weakly favorable correlation with gender and designation, indicating that older males, with more years of experience and higher job titles, possessed a greater understanding, mindset, and interpretation regarding the utilization of AI and its applications in the field of periodontology. Knowledge displayed a moderately positive correlation with attitude and a strongly positive correlation with perception, revealing that as periodontists' understanding of AI increased, their mindset and interpretation also improved, as shown in Table AI has a vast array of medical applications and has recently experienced a notable surge in its prevalence, thus necessitating a thorough exploration of its implementation in the field of dentistry, particularly periodontology. The integration of AI into the realms of Medicine and Dentistry has been facilitated by the advent of smartphones and internet connectivity, aligning it with the latest advancements in engineering and technology. Nevertheless, numerous scientists and medical practitioners remain unfamiliar with AI and its potential ramifications in both their personal and professional lives . To the The study's findings revealed that the response rate reached 78%, with the majority of the participants being female (68%). This observation can potentially be attributed to the higher number of females pursuing dentistry as their chosen profession, with a significant portion opting for higher education ,12. The Additionally, most periodontists agree with the use of AI in determining periodontal bone loss, and diagnose cases of aggressive or chronic periodontitis. The majority of them were senior lecturers who agreed, which aligns with the findings of previous studies ,7,8. PapHowever, most of the periodontists disagree with the use of AI to predict clinical attachment levels. Scott et al. conducted a scoping review on the use of AI in periodontics, and concluded that there was lot of heterogeneity in data obtained from the studies, leading to varying results . AI modeThe present study revealed that most senior lectures were aware of and had knowledge about AI compared to readers and professors. The reason for this phenomenon could potentially be attributed to the escalated level of contact that young individuals have with digitization, which encompasses their inclination to investigate and gain knowledge about novel technological advancements, such as AI .The knowledge, attitude, and perception about the use of AI in diagnosing periodontal problems was found to increase with the increase in designation or level of experience of the periodontists. However, most of them did not use AI in their dental practice or for research purposes. This finding is in agreement with previous studies ,18,19. T82% respondents felt that AI should be a part of their postgraduate curriculum and should be implemented in clinical practice. This finding is supported by previous studies ,18,19. P49% respondents disagree that AI can replace periodontists in diagnosing periodontal diseases, but they feel that AI can successfully help in taking their dental practice to a new level. This is in accordance with previous studies ,21,22. ARecommendationsTo enhance and foster the growth and motivation of periodontists, it is important to integrate AI into the dental curriculum or regular training programs. To expand our knowledge and understanding in this field, it is recommended that future research endeavors concentrate on the development of models that possess a heightened level of accuracy when it comes to diagnosing various dental ailments, as well as predicting the outcomes of diverse treatment methods. Periodontists should use AI for diagnosis, and treatment planning in their practice.\u00a0Limitations of the studyThe present study did not identify any barriers to AI use in periodontology. The sample was not representative of postgraduates or private practitioners. Closed-ended questions utilizing Likert scale\u00a0may have hindered the generation of suggestions or proposals for questions requiring multiple perspectives, thereby leading to miscommunication. Future surveys should be conducted on samples of diverse populations, including postgraduates, private practitioners, and open-ended questions, where participants can express their views on AI.Periodontists have varying opinions on AI applications. Therefore, it is important to understand that AI is not a suitable substitute for periodontists and dental professionals. Instead, it would be helpful for better diagnosis, prognosis, and treatment plans in periodontology. The prevailing number of respondents possessed knowledge regarding the advantages associated with the utilization of AI in periodontology and held the conviction that it would serve as a valuable resource. This study revealed that enhanced technological provisions within dental clinics and the instruction of practitioners at both the undergraduate and postgraduate levels could potentially overcome forthcoming obstacles pertaining to the incorporation of AI in the field of periodontology."}
+{"text": "ClinicalTrials.gov until October 2022.\u00a0We pooled the data on mortality, readmission rate, and quality of life (QoL) as primary outcomes. The certainty of evidence was evaluated by the grading of recommendations assessment, development, and evaluation (GRADE) approach. We included three studies with 390 patients with heart failure. Peer support may have resulted in a slight increase in mortality (risk ratio (RR)=1.16, 95% confidence interval (CI)=0.61-2.21; low certainty of the evidence) and in a reduction in the readmission rate . The evidence was very uncertain about the effect of peer support on QoL . Despite that the certainty is low or very low, the extant data available evidence suggests that peer support may not yield substantial improvements in critical outcomes for patients with heart failure. Consequently, endorsing peer support for patients with heart failure currently seems unjustifiable.Peer support, which is given by people with similar life experiences and experiential knowledge, has been shown to be effective for patients with diabetes and mental illness. However, the impact of such peer support on patients coping with heart failure remains indeterminate. The objective of this systematic review and meta-analysis is to scrutinize the potential benefits of peer support for patients with heart failure. We included randomized controlled trials (RCTs) evaluating the effectiveness of peer support for patients with heart failure in contrast to those without peer support. We searched the Cochrane Central Register of Controlled Trials, MEDLINE, Embase, WHO International Clinical Trials Registry Platform, and  Heart failure represents a complex syndrome presenting significant clinical and societal challenges, including escalated morbidity, mortality, and substantial strain on global healthcare systems . By the Peer support, a constituent of disease management strategies, is defined as the process of providing and receiving non-clinical aid through an empathetic relationship to surmount severe mental, psychological, or addiction challenges, thereby facilitating long-term recovery -7. Peer To our knowledge, no report has summarized the evidence for peer support in patients with heart failure. A prior systematic review unveiled salutary effects of peer support for individuals afflicted with heart disease, encapsulating heightened self-efficacy, augmented physical activity, ameliorated pain, and diminished frequency of emergency room visits . NeverthMaterials and methodshttps://osf.io/uxgam/), and in concordance with the 2020 PRISMA checklist  that assess individual randomization or cluster randomization. We did not impose limits on countries or languages. Our analysis encompassed all manuscripts, both published and unpublished articles, in addition to the abstract of conference abstracts and correspondence. Studies were not precluded based on the observational duration or year of publication. The participants in question were patients who had received a diagnosis of heart failure and were treated in either an inpatient or outpatient medical setting. Patients exhibiting severe cognitive impairment or psychiatric disorders, with higher brain dysfunction after stroke or head injury, actively using narcotics or alcohol, serious visual or hearing impairment, and those actively undergoing cancer treatment were excluded from the study. The intervention method under consideration was peer support among patients, defined as a symbiotic relationship of mutual assistance between individuals sharing analogous life experiences . Any modThe primary outcomes were mortality, readmission rate, and quality of life (QoL). The mortality and readmission rates were included within one year following the intervention . The QoLTrials.gov. Two independent reviewers checked the studies using the title and abstract. All extracted studies by the two reviewers underwent a comprehensive full-text review.\u00a0The full text was subsequently utilized to ascertain the eligibility by each reviewer. In instances where only an abstract was available and the eligibility remained ambiguous, we contacted the original author. Discrepancies between the two reviewers were discussed and resolved, with a third reviewer consulted if necessary.We conducted a thorough search of MEDLINE (PubMed), the Cochrane Central Register of Controlled Trials (Cochrane Library), EMBASE , the World Health Organization International Clinical Trials Platform Search Portal (ICTRP), and Clinical We extracted data on study characteristics and outcomes, including year of publication, author, and age. We extracted the difference between the means of the data for each outcome after the intervention period. However, if the average was not available, the difference between the average of the changed values and their standard deviation was extracted. We used the relative risk ratios (RRs) and the 95% confidence intervals (CI) as an effect measure for the following binary variables: readmission and mortality rate. We used the mean difference (MD), where appropriate, standardized MD (SMD), and the 95% CI as an effect measure for the following continuous variables: QoL and self-efficacy. We summarized adverse events based on the definition in the original article, but we did not perform the meta-analysis. For the integration of means and standard deviations of continuous variables, we followed the method of the Cochrane Handbook . We endeWe performed a meta-analysis with a random effects model using Review Manager software (RevMan 5.4.2). For continuous outcomes, we pooled MD with 95% CI. The SMD was applied if outcome measures differed across studies. For binary outcomes, we pooled RR with 95% CI. We used forest plots to visually evaluate the heterogeneity of the studies included. Consequently, we calculated I2 statistics . Cochrane Chi2test (Q-test) was performed for I2 statistics, with a p-value less than 0.10 was denoted as statistically significant.We made a summary of finding (SoF) tables based on the recommendations of the Cochrane Handbook . SOF tabOwing to data limitations, certain pre-specified analyses, such as subgroup analyses with age, EF, and peer support intervention methods, as well as sensitivity analyses, excluding studies utilizing imputation statistics and those with high overall RoB, were not executed. We had planned the time to readmission to the secondary outcome, but this was not implemented due to an inability to extract pertinent data from the included studies.ResultsWe exhaustively searched for relevant articles in October 2022. A total of 1195 articles were screened, and 24 full texts were assessed for eligibility    ; MD19.2 lower, 95%CI=40.04 lower to 1.64 higher; very low certainty of the evidence)  for analysis [To the best of our knowledge, this systematic review constitutes the first study to appraise critical outcomes, such as mortality and readmission rates, in relation to peer support for patients with heart failure. In addition, we employed a robust methodological strategy that incorporated an exhaustive literature search and a predetermined protocol (analysis ,43. HowePeer support for patients with heart failure may result in a slight increase in mortality. Peer support may result in a slightly reduced readmission rate in patients with heart failure. Peer support for patients with heart failure may have little to no effect on QoL and self-efficacy, but the evidence is very uncertain.At this point, peer support for patients with heart failure may not be recommended. The evidence for peer support for heart failure may be low or very low due to the inclusion of only three RCTs in this study, the unspecified severity of heart failure among participants, and the high deviation rate. Additional RCTs are needed to confirm our conclusions and elucidate the mortality and readmission rate of peer support for patients with heart failure. Additionally, the study analyzed here did not incorporate a time-to-readmission outcome. Future RCTs should consider including time to readmission as an evaluative metric."}
+{"text": "Repetitive mild traumatic brain injuries (rmTBI) may contribute to the development of neurodegenerative diseases through secondary injury pathways. Acetyl-L-carnitine (ALC) shows neuroprotection through anti-inflammatory effects and via regulation of neuronal synaptic plasticity by counteracting post-trauma excitotoxicity. This study aimed to investigate mechanisms implicated in the etiology of neurodegeneration in rmTBI mice treated with ALC. Adult male C57BL/6J mice were allocated to sham, rmTBI or ALC + rmTBI groups. 15 rmTBIs were administered across 23 days using a modified weight drop model. Neurological testing and spatial learning and memory assessments via the Morris Water Maze (MWM) were undertaken at 48\u00a0h and 3\u00a0months. RT-PCR analysis of the cortex and hippocampus was undertaken for MAPT, GFAP, AIF1, GRIA, CCL11, TDP43, and TNF genes. Gene expression in the cortex showed elevated mRNA levels of MAPT, TNF, and GFAP in the rmTBI group that were reduced by ALC treatment. In the hippocampus, mRNA expression was elevated for GRIA1 in the rmTBI group but not the ALC + rmTBI treatment group. ALC treatment showed protective effects against the deficits displayed in neurological testing and MWM assessment observed in the rmTBI group. While brain structures display differential vulnerability to insult as evidenced by location specific postimpact disruption of key genes, this study shows correlative mRNA neurodegeneration and functional impairment that was ameliorated by ALC treatment in several key genes. ALC may mitigate damage inflicted in the various secondary neurodegenerative cascades and contribute to functional protection following rmTBI. Traumatic Brain Injury (TBI) is a global public health problem, with an estimated 69 million (95% CI 64\u201374 million) TBIs occurring worldwide each year . The leaTBI pathology involves primary injury which induces secondary injury pathways. The primary injury encompasses the physical damage resulting from external impact transmitted to the brain. In contrast, secondary injury involves concurrent and self-exacerbating molecular and chemical changes that occur over time that amplify the damage caused by the primary injury . These sAcetyl-L-carnitine (ALC) is an endogenously produced carnitine metabolite present in tissue and plasma, and readily crosses the blood brain barrier (BBB)  unlike iIn human studies, ALC has shown the ability to improve cognitive performance and slow decline in the early stages of neurodegenerative diseases , as wellThe translational application of ALC is attractive given that it is an over-the-counter supplement that can be safely administered long-term  and is oad libitum.This study was approved by the Animal Ethics Committee of Central Queensland University (CQU AEC 0000021124) in adherence with guidelines from the National Medical Research Council of Australia. The ARRIVE guidelines were used for study design and reporting. A total of 48 male C57BL/6J mice  were used for the study, and were 10 weeks old at the initiation of the protocol. Mice were housed four per cage in a 12:12\u00a0h light-darkness cycle, in a constant temperature of 22\u00b0C \u00b1 2\u00a0\u00b0C, with food and water permitted The study design included two separate arms, acute investigation involving mice euthanized 48\u00a0h following final impact, and chronic investigation involving mice euthanized 90 days following final impact . In bothMice in the rmTBI and ALC + rmTBI groups undertook 15 rmTBI across 23 days via an apparatus engineered to mimic the head acceleration forces of human mTBI, as described previously . Prior tThe righting reflex (RR) recovery time was assessed after each mTBI to provide a measure of neurological restoration in all groups. Mice were positioned on their back and the time to adopt a prone position was recorded. The RR time was defined as from the cessation of isoflurane inhalation to the commencement of the RR.Neurological function was assessed using the 10-point neurological severity score (NSS) as undertaken previously . The NSSSpatial learning and memory were assessed via the Morris Water Maze (MWM) , as descEuthanasia was performed via isoflurane inhalation until the cessation of vital signs and absence of pedal reflex. The brain was removed, weighed, and washed in ice cold oxygenated  artificial cerebrospinal fluid (CSF) containing 118.0\u00a0mM NaCl, 3.5 mM KCl, 1.3 mM MgCl2, 26.2\u00a0mM NaHCO3, 1.0\u00a0mM NaH2PO4, 2.5\u00a0mM CaCl2, 11.0\u00a0mM glucose. The cortex and hippocampus tissues were then dissected on a frozen dissection platform, and frozen at \u221280\u00b0C for genetic analysis. Collection tubes were coded to enable blinding of the molecular analysis.The following gene expression changes were assessed - MAPT (microtubule associated protein tau), GFAP , AIF1 , GRIA1 (glutamate ionotropic receptor AMPA type subunit 1), TNF (tumor necrosis factor), CCL11 (C-C motif chemokine 11), and TDP-43 (TAR DNA-binding protein 43). mRNA was extracted from tissue homogenates of the hippocampus and cerebral cortex of rmTBI and control groups (n = 4 per group at 48\u00a0h or 90\u00a0days after injury) using the phenol-chloroform method . Sample a priori power analysis used \u03b1 of 0.05, power of 0.8, and means and SD from previous laboratory data, and determined that eight mice per group were required for cognitive and neurological tests. Statistical analyses were performed using IBM SPSS Statistics for Windows Version 25.0 . Data were evaluated for normality via Shapiro-Wilks test prior to formal testing. One-way ANOVA, or repeated-measures two-way ANOVA, with Tukey post hoc tests  was used to assess for statistically significant differences. All data are presented as means with standard deviations.An Mice in the rmTBI and the ALC + rmTBI groups showed no signs of physical stress following impacts, and no mice were withdrawn from the study. Mice demonstrated no adverse effects from the administration of ALC.p < 0.05). For all other impacts there was no difference between rmTBI and Sham groups. There were significant differences in RR time between the rmTBI and ALC + rmTBI groups following impacts two through five (p < 0.05), with no difference between these groups following impacts six through 15. There were no significant differences between the Sham and ALC + rmTBI group following any impact.Recovery of RR was measured following all impacts or Sham group anesthetizations . The rmTp < 0.05). The ALC + rmTBI group score was not significantly different from Sham group.NSS was undertaken in the acute phase of recovery . The rmTp < .05). Notably, there was a significant difference between rmTBI and ALC + rmTBI group times in trial 4 (p < .05). There were no significant differences in trial times between the Sham and ALC + rmTBI groups. In the 3-month testing period, there were group differences between the Sham and rmTBI groups at trial 2, 3, and 4 (p < .05). At trial 4, mean group times were again significantly different between the rmTBI group and the ALC + rmTBI groups (p < .05). There were significant differences between the Sham and ALC + rmTBI groups at trial 2, 3, and 4 (p < .05). Swim speed was not different between any of the groups at any of the trials, which indicates that differences seen in time to find the platform were related to cognitive deficits rather than motor impairment.p < .05). There was not a significant difference between the Sham and the ALC + rmTBI groups.Probe trials were undertaken in the chronic testing arms, with the rmTBI displaying impaired searching ability compared with the Sham and the ALC + rmTBI groups (p < .05). Assessment of acute cortex also revealed significant differences between the rmTBI and ALC + rmTBI groups in MAPT expression (p < .05), and group differences between the sham and ALC + rmTBI group in AIF1 expression (p < .05) (p < .05) (p < .05) (p < .05). All gene expression data are shown in The molecular analysis assessed expression changes between the three groups for seven genes, with differential expression seen between the cortex and hippocampus. At acute injury assessment in the cortex, MAPT, GFAP, AIF1 and TNF showed upregulation in the rmTBI group relative to Sham mice (pression . In the p < .05) . In acutp < .05) . In the p < .05) . The rmTThere is currently a lack of evidence for safe therapeutics that can be administered long-term to reduce the risk of individuals developing cognitive and neuropsychological deficits after rmTBIs. This study evaluated the role of prophylactic ALC administration on reducing the secondary injury mechanisms of axonal neurodegeneration, reactive astrogliosis and microgliosis, inflammation and glutamate excitotoxicity in the hippocampus and cortex resulting from rmTBI. The dosing profile followed a modified prevention paradigm to fully supplement the anti-inflammatory and metabolic cellular protective pathways targeted by ALC supplementation. The mTBI model in this study has been characterized to deliver repetitive, biomechanically relevant impacts, to induce CTE-like neuropathology and cognitive impairment . ThroughTDP-43 is an important protein for stabilization of RNA processes such as transcription, mRNA stability and alternative splicing . Recent A key constituent of these NFTs is tau, a microtubule-associated protein involved in axon construction . In our In the acute phase following rmTBI, microglia activate inflammation as a key secondary injury response, aimed at providing immediate neuroprotection. The response induces cytokine production, arachidonic acid conversion to prostaglandins, and excitotoxin release, intended to repair damage and return homeostasis . HoweverAstroglial cells provide another mechanism for inflammation after mTBI, as they communicate with microglia to respond to the demands of the central nervous system (CNS) . Glial fThe inflammatory cascade driven by glial cell response to injury involves the release of products such as cytokines, and our study assessed the levels of pro-inflammatory cytokine, TNF. ALC administration has been shown to decrease TNF activation in an epilepsy model . In our We sought further evidence of the neuroinflammatory response to rmTBI and ALC treatment by examining the expression of CCL11, a chemokine that has received increased attention in TBI . CCL11 cAnother important secondary injury mechanism following mTBI is excitotoxicity, involving impaired neuronal calcium regulation and glutamate release resulting in dendrite damage . UpregulWe further investigated whether the neuroprotective effects of ALC at the molecular level would translate into mitigation of behavioral and cognitive dysfunction seen following rmTBI. Time to regain RR in the rmTBI group were within the range of previous studies . ImportaSmall animal models provide advantages in the study of TBI in the ability to screen for pathology that is otherwise hard to determine. The knowledge that is gained from the findings in mice provide the stimulus to explore the concepts in higher mammals and humans. However, there are limitations to mouse models including the small size and lack of complexity of the mouse brain . This st"
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