diff --git "a/deduped/dedup_0898.jsonl" "b/deduped/dedup_0898.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0898.jsonl" @@ -0,0 +1,40 @@ +{"text": "Soilborne wheat mosaic virus (SBWMV) 19K protein is a cysteine-rich protein (CRP) and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX) viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP) expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.Amino acid sequence analyses indicate that the Cucumber mosaic virus (CMV) 2b, the Tobacco etch virus (TEV) HC-Pro, and the Tomato bushy stunt virus (TBSV) P19 . The p26SBE-2 plasmid was digested with The author(s) declare that they have no competing interests.Jeannie Te did all cloning, plant inoculation experiments, gene silencing experiments. Ulrich Melcher did the amino acid sequence alignments and phylogenetic comparisons. Amanda Howard did some gene silencing experiments, photography. Jeanmarie Verchot-Lubicz conceived the study, did some molecular analysis, and wrote the paper. Special thanks to Wenpiny Qiu at Southwest Missouri State University for assistance with studies using TBSV to express the SBWMV 19k."} +{"text": "Environmental Science & Technology now cautions that the added electric load incurred by charging PHEV batteries would increase the amount of water used by power plants. This isn\u2019t necessarily a deal-breaker, the authors say, but planners need to prepare now to meet the demand.Plug-in hybrid electric vehicles (PHEVs) are viewed as a major step in conserving oil and reducing emissions of greenhouse gases and other pollutants. So great is the hope for PHEVs that the U.S. Department of Energy announced in January 2008 it would invest up to $30 million in the development and demonstration of these vehicles, which can run on both gasoline and electricity. A study scheduled for the 15 June 2008 issue of The big difference between PHEVs and today\u2019s hybrids is the battery. Exisiting hybrids have a smaller battery that\u2019s charged by the gas engine. This is done, for example, by regenerative braking, which converts the energy from the speed of the car (kinetic energy) into electricity to be stored in the battery. A PHEV is similar, but its larger, as-yet undeveloped battery would be charged by plugging it into a household outlet, typically overnight.The authors analyzed a range of figures on water consumption and withdrawal for both oil refining and electricity generation. Water that is \u201cconsumed\u201d ends up in the atmosphere after use, whereas water that is \u201cwithdrawn\u201d typically is returned to its source. They found that, compared with gas miles, up to 3 times the water per mile is consumed to power electric miles, and up to 17 times the water is withdrawn. This water is used primarily for cooling at power plants, where electricity is generated by steam-driven turbines. The steam clouds often seen rising from cooling towers indicate waste heat being dissipated into the atmosphere through evaporation.Study coauthor Michael Webber, a University of Texas engineer who strongly favors the development and use of PHEVs, says local water policy planners need to be aware of this impact. \u201cIf they are in a place that has a water-intensive power plant, and they think that plug-in hybrids will be important, they need to be preparing for that future demand because the increased load on the plant can increase the strain on local water resources,\u201d he says. To alleviate this impact, he says, power plants could consider using reclaimed water for cooling.But Mark Duvall, project manager for electric transportation at EPRI, a nonprofit research organization funded by electric utilities, argues that PHEVs will not measurably increase electric utilities\u2019 water needs. He says utilities are certain to improve the efficiency in the way they use water over the next several decades. \u201cEven if plug-in hybrids become the next big technology and become wildly successful, [the added electricity demand] will still be very small relative to all the electric loads we have today,\u201d he says.Duvall projects that if PHEVs capture approximately 60% of the auto market by 2050, that will mean an increase in electric capacity of about 4% and a savings of 3\u20134 million barrels of oil per day. He says the electric sector has been growing by 1\u20132% annually, but the water withdrawal rate has remained constant.Comparing the Benefits and Impacts of Hybrid Electric Vehicle Options, more than half of respondents would pay 26.5% more for a hybrid than a conventional car if the hybrid could go 60 miles per charge cycle powered solely by electricity. Just over 7% would pay nearly 80% more for such a car.A crucial question in these calculations is how many PHEVs might eventually take to the roads. Duvall cautions that any estimates are purely speculative at this point and based largely on focus groups and similar tools. According to a survey cited in the 2001 EPRI report But Duvall notes that actual customer behavior may be altered by a variety of conditions that were not present when the survey was done, such as gas prices. He says about 1 million regular hybrids are on the road right now, and he expects there may be a similar number between 2010 and 2018. Meanwhile, Saturn and Toyota in early 2008 announced they plan to market PHEVs within the next two years."} +{"text": "Changes in foot and ankle posture are a normal aspect of infant and child development but may also reflect neurological conditions affecting the brain, spinal cord, peripheral nerves or muscles. Development of cavus or planus foot deformities often reflects underlying neurologic disorders and may predate development of more overt neurological signs. This session will concentrate upon signs and symptoms suggestive of occult neurologic problems in children presenting with positional or developmental abnormalities of the lower limbs."} +{"text": "Direct contact test (DCT) was used to evaluate the antibacterial and antifungal activity of products after 48 h and 7 days of incubation. The results demonstrated that all the cements presented antibacterial activity but the antibacterial activity of glass ionomer cements was more than that of zinc phosphate cements. Counts of C. albicans after 48 h were lower and statistically different in the GIC group in relation to the control groups. But no differences were observed between GIC and control groups at 7 days. Based on the results of this study, the antimicrobial and mainly antifungal effects of all the cements were so short.White spot lesions are observed in nearly 50% of patients undergoing orthodontic treatment. Long-lasting antibacterial properties of orthodontic cements can reduce this phenomenon. The aim of this research was to compare antimicrobial activity of three commercial glass ionomer cements with three commercial zinc phosphate cements, over time, against Streptococcus mutans is one of the bacteria frequently implicated in decalcification of enamel -19. The The growth inhibitory effects of some cements are considered beneficial in preventing bacterial colonization. In addition, the antibacterial activity, during the time, assumes clinical relevance. Based on these outcomes and on the lack of studies on the antibacterial activity of the Iranian orthodontic cements, the aims of this study were as follows:streptococcus mutans and candida albicans using DCT, comparing the antimicrobial activity of the cements from different companies, comparing the antibacterial activity during the time.Comparing the antimicrobial activity of two orthodontic cements against the The orthodontic cements evaluated in this study are shown in Table 1. The antibacterial activity of each material was evaluated against the Streptococcus mutans (ATCC#35668) and Candida albicans (ATCC#10231).Preparation of glass ionomer samplesSix wells (7 mm diameter and 3 mm thickness) were punched in the Muller-Hilton agar plates and filled with 6 cements. A uniform surface was achieved by using a small flat-ended dental instrument, such as a dental spatula. The material was allowed to set in accordance with the manufacturer\u203as recommendation.Antibacterial activity test\u00b0C, for 24 h. The top 4 mL of the resulting undisturbed bacterial cultures were transferred to new test tubes and centrifuged for 10 min at 3, 2 gravity. The resulting supernatant was discarded and the bacteria was resuspended in 5 mL of phosphate-buffered saline (PBS) with a pH of 7.5 and mixed gently by vortexing for 10 sec.Bacterial strain from stock cultures was cultivated in Brain Heart Infusion broth at 37We used DCT to test the antibacterial properties of the cements. The antimicrobial susceptibility profiles were determined by disk diffusion agar method according to CLSI M100-S12 protocols (2005). In each sterilized Petri dish (20100 mm), a base layer containing 15 mL of blood agar mixed with 100 \u03bcL of inoculum was prepared. After the solidification of culture medium, wells measuring 7 mm in diameter were made in each plate and the testing materials were transferred to wells. Two wells were served as the positive control without the tested cements. Plates were incubated at 37\u00b0C for 48 h and after that, diameters of zones of inhibition produced around the specimens were measured at three different points. The size of inhibition zones was calculated through subtracting the diameter of specimen (7 mm) from the average of three measurements of the halo. All measurements were performed twice by the same blinded operator. Antibacterial tests were repeated 5 times to confirm the homogeneity of the results. Moreover, diameters of zones of inhibition produced around specimens were measured after the reincubation of plates at 37\u00b0C for 5 days.Antifungal activity testCandida albicans ATCC 10231 was placed on Sabouraud dextrose agar and incubated at 37\u00b0C for 24 h. After this period, a suspension was prepared in sterile saline solution (0.85% NaCl) with the aid of a spectrophotometer , adopting the optical density of 450 nm.C. albicans suspension was inoculated in each tube. Tubes were incubated at 37\u00b0C for 48 h. Tubes without any specimen of the tested materials were incubated as positive controls. After the incubation period, each initial suspension was diluted 10, 100 and 1,000 times in sterile saline solution, and 0.1 mL of each suspension was plated in duplicate on Sabouraud Dextrose agar and incubated in 37\u00b0C for 48 h. After this period, the number of colony-forming units per milliliter (CFU/mL) was obtained. Similar procedure was performed after 7 days of incubation.The samples of cements were transferred to tubes containing 3 mL of Sabouraud Dextrose broth . Then, 0.5 mL of the standardized Statistical analysisThe mean diameter of inhibition zone values for each material was used for statistical analysis by using General Linear Models to compare the inhibition zones of bacteria around each cement. Tukey\u2019s studentized post-hoc tests were performed to identify the differences between the cements, the level of significance set at p < 0.001. A Tukey\u2019s complementary test was also used to determine if there was a significant difference between the inhibitory effects of 2 and 7 days specimens (p < 0.001).The mean values and standard deviations of the inhibition zones and CFU for each material according to the bacteria strain, at different days, are shown in Table 2. There was a significant difference between the type of cements, brand of cements, time of evaluation and the antibacterial activity . The antin-vitro methods have been used to study the antibacterial activity of dental materials. Boeckh et al. throughout their experiments using strains of S. mutans, showed the important role of this microorganism in caries etiology in the GIC group in relation to the control groups. But no differences were observed between GIC and control groups at 7 days. Another study reported that GICs had no antifungal effect in 2 days and GIC (B) were the most active antibacterial cements and ZPC (A) and ZPC (B) were the most active antifungal cements. Combined with the mechanical and biological properties, these differences should be taken into account when one is choosing cement for orthodontic clinical use."} +{"text": "Advances in lifesaving technologies and treatments make it possible for children with profound physical and cognitive impairments to survive into adulthood. Questions regarding how and where they should live are discussed rarely and, when they are, primarily focus on safety and/or containing costs. Since models of long-term care provision are age-based, children who reside in institutions are \u2018discharged\u2019 to adult facilities when they reach an arbitrary age. Such transfers may not be in the best interests of these young people or their families. Our aim in this debate is to highlight why age is a problematic criterion for placement decisions, with the goal of stimulating further research and inquiry.Transfers from pediatric to adult institutions are driven primarily by funding arrangements and underpinned by stage-based theories of human development. Arguments supporting such transfers point to the value of communal living with same age peers, and engagement in age-appropriate activities. These goals are questionable for individuals who are minimally interactive and/or where equally worthy interactions are feasible in intergenerational settings. Instead their accommodation needs might more closely align with palliative care principles of supporting individuals and families to enjoy what they bring to each other\u2019s lives and minimize suffering. Innovative models of \u2018vertical care\u2019 and \u2018lifetime homes\u2019, which enable continuous flexible services across the lifespan, are discussed as examples of alternative approaches requiring further debate and research.Entrenched funding and service models that require the transfer of profoundly impaired young people from pediatric to adult facilities need to be re-examined with considerations of best interests, needs, and preferences of individuals and their families. Questions of what constitutes a \u2018good life\u2019 for these individuals are tenacious and require further thought and research. Nevertheless, they need to be regarded as citizens of our human community deserving of a good life in whatever form that may take, in settings that enable them to flourish. Although Western industrialized countries are focused on the health care implications of the growing numbers of older adults, there is another rising population with burgeoning health care needs \u2013 young people who have survived formerly fatal anomalies, injuries, or diseases -3. AdvanPersons with profound impairments who have survived previously fatal childhood conditions thus constitute another \u2018aging population\u2019. However, this population has received relatively little attention from policy makers and health researchers. Their ongoing survival and growing numbers are raising pressing questions regarding where and how they should live, and society\u2019s obligations to this vulnerable group. Lives are saved through impressive technological advances but little attention is given to determining how best to support them throughout increasingly longer life spans. When quality of life issues are raised, the debate is focused almost exclusively on the ethics of using particular life saving measures . What iIn this paper we focus on individuals who appear to have little understanding of verbal language, have ongoing high care needs, and have no capacity for self-support. The severely compromised physical repertoire of these individuals renders them minimally interactive. They require twenty-four hour care that includes combinations of assistance with bodily maintenance, reliance on life sustaining technologies, and/or skilled professional care. Diagnostic groups include, for example, severe cerebral palsy, traumatic brain injury, or brainstem stroke which have rendered individuals unable to move, gesture or speak; and individuals who are \u2018locked in\u2019, \u2018minimally conscious\u2019 or in a \u2018persistent vegetative state\u2019. In what follows we refer to these individuals collectively as \u2018profoundly impaired\u2019.a decisions are made. Rather than provide definitive answers to these complex questions, we modestly aim to signpost some key parameters for discussion and further inquiry, and stimulate ethical reflection on how to accommodate (in the many senses of this word) these vulnerable persons.Current models of long-term care provision are age-based such that children receiving long-term institutional care are inevitably \u2018discharged\u2019 from pediatric institutions and moved to adult facilities when they reach adulthood. This move, which has been likened to an eviction, can be disruptive and upsetting for all involved \u2013 parents, young people and institutional care providers alike. We believe that such transfers neither reflect the best interests of young people nor family-centered care principles. Rather they seem driven primarily by funding arrangements that disallow adults to reside in facilities designated for the care of children (and vice versa), and are underpinned by traditional stage-based theories of human development . Our aimIn the last decade, the rhetoric of \u2018transition\u2019 has been used to frame discussions pertaining to transferring young people from pediatric to adult services and settings. Traditional transition initiatives typically focused on health services; however, they are increasingly concerned with \u2018life transitions\u2019 and support services enabling young people to take up adult social roles, thereby moving from one life stage (childhood) to another (adulthood) . ConceptWe concur with Priestley that aduThere is little research or scholarship examining institutional placement and transfer for profoundly impaired young people who cannot assume adult social roles ,15, withet al. [Research has aimed to address the wellbeing of affected young people and their families, but in most cases has not directly considered the needs of the most profoundly impaired young people. Moreover, we have found no research questioning or investigating age-based criteria for institutional placement or transfer for this group. Across the literature there is a prevalent assumption that disabled youth share similar transition needs vis-\u00e0-vis transferring to adult services. For example, Doug et al. conducteb but closely align with Western notions of individualism and progress [Critiques of the use of age criteria and their role in structuring health and social services have come primarily from the social sciences ,26-28 anprogress . Meyer [progress , for exaprogress ,35 has sprogress -41). PriThe lives of profoundly impaired individuals do not conform to a developmental trajectory of progressive self-sufficiency from childhood to adulthood. Their impairments render it impossible to live or work independently, and they require ongoing intense services and supports throughout their lives. Moreover, except for physical/biological changes, a developmental perspective to human growth and adaptation over the lifespan has minimal relevance. The development of self-regulative capacity to adapt to different conditions and contexts has littIt follows that moving profoundly impaired young people from one institutional setting to another solely on the basis of age may be ill-advised and even harmful to them and others. Such moves may needlessly disrupt their lives and those of their families and seveBecause most profoundly impaired persons continuously live in institutional settings, moving from one setting to another may be inimical to their wellbeing. These facilities are \u2018homes\u2019 and comprise stable venues for ongoing family engagement. Long term relationships also develop with staff members who know the person\u2019s needs and responses, have developed effective routines and uses of familiar devices, equipment and adaptations, and may be best positioned to \u2018read\u2019 non-verbal cues as rhythms of daily life become established over time ,45. InstGiven the significance of home to human wellbeing, questions of placement, discharge or transfer of profoundly impaired people from one institutional setting to another are clearly important. Changing homes is not just a change in locale, and involves more than procuring a bed space or providing routine health and care services. Housing is central to the health and wellbeing of all individuals and their families . Homes aIf age is not taken for granted as the cardinal criterion for placement decisions, other alternatives can be considered. As noted above, very little research has investigated the institutional care needs of profoundly impaired children and young people. Thus, rather than provide definitive solutions, in this section we signpost some possible considerations and emerging models that could ground further inquiry.In previous work we have suggested that, at a minimum, all homes should promote individual dignity beyond the provision of safety and basic care . FurtherSupport for vertical care is further reflected in notions of lifetime homes for disabled people . LifetimDecisions regarding where and how profoundly impaired individuals should live inevitably rest on questions of what constitutes a \u2018good life\u2019 for those unable to articulate their preferences. Recent neuro-imaging research has shown it is possible to communicate with some \u2018behaviorally non-responsive\u2019 individuals who have the ability to respond to commands through wilfully modulating their brain activity . Such reet al. [Parents\u2019 priorities provide some direction in establishing placement/transfer criteria as alternatives to age. Research conducted by Rabiee et al. found thet al. . Identifet al. ,44,55.qua human beings [To conclude, we suggest that decisions regarding placement for profoundly impaired persons should not be made using the limited criteria of age, physical safety and the provision of bodily care. The latter two provisions are necessary but insufficient conditions that reduce persons to objects of care and fail to address their inherent dignity n beings . Second,n beings . We suggn beings , care foFor the foreseeable future, at least some profoundly impaired young people will continue to require institution-based care. Given this reality, there is a pressing need for interdisciplinary research to develop and evaluate alternative service models. We envision partnerships between child health researchers, social scientists and policy makers working closely with care providers and families to determine what best meets their needs. A useful starting place could be targeted pilot case study research with families who wish to remain in pediatric facilities, working in partnership to develop models of care delivery and family supports.In this debates paper, we have suggested that age is an arbitrary and inappropriate criterion for discharging and displacing profoundly impaired individuals from pediatric to adult institutional long-term care settings. Entrenched funding and service models that require such dis-placements need to be re-examined with considerations of best interests, needs and preferences of individuals and families. Innovative models such as vertical care and life time homes merit serious deliberation and further research as increasing numbers of children are surviving previously fatal conditions. Questions of what constitutes a \u2018good life\u2019 for these individuals are tenacious and require further thought and innovative research. Nevertheless, profoundly impaired persons must be regarded in terms other than \u2018bed blockers\u2019 or \u2018tragedies\u2019. They are citizens of our human community deserving of a good life in whatever forms that may take. To that end we welcome further dialogue and debate.a By \u2018placement\u2019 we are referring to the common rhetoric of health professionals charged with finding appropriate living and care arrangements for people who need health and/or attendant care services and, for whatever reason, need to leave their current arrangements.b See for example the World Health Organization Life Course Model used to train health and social care professionals [ssionals . The modThe authors declare that they have no competing interests.BG drafted the manuscript. PMcK conceived of the paper. All authors contributed to the development of the ideas and to the writing. All authors read and approved the final manuscript.BG is an Associate Professor in the Department of Physical Therapy, University of Toronto, and a Senior Scientist at the Bloorview Research Institute at the Holland Bloorview Kids Rehabilitation Hospital in Toronto, Canada, where she holds the Bloorview Children\u2019s Hospital Foundation Chair in Childhood Disability Studies. Her research examines the social and ethical dimensions of childhood disability and rehabilitation, particularly the norms embedded in rehabilitation practices and the effects on disabled children and young people. GK is a Senior Scientist at the Bloorview Research Institute. She has a PhD in Social Psychology and conducts research on psychosocial issues of children with disabilities and their families. She has published articles on resiliency, quality of life, meaning in life, family-centered service, youth transitions, and child and family wellbeing and adaptation. SK is a Clinical Study Investigator at the Bloorview Research Institute and Lead of Evidence to Care at the Teaching & Learning Institute at the Holland Bloorview Kids Rehabilitation Hospital in Toronto, Canada. Building on her background in Developmental Psychology, her research interests focus on enhancing the participation and inclusion of youth with physical disabilities and/or medical complexity, including their transition to adulthood, impact of alternative and complementary therapies and service models supporting continuity of care. PMcK is a Senior Scientist, Bloorview Research Institute, Holland Bloorview Kids Rehabilitation Hospital, and Professor, Lawrence S. Bloomberg Faculty of Nursing, University of Toronto. Her interdisciplinary program of research focuses on disabled children, their sense of embodiment, the technologies they use, their care providers and the places where they live, attend school, and/or receive care. Her research addresses the physical, social and policy barriers to inclusion that disabled children and their families encounter."} +{"text": "N-linked glycosylation were mutated , and chemical inhibitors that prevent glycosylation at specific stages of oligosaccharide were added or modified. The single N-linked glycosylation mutants, N65A and N92A, efficiently inhibited the release of Vpu-defective human immunodeficiency virus type 1 (HIV-1). In contrast, the non-glycosylated double mutant, N65,92A, lost its ability to block HIV-1 release. The inability of the N65,92A mutant to inhibit HIV-1 release is associated with a lack of cell-surface expression. A role for glycosylation in cell-surface tetherin expression is supported by tunicamycin treatment, which inhibits the first step of N-linked glycosylation and impairs both cell-surface expression and antiviral activity. Inhibition of complex-type glycosylation with kifunensine, an inhibitor of the oligosaccharide processing enzyme mannosidase 1, had no effect on either the cell-surface expression or antiviral activity of tetherin. These results demonstrate that high-mannose modification of a single asparagine residue is necessary and sufficient, while complex-type glycosylation is dispensable, for cell-surface tetherin expression and antiviral activity.Tetherin is an interferon-inducible antiviral protein that inhibits the release of a broad spectrum of enveloped viruses by retaining virions at the surface of infected cells. While the role of specific tetherin domains in antiviral activity is clearly established, the role of glycosylation in tetherin function is not clear. In this study, we carried out a detailed investigation of this question by using tetherin variants in which one or both sites of N-terminal cytoplasmic tail (CT), a transmembrane (TM) domain, a rod-like coiled-coil (CC) ectodomain, and a C-terminal glycosylphosphatidylinositol (GPI) anchor [N-linked oligosaccharides [The innate immune response is the first line of defense against invading viral pathogens. Mammalian cells encode a large number of incompletely characterized factors that impede virus replication at various stages of the virus replication cycle. These inhibitory, or \u201crestriction\u201d, factors are either expressed constitutively or are induced by type-I interferon (IFN). One such restriction factor that interferes with a late stage of the viral replication cycle is tetherin , cluster of differentiation 317 (CD317) or HM1.24), which inhibits the release of human immunodeficiency virus type 1 (HIV-1) and is counteracted by the HIV-1 accessory protein Vpu ,11,12,13) anchor ,14,15. T) anchor ,17,18,19) anchor . Du Pont) anchor . The ectcharides ,22. Delecharides .vpu gene, antagonize tetherin through their Nef and envelope (Env) proteins, respectively, in part through an intracellular sequestration mechanism [Tetherin inhibits the release of not only HIV-1 but also that of a broad spectrum of other enveloped viruses, including alphaviruses, filoviruses, rhabdoviruses, arenaviruses, herpesviruses, paramyxoviruses, flaviviruses, orthohepadnaviruses, orthomyxoviruses, and other retroviruses . echanism ,46,47,48N-linked glycosylation sites in the extracellular CC domain. The requirement for tetherin glycosylation in the inhibition of virus release remains controversial. Early studies reported that N-linked glycosylation of Asn 65 (N65) and 92 (N92) is important for the inhibition of HIV-1 release [As mentioned above, human tetherin contains two putative release ,52,53,54 release ,55,56. S release ,50, whil release ,53,54,57 release ,50,53,54 release ,54, prot release , bovine N-linked) glycosylation is a highly regulated, post-translational modification that is important for the structure and function of eukaryotic proteins. N-linked glycosylation is initiated in the lumen of the endoplasmic reticulum (ER) with the cotranslational transfer of a high-mannose oligosaccharide moiety to Asn residues in Asn-X-Ser/Thr motifs (where X is any amino acid except Pro) in the target protein. Trimming of the high-mannose side chains occurs in the ER and Golgi apparatus, and additional sugars are attached to generate complex side chains. The glycoprotein is then exported to the PM through the secretory pathway [Asn-linked and chemical inhibitors of the glycosylation pathway. We observed that oligosaccharide modification of one of the two sites of tetherin N-terminal hemagglutinin (HA) epitope tag or an HA tag inserted at residue 154 in the extracellular CC domain of tetherin have been described previously [N-linked glycosylation. Anti-HA antiserum, kifunensine, and tunicamycin were purchased from Sigma . Alexa Fluor 488 or 594-conjugated secondary antibodies were from Invitrogen . Anti-Vpu, anti-tetherin, and anti-HIV-1 immunoglobulins (Ig) were obtained from the NIH AIDS Research and Reference Reagent Program.The full-length, infectious HIV-1 molecular clone pNL4-3 and the Vpu-defective counterparts pNL4-3delVpu and pNL4-3Udel have been described previously ,61,62. pHeLa and 293T cell lines were maintained in Dulbecco-modified Eagle\u2019s medium (DMEM) containing 5% or 10% fetal bovine serum (FBS), respectively. One day after plating, cells were transfected with appropriate plasmid DNA using Lipofectamine 2000 according to the manufacturer\u2019s recommendations. Eight hours after transfection, cells were either untreated or treated overnight with tunicamycin or kifunensine; cells and virus were harvested 24-h post transfection and used for further analysis. To knock down tetherin expression in HeLa cells, one day after plating HeLa cells were treated with 100 nM tetherin small interfering RNA (siRNA) with Oligofectamine transfection reagent (Invitrogen) for 24 h.HeLa or 293T cells were transfected with HIV-1 molecular clones, virions were collected after 24 h, pelleted in an ultracentrifuge, cell and virus pellet were lysed in a buffer containing 50\u2009mM Tris-HCl (pH 7.4), 150\u2009mM NaCl, 1\u2009mM EDTA, 0.5% Triton X-100, and protease inhibitor cocktail . After denaturation by boiling in sample buffer, proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and incubated with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and chemiluminescence signal was detected by using West Pico or West Femto Chemiluminescence Reagent . The protein bands were quantified by using Imagelab-Chemidoc .One day after plating, HeLa or 293T cells were transfected with wild-type (WT) or Vpu-defective pNL4-3 molecular clones; one-day post transfection, virions were pelleted in an ultracentrifuge and cell and virus pellets were lysed . Viral p293T cells were transfected with WT or mutant human tetherin expression vectors in which an HA tag was inserted in the CC extracellular domain of tetherin and green fluorescent protein (GFP) expression plasmid. HeLa and transfected 293T cells were either untreated or treated with glycosylation inhibitors tunicamycin or kifunensine. Twenty-four hours after transfection or treatment with inhibitors, cells were harvested by adding a solution of 5 mM EDTA in phosphate buffered saline (PBS) and washed in ice-cold 1% bovine serum albumin (BSA)-PBS. The cells were then incubated with anti-HA (mouse) or anti-tetherin (rabbit) antiserum in 1% BSA-PBS for 1 h at 4 \u00b0C. The cells were then washed twice in 1% BSA-PBS and stained with Alexa Fluor 594-conjugated anti-mouse (293T cells) or Alexa Fluor 488-conjugated anti-rabbit (HeLa cells) IgG secondary antibody in 1% BSA-PBS for 1 h at 4 \u00b0C. The cells were washed three times with PBS, fixed in 1% paraformaldehyde and 5000 gated events were collected using a Becton Dickinson fluorescence-activated cell sorting (FACS) Calibur flow cytometer . Analysis was performed using FlowJo .For microscopy studies, HeLa cells were cultured in chamber slides. One day after plating, cells were untreated or treated with kifunensine or transfected with siRNA targeting tetherin. After 24 h, cells were rinsed with PBS and fixed in 3.7% paraformaldehyde in PBS for 30 min. The cells were rinsed with PBS three times, blocked with 3% BSA-PBS for 30 min, and incubated with anti-tetherin antibodies appropriately diluted in 3% BSA-PBS for 1 h. The cells were washed with PBS three times and then incubated with secondary antibody conjugated with Alexa Fluor 488 diluted in 3% BSA-PBS. After washing with PBS three times, cells were mounted with Vectashield mounting media with 4\u2032,6-diamidino-2-phenylindole (DAPI) and examined with a Delta-Vision RT deconvolution microscope .N-linked glycosylation in tetherin antiviral function remains to be clearly established. To define the role of N-linked glycosylation of human tetherin in HIV-1 release we used two molecular clones that lack a functional Vpu: a Vpu-deletion mutant pNL4-3Udel [As discussed in the Introduction, the requirement for L4-3Udel , and pNLL4-3Udel , which hL4-3Udel ,30,31,34L4-3Udel ,20, the N-linked glycosylation by blocking the transfer of N-acetylglucosamine-1-phosphate to dolichol phosphate [To further investigate the role of glycosylation in tetherin function, we overexpressed WT and N65,92A tetherin in 293T cells and treated the cells with tunicamycin, a nucleoside antibiotic that specifically inhibits the first step of hosphate . As showAs discussed earlier, there are conflicting reports in the literature on the surface expression of glycosylation-defective tetherin mutants. Some authors reported that the cell-surface expression of the N65,92A mutant was significantly reduced ,20, sligAs discussed in Introduction, tetherin is expressed in several forms: a 23-kDa, non-glycosylated species, and species containing a single high-mannose side chain at either Asn 65 or 92 (\u224824.5 kDa), high-mannose side chains at both Asn residues (\u224826 kDa), or complex-type side chains at either or both positions (\u224832 to 40 kDa) A. Next, The above results demonstrate that complex-type glycosylation of tetherin is not required for its inhibitory function. Since cell-surface expression of tetherin is necessary for inhibition of virus release, these observations would suggest that complex-type oligosaccharide modifications are not required for cell-surface tetherin expression. To directly examine this question, HeLa cells were treated with kifunensine for 24 h and tested for cell-surface expression of endogenous tetherin by both microscopy and flow cytometry. As shown in As shown in N-linked glycosylation in tetherin function remains poorly defined. In this study, we carried out a detailed investigation on the role of N-linked glycosylation in the antiviral activity of human tetherin by using not only non-glycosylated tetherin mutants but also chemical inhibitors of the glycosylation pathway.The unique topology of tetherin allows it to bind and restrict the release of virions from the surface of infected cells. Several structural domains of tetherin, including the CT, TM domain, GPI anchor, and dimerization motif in the CC region of the ectodomain, are critical for tetherin\u2019s antiviral activity ,15,20,30N-linked glycosylation of at least one Asn residue in tetherin is required for inhibition of HIV-1 release, as mutating a single site of N-linked glycosylation (N65 or N92) has no impact on the antiviral activity of human tetherin. These single mutants are efficiently expressed on the cell surface, suggesting that N-linked glycosylation at a single Asn is sufficient for trafficking to the PM. In contrast, the double mutant, N65,92A, is defective in both cell-surface expression and inhibition of HIV-1 release. This loss of cell-surface expression and antiviral activity could not be rescued by increasing the total expression of the N65,92A mutant. Consistent with our data, Perez-Caballero et al. showed that N-linked glycosylation of tetherin is essential for its cell-surface expression [N-linked glycosylation is essential for inhibition of HIV-1 release [N-linked glycosylation in the antiviral activity of feline tetherin was also reported [N-linked glycosylation in tetherin activity. In contrast, several other groups reported that glycosylation of human tetherin is not essential for restricting the release of HIV-1 [Our data indicate that pression . Other l release ,52,53,54 release . Taylor release . It was release . Interes release ,67 and a release . A requireported ,54. A slreported . Tetherireported . These sof HIV-1 , foamy vof HIV-1 , Lassa, of HIV-1 . The difN-linked glycosylation, blocked the glycosylation, cell-surface expression, and antiviral activity of tetherin in a manner similar to the N65,92A double mutant. We also evaluated whether complex glycosylation of tetherin is essential for its antiviral activity. Treating cells with the mannosidase I inhibitor kifunensine resulted in the loss of complex glycosylation of WT tetherin but no loss in its cell-surface expression or antiviral function. Similarly, the cell-surface expression and antiviral activity of the single Asn mutants, N65A and N92A, were not diminished by kifunensine treatment. These results indicate that the mannose trimming activity of mannosidase I, which is required for conversion of high-mannose to complex oligosaccharide side chains, is not required for the cell-surface expression or antiviral activity of tetherin.The studies discussed above were carried out with Asn mutants of tetherin that are defective in glycosylation. In contrast to previous studies, we also adopted the complementary approach of using chemical inhibitors of the glycosylation pathway. Treating cells with tunicamycin, which inhibits the first step of In conclusion, here we show that glycosylation of at least one Asn is essential, but complex side chain modifications are dispensable, for the cell-surface expression and antiviral activity of human tetherin. This study provides new insights into not only the number of oligosaccharide side chains required for tetherin transport and activity, but also the role of oligosaccharide modifications in these functions."} +{"text": "The pathogenesis of cardiovascular diseases is a multifunctional process in which the mineralocorticoid receptor (MR), a ligand-dependent transcription factor, is involved as proven by numerous clinical studies. The development of pathophysiological MR actions depends on the existence of additional factors e.g. inflammatory cytokines and seems to involve posttranslational MR modifications e.g. phosphorylation. Casein kinase 2 (CK2) is a ubiquitously expressed multifunctional serine/threonine kinase that can be activated under inflammatory conditions as the MR. Sequence analysis and inhibitor experiments revealed that CK2 acts as a positive modulator of MR activity by facilitating MR-DNA interaction with subsequent rapid MR degradation. Peptide microarrays and site-directed mutagenesis experiments identified the highly conserved S459 as a functionally relevant CK2 phosphorylation site of the MR. Moreover, MR-CK2 protein-protein interaction mediated by HSP90 was shown by co-immunoprecipitation. During inflammation, cytokine stimulation led to a CK2-dependent increased expression of proinflammatory genes. The additional MR activation by aldosterone during cytokine stimulation augmented CK2-dependent NF\u03baB signaling which enhanced the expression of proinflammatory genes further. Overall, in an inflammatory environment the bidirectional CK2-MR interaction aggravate the existing pathophysiological cellular situation. In epithelial tissues aldosterone-activated MR mediates sodium and water retention and thereby long term blood pressure regulation. Independently of its hemodynamic effects, inappropriate MR activation promotes pathophysiological effects in the cardiovascular system like inflammation, fibrosis and hypertrophy, leading to endothelial dysfunctions and heart failure2. However, the molecular mechanisms involved are incompletely understood.The mineralocorticoid receptor (MR) belongs to the nuclear receptor superfamily which includes the progesterone (PR), estrogene (ER), androgene (AR), and glucocorticoid receptor (GR), representing a family of ligand-activated transcription factors (TFs)3 as homodimers to modulate gene transcription4.The human MR contains a regulatory N-terminal\u00a0AB domain (NTD) followed by a DNA-binding domain C, a hinge region D and the ligand-binding domain EF (LBD)5. Incubating aldosterone-stimulated cell lysates with a phosphatase abolished the aldosterone-induced shift6 indicating that MR represents a phosphoprotein as has been reported for other steroid hormone receptors (SHR)1. Aldosterone-induced MR phosphorylation can modify its binding affinity for hormone response elements (HRE), its nuclear translocation, its interaction with co-regulators1 and its ligand binding ability7.Subsequent to activation of the MR, aldosterone-induced posttranslational modifications (PTMs) like acetylation, oxidation, phosphorylation, sumoylation and ubiquitylation could be detected by an increase in the apparent molecular weight8 which is composed of two catalytic (\u03b1 or \u03b1\u2019) and two regulatory (\u03b2) subunits and possesses three isoforms: \u03b1\u03b1\u03b2\u03b2, \u03b1\u03b1\u2019\u03b2\u03b2, \u03b1\u2019\u03b1\u2019\u03b2\u03b2. CK2\u03b2 mediates the interaction between the catalytic subunits and recruits CK2 substrates and regulators, modulating substrate selectivity and catalytic activity9. CK2 utilizes ATP and GTP as phosphate donors and phosphorylates S/T residues within clusters of acidic amino acids possessing the minimal consensus sequence S*/T*-X-X-D/E. CK2 phosphorylates more than 300 substrates and is involved in many cellular processes like proliferation, apoptosis, differentiation, tumorigenesis and response to cellular stress and DNA damage10.Casein kinase 2 (CK2) represents an ubiquitously distributed multifunctional tetra-heteromeric serine (S)/threonine (T) kinase12. Although CK2 itself does not have molecular chaperone activity, it represents a necessary member of the molecular chaperone system11. Thus, it is conceivable that certain HSP90-binding proteins, like the MR, may also be phosphorylated by CK2. Besides, it has been shown that CK2 modulate the genomic activity of other SHR like AR, PR and ER13. Formerly, CK2 was described as a constitutively active, non-regulated protein kinase. Recent evidence indicates that CK2 activity and expression are regulated under pathophysiological conditions like inflammation, fibrosis and hypertrophy15 that also facilitate pathophysiological MR activation. Therefore, in this study we investigated i) the influence of CK2 on MR transactivation activity under control conditions, ii) the existence of directly CK2 phosphorylated MR residues, iii) the protein-protein interaction between MR and CK2 and iv) the influence of CK2 on transcriptional MR activity under inflammatory conditions.HSP90 is also associated with CK2 and facilitates CK2 activity. Thereby HSP90 enables phosphorylation of CK2 substrates whereas CK2-dependent phosphorylation of HSP90 is required to obtain HSP90 chaperone activity towards client proteins50\u2009=\u20098.1\u2009\u00b5M) \u22123-(2.3.4.5-tetrabromophenyl)acrylic acid) concentration-dependently diminished basal and aldosterone-induced genomic MR activity after 6\u2009h Fig.\u00a0. Additio\u00b5M) Fig.\u00a0 and CX-4\u00b5M) Fig.\u00a0.Figure 1To exclude toxic effects as a cause for reduced transcriptional MR activity, LDH release and protein content were measured but revealed no alterations . Aldosterone-activated MRfull length was more sensitive to inhibition by TBCA than MRCDEF after 6\u2009h all nine peptide spots of one MR peptide had to be phosphorylated; (ii) clear signal intensity of peptide spots; (iii) existence of a phosphorylation trend i.e. serine of interest was phosphorylated in more than one overlapping peptide. We identified five MR peptides containing CK2 consensus sequences on the peptide microarray. The two sites with the most consistent results in the in silico and in vitro analysis, S111 and S459, were analyzed further.To identify the exact serine/threonine residue(s) of the MR which are phosphorylated by CK2 we utilized s/) Fig.\u00a0 and inves/) Fig.\u00a0. SynthetTo verify their functional relevance, we converted S111 and S459 by site-directed mutagenesis into alanine (A) or aspartate (D) to acquire phospho-deficient and phospho-mimetic MR mutants . Equal protein expression of the MR mutants compared to wild type MR (WT-MR) was verified by Western blot Fig.\u00a0. StimulaCoIP experiments were performed to test whether MR and CK2 assemble in a protein-protein complex. All three CK2 subunits coimmunoprecipitated with the EGFP-MR specifically Fig.\u00a0. We obtaCDEF on MR-CK2 crosstalk and analyzed MR and CK2 mRNA and protein expression and MR activity.An inflammatory environment has been reported to aggravate pathophysiological MR effects and enhance CK2 activityCytokine exposure led to a time dependently increased MR mRNA with maximum after 6\u2009h Fig.\u00a0. WesternTo assess the crosstalk between an inflammatory milieu and MR activity we analyzed transcriptional activity of MR in the presence of aldosterone and/or cytokines using a classical GRE- and a NF\u03baB reporter gene assay, known to be responsive to inflammatory stimuli. As expected, aldosterone increased the GRE-reporter activity time dependently with a maximum after 24\u2009h. Cytokines by themselves induced GRE activity after 6\u2009h and strongly reduced GRE activity after 24 and 48\u2009h. Stimulation of cells with aldosterone and cytokines for 6\u2009h resulted in enhanced GRE-SEAP activity while during longer incubation periods cytokines inhibited aldosterone-induced MR activity Fig.\u00a0. For theApplication of TBCA inhibited NF\u03baB activation by aldosterone completely and reduced the cytokine effect partly after 24 and 48\u2009h. The additional NF\u03baB activation by combined incubation of aldosterone and cytokine was completely abolished by TBCA Fig.\u00a0. These rExpression of inflammation-associated genes containing NF\u03baB binding site(s) in their promoter was analyzed time dependently after cytokine and aldosterone stimulation\u00a0in MR transfected HEK cells Table\u00a0. As expeThe influence of an aldosterone-intensified inflammatory environment was analyzed in human endothelial cells (TIMEs), a cell model with closer proximity to pathophysiological vascular MR effects. Analogous to the results obtained by HEK cells, we observed an induction of the mRNA levels of MCP-1, COX-2, EGFR, VCAM-1, E-selectin and NOX-4 by cytokine treatment 22. PTM of residues especially in the NTD of the MR can alter the receptor DNA binding ability due to conformational changes or altered cofactor recruitment. Besides, in AF1b (450\u2013460) exists a highly conserved binding motif for the scaffolding protein caveolin which enables a crosstalk between SHR and other signaling pathways23.The NTD of the MR, a highly disordered region, consists of 3 subdomains: activating function AF1a (1\u2013163), inhibitory domain and AF1b (445\u2013602) with serval putative phosphorylation sites. S459 is located in the NTD within AF1b which mediates the interaction with the transcriptional apparatus25. So far, it is not known whether i) CK2-induced phosphorylation of MR-S459 alters the interaction with MR co-regulators, ii) MR-caveolin signaling and/or iii) whether known MR co-activators are regulated by CK2-induced phosphorylation directly which will be investigated in further experiments.Additionally, the change in genomic MR activity induced by mutation of S459 was smaller than the pharmacological CK2 inhibition, indicating that CK2 can phosphorylate additional S/T residues i) of the MR in its CDEF domain Fig.\u00a0 and/or in the cytosol or by MR-CK2\u03b1/\u03b1\u2019 complexes during cytosolic nuclear MR shuttling since aldosterone treatment reduces MR-CK2\u03b2 interaction direct MR phosphorylation e.g. on S459 and ii) probably by indirectly phosphorylation of MR co-regulators under control conditions. Additionally, activated MR aggravates an existing inflammatory situation via CK2-dependent mechanisms by augmenting NF\u03baB activity and consequently the expression of proinflammatory genes\u00a0Figure .2. 24\u2009h prior to the experiments, cells were cultivated in serum-free medium for 24\u2009h and treated with vehicle (DMSO 0.1%), aldosterone or a cytokine mixture composed of IL-1\u03b2 (10 ng/ml), IL-6 (20 ng/ml), TNF\u03b1 (20 ng/ml). Single exception represents the TIMEs which made quiescent by utilization of charcoal-stripped steroid-free medium with a reduced FCS content (2%) for 24\u2009h.HEK-293 cells, A7r5, EA.hy926 and TIME cells (American Type Culture Collection) were cultivated in the recommend media as indicated in Supplemental Table\u00a0CDEF\u200942, pEGFP-C1 (Clontech), pMR-DsRed2-N1 (RFP)-MR26 or pcDNA3.1-His-LacZ was performed as described before42 with Polyfect reagent (Qiagen).Transfection of HEK cells with MR expression vector pEGFP-C1-MR , pEGFP-MR\u00ae (Biorad).Cells were lysed with RIPA buffer for 1\u2009h or geldanamycin (2\u2009\u00b5M) for 2\u2009h. CoIPs were performed with anti-GFP-coupled magnetic beads and \u00b5 columns from Miltenyi (Germany) as recommended by the manufacturer followed by Western blot analysis.Cells were transfected with pEGFP-C1, pEGFP-hMR, pEGFP-hMRCDEF protein expression compared to MRfull length and normalized to the transfection control \u03b2-galactosidase.Transactivation of EGFP-MR and EGFP-MR mutants was assessed by the Mercury Pathway Profiling reporter gene assay system (Clontech) using secretory alkaline phosphatase (SEAP) as reporter4. The nuclear-cytoplasmic distribution after incubation with vehicle or TBCA (25\u2009\u00b5M)\u2009\u00b1\u2009aldosterone (10\u2009nM) was monitored for 40\u2009minutes and evaluated with ImageJ software .Time lapse experiments were performed with a digital BZ-8000 fluorescence microscope from Keyence with an incubation chamber as described earlier42.Cytosolic extract of EGFP or EGFP-MR transfected HEK cells which were treated with vehicle or TBCA (25\u2009\u00b5M)\u2009\u00b1\u2009aldosterone (10\u2009nM) for 1\u2009h were isolated with the Nuclear Extract Kit (Active Motif) as recommended by the manufacturer. Protein content was determined using Bradford reagent (Biorad). For Tf-ELISA experiments biotinylated DNA-(GRE) probes are captured in streptavidin-coated wells and the TFs bound were detected by specific antibodies as described earlierPeptide microarrays were produced by JPT peptide Technologies GmbH . Every MR peptide was composed of 15 aa with 12 aa overlap resulting in 341 peptides. Each peptide was spotted three times in triplicates resulting in 9-fold presentation of each individual peptide.2 10, KCl 50, ATP 0.25\u2009\u00b5M, 0.36 MBq \u03b3-33P-ATP) for 2\u2009h at 25\u2009\u00b0C in a humid chamber as described by43. The incorporated radioactivity was detected after rigorous washing by exposure of the peptide microarrays for 26\u2009h to imaging plates followed by readout with a FLA 3000 phosphor imager (Fuji). Control experiments with radioisotopically labeled ATP in buffer without kinase did not yield any signal after phosphor imaging.Microarrays were incubated with recombinant CK2 (500 U) in a kinase buffer as recommended by the manufacturer. Cloning results were confirmed by sequencing (Eurofins MWG Operon).\u2206\u2206Cq method, using the 18\u2009S signal for normalization. Details of the utilized primers are listed in Supplemental Table\u00a0For mRNA expression analysis HEK and TIME cells were incubated with vehicle, aldosterone (10\u2009nM)\u2009\u00b1\u2009cytokines: IL-1\u03b2, IL-6, TNF\u03b1 for the indicated time points Fig.\u00a0. Total RData are presented as mean\u2009\u00b1\u2009standard error mean (SEM). Significance of difference was tested by unpaired Student\u2019s t-test or One-way ANOVA with p\u2009\u2264\u20090.05 considered statistically significant. N represents the number of individual experiments and n the number of wells or culture dishes investigated per experiment.Supplementary dataset"} +{"text": "The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs) is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer\u2019s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer\u2019s disease, chronic stress and epilepsy. The mammalian genome encodes four AMPAR subunits, GluA1\u20134. These combine as a dimer of dimers to form functional tetramers that are generally permeable only to NaC. elegans and activates a Ca2+-dependent signaling cascade that involves the activation of Ca2+/calmodulin-dependent kinase II , Nedd4-2, RNF167 and APC2+/phospholipid-binding domain, multiple WW protein-protein interaction domains and a C-terminal HECT domain. These ligases preferentially form K63-linked polyubiquitin chains on their substrates domain, which first accepts ubiquitin from an E2 ligase onto its catalytic cysteine residue, prior to transferring it to the substrate has also been demonstrated to facilitate GluA1 ubiquitination in neurons 167 is a member of the really interesting new gene (RING) E3 ligase family that comprises over 600 proteins in the human genome. Unlike the HECT domain, the RING domain lacks a catalytic cysteine and merely acts as a scaffold to facilitate the direct transfer of ubiquitin from E2 to the substrate. RNF167 is an integral membrane protein that is localized to the plasma membrane, endosomes and lysosomes, and has a role in regulating endosomal trafficking and the degradation of substrates in lysosomes is a large multisubunit E3 ligase that targets key cell cycle regulatory proteins for proteasomal degradation catalyze the removal of covalently attached ubiquitin from proteins, thereby controlling ubiquitin signaling and maintaining the free ubiquitin pool in cells. In contrast to the diversity of ubiquitin E3 ligase, the human genome encodes only ~95 DUBs , ubiquitin-specific protease 8 (USP8) is a key regulator of the endosomal sorting complex required for transport (ESCRT) pathway that is essential for the formation of multivesicular bodies and lysosomes overexpression of Nedd4-1 in neurons, which enhances GluA1 ubiquitination, causes a reduction in the number of AMPARs on the plasma membrane peptides and intracellular filaments composed of hyperphosphorylated tau. Strong evidence from human genetics and transgenic mouse models has indicated a role for A\u03b2 in the etiology and pathogenesis of Alzheimer\u2019s disease Selkoe, . It is wRecent work from our own and the Patrick laboratory have directly demonstrated the direct involvement of AMPAR ubiquitination as a critical pathway in mediating the A\u03b2-induced synaptic depression in neurons are controversial and debatable (Goo et al., 2+ subsequently activates E3 ligases through Ca2+-dependent translocation of Nedd4-1 to the plasma membrane and/or direct phosphorylation of Nedd4 and RNF167 by CaMKII. In one scheme, surface AMPARs are internalized without ubiquitination but are subsequently ubiquitinated in endosomes. In another scheme, ubiquitination of surface receptors recruits the binding of an endocytic adaptor, Eps15 and facilitates the internalization of AMPARs. Ubiquitinated AMPARs are then sorted to late endosomes and degraded in lysosomes. The activation of NMDARs can recruit USP8, and potentially USP46, to deubiquitinate AMPARs and promote their recycling back to the plasma membrane. Through an unknown mechanism, ubiquitinated AMPARs may also be degraded through the proteasome system.In summary, we propose the following working model Figure . First, Similar to kinases, components of the ubiquitination systems are often dysregulated in disease. Several recent findings have started to implicate dysregulation of AMPAR ubiquitination in the pathophysiology of Alzheimer\u2019s disease, epilepsy and chronic stress (Yuen et al., JW, SG, SEJ and VA wrote, revised and finalized this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The addition of various Mg salts revealed that anions influence the solubility of proteins in both USPs and UIPs. Moreover, addition of MgCl2/NaCl mixtures, in which the Cl\u2212 concentration was held constant, demonstrated that Mg2+ was essential for UIP formation but not for USP formation and that Cl\u2212 was inconsequential for both USP and UIP formation. NaCl addition showed that the driving force for USP formation was salting-out due to the presence of cations. Overall, our data indicated that the Mg2+ concentration determined the separation of USPs and UIPs. These results will help elucidate the molecular mechanisms mediating the separation of silken tofu and regular tofu.Different coagulant concentrations can induce urea-soluble precipitates (USPs) and urea-insoluble precipitates (UIPs) during tofu-like precipitate formation. In this study, MgCl The tra2 .Accordingly, in the present study, we investigated and quantified MgCl22.1Plain soymilk (40 mg protein/mL) was purchased from Nagoya Seiraku Co., Ltd. . Protein assay dye reagent concentrate was purchased from Bio-Rad Laboratories . Other reagents were purchased from Wako Pure Chemicals .2.22, MgSO4, MgBr2, Mg(NO3)2, and Mg(ClO4)2) and NaCl were dissolved in distilled water at various concentrations. In addition, MgCl2 was dissolved with NaCl in distilled water to prepare various MgCl2/NaCl mixtures containing different Mg2+ concentrations and constant Cl\u2212 concentrations. In these mixtures, the Na+ concentration was decreased by increasing the Mg2+ concentration.Mg salts . The ratio of the residual protein concentration was expressed as the protein concentration in the supernatant after coagulant addition to that of soymilk diluted 10:1 by distilled water. The ratio of the urea-soluble protein concentration was expressed as the concentration of urea-soluble proteins to the protein concentration of soymilk diluted 10:1 by distilled water. The data shown represent the average \u00b1 standard deviation of three independent experiments.2.6rn) was calculated using the following equation:r, rmin, and rmax are the percentage of each urea-soluble protein concentration, the minimum percentage, and the maximum percentage, respectively, as determined by adding the Mg salt solution or MgCl2/NaCl mixture, as described above.To compare changes in urea-soluble protein concentrations, the normalized ratio 2 (4)2 . The Cm values for USP and UIP formation also approximated the order of the Hofmeister series 2 D, and Mg3)2 (4)2 E, were a3)2 (4)2 . The obsr series : SO42\u2212 100 mM Cl\u2212) were added. With NaCl addition, a finer resolution of the Cl\u2212 concentration (every 2 mM Cl\u2212) was also analyzed in the range of 0\u201340 mM of Cl\u2212 (Data not shown). These results also indicated that NaCl addition clearly produced no USPs in the range of 0\u201340 mM Cl\u2212. If the Cl\u2212 concentration is an essential factor for USP formation, then USP formation should occur when only NaCl is added at a concentration of 14 mM. However, no USP formation occurred at this Cl\u2212 concentration (14 mM) with only NaCl addition (Data not shown). Our results also demonstrated that the anion concentration was an inconsequential factor for USP formation. As described in Figs. As shown in 2 4 mM Cl\u2212.2+ (but not Na+), suggesting that Mg2+ addition was essential for UIP formation. In contrast, USP formation was induced by adding only Mg2+ or only Na+. Based on these differences, we assumed that salting-out of soy proteins occurred during USP formation by cation interactions with the surface carboxyl groups of soy proteins and that the linkage between soy proteins through salt bridges was an essential factor for UIP formation. Cations would have different functions during separation into USPs and UIPs during tofu-like precipitate formation. Interestingly, Moreover, from the data of urea-soluble proteins described above, it was noted that UIP formation was induced by adding only Mg2 and CaSO4 at concentrations of approximately 10 mM during the preparation of silken tofu facilitated a suitable protein yield and texture was most suitable for detection of the separation of SP and RP, suggesting that the concentration of urea may be useful for breaking the molecular interactions of USPs, but not breaking the molecular interactions of UIPs. Thus, studying USP and UIP formation may help elucidate the molecular mechanisms relevant to tofu preparation.In our previous study, USPs and UIPs were detected in tofu-like precipitates with SP and RP, respectively, by suspension in 2 M urea . This cotrations . Moreovetrations . We spec3.72 concentration (Figs. The sum of the residual proteins and urea-soluble proteins exceeded 100% at the same MgClon Figs. . This exon Figs. . In soymon Figs. . A fract42+ and Na+ induced USPs, strongly suggesting that the driving force for USP formation was salting-out due to the interactions of cations with the surface carboxyl groups of soy proteins. Mg2+ was essential as an initiation factor for UIP formation. Anions were found to be inconsequential for USP and UIP formation but clearly influenced the degree of protein denaturation. The different urea solubilities observed between USPs and UIPs may have been induced by different anion concentrations. Based on these data, we concluded that the different roles of cations determined the outcome of USP and UIP formation (In this study, we found that both Mgormation . These mYasuhiro Arii: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Kaho Nishizawa: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.Tojuro Iijima Foundation for Food Science and Technology under Grant Number 2016-37.This work was supported by the The authors declare no conflict of interest.No additional information is available for this paper."} +{"text": "Phormidium biofilms in rivers is becoming a worldwide sanitation problem for humans and animals, due to the ability of these bacteria to produce anatoxins. To better understand the environmental conditions that favor the development of Phormidium biofilms and the production of anatoxins, we monitored the formation of these biofilms and their toxins for two years in the Tarn River, biofilms from which are known to have caused the deaths of multiple dogs. As previously observed in New Zealand, Phormidium biofilm development occurred in riffle areas. The coverage of these biofilms at the bottom of the river exhibited strong spatial and temporal variations, but was positively correlated with water temperature and depth. Anatoxin-a was detected in less than 50% of the biofilms. The concentrations of these toxins in the biofilms exhibited high spatiotemporal variability, with the highest concentrations being recorded at the end of the summer period at the upstream sampling sites. These findings suggest that the maturity of the biofilms, combined with the local environmental conditions, have an impact on the production of anatoxin, making risk assessment for these benthic proliferations challenging.Proliferation of Phormidium has been described as a potential risk for humans and animals, due to the production of neurotoxins by these microorganisms [Phormidium strains are able to synthesize mainly anatoxin-a (ATX) and homoanatoxin-a (HTX) [a (dhATX) and dihydrohomoanatoxin-a (dhHTX) [In recent years, the proliferation in rivers of biofilms dominated by filamentous cyanobacteria belonging to the genus r et al. ). This pand (NZ) ,3, the Uand (NZ) ,5, and Fand (NZ) ,6. It ha-a (HTX) ,8,9,10, (dhHTX) . ATXs ar (dhHTX) ). Among (dhHTX) ,8. Phormidium since the beginning of the 2000s [Phormidium (hereafter referred to as Phormidium biofilms) has frequently been suggested, but has not been demonstrated. It is well established that ATXs are not secreted by cells and, consequently, that the unique method of intoxication is the ingestion of biofilms ,10. To dhe 2000s ,13. No ePhormidium biofilms depends, in part, on the importance of the development of these biofilms in rivers, which can be estimated by the percentage of biofilm coverage at the bottom of the river. Biofilm coverage is greatest during the summer period, when water temperatures are warm and the river flow relatively stable [Phormidium cells produce ATXs, while others do not [It has been shown, in NZ, that the potential toxicity of y stable ,14,15,16s do not . Phormidium biofilms in the Tarn and Loue rivers [Phormidium biofilms in the Tarn River during the summer season in 2013 and 2014 with the following goals: (1) to identify the favorable biotopes and environmental conditions for the development of Phormidium biofilms in this river, and (2) to characterize the potential toxicity of these biofilms and the spatiotemporal variations in this potential toxicity. In France, only two studies have been published on the toxicity of e rivers ,18, and Phormidium biofilms occurs almost exclusively in the riffle areas and never in pools with low flow. The five riffle areas per cm2) and in the Phormidium coverage during the two sampling years and that the genus Phormidium represents more than 97% of the total biovolume of cyanobacteria. Based on the sequencing of the 16S rRNA gene fragment using Illumina MiSeq, the dominant Phormidium operational taxonomic unit (with a 97% cut-off) of collected biofilms from the Tarn River displayed a 100% sequence similarity with Phormidium uncinatum [In these riffle areas, significant spatial and temporal variations were found in the biofilm biomasses (expressed in \u00b5g of chlorophyll- < 0.05) . In part < 0.05) . When bincinatum . Phormidium coverage observed between the biofilm biomass per surface unit and the proportion of Phormidium (Phormidium biofilms (The positive correlation (ormidium G confirmbiofilms H.3 s\u22121 in May 2013), followed by a consistent decrease in the flow rate throughout the summer period were screened by PCR for the presence of selected gene fragments involved in the biosynthesis of ATXs, microcystins, and saxitoxins. No positive PCR signal was detected for the microcystin and saxitoxin gene fragments. On the other hand, the ks2 ATX gene fragment was detected in 15 of the 66 samples . By LC-MThe overall congruence between the findings provided by the PCR and LC-MS/MS analyses was good (81%). All the positive results from the PCR analysis were confirmed by LC-MS/MS analysis, but some negative results from the PCR analysis were positive by LC-MS/MS analysis. Regarding the latter data, notably, the ATX concentrations were not quantifiable or very low in these samples. \u22121 FDW (freeze-dried weight) in biofilms, and all the biofilms that produced ATX were collected in August and September in 2013 and 2014. The highest ATX concentrations were always recorded in September in sites T1 and T2, in the upstream part of the river. As shown in Phormidium coverage was observed between the two parameters, nor with biomass (data not shown). Regarding water parameters, no significant correlation was observed between ATX concentration and depth or flow velocity was positively correlated with biofilm coverage, but negatively correlated with the biofilm biomass (estimated per surface unit and thus expressing the thickness of the biofilm). We assume that light availability might explain this phenomenon; no acids ), could \u22121 and 0.02 mg L\u22121, respectively, while severe proliferation of Phormidium biofilms was observed in the rivers in NZ with average nitrate concentrations between 0.06 and 0.3 mg L\u22121, and an average phosphorus concentration of approximately 0.01 mg L\u22121. Moreover, high nutrient concentrations were recorded in Spain during benthic cyanobacterial development , during the study period in the Tarn River, were 1.46 mg Losphates ,23). Lozosphates observedr bodies ,26,27. Phormidium biofilms was observed, for the first time, in several rivers in central France, including the Loire River (the longest French river), after a prolonged low-flow period (data not published). This is interesting to consider in the context of global climate change and effect of anthropogenic perturbations on aquatic ecosystems. Only a limited number of works were devoted to the study of toxic benthic cyanobacteria, despite the serious risk these microorganisms poses to animal and human health. We hypothesized that this may be explained by a recent and rapid increase of benthic proliferations. This is supported by the studies reporting toxic Phormidium proliferations over the last years in other geographic locations, including the Munda\u00fa River in Pernambuco (Brazil) [The low impact of trophic conditions on benthic cyanobacterial proliferations suggests that these events probably occur in a variety of rivers exhibiting physical and hydrological conditions suitable for the development of these biofilms. For example, during the summer of 2017, the proliferation of (Brazil) ; the Eel(Brazil) , and oth(Brazil) .Phormidium biofilms in the Tarn River was assessed first by a PCR-based method, then by LC-MS/MS, to detect ATX. Only the ks2 gene fragment, involved in the biosynthesis of ATX, was detected by PCR, while negative signals were observed for gene fragments involved in microcystin and saxitoxin biosynthesis. These latter gene fragments (associated with microcystin and saxitoxin synthesis) were screened because other cyanobacterial species , that are known to produce these toxins, can be found in association with Phormidium in biofilms, and because microcystins [Phormidium extracts.The potential toxicity of ocystins ,29,30,31With regard to ATX, all the positive results obtained by PCR were confirmed by mass spectrometry, but false negative results were also obtained by PCR. Previous studies have shown that the existence of polymorphisms in the ATX gene cluster ,32 can rPhormidium biofilms [Unlike the results from NZ, where HTX and di-hydro derivatives were the dominant toxins in biofilms , but conbiofilms in benthbiofilms , but werbiofilms . As showPhormidium coverage nor biomass, which was consistent with the results previously obtained in NZ rivers [Phormidium cover and toxin concentration were nearly linear at coverages above 10%. In order to better understand this relationship, further studies should be conducted. This lack of correlation between biofilm biomass and ATX concentration is interesting to consider in the context of the implementation of monitoring programs for these benthic cyanobacteria, by showing that biofilm biomass cannot be used as a proxy for ATX concentration. Our results revealed no correlation between ATX concentration and Z rivers . A recenZ rivers showed tPhormidium populations contain a mix of ATX-producing and non-ATX-producing filaments; therefore, it is possible that variations in environmental conditions from upstream to downstream may lead to differential selection of these two types of filaments in these areas. Interestingly, our data have revealed the existence of (i) an upstream\u2013downstream negative gradient in these ATX concentrations in the Tarn River, and (ii) an increase in ATX concentrations during the summer season (from June to September). While significant temporal shifts occurring on a weekly basis were observed in rivers in NZ (see review by Wood and Puddick ), such aPhormidium biofilms were detected . Sites TPhormidium biofilm was sampled using an underwater viewer. Biofilms were removed from the cobble using sterile tweezers, and subsamples were obtained for DNA and toxin analyses. In addition, a known area of the biofilm (7.07 cm2) was sampled for chlorophyll-a quantification. All these samples were placed in a 15 mL Falcon tube. Finally, 1 cm2 of the biofilm was also fixed with Lugol\u2019s iodine solution for subsequent microscopic identification and enumeration. All the samples were stored chilled in the dark and subsequently frozen (\u221220 \u00b0C), except for the samples used for microscope analysis, which were stored at 4 \u00b0C. On each site (except for site T1 in June 2013) a grid of 10 m \u00d7 10 m was set up from the shoreline and, within this grid, ten sampling points were randomly selected . At eachPhormidium biofilms was very low (<5% of the river substrate), three sampling points, located on a transect parallel to the water\u2019s edge and positioned 1.5 m from the shoreline, were sampled. At each point, all cobbles (5 to 20 cm length) visible in a single field of view of the underwater viewer (707 cm2) were collected. The cobbles were scrubbed, and the biomass was collected in 150 mL of river water. Aliquots (5 mL) were filtered for Chl-a (GF/C Whatman) and DNA extraction . Subsamples (1 mL) were fixed with Lugol\u2019s iodine solution. The remainder of the procedure was the same as that used for the other samples.For site T1 in June 2013, as the coverage of the substrate by Phormidium biofilms at the site during the sampling campaign.Rivers are very dynamic systems and, thus, biofilms that develop in these ecosystems are subjected to changes in water flow/velocities. The missing data points in Phormidium biofilms at the five different sites was determined by estimating the percentage of substrate covered by Phormidium biofilms (biofilm coverage) and the biomass of the biofilms per surface unit, using chlorophyll-a as a proxy. The extent of the development of Phormidium biofilm coverage was estimated at ten randomly selected points within the grid. At each point, the coverage was visually estimated through an underwater viewer independently by two operators. In this paper, the Phormidium biofilm coverage of a site represents the average value of the ten sampling points.a was extracted from the biomass samples or glass fiber filters (from biofilms collected at site T1 in June 2013) with methanol (10 mL) in Falcon tubes (15 mL) covered with aluminum foil, following a previously described protocol [a per cm2, according to a protocol described previously [Chl-protocol . Biofilmeviously . http://hydro.eaufrance.fr). The average concentrations of nitrates and TP measured at Qu\u00e9zac (~6 km upstream from Montbrun) were 1.46 mg L\u22121 (NO3-N) and 0.017 mg L\u22121, respectively, during the sampling period . At each sampling site water temperature and pH were measured using a multi-parameter probe . Then, at each sampling point, flow velocity and depth were measured with a Flow Probe FP111 . In addition, the flow rate was obta\u22121) to break up the filaments but avoid cell damage. The samples were diluted in Milli-Q water, and identification and enumeration were performed using a light microscope and a Malassez chamber . Counting, identification, and biovolume estimation of the samples were performed as previously described [Samples preserved with Lugol\u2019s iodine were homogenized briefly following the manufacturer\u2019s instructions. One ATX-producing strain, Phormidium favosum, isolated from the Loue River (PMC 240.04) was also analyzed as a control strain. Nucleic acid concentrations were determined by spectrophotometry , and the samples were stored at \u221220 \u00b0C. The ATX production potential of the environmental samples was assessed by PCR-based targeting of the ks2 DNA fragment . Three biofilm samples from each sampling site were lyophilized . Lyophilization was performed directly from frozen biofilm material (\u221220 \u00b0C) contained in Falcon tubes (15 mL). Falcon tubes were kept protected from light and biofilm samples were freeze-dried (\u2264\u221250 \u00b0C and 0.13 mBar vacuum pressure) for 24 to 48 h, depending on biomass and water content. DNA was extracted from ca. 50 mg of lyophilized biofilm material using a Power Biofilm region; ). In add region; ) and saxver PMC 2.04 was ag, 30 min at 4 \u00b0C). The supernatants were concentrated by lyophilization and rehydrated with water, and the pH was adjusted to 9.0 with aqueous NH4OH. Solid-phase extraction was performed using a C18-SD column , previously washed with 250 \u03bcL of MeOH and then with 500 \u03bcL of H2O. Each sample was loaded by gravity, and the column was washed successively with 500 \u03bcL of H2O and 500 \u03bcL of 10% MeOH/H2O (v/v). The toxins were eluted with 300 \u03bcL of MeOH. After solvent evaporation under reduced pressure , the residues were dissolved in 50 \u03bcL of 0.1% formic acid/H2O (v/v). To calculate the percentage of ATX loss during the extraction procedure, two replicate samples were enriched with a known ATX concentration, and these samples were subjected to all the steps of extraction until quantification. The ATX recovery percentage was 35% \u00b1 3.5 (average of two enriched samples), implying that the ATX concentration was probably underestimated in our study.Triplicates of the lyophilized biofilm material (100 mg) were resuspended in 10 mL of distilled water (Milli-Q) containing 0.1% formic acid. Strain isolates (~15 mg) were resuspended in ~1.5 mL of distilled water (Milli-Q) containing 0.1% formic acid. Samples were extracted according to a previously described protocol with som\u22121, and the injection volume was 10 \u00b5L. Collision-induced dissociation (CID) in the ion trap MS was performed. The ESI source of QTRAP was operated with the following optimized values of source-dependent parameters: ion spray voltage (IS), 5500 V; curtain gas (CUR), 20; collision gas (CAD), high; declustering potential (DP), 20; entrance potential (EP), 10; collision energy (CE), 20; and collision cell potential (CXP), 15. The mass spectrometer was operated in multiple reaction monitoring mode, detecting in positive mode, to analyze the following transitions: ATX , HTX , dhATX (m/z 168.0 > 56), dhHTX (m/z 182 > 57), EpoxyATX (m/z 182 > 98) and EpoxyHTX (m/z 196 > 140). The instrument was calibrated with dilutions of authentic standards of ATX and HTX in 0.1% formic acid. The LC-MS/MS analyses were performed by a combination of HPLC and mass spectrometry. The HPLC system was an Ultimate 3000 from Dionex coupled to a QTRAP LC/MS/MS system from ABI Sciex , which integrates a hybrid quadrupole-linear ion trap mass spectrometer with an electrospray ionization (ESI) source. The analytical LC column used for cyanotoxin separation was a reversed-phase C12 Jupiter 4U Proteo 90A 4 micron (250 mm \u00d7 1.0 mm i.d.) from Phenomenex . The temperature was set at 20 \u00b0C. The composition of the mobile phase was as follows: water (A) and acetonitrile (B), both containing 0.1% of formic acid. Chromatographic separation was performed by gradient elution (50 min): isocratic for 15 min at 100% A, followed by a gradient to 60% B over 10 min and, finally, at 100% A. The mobile phase flow rate was 70 \u00b5L mina, cyanobacterial biovolume proportions and percentage Phormidium spp. coverage) were analyzed using two-way ANOVA, followed by multiple comparisons using Tukey\u2019s honestly significant difference (HSD) post hoc test. Statistical analyses were performed using the statistical software package R 3.1.0 . Normalr2 values) presented throughout the manuscript correspond to squared correlation coefficients from type II regression analysis. Differences were considered significant when p < 0.05. The relationship analysis by type II linear regression was per"} +{"text": "Specifically, activation differences in ventral precuneus has been linked to an exemplar-based judgment process, where judgments are based on memory for previous similar cases. Ventral precuneus is implicated in various episodic memory processes, notably such that increased activity during learning in this region as well as in the ventromedial prefrontal cortex (vmPFC) and the medial temporal lobes (MTL) have been linked to retrieval success. The present study used fMRI during a multiple-cue judgment task to gain novel neurocognitive evidence informative for the link between learning-related activity changes in ventral precuneus and exemplar-based judgment. Participants (N = 27) spontaneously learned to make judgments during fMRI, in a multiple-cue judgment task specifically designed to induce exemplar-based processing. Contrasting brain activity during late learning to early learning revealed higher activity in ventral precuneus, the bilateral MTL, and the vmPFC. Activity in the ventral precuneus and the vmPFC was found to parametrically increase between each judgment event, and activity levels in the ventral precuneus predicted performance after learning. These results are interpreted such that the ventral precuneus supports the aspects of exemplar-based processes that are related to episodic memory, tentatively by building, storing, and being implicated in retrieving memory representations for judgment.The brain networks underlying human The act of multiple-cue judgment \u2013 to estimate a continuous criterion based on multiple cues \u2013 is something people engage in repeatedly in life. Consider for example how a teacher grades an essay, a car dealer estimates the price of a used car, or judges sentencing a criminal. Behavioral multiple-cue judgment research has traditionally focused on how people rely on rule-based processes, with weighted integration of each cue value, in order to make inferences. Linear regression models tend to describe such behavior well e.g., . HoweverN = 74), during both rule-based and similarity-based judgment processes. Interestingly, activity in this region predicted how well an EBM, but not a rule-based model, fit judgment data, raising the intriguing possibility that the role for ventral precuneus in human multiple-cue judgment is related to similarity-based processes . For instance, some recent studies have used model-based fMRI with EBMs and have found the MTL to be involved in concept learning have focused on the logic of contrasting strategies against each other. Thus, to the extent that similarity-based processes are at play also when other strategies are executed, common activations in this brain region might have been canceled out. Similarity-based processes have instead by the logic of contrasting strategies for example been found to evoke higher activity than rule-based processes in the anterior prefrontal cortex and the inferior parietal cortex, whereas rule-based strategies instead evoke higher activity than similarity-based processes in the anterior cingulate cortex, the dorsolateral prefrontal cortex, and the posterior parietal cortex e.g., . Anotherlearning , memory learning , and typlearning . Others ganglia and cort ganglia . Finally ganglia implyingmnemonic representational aspects necessary for exemplar-based processes in human multiple-cue judgment. The ventral precuneus has been extensively linked to episodic memory during judgment learning to investigate how ventral precuneus plays a role in exemplar-based processes and whether this role should be characterized as reflecting mnemonic representational aspects, or visuo-spatial attention. If an exemplar-based process in the ventral precuneus is key for human judgment, we should see changes in this region during the learning phase. If so, we hypothesized that, if the ventral precuneus is involved in building memory representations for exemplar-based judgment, an ss e.g., . On the During scanning, participants spontaneously learned a multiple-cue judgment task based on a biological scenario. A multiplicative relationship between cue and criterion was applied, successfully used to induce exemplar-based processes in past behavioral studies e.g., and two The experimental set up was designed to yield large differences in cognitive model predictions on the final test phase judgments between rule-based and EBMs, in order to confirm that the task manipulation described above had been successful. Some items were introduced during learning (training items) and a set of new items at test, and the behavioral responses were used to verify the models predictions. An EBM predicts that stored learning items will be retrieved from memory to make a judgment of new test items, with better performance for old than for new items and an inability to extrapolate to criterion values outside the acquired learning range . A lineaa priori predictions included three post hoc tests to explicitly test for changes as a function of learning, and a correlation test to establish a link between the ventral precuneus activity and performance on training items .The critical comparison with regards to the brain imaging data concerned BOLD signal differences between late and early learning. Further characterization of activity changes in the brain regions for which we had Mage = 24.9; Range = 20\u201338 years, SDage = 4.54) were recruited at Ume\u00e5 University through public advertisement. Participants were right-handed by self-report, met the criteria for MR-scanning , were neurologically healthy, had no dyslexia or dyscalculia, and had not participated in previous similar studies. All participants provided written informed consent in accordance with the declaration of Helsinki. All procedures were approved by the Regional Ethical Review Board in Ume\u00e5, Sweden. Participants received 500 SEK for participation in the experiment. An extra bonus of maximally 200 SEK could be earned based on performance .A total of 34 participants .In a within-subjects design, the task was to learn to judge the toxicity of fictional \u201ctropical death-bugs\u201d on a pseudo-continuous scale with outcome feedback, a task used in several previous studies on multiple-cue judgment to study rule-based and exemplar-based strategies ; judging whether a bug with similar resemblance to the death-bug had gray dots located on its body. The stimuli used in the familiarization session was unrelated to the main task in order to facilitate experience of the trial design and response mode but at the same time maintain novice to the to-be-learnt material before the start of the actual data collection. Participants were then informed about the fMRI judgment session where they should learn to judge the toxicity of a number of death-bugs with outcome feedback while being in the scanner. The death-bug judgment task was randomly alternated with the dot-bug judgment task (five items in each learning block) where participants were asked where on a bug dots were located; at the top, the bottom, or both. The structure of the dot-bug judgment task was identical to the death-bug judgment task on all aspects except that no outcome feedback was provided. Participants received no explicit instructions on how to solve the task.The experiment consisted of four sessions that were administered the same day. Participants began with a behavioral judgment session, including a final test phase, where participants continued to make judgments of death-bugs but outside of the scanner. Lastly, there was a questionnaire session, where participants answered questions regarding participants background, motivation, task difficulty, and knowledge on how the bodily features of the death bugs affected the toxicity.Following the fMRI judgment session was a learning blocks, wherein each of the 10 training items (death-bug judgment task) were presented twice in random order, in addition to the dot-bug judgment task . In between each learning block was an intermediate test phase, used for cognitive modeling. See The task was displayed on a 32 inches computer screen inside the scanner. The participants viewed the screen through a mirror attached to the head coil. E-prime 2.0 was used to control stimuli presentation and logging of responses. A Lumitouch fMRI optical response four button keypad was used to collect responses. Participants entered the scanner where they learned to estimate the toxicity of the items from the training set see with outdeath-bug or dot-bug (2\u20138 s). Next, the stimuli to-be-judged was presented for a maximum of 10 s. Participants were asked to make a judgment while the stimuli were displayed on the screen: either judging the toxicity of the displayed death-bug, or determining where dots were located, and press a button when they had formed their judgment. A jittered cross hair appeared when the button had been pressed or, if no button was pressed, after 10 s, and stayed on the screen for 2\u20136 s. That was followed by a scale where participants used their right-hand fingers to step to the location on the scale that matched their judgment. The scale was either ranging from 0 to 30 or with three alternatives . When a scale confirmation button had been pressed, or after 10 s, the pictorial death-bug became visible on the screen again together with the participants response and the correct numerical criterion as feedback (for 5 s). This was followed by the next trial . The learning criterion was employed to ensure that all participants mastered the task about equally well at the end of learning.final test phase, where participants judged all training and all new items twice in random order without outcome feedback .Lastly, participants answered a questionnaire with follow-up questions in the An EBM and a rule-based model were implemented in order to infer what strategy participants had relied on and si [four free parameters in the interval ] if there is a mismatch.where With CAM it is assumed that the impact of each cue on the criterion is combined to form a judgment in a linear and additive way, captured by:Ci.k (the intercept) and \u03c9i (the cue-weights) are free parameters . Goodness-of-fit was measured as the root mean squared deviation (RMSD) between the model\u2019s predictions and the participant\u2019s responses.The models\u2019 were fitted to participants test-phase data using a rocedure . For eact-test.To confirm that participants adhered increasingly to an exemplar-based strategy over the course of the four scanned learning blocks, a 2 (CAM vs. EBM) \u00d7 4 (intermediate tests 1\u20134) repeated measurements ANOVA was done with RMSD of the EBM and the CAM, respectively as dependent variables. If the sphericity assumption was violated, corrected statistics using Greenhouse Geisser was applied. Finally, to confirm that participants were better fit by the EBM than the CAM during the final test phase, that is, after the learning criterion had been met, RMSD of the EBM, and the CAM was compared with a paired-samples Performance was measured as root mean squared error (RMSE) between the criterion values and participants judgments on the death-bug task. To confirm that learning took place during the four blocks that were scanned with fMRI, RMSE of the four blocks (1\u20134) was entered into a repeated measurements ANOVA. If the sphericity assumption was violated, Greenhouse Geisser corrections were applied.t-test to accommodate the assumption of performance differences between items acquired during learning and novel test items (c.f. Differences in performance (RMSE) on training and new items during the final test phase was investigated with a paired samples ems c.f. , 2008.extrapolation index (EI). EI was calculated by (5):To further confirm that the behavioral responses during the final test phase was in line with what can be expected by participants adhering to a similarity-based strategy, we calculated an x28 and x2 are a participant\u2019s response on items with the highest and lowest toxicity level, and p28 and p2 are the predicted judgments by linear extrapolation to the same items, where the prediction was based on regressing participants judgments of the ten training items against the criteria , implemented within an in-house program (DataZ). Images were corrected for slice-timing, head movement were corrected through unwarp, and realignment. Images were spatially normalized with Dartel with an attached 32 channels head coil. A GE-EPI sequence with the following parameters was used: echo time (TE) 30 ms; repetition time (TR) 2000 ms; flip angle 80\u00b0; field of view (FOV) 25 cm; matrix = 96 \u00d7 96; 3.4 mm slice thickness (37 slices acquired). High-resolution T1-weighted structural images were obtained for each participant. The EPI sequence used for BOLD imaging was applied to acquire T2h Dartel , transfoThe primary analyses focused on regions with a different BOLD signal during late learning compared to early learning and was identified by comparing activity from the fourth (last) scanned learning block with the first scanned learning block and vice versa .t-tests based on the contrasts defined at the first-level, in a whole-brain analysis.In the first level analysis, individual general linear models (GLM) were estimated for each participant where the death-bug judgment task events and the dot-bug events during blocks one, two, three, and four were modeled as regressors of interest, and four regressors of no interest; rest > (death-bugs block 1 \u2013 dot-bugs block 1)] was defined at the first level, and evaluated at the second level in a one-sample t-test. Second, the difference between block 1 and 4 for the death-bug judgment task and the dot-bug judgment task [defined as (Task block 4 \u2013 Rest block 4) \u2013 (Task block 1 \u2013 Rest block 1)], respectively, was calculated. The purpose was to evaluate if the learning-related changes in the regions of interest were linked to the death-bug judgment task, and not the dot-bug judgment task, as evaluated with a paired samples t-test. Third, in order to investigate whether the effects would change by modeling additional events of no interest we defined a model where three additional regressors of no interest were included: the probe events, the scale events and the feedback events for all learning blocks (see t-contrast (block 4 > 1) was defined at the first level, and evaluated at the second level in a one-sample t-test. Note that both models in all other aspects were identical to the primary model.Three control analyses were performed to test the specificity of results obtained from the primary model for the contrast block 4 > 1. First, a model with the purpose to test if a tighter baseline comparison yielded results consistent with the primary model was defined. The five dot-bug judgment task events from the first learning block and the five dot-bug judgment task events from the last learning block were included as separate regressors in the model. A ocks see . A t-conp \u2264 0.05 (FDR corrected) at the voxel level and k > 0 at the cluster level for the whole brain analyses.The statistical threshold was set to post hoc tests were performed to further characterize the activation pattern observed in the primary analysis where the activation map identified in the former step was used as mask to define our regions of interests .Next, a set of t-tests.First, block-wise comparisons were performed to test activity changes between each block , with paired-samples Second, to explicitly test whether there was a link between activity levels in the ventral precuneus and retrieval success that might be missed when averaging activity within blocks. The purpose of this approach was related to that repeated judgment continuously improve learning, such that more learning lead to better memory representations that can be retrieved for similarity comparison. First, exponential and linear adaption of the BOLD signal was evaluated separately, and the individual judgment task events (duration modeled as response time), and linear or exponential functions included as a separate regressor in the GLM see . BOLD sip < 0.05 and k > 0 at the cluster level.Statistical threshold for the post-hoc tests were set to t-test at the second level was used to evaluate the contrast in a whole-brain analysis.Finally, it was evaluated whether ventral precuneus activity was significantly higher than baseline across all scanned blocks during death-bug judgments, and in addition if this activity was related to the fit of an EBM during the final test. t-test with the fit of the EBM was used as covariate of interest to test for a functional relationship between model fit and activity levels in the regions of interest that was consistently engaged during learning . Extracted beta values (5 mm radius around the peak voxel) was correlated with RMSD of the EBM on final judgment test-phase data.Finally, the activation map identified in the whole-brain analysis was again used as mask, and a one-sample t-test did importantly confirm that EBM fit participants data better than CAM, t (26) = \u221211.21, p < 0.001 = 100.91; MSE = 3.69; p < 0.001, F = 8.97; MSE = 8.46; p < 0.001, F = 2.36; MSE = 3.16; p = 0.094, The model fit during the four intermediate tests revealed that participants adhered increasingly to an exemplar-based strategy over the course of the four scanned learning blocks see : a repeaF = 99.67; MSE = 1.48; p < 0.001; = 0.79]. Paired samples t-tests moreover confirmed significant improvements between each of the first four blocks . On average, participants reached the learning criterion (\u22641.5 RMSE) after seven blocks .Performance (RMSE) as a function of blocks is shown in t-test on RMSE demonstrated that participants were better at judging training exemplars compared to new exemplars in the final test phase, Mold = 1.31, SD = 1.1, Mnew = 6.8, SD = 1.95, t(26) = \u221213.3, p < 0.001, a typical pattern predicted by EBMs = \u22122.48, p = 0.02.A paired samples Ms e.g., , 2008. TEI based on judgments of the most extreme novel test items was negative and the confidence interval did not include zero , demonstrating that participants were poor at extrapolating outside the acquired learning range. Again, this is a typical pattern predicted by EBMs . The contrast (block 4 > 1) yielded effects in a number of large clusters that were more engaged during late learning than early learning . Significant increases in the ventral precuneus, the vmPFC, and the bilateral MTL were observed between the second and the third block and the first and the fourth block , the vmPFC , and the bilateral MTL . Second, the difference in BOLD activity between blocks 1 and 4 [defined by (Task4 \u2013 Rest) \u2013 (Task 1 \u2013 Rest)] was calculated for the death-bug judgment task and the dot-bug judgment task, respectively. The observed patterns in the regions of interest strongly suggested that the learning-related changes between block 1 and 4 were selective (or considerably stronger) in the death-bug judgment task than in the dot-bug judgment task .In support of the findings from the primary statistical model, all three control analyses yielded overlapping results. First, when contrasting the death-bug judgment task with the dot-bug judgment task, the group-level comparison revealed an overlapping activation pattern, albeit under a less conservative statistical threshold see . Importatask see . Third, Reversing the contrast (block 1 > 4) yielded marked BOLD signal differences in one large cluster, including frontoparietal regions and the basal ganglia, distributed over both hemispheres, with the strongest peak in the visual cortex see . Similarr (24) = \u22120.46, p = 0.019], suggesting that the higher the activity level in the ventral precuneus toward the end of learning, the better performance on those same training items during the final judgment test phase with performance on training items (RMSE) at the final test revealed a strong negative correlation and also in the vmPFC but not in the MTL.In terms of a functional relationship between BOLD activity and model fit of the EBM during the final test, the results revealed negative correlations between BOLD level activity that was consistently engaged during learning and model fit of the EBM in the ventral precuneus [Exemplar-based processes, as captured by EBMs, are important for adaptive human judgment, decision making, and categorization. The ventral precuneus was recently identified as a key brain region for human multiple-cue judgment and brain activity in this region could be linked to how well an EBM fit novel test-phase data . HoweverNeural correlates traditionally expected during episodic memory processes are frequently reported also in research on human judgment, decision making, and categorization . For exaHow could the link between ventral precuneus and exemplar-based processes be understood? The ventral precuneus, together with the vmPFC and the MTL, has been linked to a network that enables episodic memory retrieval e.g., . It has It should be noted that the reduction of the BOLD signal between the first and the second learning block suggest that this process might not have been equally engaged during the entire fMRI judgment learning phase. Human judgment and decision making are known to be adaptive, with suggested contingent strategy shifts based on the content of the task suggesting that two competing neural systems underlie human categorization . COVIS dOne assumption in categorization and judgOne limitation of the present study is that it was not practically possible to scan the entire learning session, due to the fact that each trial in a multiple-cue judgment task require rather long events. Nevertheless, as was evident in the behavioral data, a large proportion of learning occurred during the first four learning blocks.The dot-bug judgment task was included in the experiment to be able to control for sensory-motor activity related to inspecting the visual stimuli and conducting a response. This control task included a limited number of events . The reasons for including fewer events in the dot-bug judgment task were (i) that the experiment was quite long, and (ii) no learning-related changes for the dot-bug judgment task with unique (not re-presented) items were expected. Still, ideally, the experiment should have included the same number of events (80) in both the death- and dot-bug judgment task. We note this as a limitation of the design, but highlight that the results pattern when the dot-bug events from blocks 1 and 4 were subtracted from the death-bug events indicated that learning-related changes were selective (or considerably stronger) in the death-bug judgment task.Finally, the main purpose of the present study was to investigate how a similarity-based process in the ventral precuneus, critically suggested to be a key region for similarity-based and rule-based processes in human judgment see should bWe investigated how the ventral precuneus contribute to exemplar-based processes in human judgment. Focusing on differences in the BOLD signal between early and late learning during similarity-based judgment learning, we investigated implications for this region in mnemonic representational processes. The results showed an increase of the BOLD signal in a parietal memory network related to retrieval success, including the ventral precuneus. Moreover, activity in the ventral precuneus toward the end of learning predicted retrieval success after learning. Thus, our findings indicate that activity levels in the ventral precuneus reflect gradual accumulation of exemplars in memory that could guide behavior, presumably by building memory representations for exemplar-based judgments.This study was carried out in accordance with the recommendations of the Regional ethical Review board in Ume\u00e5, Sweden. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Regional ethical Review board in Ume\u00e5, Sweden.All authors conceived and designed the study, wrote sections of the manuscript, and revised, read, and approved the submitted version of the manuscript. SS was responsible for data collection and wrote the first draft of the manuscript. SS and LKW performed the statistical analyses.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": "Solanum lycopersicum) PPO family . Here we report the complex spatial and temporal expression of one of the members, PPO B. The PPO B promoter was sequenced and subjected to homology analysis. Sequence similarities were found to nucleotide sequences of genes encoding enzymes/proteins active in the following systems: phenylpropanoid biosynthesis, signal transduction and responsiveness to hormones and stresses, fruit and seed proteins/enzymes, and photosynthesis. Chimeric gene fusions were constructed linking PPO B 5\u2032 flanking regions to the reporter gene, \u03b2-glucuronidase (GUS). The resultant transgenic plants were histochemically analyzed for GUS activity in various vegetative and reproductive tissues, and evaluated for PPO B responsiveness to ethylene induction. It was shown that PPO B expression was tissue specific, developmentally regulated, ethylene induced, and localized predominantly to mitotic or apoptotic tissues.Plant polyphenol oxidases (PPOs) are ubiquitous plastid-localized enzymes. A precise analysis of PPO function in plants has been complicated by the presence of several family members with immunological cross reactivity. Previously we reported the isolation of genomic clones coding for the seven members of the tomato ( The quinonoid reaction products are strong electrophiles which can participate in two secondary, nonenzymatic reaction pathways: the covalent 1,4 addition of o-quinones to cellular nucleophiles, and the reversed disproportionation of quinones to semiquinone radicals. Semiquinone radicals can either add covalently to other molecules or generate the superoxide anion radical (O2-), which may then give rise to other reactive oxygen species EC 1.10..2). The ies . Most partial sequence matches (ca. 60-90 % homology in 30-130 bp nucleotide sequences) were primarily to the 5\u2019 or 3\u2019 flanking DNA sequences or introns of genes encoding enzymes/proteins which are active in one of the following systems: phenylpropanoid biosynthesis, signal transduction pathways and responsiveness to hormones and stresses, fruit and seed proteins/enzymes, and photosynthesis. These matched sequences did not necessarily contain putative cis-acting elements.Arabidopsis chalcone synthase (CHS), chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR ). The buffer was a modification of McCabe et al. [The plant sections described above were immersed in GUS buffer plants were harvested for histochemical GUS staining and fluorometric GUS assay. Histochemical GUS staining was performed as described above with the following modification. Prior to incubation with GUS buffer, plant sections were incubated in 20% (v/v) methanol for 30 min. The GUS buffer contained 10 mM NaPO4 pH 7.0, 10 mM EDTA pH 8.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% (v/v) Triton X-100, 1% (v/v) DMSO, 20% (v/v) methanol and 0.5 mg/mL X-Gluc. Samples were incubated in GUS buffer for 14 h.BG and BxG shoots containing apical leaf nodes through node 8 leaves were cut and put in a solution of 1 mM ethephon, an ethylene releasing compound, in 0.1 M NaPO2. Frozen tissues were ground to a fine powder with a pestle and extracted at a ratio of 300 mg fresh weight to 1 mL extraction buffer SDS and 0.1% (v/v) Triton X-100). The homogenates were centrifuged twice at 12,000 g for 15 min.Leaf and stem tissues were weighed and frozen in liquid ND-glucuronide (4-MUG) and incubated for 0, 1 and 2 h at 37 \u00b0C. The reaction was stopped by adding 1.6 mL of 0.2 M sodium bicarbonate and the fluorescence was measured using 4-methylumbelliferone (4-MU) as a standard.GUS activity was assayed fluorometrically by measuring the 456-nm emission from the 365-nm excitation. Leaf homogenates containing 5 \u00b5g total protein were added to 400 \u00b5L 2 mM 4-methylumbelliferyl \u03b2-cis-elements related to hormonal and environmental responses were identified in the PPO B promoter region. Promoter sequence homologies and PPO B-driven GUS expression patterns suggested a role for PPO B in programmed cell death and/or plant defense. However, these putative roles required further evaluation by functional analysis using RNAi to inhibit individual PPO B gene specifically. The knowledge will provide insight into how PPO could be manipulated to allow maximal benefits of reduced browning and enhanced pest resistance without deleterious effects on plant growth and development.PPO B, one of the seven PPO gene family members in tomato, was spatially and temporally regulated in vegetative and reproductive tissues under normal growth and differentiation and in response to ethylene. The 5\u2019 flanking sequence of PPO B was partially homologous to sequences of genes encoding enzymes/proteins active in the following systems: phenylpropanoid biosynthesis, signal transduction and responsiveness to hormones and stresses, fruit and seed proteins/enzymes, and photosynthesis. Several putative"} +{"text": "Gemcitabine (GEM) is the standard chemotherapy drug for pancreatic cancer. Because of widespread drug resistance, the effect is limited. Therefore, it is urgent to reveal the underlying mechanism. Glycolysis is the most remarkable character of tumor aberrant metabolism, which plays vital roles on tumor drug resistance. Hexokinase 2 (HK2), as the key enzyme regulating the first\u2010step reaction of glycolysis, is overexpressed in many kinds of tumors. The putative role of HK2 resisting GEM therapy was investigated in this study. We found that HK2 was overexpressed in pancreatic cancer and associated with poor prognosis. HK2 knockdown decreased pancreatic cancer cell proliferation, migration viability, and promoted cell apoptosis in vitro. HK2 high expression in pancreatic cancer showed GEM resistance. HK2 knockdown increased the sensitivity of pancreatic cancer cell to GEM, the growth of xenograft tumor with HK2 knockdown was also further decreased with the GEM treatment compared with control in vivo. GEM\u2010resistant pancreatic cancer showed the increase of HK2 dimer rather than HK2 mRNA or protein. Our study revealed that the ROS derived from GEM promoted HK2 dimerization combining with voltage\u2010dependent anion channel, which resulted in the resistance to GEM. Meanwhile, our study established a new sight for GEM resistance in pancreatic cancer. Hexokinase 2 (HK2) overexpression promoted pancreatic cancer growth and gemcitabine (GEM) resistance. ROS mediated the HK2 dimerization induced by GEM. VDAC controls the entry and exit of ions and metabolites between the cytosol and mitochondria through the \u201copen\u201d and \u201cclosed\u201d states.The study described in this report established the critical role of HK2 overexpression in resistance to GEM\u2010induced cell apoptosis and elucidated the underlying mechanism in pancreatic cancer, which demonstrated that HK2 deletion increases sensitivity to GEM therapy.22.12.Human pancreatic cancer cell lines BxPC\u20103, PANC\u20101, Capan\u20101, MIAPaCa\u20102, and SW1990 were purchased from the American Type Culture Collection. All cells were cultured with DMEM or 1640 medium with 100\u00a0U/mL penicillin, 100\u00a0\u03bcg/mL streptomycin, 10% fetal bovine serum at 37\u00b0C under 5% CO2.2The pancreatic cancer tissue microarray (TMA) and samples were obtained from Pancreas Surgery of Fudan University Shanghai Cancer Center from 2010 to 2012. Pancreatic cancer samples were analyzed. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained.2.3HK2 and GAPDH antibody , VDAC antibody were purchased from Abcam (Abcam), GEM was bought from MedChemExpress .2.4Cells were prepared, treated, and collected for protein extraction. Briefly, cells in the dish were washed once with phosphate buffer saline (PBS) and lysed at 4\u00b0C with radio\u2010immunoprecipitation assay (RIPA) lysis. The mixture was centrifuged and the supernatants were collected as protein samples. The protein concentration was measured by the bicinchoninic acid (BCA) protein concentration assay kit . Protein samples with same mass were prepared, separated via SDS\u2010PAGE, and transferred onto Polyvinylidene Fluoride membranes. Then the membrane was blocked with 5% nonfat milk and incubated with the specific primary antibody at 4\u00b0C. Next, the membranes were washed with phosphate buffered saline tween\u201020 (PBST) and incubated with the corresponding horseradish peroxidase (HRP)\u2010conjugated secondary antibody. The membranes were washed with PBST. The target protein was finally visualized using an enhanced chemiluminescence system.2.5Cells were treated and total RNA was isolated by Trizol according to the manufacturer's instructions. Next, reverse transcription was performed using the Takara reverse transcription kit . Real\u2010time PCR was performed using SYBR Green reagent according to the manufacturer's instructions. The reaction system consisted of cDNA 4.5\u00a0\u03bcL (100\u00a0ng/\u03bcL), 2\u00d7 SYBR mix 5\u00a0\u03bcL, forward primer and reverse primer 0.2\u00a0\u03bcL each, and ROX 0.1\u00a0\u03bcL; the total volume was 10\u00a0\u03bcL. Finally, the HK2 mRNA expression level was calculated. Real\u2010time PCR detection was performed in at least three independent experiments. The HK2 forward primer sequence: TCAATATTAGAGTCTCAACCCCCA and reverse primer sequence GAAGGCGCTTGTGGAGAAGG.2.6Immunohistochemistry (IHC) was performed as previously described.2.7g to remove cell debris and filtered through a 0.45\u2010\u03bcm filter (Merck Millipore). MIAPaCa\u20102 cells were transfected with lentivirus particles expressing shHK2 or scrambled nontarget shNC. For screening the stable cells, puromycin (2\u00a0\u03bcg/mL) was added into cells after lentivirus infection. For control, the nontarget shNC was transfected, and cells were selected with puromycin. The HK2 knockdown efficiency was verified by western blot analysis.The target sequence of shHK was given below: shHK2f: CCGGTCCAAAGACATCTCAGACATTGTTCAAGAGACAATGTCTGAGATGTCTTTGGTTTTTG, shHK2r:AATTCAAAAACCAAAGACATCTCAGACATTGTCTCTTGAACAATGTCTGAGATGTCTTTGGA, shNC\u2010f:CCGGTTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG, shNC\u2010r:AATTCAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAA. The sequences including 62\u00a0bp were synthesized and constructed into pLKO.1\u2010puro vector at AgeI and EcoRI sides. The constructed plasmids were sequenced, and the sequence was confirmed to be correct. Lentiviruses were generated by cotransfection of pGAG, pVSVG, and pLKO.1\u2010shHK2 according to the ratio 3:3:5 into 293T cells with NanoEnter . To collect lentivirus particles, the supernatant was centrifuged at 300 2.86 cells/mL. Next, the cells were plated into a 96\u2010well plate with 2000 cells per well for cell viability assay and a 6\u2010well plate with 1000 cells per well for clone formation assay. Cell viability assay was completed by CCK8 and the proliferation curve was calculated. Cell clones were counted by crystal violet staining.Cells were digested and the cell concentration was adjusted at 102.96 cells/mL. The transwell chamber was placed into the wells of a 24\u2010well plate, and medium containing 10% serum was added into the bottom of the chamber. Cell suspension with approximately 105 cells was added into the chamber and cultured for approximately 24\u00a0hours. Finally, the chamber was stained with crystal violet, and the number of transferred cells was calculated.Cells were digested and suspended in serum\u2010free medium at a concentration of 102.10Cells for apoptosis detection were plated into a six\u2010well plate. Following treatment completion, the cells were digested and washed once with PBS. The cell staining was completed according to the manufacturer's instructions. Briefly, the sample was resuspended with 195\u00a0\u03bcL of apoptosis staining buffer, 5\u00a0\u03bcL of Annexin V\u2010fluorescein isothiocyanate was added and mixed, and 10\u00a0\u03bcL of PI was added and mixed. The mixture was incubated away from light at room temperature for 20\u00a0minutes. Finally, the cell suspension was assayed via flow cytometry.2.117\u00a0cells/mL. Approximately, 100\u00a0\u03bcL of cell suspension with 106 cells was injected subcutaneously into 4\u2010 to 5\u2010week\u2010old female or male Balb/C nude mice in the right and left abdomen. The xenograft tumor growth was monitored by the measurement of long diameter and short diameter, and tumor volume was calculated as described, V (mm3)\u00a0=\u00a0width2 (mm2)\u00a0\u00d7\u00a0length (mm)/2. Finally, the tumors were harvested and analyzed.Animal experiments were conducted with the approval of the animal ethics committee of Fudan University. Pancreatic cancer cells were digested and suspended with cold PBS at a density of 102.12Glutaraldehyde cross\u2010linking assay was employed to analyze HK2 dimerization Figure .2O as stock solution. Cells from 6\u2010cm dishes with about 90% cell density were scraped and lysed in 400\u2010\u03bcL 0.1% NP\u201040 lysis buffer with phenylmethanesulfonyl fluoride (PMSF) at 4\u00b0C for 30\u00a0minutes, and centrifuged at 12000 g for 20\u00a0minutes at 4\u00b0C Then 200\u2010\u03bcL lysate of each sample was drawn into two tubes. One tube was as control. The other one was added 10\u2010\u03bcL 0.5% glutaraldehyde stock solution to the lysate and incubated on ice for 5\u00a0minutes. About 10\u2010\u03bcL 1\u00a0mol/L glycine was added for 15\u00a0minutes at room temperature for quenching glutaraldehyde. Each tube was added 50\u2010\u03bcL 5\u00d7 loading buffer and boiled at 100\u00b0C for 10\u00a0minutes. Finally, the sample was detected by western blot.Glutaraldehyde solution was firstly prepared. About 10\u2010\u03bcL glutaraldehyde solution 50\u00a0wt.% (Sigma Aldrich #340855) was added to 990\u2010\u03bcL double\u2010distilled H2.13t test. The statistical results were presented as the mean\u00a0\u00b1\u00a0SD from experiments conducted in at least triplicates. Differences were considered to be significant when P\u00a0<\u00a0.05 for all statistical analyses.The significant differences between the mRNA and protein level were assessed via Student's 33.1P\u00a0<\u00a0.05) (Figure To establish the expression of HK2 in pancreatic cancer, we analyzed the GEO database. From GSE15471 with 39 pairs of pancreatic tumor and peritumor tissues, we found that HK2 expression was higher in pancreatic tumors compared with the corresponding peritumor tissue (3.2To confirm the role of HK2 in pancreatic cancer cells, we analyzed HK2 expression in five pancreatic cancer cell lines via western blot analysis. HK2 was commonly expressed (Figure 3.3Given that HK2 promoted cell proliferation and resistance to apoptosis in pancreatic cancer cells, we analyzed the response to GEM therapy when HK2 was knocked down. Cells were divided into four groups with different treatments: shNC, shHK2, GEM, and shHK2 with GEM. Compared with shNC group, the cell viability was decreased by approximately 40% in the shHK2 group, 52% in GEM treatment group, and 63% for the shHK2 with GEM treatment in the SW1990 cells (Figure 3.4To explore the underlying mechanism, we detected the mRNA and protein expression of HK2 after GEM treatment. The mRNA was not significantly changed after GEM treatment in SW1990 and MIAPaCa\u20102 cells Figure A,B. Simi3.5HK2 exists the monomer and homodimer formation. Next, we analyzed whether GEM had an effect on HK2 dimerization. The western blot results showed that HK2 dimer was significantly increased after GEM treatment in SW1990 and MIAPaCa\u20102 cells at concentration\u2010 or time\u2010dependent way Figure A,B. HK2 3.6It has been reported that HK2 combining with VDAC inhibited cell apoptosis.3.76\u00a0cells. GEM was administered via tail intravenous injection every other day in the groups of GEM treatment and shHK2 with GEM treatment. The shNC and shHK2 groups were injected saline as control. The xenograft tumors were monitored and measured every other day for evaluating the xenograft tumor growth. After 30\u00a0days, the xenograft tumors were harvested for further analysis (Figure To further explore the role of HK2 in GEM treatment in vivo, a mouse subcutaneous xenograft tumor model was completed. The mice were divided into four groups including shNC, shHK2, GEM treatment, and shHK2 with GEM treatment. Nude mice\u00a0were injected\u00a0subcutaneously with10s Figure A. The tus Figure B and weis Figure C were cas Figure D. These 4Metabolism reprogramming has been identified as a hallmark of cancer, this effect results in a high flux of glucose utilization in tumors to support rapid tumor growth.Overexpression of HK2 protects cancer cells against apoptosis induced by oxidants or other stimuli.It has been reported that GEM resistance is common in primary or acquired pancreatic cancers.Previous studies have demonstrated that the antiapoptosis role of HK2 was responsible for the interaction with VDAC. They have reported that AKT, as an important regulator, promotes HK2 translocation from the cytoplasm to mitochondria; therefore, targeting AKT inhibition was believed to be feasible. GEM treatment promoted HK2 dimerization rather than HK2 mRNA and protein expression. It has been reported that HK2 interacts with VDAC in polymer form.It is known that embryonic and cancer cells preferentially use aerobic glycolysis to support proliferation.The authors declare no conflict of interest.\u00a0Click here for additional data file."} +{"text": "Scientific Reports 10.1038/s41598-019-53167-5, published online 13 November 2019Correction to This Article contains errors.After publication of the Article it was brought to Authors' attention that since the quantifier they were proposing for networks consider the node indices to build the random-walk-based distribution, if these indices are permuted without altering the network structure, the results for Network Fisher may change. This is a limitation of the original study, which should be acknowledged.1.In order to overcome this limitation, we recommend using a matrix reordering algorithm, e.g., Optimal Leaf Ordering, that enhances the presence of block-patterns in the matrix, allowing the quantifier to characterize the network structure based on the adjacency matrix representation. For more information, readers can refer toThese limitations do not affect the overall conclusions of the Article."} +{"text": "A 22-year-old male with underlying end-stage renal failure was referred to our center for a malpositioned dialysis catheter. Imaging showed the tip of the catheter to be placed in the brachiocephalic trunk; the patient was, however, asymptomatic. Surgical removal of the malpositioned catheter followed, with no postoperative complications. A 22-year-old male with underlying end-stage renal failure was referred to our center for a malpositioned dialysis catheter. He has been on regular continuous ambulatory peritoneal dialysis via a Tenckhoff's catheter; however, suspicion of a blocked catheter necessitated its removal.Right internal jugular dialysis catheter was inserted via an anatomical landmark technique.After insertion, pulsatile flow, as well as an appearance of oxygenated blood, raised suspicion of an inadvertent arterial injury. No obvious hematoma was clinically observed, and the patient did not report neurological or obstructive symptoms. He was clinically stable.An urgent computed tomography angiography (CTA) revealed the catheter puncturing through the medial aspect of the right internal jugular vein, and entering the right subclavian artery (black arrow,A partial sternotomy, exploration of the subclavian and jugular vessels and surgical catheter removal were done. Intraoperatively, the catheter was seen exiting the right internal jugular vein at its medial aspect, penetrating the right subclavian artery at its superior aspect. The catheter could be felt within the right subclavian artery and the brachiocephalic trunk. The catheter was then removed, and the right subclavian artery was clamped both proximally and distally. The injured vessels were repaired using prolene 5/0. No overt hematoma was seen.The patient recovered well postoperatively and was discharged on day 3. Postoperative radiograph did not show any complications .Central venous catheters are used routinely in clinical practice for a multitude of reasons as follows: venous access, prolonged antibiotics administration, nutritional support, as well as in managing perioperative fluid status. However, insertion of these catheters poses associated risks, among them being inadvertent arterial puncture, which can occur in up to 6% of patients when using external landmark techniques.Central venous catheter insertion via ultrasound guidance has been shown in multiple studies to reduce catheter-related complications by up to 71%.When arterial injuries do occur, management has to be tailored on a case-by-case basis, taking into account the expertise and services available in a particular institution. The catheter is secured to prevent movement or dislodgment, followed by cross-sectional imaging via CT or magnetic resonance imaging, to better delineate the position of the catheter, guiding subsequent treatment approach.In our center, the cardiothoracic surgery service is readily available, hence the surgical approach was conducted.An alternative technique would be via an endovascular approach, utilizing covered stents, balloon tamponade, or vascular closure devices."} +{"text": "There is a lack of published patient reported outcome measures (PROMs) for the use of acellular dermal matrix (ADM) based breast reconstruction. This cohort study reviewed our clinical outcomes and PROMs.TM pre and post-operative questionnaires.All patients undergoing mastectomy with ADM assisted immediate breast reconstruction under a single surgeon between June 2013 and June 2017 were included. A prospectively kept database, clinic letters and operation notes were analysed. All patients received BREAST-QSixty-two consecutive patients with 77 reconstructions were included. Mean hospital stay was 3.3 days. All patients received 48\u00a0h of intravenous antibiotics, followed by a two-week course of oral antibiotics. Mean post-operative follow up was 17 months. There were 8 cases of skin necrosis (10.4%), and 1 infection (1.3%). These resulted in 4 explantations (5.2%); 3 following skin necrosis and 1 following infection. There was no observed \u2018red skin\u2019 syndrome. Post-operative mean score for \u2018satisfaction with outcome\u2019 was 83.1%. Mean score for \u2018Psychosocial well-being\u2019 was 70.7% and the mean score for \u2018physical well-being\u2019 was 77.9%.Our complication rates were comparable to those published, and PROMs were consistently good. The skin necrosis rate was potentially due to earlier practice of performing single-stage immediate reconstruction using fixed volume breast implants. We have modified our patient selection criteria and ADM based reconstructive techniques with experience. Longer term clinical and patient reported outcome should be sought. Breast cancer represents the commonest malignancy in the UK, with around 55,200 new cases in 2014, accounting for 31% of cancers in women.3Options for immediate reconstruction remain varied but implant based reconstruction remains the most popular with 37% of immediate breast reconstructions in the UK being implant based.Breast surgery is fundamentally deforming and can have negative effects on body image and self-esteem which can result in depression, anxiety, shame and even suicide.The aims of this study are firstly to examine our own surgical outcomes and secondly to collect PROMs data in order to fully assess our units\u2019 quality of care and to add to the growing body of evidence on the use of ADM.This prospective cohort study recruited 62 consecutive patients with 77 reconstructions from June 2013 to June 2017. Included were all patients who underwent mastectomy and immediate reconstruction with implant and ADM under a single surgeon. All patients underwent consultation with both an oncoplastic trained surgeon and specialist breast care nurse to discuss the full range of reconstructive options before deciding on this form of reconstruction. There were no exclusion criteria and no pre-selection. All patients were discussed at breast MDT and underwent adjuvant and neo-adjuvant therapy as advised. The choice of reconstructive procedure had no impact on this decision or the timing of this treatment. At pre-operative consultation, advice was given to smokers on the advantages of quitting smoking pre-operatively but smokers were not selected against. With permission, our unit utilised the BREAST-Q reconstruction module and the registered BREAST-Q scoring software to analyse the results. At a set time point all patients were invited to fill in pre-operative and post-operative BREAST-Q PROMs questionnaires via post.Data collected included basic patient demographics, smoking status, BMI, neo-adjuvant treatment, adjuvant treatment, tumour characteristics, length of stay, antibiotic duration, drain duration, post-operative contact, post-operative complications, further interventions and final follow up date. A database was kept prospectively and data was collected via electronic notes, MDT records and operation notes.TM) fixed inferiorly using 2.0 PDS and along the inferior border of pectoralis major using 2.0 monocryl. The ADM was fenestrated with a 4mm punch biopsy needle at 10\u201315 places. 2 drains were placed outside of the ADM/pectoral pocket, one superior and one inferior (in front of the ADM). One adjustment was the reduction in the use of large (more than 550cc), fixed volume implants in favour of the use of expanders in cases where the mastectomy pocket was felt to be under tension with a fixed volume implant of the most appropriate size for the patient. Where a fixed volume implant was felt appropriate, Mentor CPG \u00ae anatomical implants were used, and where an expander was needed, Becker-35 \u00ae expandable implants were used. All patients had surgical follow up at a week and two weeks post-operatively and thereafter as required.Patient care was administered as standard for that operating surgeon and a consistent, reproducible approach was taken. All patients were admitted on the day of surgery, received 1.2g co-amoxiclav at induction, 48\u00a0h of IV antibiotics (1.2g TDS) and two weeks of oral co-amoxiclav (625\u00a0mg TDS). A single surgeon performed all procedures with a standardised technique, although experience led to some minor adjustments. A nipple sacrificing mastectomy using sharp dissection was performed on all. The implant was placed in the sub-pectoral plane with the ADM identified, three resulted in implant loss, two underwent superficial debridement only and three required no surgical intervention. There were four haematomas identified (5.2%), only one of which required evacuation. There was a single case of seroma (1.3%) that did not require drainage and a single case of infection (1.3%) that did lead to implant loss. Overall there were four cases requiring implant removal (5.2%), one following infection and three following skin necrosis. .Table 3PPROMs outcomes were analysed via the registered PROMs Breast-Q scoring system and results are given as a score out of one hundred for each of the domains. Results are presented below as the mean score for each domain and the respective standard deviation. .Table 4MPre- and post-operative photos were obtained where possible and with written consent. Since their introduction for use in burns victims in the 1990s the use of ADM has been increasing, the first published use of ADM for breast reconstruction was in 2001 to correct implant rippling In the wake of a cosmetically deforming operation that is well recognised to have potential negative effects on body image and self-esteem, patient satisfaction and quality of life must be recognised as a significant outcome when evaluating surgical success. Unfortunately, PROMs data has no standardised format and is inconsistently reported. This makes it difficult to accurately make comparisons and parallels between different publications. Even without comparison, our units results are excellent with a mean score of 82.5/100 for overall \u2018satisfaction with outcome\u2019, a mean score of 71.3/100 for \u2018Psychosocial well-being\u2019 and a mean score of 77.9/100 \u2018physical well-being\u2019. These compare favourably in all domains when compared to a similar study published in 2016 by Vu et al. that collected PROMs data for 72 reconstruction using ADM. However, despite a lack of comparative data within ADM based reconstruction, the information gleaned from PROMs is important in enabling patients to make an informed decision about the type of breast reconstruction they wish to undergo. With PROMs data becoming more widely collected across the different reconstructive techniques this should become increasingly possibleThis study reflects high levels of satisfaction across most domains of the BREAST-Q, especially with regard to overall satisfaction. Minimal problems with infection, seroma and haematoma are re-assuring, however the relatively high rates of skin flap necrosis, is still of concern. An evolving surgical technique to prevent undue thinning of and tension across the skin flaps may help to bring this figure down. In an era of evolving use of ADM it is imperative to ensure the continued evaluation of outcomes, including prospective PROMs data collection to enable comparison of different techniques and the maintenance of high standards.NoneNone"} +{"text": "Case-fatality from COVID-19 has been reported to be relatively high in patients age 65\u2009years or older. We sought to determine the age-specific rates of COVID-19 mortality at the population level.We obtained information regarding the total number of COVID-19 reported deaths for six consecutive weeks beginning at the 50th recorded death, among 16 countries that reported a relatively high number of COVID-19 cases as of April 12, 2020. We performed an ecological study to model COVID-19 mortality rates per week by age group and sex using a Poisson mixed effects regression model.Over the six-week period of data, there were 178,568 COVID-19 deaths from a total population of approximately 2.4 billion people. Age and sex were associated with COVID-19 mortality. Compared with individuals ages 54\u2009years or younger, the incident rate ratio (IRR) was 8.1, indicating that the mortality rate of COVID-19 was 8.1 times higher among those 55 to 64\u2009years, and more than 62 times higher among those ages 65 or older. Mortality rates from COVID-19 were 77% higher in men than in women .In the 16 countries examined, persons age 65\u2009years or older had strikingly higher COVID-19 mortality rates compared to younger individuals, and men had a higher risk of COVID-19 death than women. After the emergence of a novel coronavirus (Severe Acute Respiratory Syndrome-CoronaVirus-2 [SARS-CoV-2]) in China, a pandemic has spread worldwide . ClinicaData from China and Italy suggest a case-fatality of 2.3% in patients with COVID-19, with more than 50% of the fatalities occurring in patients 50\u2009years of age or older denotes the mean number of COVID-19\u00a0deaths by age (Age) and sex (Sex) groups. We incorporated a normal, mean zero country-level random effect, bi; to account for correlated data at the country level. The model indices denote the country , age groups and sex ; the country population sizes by age and gender groups, Nijk, were modeled as an offset. The exponentiated regression coefficients are interpreted as incidence rate ratios (IRR\u2019s) of COVID-19 mortality. Six-week total COVID-19 deaths were disaggregated for each country using their respective estimated age and sex distributions of COVID-19 deaths. Our analyses accounted for this source of variation using a non-parametric bootstrap procedure. We selected 10,000 bootstrap samples from the estimated age and sex distributions of COVID-19 deaths to generate 10,000 disaggregated estimates of the COVID-19 death totals by age and sex for each country. Bootstrap standard errors were used in testing whether there were differences in the COVID-19 mortality rates between age groups and for sex. Specifically, Wald statistics were used to test for associations between the age groups and for sex. We provide 95% bootstrapped confidence intervals using the tolerance interval method on the 10,000 bootstrap estimates. Analyses were conducted using the Stata statistical package .where Of the 178,568 COVID-19 deaths reported in our six-week sample from a total population of approximately 2.4 billion people, 153,923 deaths (86.2%) were in persons age 65\u2009years or older. Table\u00a0We observed COVID-19 mortality rates for women in Fig.\u00a0Estimates for our formal analysis compared COVID-19 mortality rates by age group and sex. We observed that individuals ages 55 to 64\u2009years had 8.1 times higher COVID-19 mortality rate than individuals younger than 55\u2009years of age , and that those age 65 or older had a 62 times higher rate compared to the youngest group . Persons age 65 or older had 7.7 times higher COVID-19 death rates than those between the ages of 55 and 64\u2009years . Finally, we observed that men had 1.77 times higher COVID-19 mortality rates than did women .In this study of COVID-19 in 16 countries, we found that COVID-19 mortality rates were strongly associated with older age and (to a lesser extent) with sex, with men having 77% higher mortality. Estimated IRRs were highest in the elderly. These observations suggest that the previously observed high COVID-19 case-fatality among older persons translates into a similarly high mortality rate at the population level.Although case fatality rates have been initially reported to be similar across countries , they haConsistent with our findings, in a recent meta-analysis of all-cause mortality from four countries , and New York State, the percentage of octogenarians was found to be different across the regions with the lowest cohort being in China and the highest in New York State. Similarly, the lowest mortality was observed in China (3.1%) and the highest in New York State (21%) . The larWhile determining the incidence of COVID-19 in a given population depends in part on the prevalence and quality of testing for the presence of this infection, reporting of mortality should be less affected by these factors. Nonetheless, COVID-19 deaths will have been underreported to the extent that persons may die without being identified as positive for SARS CoV-2. To the extent that this underascertainment of COVID-19 mortality is relatively greater in the elderly, we would have underestimated the magnitude of the relative mortality rate among persons in this age group. However, we recognize that the major limitations of this study is related to the possible differences in reporting of attributable deaths across countries, which would affect the rates reported by each country. Another limitation is the lack of available data on confounding factors e.g. ethnicity and patient co-morbidities known to be risk factors for COVID-19 mortality .As policy-makers prepare to make decisions on how to curb the outbreak, while reducing the pressures on the healthcare system and reopening the economy, it will be important that future choices be tailored to account for the demographics of the population and specifically consider the prevalence of people ager 65 or older in the population in specific regions , or comm"} +{"text": "Curcuma longa L. leaves; TL) are known to have antioxidant properties, including 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2\u2032-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and Hydrogen peroxide (H2O2) radical scavenging activity in several studies. The antioxidant effects of TL come from distinct bioactive compounds, such as curcumin, total phenolic compounds, and flavonoids. Therefore, in this study, the antioxidant effects of a water extract of TL (TLE) against H2O2 treatment were assessed in vitro Vero cells and in vivo zebrafish models. The intracellular ROS generation and the proportion of sub-G1 phase cells were evaluated in H2O2- or/and TLE-treated Vero cells to measure the antioxidant activity of TLE. TLE showed outstanding intracellular ROS scavenging activity and significantly decreased the proportion of cells in the sub-G1 phase in a dose-dependent manner. Furthermore, cell death, ROS generation, and lipid peroxidation in the H2O2-treated zebrafish model were attenuated as a consequence of TLE treatment. Collectively, the results from this study suggested that TLE may be an alternative material to relieve ROS generation through its antioxidant properties or a suitable material for the application in a functional food industry.Oxidative stress, caused by the excessive production of reactive oxygen species (ROS), results in cellular damage. Therefore, functional materials with antioxidant properties are necessary to maintain redox balance. Turmeric leaves ( Reactive oxygen species (ROS) refers to a number of different reactive oxygen-containing molecules that are generated during cellular metabolism . ROS cauCurcuma longa L. (Zingiberaceae), commonly called turmeric, is an herbaceous plant and cultivated in many Asian countries including India and China [nd China . Traditind China ,12. Thesnd China . Curcumind China . Similarnd China . Howevernd China . Accordind China , turmerind China . However2O2)-induced oxidative stress. To demonstrate the effects of turmeric leaf extract, the present study investigated the composition of functional compounds in turmeric leaf extract and their effects on cellular ROS generation and apoptosis in in vitro Vero cells and in vivo zebrafish model.In this study, we aimed to evaluate the effects of turmeric leaf extract (TLE) on hydrogen peroxide (H2O2), RNase A, 2,7-dichlorofluorescein diacetate (DCFH-DA), propidium iodide (PI), Hoechst 33342, dimethyl sulfoxide (DMSO), and diphenyl-1-pyrenylphosphine were purchased from Sigma . 19 flavonoids and 11 flavonoids were purchased from Sigma-Aldrich and Tokyo Chemical Industry (TCI), respectively. Five flavonoids and three flavonoids were purchased from Chemfaces and HWI group, respectively. Formic acid (FA) and fluorescein were purchased from Sigma-Aldrich. LCMS-grade solvents were used as the solvents of HPLC-MS experiments. All flavonoids were carefully weighed and diluted using HPLC-grade MeOH or DMSO or acetone . The concentration of the flavonoid standard stock was set as 4 mg/mL. Fluorescein was utilized as the internal standard for the flavonoid analysis.Turmeric leaves were obtained from the Jindoulguem Corp. . Dulbecco\u2019s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, Dulbecco\u2019s phosphate buffered saline (DPBS) and trypsin-EDTA were purchased from Gibco . Hydrogen peroxide (HThe turmeric seeds were sown in April and fresh turmeric leaves were harvested in November at Jindo-gun (South in Korea). They were washed three times to eliminate impurities, and hot-air dried at 50 \u00b0C for 24 h. Then, the leaf powder was extracted with distilled water at 85 \u00b0C for 150 min with 1:25 extraction ratio as an optimal extraction condition. After filtration of the turmeric leaf extract (TLE) with 0.22 \u03bcm PVDF filter, the leaf extract was diluted with distilled water at 10 mg/mL and stored at \u221220 \u00b0C until use.m/z values of precursor ion and product ions and retention factor (Rf) ratio between flavonoid and internal standard obtained from the HPLC-MS/MS experiments of the flavonoid standards. The contents of the flavonoids were estimated using the internal standard method.All MS experiments were performed in negative ion mode using an AB Sciex X500R QTOF equipped with an ExionLC HPLC. ACQUITY UPLC BEH C18 column was utilized for the separation of the flavonoids in the extract. 0.1% FA water and 0.1% FA ACN were used as HPLC solvent A and B, respectively. The flow rate and the column oven temperature were set as 0.3 mL/min and 40 \u00b0C, respectively. A gradient of the two solvents was set as follows: 0\u20133.0 min, linear increase from 0% to 10% B; 3.0\u20138.0 min, linear increase from 10% to 35% B; 8.0\u201322.0 min, linear increase from 35% to 80% B; 22.0\u201323.0 min, 80% B; 23.0\u201323.5 min, linear decrease from 80% to 0%; 23.5\u201328.0 min, 0% B. We inject the sample solution of 5 \u03bcL for obtaining the single HPLC-MS data. Spray voltage, CAD gas, curtain gas, and source temperature were set as \u22124500 V, 7, 25, and 400 \u00b0C, respectively. Collision energy and collision energy spread were set as \u221235 V and 20 V, respectively. Flavonoids in the extract were identified using 2 humidified atmosphere. The cells were cultured using DMEM supplemented with 10% heat-activated fetal bovine serum (FBS), penicillin (100 unit/mL), streptomycin (100 \u03bcg/mL), and sodium pyruvate (110 \u03bcg/mL). The cells were cultured for 2 days after they reached 80% confluence. For the experiment, the cells were seeded on a 96-well plate with 1 \u00d7 104 cells/well density.Vero cells, kidney cells from African green monkey, were grown at 37 \u00b0C in a 5% CO4 cells/well and incubated for 24 h. After change the medium to the serum-free medium, different concentrations of TLE and 600 \u03bcM hydrogen peroxide (H2O2) were treated to each well. After 24 h, aspirate the medium and treat the MTT containing medium (100 \u03bcg/mL). After 3 h, remove the medium and add 100 \u03bcL of DMSO to the wells. Measure absorbance at 570 nm using Microplate readers .Vero cells were seeded on a 96-well plate with 1 \u00d7 104 cells/well and incubated for 24 h. After change the medium to the serum-free medium, TLE in different concentrations was pre-treated to the cells for 1 h. Then, 600 \u03bcM H2O2 was treated to the cells followed by an hour incubation. Then, 10 \u03bcg/mL of ROS-specific fluorescent dye DCFH-DA solution was added to the cells. After 30 min, fluorescence was measured at a wavelength of 485 nm (excitation)/535 nm (emission) using Microplate reader .Vero cells were seeded on a 96-well black plate with 1 \u00d7 102O2 treatment. The cells were seeded on a 6-well plates with 2 \u00d7 105 cells/well. After 24 h, TLE in different concentrations was pretreated to the cells for 30 min. Then, 600 \u03bcM H2O2 was added to the cells followed by 1-h incubation. After 6 h, centrifuge the cells and supernatant, and wash the cells with ice-cold DPBS. Then, fix the cells with 70% ethanol, and add 5 \u03bcg/mL of RNase A and 10 \u03bcg/mL of PI. Incubate the cells for 30 min on ice and analyze proportion of sun-G1 phased cells using flow cytometry .Cell cycle analysis was carried out to determine the proportion of sub-G1 phased cells after TLE and H4 cells/well. After 24 h, cells were pretreated with TLE at 10 \u03bcg/mL and 100 \u03bcg/mL concentration, and incubated for 24 h. Then, 600 \u03bcM H2O2 was added to the cells for 30 min. The culture medium was removed and the cells were stained with 1 \u03bcg/mL Hoechst 33342 for 30 min and 10 \u03bcg/mL PI for 1 h. Stained nuclear was observed with excitation at 350 nm and emission at 460 nm under a fluorescence microscope .Vero cells were seeded on 24-well cell imaging plates with of 5 \u00d7 10Adult zebrafish was obtained from commercial dealer . Zebrafish were kept in a 3-L acrylic tank at 28.5 \u00b1 0.5 \u00b0C with a 14/10-h light/dark cycle. The zebrafish were fed two times a day, 6 days in a week with Tetramin flake food supplemented with brine shrimp . Embryos of zebrafish were obtained by natural mating and spawning within 30 min. After spawning, the embryos were moved to a Petri dish containing 1 mg/mL methylene blue solution. After disinfection for 1.5 h, the methylene blue solution was changed to fresh embryo medium.2O2 was added to the medium, and the embryos were incubated until 24 hpf. After counting survival rate, the surviving fish were used for the analysis.After 7\u20139 h post-fertilization (hpf), embryos were individually transferred to a 12-well plate (15 embryos per group) and maintained in medium containing TLE . After 1 h of incubation, 5 mM HROS production, lipid oxidation, and cell death in zebrafish embryos were investigated according to a method proposed by Kang et al. . Dichlorp < 0.05, ** p < 0.01, and *** p < 0.001.All experiments were conducted in triplicates. All data were presented as means \u00b1 standard deviation. One-way ANOVA analysis was used to compare the mean values of each treatment in GraphPad prism software version 8.0. Significant differences between the means were identified by the Tuckey\u2019s regression test. Significance was established as * The flavonoids in TLE were analyzed with HPLC-MS method and flavonoid contents were expressed as ng flavonoid per dry weight of TLE (ng/mg). 10 flavonoids were identified in TLE as diosmetin (525.40 \u00b1 0.23 ng/mg), quercitrin (304.94 \u00b1 7.95 ng/mg), rutin (118.33 \u00b1 6.72 ng/mg), miquelianin (111.38 \u00b1 6.57 ng/mg), taxifolin (92.14 \u00b1 5.06 ng/mg), myrictrin (63.73 \u00b1 2.31 ng/mg), puerarin (55.30 \u00b1 4.56 ng/mg), narirutin (25.81 \u00b1 1.44 ng/mg), naringin (25.81 \u00b1 1.44 ng/mg), quercetin (23.31 \u00b1 0.44 ng/mg), among 30 flavonoids analytic samples .2O2-treated Vero cells was determined by MTT assay. As shown in 2O2-treated group, cell viability was reduced to 78.4%. However, the cell viabilities in all TLE treatment groups were higher than that of H2O2-treated group and showed a viability of 86.3% compared with the untreated group. These results indicated that TLE was not cytotoxic and inhibited cell death induced by H2O2 treatment in Vero cells.The effect of TLE treatment on H2O2-treated Vero cells. After 600 \u03bcM H2O2 treatment, ROS generation was increased more than approximately 7.4 times in the H2O2-treated group compared with that of the untreated group (2O2-treated cells (100%). These results indicated that TLE treatment inhibited intracellular ROS generation in H2O2-treated Vero cells.The DCFH-DA assay was performed to evaluate the intracellular ROS scavenging activity of TLE in Hed group . However2O2 treatment. In comparison with the sub-G1 population in the control group, dose-dependent decreases were observed in the TLE treatment groups, of 19.7%, 10.6%, 9.3%, and 6.3% -stained cells. The population in the untreated group was 0.4%, and the population in the control group had significantly increased, to 24.7%, after Hand 6.3% . These r2O2-induced cell death, apoptosis, and nuclear DNA fragmentation were assessed via nuclear staining with Hoechst 33342 and PI. As shown in 2O2-treated cells were associated with increased nuclear condensation and cell death . However, 10 and 100 \u03bcg/mL TLE treatment led to significant reductions in nuclear condensation and cell death in H2O2-treated cells. These results suggested that TLE treatment was able to inhibit oxidative stress-induced apoptosis in H2O2-treated Vero cells.The effects of TLE on H2O2-induced oxidative stress, we analyzed cell death, ROS production, and lipid peroxidation in zebrafish. The protective effect of TLE against H2O2-induced cell death in zebrafish was evaluated by acridine orange fluorescence staining. The cell death percentage in H2O2-treated zebrafish was considered to be 100%. TLE treatment at 100 and 200 \u03bcg/mL significantly reduced the H2O2-induced cell death in zebrafish, to 40.0% and 16.7%, respectively -induced cellular model [Turmeric leaves, the aerial parts of turmeric, are considered as by-products of turmeric plant after harvesting . Howeveroxidants . The antoxidants ,25. Espear model and amelar model . Quercitar model ,29. Base1O2), superoxide radical anion (O2\u25cf\u2212), H2O2, hydroxyl radical (\u25cfOH), and the hypochlorite ion (OCl\u2212) [ROS are reactive oxygen-containing molecules, mostly radicals, including singlet oxygen (n (OCl\u2212) . Intracen (OCl\u2212) . Howevern (OCl\u2212) ,33,34. In (OCl\u2212) ,36. Inden (OCl\u2212) . Therefo2O2 stimulation under both in vivo and in vitro condition as a distinct ROS species. H2O2 is an important metabolite in redox signaling, redox regulation, and oxidative stress-mediated cell death, where it acts as a messenger molecule [2O2, a stable molecule, diffuses across the cellular membrane with a specific carrier and is converted into a highly reactive hydroxyl radical [2O2 as an intracellular stimulant for oxidative stress. To investigate the ROS scavenging activity of TLE in vitro, intracellular ROS generation in Vero cells was detected by DCFH-DA staining. The DCFH-DA stain is taken up through the cellular membrane and is converted to DCFH via cellular esterase. Then, intracellular ROS converts DCFH into the fluorescent active DCF, which can be detected with a fluorimeter [2O2 treatment led to a significant stimulation of intracellular ROS generation, whereas all concentrations of TLE remarkably decreased ROS in Vero cells, and a dose-dependent effect was observed. These results indicated that TLE treatment may effectively regulate the ROS production induced by H2O2 treatment owing to its prominent radical scavenging activity. Therefore, cell cycle analysis was performed by PI staining to confirm the effect of TLE on ROS scavenging and apoptosis.This study focused on the cellular Hmolecule ,39. H2O2 radical . This ra2O2, also regulate various signaling mechanisms, dysregulation of cell cycle progression, growth, and proliferation [2O2-treated Vero cells. The increased proportion of sub-G1 phase cells is considered as a blockade of cell cycle kinetics, caused by DNA damage; if this damage is not repaired by the DNA repair system [2O2 treatment. Our results showed that the proportion of sub-G1 cells in H2O2-treated cells increased owing to DNA fragmentation and apoptosis. However, different concentrations of TLE significantly reduced the proportion of sub-G1 phase cells, illustrating the antioxidant effects of TLE.As many studies have reported, ROS, including Hferation . Apoptosferation . The celferation . In thisr system , cell de2O2-induced apoptosis in Vero cells, nuclear condensation and cell death were examined by fluorescence microscopy of Hoechst 33342 and PI dual stained cells. Hoechst 33342 exhibits blue fluorescence, and indicates cells in early apoptosis by staining the condensed nucleus, whereas PI exhibits red fluorescence, and indicates dead cells by binding permanently to their DNA [2O2-induced cells compared with the untreated cells. In contrast, nuclear condensation was decreased in a dose-dependent manner in the TLE-treated groups. The proportion of cell death in the TLE-treated groups was also significantly lower than that in the H2O2-treated group, indicating that the antioxidant effects of TLE occurred through inhibition of the progression of nuclear condensation and cell death.Nuclear condensation and cell death are major features of ROS-induced cellular apoptosis. To measure the protective effects of TLE treatment on Hheir DNA . As show2O2 has been successfully used to investigate the antioxidant effects of natural products in many studies [2O2 was selected as for the in vivo investigation of the antioxidant properties of TLE. Cell death, ROS generation, and lipid peroxidation of H2O2-treated zebrafish were measured to identify the antioxidant effects of TLE treatment in this study. The results indicated that TLE dramatically decreased H2O2-induced cell death, ROS generation, and lipid peroxidation in the zebrafish model.To confirm the results of TLE treatment in vitro, zebrafish was used as an in vivo model. Zebrafish models are a popular experimental tool in many research areas, such as biology, pharmacology, and toxicology . As a sp studies . Based o2O2-induced cellular damage via its own ROS scavenging properties. We believe that these findings provide supporting evidence for the use of turmeric leaves as a functional material in food industry.Collectively, the current study results showed that the water extract of turmeric leaves has strong antioxidant activity. The in vitro and in vivo experiments performed in this study also helped to determine the effects of TLE on ROS scavenging activity and oxidative stress inhibition. To the best of our knowledge, this study has provided the first evidence to show that TLE attenuated H2O2-induced ROS generation in vitro and in vivo model. Therefore, the findings of this study suggest that TLE may be a food source of antioxidants, suitable for use in functional foods or as an alternative treatment for oxidative stress-induced cellular damage.The study results suggest that the turmeric leaf extract has a strong antioxidant effect that is exerted through the suppression of H"} +{"text": "ZIP8 (SLC39A8), ZIP14 (SLC39A14), and ZnT10 (SLC30A10) have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters The mos species ,5,6.As an essential nutrient, manganese is required for the function of numerous enzymes, including arginase, glycosyltransferases, manganese superoxide dismutase (MnSOD), phosphoenolpyruvate carboxykinase, prolidase, and pyruvate carboxylase ,8,9,10. Deficiency in manganese has been associated with a number of health consequences, including impaired cognitive function, asthma, osteoporosis, and dyslipidemia. Since manganese is present in a broad range of foods and sufficient manganese can be obtained through diet under normal conditions, reports of dietary manganese deficiency are rare . ManganeZIP8 (SLC39A8), ZIP14 (SLC39A14), and ZnT10 (SLC30A10) have been reported to cause dysregulated manganese homeostasis [Tight homeostatic control is required to meet the dual challenge of avoiding manganese deficiency and preventing manganese overload. At the systemic level, this control is maintained mainly by the intestine and the liver ,27,28,29ZnT10 SLCA10 have eostasis ,32,33, deostasis . In thisZIP8 gene encodes a multi-transmembrane protein capable of transporting several divalent cations, including cadmium, zinc, iron, and manganese [ZIP8 mutations promoted the understanding of the physiological function of this transporter. In 2015, two clinical studies identified ZIP8 mutations in humans [ZIP8 mutations that led to manganese deficiency in a group of children [ZIP8 mutations in two unrelated patients with a congenital disorder of glycosylation (CDG) [ZIP8 was identified in two siblings to cause severe manganese deficiency, presenting as CDG and Leigh-like syndrome, with standard features of manganese deficiency that included developmental delay, brain atrophy, hypotonia, and seizures [ZIP8 is a member of the ZIP metal-ion transporter proteins. The ZIP family takes the name from zinc-regulated transporter (ZRT), iron-regulated transporter (IRT)-like proteins. The anganese ,36,37. Tn humans ,31. One children . The anaseizures .ZIP8 mutations stem from consanguineous families, with both parents being heterozygous carriers of the same mutation as that identified in their children. The disease onset occurs at birth or very early during childhood. The detailed information about identified cases of human ZIP8 mutations are listed in So far, a majority of the identified Xenopus oocytes injected with ZIP8 cRNA, manganese serves as a strong inhibitor for Zn uptake, suggesting a primary role for ZIP8 in mediating manganese uptake into those cells [ZIP8 is ubiquitously expressed throughout the body ,40,41,42se cells . These iZip8 is embryonic lethal in mice [Zip8 knockout (Zip8-iKO) mice were generated, and metal concentrations in different organs of these mice were compared to that of wild-type (WT) animals. Zip8-iKO mice had markedly reduced manganese in multiple organs, including the liver, brain, kidney, and heart, and presented with decreased manganese-dependent arginase and \u03b2-1,4-galactosyltransferase activities [ZIP8 mutations develop severe systemic manganese deficiency. Despite the ability of ZIP8 to transport iron and zinc in cell culture studies, the levels of tissue iron and zinc were not different in Zip8-iKO mice compared with that of the control animals, proving that the primary function of ZIP8 is to regulate manganese metabolism. Immunofluorescence analyses determined that ZIP8 is localized to the hepatocyte apical canalicular membrane, suggesting that hepatic ZIP8 may function to mediate manganese transport from the bile into hepatocytes.Conventional whole-body knockout of in mice ; therefotivities , which iZip8 knockout (Zip8-L-KO) mice were created [Zip8-iKO mice, Zip8-L-KO mice developed manganese deficiency in the liver, brain, kidney, and heart, suggesting that hepatic ZIP8 is required to regulate whole-body manganese homeostasis. Despite reduced whole-body manganese, Zip8-L-KO mice had a 55% increased level of manganese in the bile, supporting the role for ZIP8 in mediating transport of manganese from the bile into hepatocytes. Furthermore, liver-specific overexpression of ZIP8 through adeno-associated virus (AAV)-mediated gene delivery into WT mice resulted in a 76% decrease of bile manganese, an 87% increase of liver manganese, and a 94% increase of whole blood manganese, corroborating the view that higher than normal levels of ZIP8 will increase manganese accumulation and possibly lead to manganese overload, presumably through increased manganese reabsorption from the bile [ZIP8. Studies to examine ZIP8 expression under manganese deficiency and overload conditions are needed to further elucidate ZIP8\u2019s function in regulating manganese homeostasis.To determine the significance of hepatic ZIP8 in the regulation of whole-body manganese homeostasis, hepatocyte-specific created . Similarthe bile . Taken tXenopus oocytes determined that ZIP14 can mediate the cellular influx of a broad scope of metals, including cadmium, zinc, iron and manganese [ZIP14 include the liver and small intestine [ZIP14 (SLC39A14) was first identified as a gene responsible for cellular zinc influx based on the observation that overexpression of ZIP14 in Chinese hamster ovary (CHO) cells stimulates the uptake of zinc . Later sanganese ,53,54. Nanganese . Human mntestine . A recenntestine . In micentestine ,57.ZIP14 mutations developed manganese toxicity and early-onset dystonia [ZIP8 mutations, a majority of ZIP14 mutation cases stem from consanguineous families. The onset of the disease occurs at infancy or during early childhood and are evident with loss of developmental milestones, progressive dystonia, bulbar dysfunction, spasticity, limb contractures, scoliosis, and loss of independent ambulation, with some showing parkinsonian features of hypomimia, tremor, and bradykinesia. The detailed information about identified human ZIP14 mutations are listed in The physiological function of ZIP14 became clear with the identification of human mutations. Patients carrying dystonia ,59,60,61Zip14 knockout mice (Zip14\u2212/\u2212) had markedly increased manganese levels in the blood and brain [Significant insights regarding the disease mechanisms underlying manganese toxicity induced by ZIP14 loss have been gained from animal models with ZIP14 deficiency. ZIP14-deficient zebrafish hyper-accumulated manganese in the brain, but not in the liver, and presented with reduced locomotor activities ; whole-bnd brain ,63,64,65nd brain ,64,65. Tnd brain ,62,63,65Zip14 knockout mice (Zip14-L-KO) had significantly decreased manganese in the liver, confirming the essential function for ZIP14 to import manganese to hepatocytes; however, even with reduced manganese uptake into the liver, Zip14-L-KO mice had normal manganese levels in the blood and other tissues [Under normal dietary conditions, hepatocyte-specific tissues ,66. Thes tissues ,66.Zip14 knockout mice (Zip14-In-KO) were generated. In contrast to Zip14-L-KO mice that did not develop manganese overload in the body, Zip14-In-KO mice developed increased manganese in both the liver and brain under normal dietary conditions, verifying the importance of intestinal ZIP14 in maintaining systemic manganese homeostasis [In addition to the liver, ZIP14 is highly expressed in the small intestine. To examine ZIP14\u2019s function in enterocytes, ZIP14 knockout Caco-2 cells were created . Caco-2 eostasis ,67. BaseZnT10 mutations.ZnT10 is a member of the Zinc transporter (ZnT) family proteins. In contrast to the function of ZIP members that increase cytosolic metal levels, ZnT proteins function to efflux metals from the cytoplasm either out of the cell or into intracellular organelles ,69. As tZnT10 when two additional patients, presented with a similar clinical phenotype, were reported in 2012 [ZnT10 mutations as the causal gene defect in two consanguineous families with neurological disorders and juvenile-onset severe manganese overload [ZnT10 mutations are evident with phenotypical features of hypermanganesemia with dystonia. Genetic evaluations revealed that in the majority of identified cases, both parents of the affected individuals were heterozygous carriers of the same mutations found in their children. Neurological symptoms can occur during childhood (early-onset) or adulthood (adult-onset). In early-onset cases, children will experience symptoms that include dystonia in the arms or legs, involuntary trembling, unusually slow movement, and slurred speech. In adult-onset, symptoms include movement abnormalities, tremor, extraordinarily slow motion, muscle rigidity, and postural instability [ZnT10 mutations are listed in In 2008, a clinical case report first described a 12-year old girl with severe dystonia and significantly elevated blood manganese . No hist in 2012 . At the overload . Individtability . Additiotability ,79,80. DZnT10 mutations indicated that ZnT10 plays a pivotal role in regulating manganese homeostasis. While blood manganese concentrations are notably higher in individuals carrying homozygous ZnT10 mutations, zinc and iron concentrations in the blood either remain normal or show subtle changes compared to the standard values [ZnT10 mutations also present with high levels of hepatic manganese [Znt10 knockout (Znt10\u2212/\u2212 mice) developed markedly elevated manganese in the liver, brain, and blood, with no changes or minor changes in body iron and zinc contents [The identification of human d values ,33,81, fanganese . Consistanganese ; mice wicontents ,85,86.Znt10 knockout mice (Znt10-L-KO) were generated [Znt10-L-KO mice had almost no manganese excretion through the bile, confirming that ZnT10 is essential for biliary manganese excretion [Znt10\u2212/\u2212 mice that had 20\u201360 times more manganese in different tissues of the body, Znt10-L-KO mice only developed minimal excess of tissue manganese (about 2-fold increase compared to controls), indicating that ZnT10 in other organs may compensate for the loss of hepatic ZnT10 [Because immunohistochemistry analysis of human liver tissues found that ZnT10 was abundant in hepatocytes and localized both intracellularly and on the plasma membrane facing the bile duct, it was initially postulated that the primary function of ZnT10 is to remove manganese from hepatocytes for biliary excretion and that the systemic manganese hyperaccumulation observed in individuals lacking ZnT10 is secondary to the defect of hepatic ZnT10 . To deteenerated ,87. Aftexcretion and mice with Znt10 deletion in both hepatocytes and enterocytes (Znt10 double knockout (Znt10-DKO)) were created [Znt10-L-KO mice, Znt10-In-KO mice only had moderate manganese accumulation in the body, despite reduced manganese export into the intestinal lumen. With a combined deletion of ZnT10 in hepatocytes and enterocytes, Znt10-DKO mice developed manganese overload that was more severe than that seen in Znt10-L-KO mice or Znt10-In-KO mice, but less severe than the manganese loading observed in the whole-body Znt10\u2212/\u2212 mice. Together, these results demonstrated that hepatic and intestinal ZnT10 both contribute to the regulation of manganese homeostasis and that in addition to the liver and intestine, other organs with ZnT10 expression also contribute to the regulation of systemic manganese homeostasis. Future studies are required to determine ZnT10\u2019s function at other sites.Besides the liver, the intestine also expresses high levels of ZnT10. To investigate the function of ZnT10 in enterocytes, polarized Caco-2 cells grown on Transwell inserts were used as a model to examine manganese transport. Immunofluorescence analysis and manganese transport assay indicated that ZnT10 localizes to the apical domain of Caco-2 cells and mediates the efflux of intracellular manganese at the apical membrane . This fu created . SimilarMaintaining manganese balance within the body is essential for health. The discoveries of ZIP8, ZIP14, and ZnT10 as crucial manganese transporters have opened new paths towards a better understanding of manganese homeostatic regulation. Experiments using genetically modified animal models with global and tissue-specific knockouts of these genes have provided significant insights into the molecular basis of how deficiency in these genes leads to disorders of manganese metabolism.The systemic manganese homeostasis is regulated mainly by intestinal absorption and hepatobiliary excretion. In enterocytes of the intestine, ZIP14 localizes to the basolateral membrane and imports extracellular manganese from the circulation into enterocytes, while ZnT10 functions at the apical membrane to export intracellular manganese into the lumen of the intestine. These two transporters function in the secretory direction to restrict the amount of manganese being absorbed into the blood A. With iIn hepatocytes of the liver, ZIP14 is expressed at the basolateral membrane to import circulating manganese into hepatocytes, while ZnT10 localizes at the apical canalicular membrane to export manganese into the bile. Both transporters work in the same direction to facilitate manganese removal through biliary excretion B. As a t"} +{"text": "Aspergillus oryzae, commonly known as koji mold, has been widely used for the large-scale production of food products and can accumulate a high level of lipids. In the present study, we showed the dynamic changes in A. oryzae mycelium growth and conidia formation under nitrogen and phosphorus nutrient stress. The fatty acid profile of A. oryzae was determined and the content of unsaturated fatty acid was found increased under nitrogen and phosphorus limitation. Oleic acid (C18:1), linoleic acid (C18:2), and \u03b3-linolenic acid (C18:3) production were increased on five nitrogen and phosphorus limitation media, especially on nitrogen deep limitation and phosphorus limitation group, showing a 1. 2\u2013, 1. 6\u2013, and 2.4-fold increment, respectively, compared with the control. Transcriptomic analysis showed the expression profile of genes related to nitrogen metabolism, citrate cycle, and linoleic acid synthesis, resulting in the accumulation of unsaturated fatty acid. qRT-PCR results further confirmed the reliability and availability of the differentially expressed genes obtained from the transcriptome analysis. Our study provides a global transcriptome characterization of the nitrogen and phosphorus nutrient stress adaptation process in A. oryzae. It also revealed that the molecular mechanisms of A. oryzae respond to nitrogen and phosphorus stress. Our finding facilitates the construction of industrial strains with a nutrient-limited tolerance. Fatty acids (FAs) are carboxylic acids with long aliphatic chains, which may be straight or branched, saturated or unsaturated, and the majority of FAs generally found in nature have 6\u201318 carbon atoms . FAs are16 and C18, containing oleic, palmitic, linoleic, and stearic acid , linoleic acid, and linolenic acid in the biomass of Trichoderma viride NRC 314, up to 30, 23, and 13% of total fatty acids (TFAs), respectively, over other fungal strains required to improve the lipid production by Penicillium brevicompactum were also obtained , followed by alpha-linolenic acid and arachidonic acid and adenosine mono-, di-, tri-phosphate . Both of them are directly involved in FA synthesis, as reductant and energy transfer molecules, separately was attained at the lipid accumulation stage under medium nitrogen treatments , both of which have essential functions in the prevention and treatment of the brain or cardiovascular disease. Similarly, Porphyridium cruentum, nitrogen sufficiency and phosphorus deprivation, nitrogen sufficiency, and phosphorus limitation, respectively. Additionally, several exploitations have successfully increased the content and proportions of FAs by adopting distinct concentrations and ratios of nitrogen and phosphorus, such as green microalga Dunaliella tertiolecta, Isochrysis zhangjiangensis, and Porphyridium cruentum , and stearic acids in the ALE_70 and ACA DC 50109 strain revealed that Se seems to influence the activity or the expression of saturases, desaturases, and elongase in both strains.Culturing oleaginous microorganisms under the condition of these nutrients are sufficient or limited and are the main part of nutrient stress strategies for the increment of FA production. In yeast strains, UFAs production has also been studied for over 80 years. eatments . Nitrogecruentum . Except sources . Concomimulation . MoreoveA. oryzae, commonly known as koji mold, is a safe production filamentous fungus identified by the FDA and WHO, which has been used in the manufacturing of oriental fermented foods and fungal secondary metabolites for thousands of years , bean curd seasoning, vinegar, miso (soy bean paste), and shochu (distilled spirits) to break down the bigger molecules such as starches, carbohydrates, proteins, and fats, and they are being reduced into smaller monosaccharides, amino acids, fatty acid chains, etc . Additioins, etc . These eins, etc . Moreove enzymes .A. oryzae metabolism and related regulations during koji fermentation remain only partially understood, inhibiting targeted strain improvement, and effective strain maintenance against industrial strain degeneration. Therefore, precise physiological knowledge of A. oryzae metabolism and regulations is needed. Compared with the detailed experimental evidence for oleaginous microorganism nutrient demands for improving the content of UFAs, such as carbon, nitrogen, and phosphorus, there is still no consistent theoretical framework for the relationship between A. oryzae nutrient limitations or responses and FA biosynthesis. To elucidate the adaptive mechanisms of A. oryzae toward nutrient stress to improve the nutrient stress tolerance of A. oryzae strains, obtain highly stable engineered strains support for the industrial production and application, and methods that can maintain stable FA production in industrial microbial processes, here, we presented a case study of the effect of nutrient stress on FA content in oleaginous filamentous fungi A. oryzae.Until now, the molecular mechanisms of Aspergillus oryzae strain 3.042 (CICC 40092) was obtained from the China Center of Industry Culture Collection and used to check the response to nitrogen (N) and phosphorus (P) limitation. The basal Czapek\u2013Dox (CD) medium was used. First of all, A. oryzae strain was pre-cultivated on a CD plate at 30\u00b0C for 3 days. According to the previous methods, spores were then harvested after 3 days of culture for follow-up experiments, and the conidia concentration was determined by using a hemocytometer . All of these media were based on the CD medium, and the basal CD culture medium was used as the CK. Sodium nitrate and potassium dihydrogen phosphate were used for N and P limitation, and their initial concentrations in all media are shown in 7 conidia was inoculated and cultured on distinct treatment plates covered by cellophane fresh at 30\u00b0C for 3 days. Each treatment was performed in three groups for the determination of biomass, spore density, and extraction of intracellular fatty acid. At least three replicates were carried out for each nutrient-limited group. It was followed by scraping, and then the fungal mycelia was dried overnight at 60\u00b0C for the biomass determination, and the density of spores was determined by a hemocytometer.Seven media recipes were used in this study, including both N and P sufficiency , N limitation and P sufficiency (N-), N deep limitation and P sufficiency (N- -), N sufficiency and P limitation (P-), both N and P limitation (N-P-), N deep limitation and P limitation (N- -P-), and both N and P deprivation . FAME components were separated and analyzed using coupled QP2010 gas chromatography-mass spectrometry (GC-MS) . The system was equipped with Supelco SP-2340 fused silica capillary column . FAMEs were identified by comparing their mass spectra with a spectrum database. FA peaks were identified based on comparisons of their retention times to the external standards or similarity search. The relative amounts of individual FA components were performed by using the peak area of the most intensive ion of each peak, and the percentage of UFAs was also calculated database to get the final clean reads, which were further employed for assembly and transcriptome analysis according to the protocol of the manufacturer, coupled with DNA digestion. NanoDrop ND-1000 spectrophotometer and Bioanalyzer 2100 were employed to analyze RNA concentration and integrity. To ensure reliability and reproducibility, equal quantities of RNA of each pool from three individual cultures were used for cDNA library construction. Thereafter, mRNA was enriched by Oligo(dT) beads. The enriched mRNA was then fragmented into several short fragments by fragmentation buffer at 94\u00b0C for 5 min and then reversely transcripted into cDNA using random primers. Using these mRNA fragments as templates, the first-strand and second-strand cDNA were synthesized through our previously described methods . The cDNanalysis . RNA-SeqA. oryzae 3.042 reference genome using Tophats2 (v2.0.3.12) . The RSE.0.3.12) . Differe.0.3.12) . The log.0.3.12) . Then thA. oryzae, were selected for real-time RT-PCR validation. Total RNA was extracted with E.Z.N.A. Fungal RNA Kit according to the protocols of the manufacturer. Reverse transcription of each RNA sample was performed to get the first-strand cDNA using the PrimeScript RT reagent Kit with gDNA Eraser . Real-time RT-PCR was performed using Real-Time PCR System (Bio-Rad). GAPDH served as the reference gene for normalization of the target gene expression and to correct for variation between samples. The thermal cycle for RT-PCR was as follows: 95\u00b0C for 2 min, followed by 40 cycles of 95\u00b0C for 10 s, 60\u00b0C for 15 s, and 72\u00b0C for 20 s. Melting curve analyses of the amplification products were performed at the end of each PCR reaction to ensure that only specific products were amplified. Primers used for the candidate genes are designed according to the Illumina sequencing data by using Primer Premier 5 and listed in \u2013\u0394\u0394CT method was employed to calculate the relative expression between the target genes.To validate the transcriptional level results from RNA-Seq data analysis, four genes including D9D1, D9D2, D12D1, and D12D2, which are involved in the linoleic acid biosynthesis in p < 0.05. Moreover, all the difference analysis performed in this study was targeted between the control and nutrient-treated groups.Three independent experiments were performed, and all of the data in this study are presented as mean \u00b1 SE of three replicates. Data from the same period were evaluated by one-way nested analysis of variance (ANOVA), followed by the least significant difference test (LSD) for mean comparison. All statistical analysis was performed with SAS 9.20 software at A. oryzae, a preliminary study of different concentrations of nitrogen and phosphorus treatments was conducted. After incubation for 48 h, the dry biomass of A. oryzae mycelioid colonies of the nitrogen and phosphorus-treated group were different from the control is present in A. oryzae strain was not growing on the group, and thus, this group was excluded from the follow-up analysis. Overall, nitrogen and phosphorus limitation inhibited the dry biomass of A. oryzae to varying degrees, and thus, repressed cell growth. The density of spores of A. oryzae showed significant differences between the nitrogen and phosphorus limitation and the control , and palmitic acid (20:0), and 2-decyltetradecanoic acid (C24:0) were decreased in varying degrees. Intriguingly, all these four FA contents on P- medium surpassed another four media and less than the control. This result showed that the effect of inhibition on FA contents caused by single phosphorus limitation is weaker than single nitrogen and other coupled limitation. Contrary to four FAs, the contents of oleic acid, linoleic acid, and \u03b3-linolenic acid were increased on five nitrogen and phosphorus limitation media compared with the control. Especially on nitrogen deep limitation and phosphorus limitation group, the productions of these three UFAs were increased by 1. 2\u2013, 1. 6\u2013, and 2.4-fold, respectively, compared with the control. In addition, it is worth noting that the contents of these three FAs on P-medium are below other media. Additionally, a major change was observed in the degree of fatty acid unsaturation ; P1, the single P limitation (P-); NP, N deep limitation and P limitation (N- -P-)], were 934, 544, 861, 1,094, 1,093, and 785, respectively . There wstructed . It was Based on the KEGG enrichment analysis, nitrogen metabolism and citrate cycle is a general metabolic pathway involved in FA formation. A total of 18 DEGs encoding 11 enzymes were identified for the different steps in nitrogen metabolism. There were 3, 3, 2, 2, and 2 genes encoded by acetylcholinesterase (EC 1.7.7.2), carbamoyl-phosphate synthase (EC 6.3.4.16), glucokinase (EC 1.7.2.5), glutamate dehydrogenase (EC 1.4.1.2), and glutamine synthetase (EC 6.3.1.2), respectively; all of the other enzymes are encoded by single genes . Apart fThe TCA cycle is an important aerobic pathway for the final steps of the oxidation of carbohydrates and FAs. A total of 22 DEGs encodings 10 enzymes were identified for the different steps in carbohydrate metabolism. Except for pyruvate dehydrogenase, citrate synthases, isocitrate dehydrogenase, and succinate dehydrogenase, which are encoded by 4, 3, 3, and 3 genes separately, the other enzymes are encoded by single or two genes . At the A. oryzae linoleic acid biosynthesis were identified. They encoded delta 9 fatty acid desaturases (D9D) and delta 12 fatty acid desaturases (D12D), which were rate-limiting enzymes in the biosynthesis of UFA and PUFAs, respectively. Analysis of gene expression levels for D9D and D12D revealed that the four genes involved in linoleic acid biosynthesis were upregulated in response to nitrogen and phosphorus limitation, indicating that UFA synthesis was enhanced in A. oryzae cells in response to different levels of nitrogen and phosphorus limitation were significantly higher than nitrogen and phosphorus limited media and decreased with the decrease in nitrogen concentration which play a buffering role in the growth media, and their low levels led to the decrease in pH. Low pH as a consequence of the low phosphorus source, thus, negatively affected the growth of oleaginous Mucoromycota fungi were identified on the condition of phosphorus limitation, the lowest among all treatments, among which, 304 were upregulated genes and 240 were downregulated genes of stearic acid was converted into linoleic acid in control . Consistae fungi . Generalae fungi . The oveae fungi . It was revisiae . Furtherturation under th control . It is an levels , which is active .A. oryzae mycelium growth and conidia formation over time under nitrogen and phosphorus nutrient stress. The improvement of linoleic acid synthesis was characterized, and the understanding of the mechanisms involved in the nutrient stress of A. oryzae was also improved. Transcriptomic analysis showed high expression levels of the genes related to linoleic acid synthesis, resulting in a corresponding intracellular accumulation of linoleic acid. Our study provides a global transcriptome characterization of the nitrogen and phosphorus limitation adaptation process in A. oryzae. It also laid a solid foundation for further investigation to explore the relationship of UFA accumulation and nitrogen and phosphorus limitation in A. oryzae.In summary, we successfully characterized the transcriptomic profiles, which uncovered new aspects of the dynamic changes in https://www.ncbi.nlm.nih.gov/sra, Bioproject: PRJNA748192; Biosamples: SAMN20309416-SAMN20309419).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI/SRA database (GL conceptualized the study and wrote and prepared the original draft. YX conceptualized the study. XC and YT were in charge of the project administration. BZ supervised the study and acquired the funding. JH supervised the study. BH wrote reviewed, and edited the manuscript, and acquired the funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Hyperthermic intrathoracic chemotherapy (HITOC) is an additive, intraoperative treatment for selected malignant pleural tumors. To improve local tumor control, the thoracic cavity is perfused with a cisplatin-containing solution after surgical cytoreduction. Since cisplatin is probably carcinogenic to humans, potential contamination of surfaces and pathways of exposure should be systematically investigated to enable risk assessments for medical staff and thus derive specific recommendations for occupational safety.Wipe sampling was performed at pre-selected locations during and after ten HITOC procedures, including on the surgeon's gloves, for the quantitation of surface contaminations with cisplatin. After extraction of the samples with hydrochloric acid, platinum was determined as a marker for cisplatin by voltammetry.2, IQR: 9.36\u00a0pg Cis-Pt/cm2) and perfusionists\u2019 gloves. The display of the perfusion device showed partially elevated levels of cisplatin up to 4.92\u00a0pg Cis-Pt/cm2 and thus could represent an origin of cross-contamination. In contrast, cisplatin levels on the floor surfaces in the area of the surgeon and the perfusion device or in the endobronchial tube were relatively low.High median concentrations of cytostatic drugs were detected on the surgeons\u2019 (1.73\u00a0pg Cis-Pt/cmWith a correct use of personal protective equipment and careful handling, intraoperative HITOC appears to be safe to perform with a low risk of occupational exposure to cisplatin. In Germany, about 350 of these procedures have been performed in 10\u00a0years by 17 specialized clinics, and the number is increasing were excluded. The primary endpoint of the study was the potential exposure of the surgical staff to cisplatin via surfaces during HITOC. For this purpose, a quantitative detection of platinum as a marker for the cytostatic drug cisplatin on defined surfaces in the OR was performed. The approval of the ethics committee of the University Regensburg was obtained (Reference number: 20-1698-101). Informed consent was waived, as the examinations do not affect the patient or change the procedure of the HITOC and only serve quality assurance and safety at work.This prospective observational study considered all patients who received HITOC after surgical cytoreduction between January 2020 and November 2020 in the University Hospital Regensburg and the Hospital of Barmherzige Br\u00fcder Regensburg. Patients with less than 60\u00a0min of intrathoracic perfusion or an intrathoracic dose of cisplatin other than 175\u00a0mg/m2 body surface area in all patients. After completion of the 60-min HITOC, the perfusion volume was passively released and the drainages were connected to the chest drainage units. The tube and reservoir system were disposed in dedicated containers for cytostatic drugs and the perfusion device was cleaned. The patient was regularly extubated in the OR. The duration between completion of the HITOC and subsequent cleaning of the entire OR usually is about 20\u00a0min.The HITOC was conducted after the resection of the pleural tumor manifestations, which was performed always with the aim of a macroscopic complete tumor resection. The thoracotomy was closed after insertion of one inflow and two to three outflow drains. After connecting the drainages to the perfusion system, the patient's thorax was evacuated by adding a priming volume of 3\u20134\u00a0l isotonic (0.9%) sodium chloride solution to remove air until a stable perfusion circuit was obtained. Once a temperature of 42\u00a0\u00b0C was reached at the outflow drainage, the chemotherapeutic agents were injected into the circulation as a bolus. The total volume of solution in the circulation varied depending on the individual dimensions of the thoracic cavity. The dosage of cisplatin was 175\u00a0mg/m2), the floor area below the perfusor (900 cm2), the floor area below the surgeon (900 cm2), the inside of the breathing tube and the outside surface of both gloves of the surgeon and the perfusionist directly after the end of the perfusion of the perfusion device were added directly to the sample after initial the voltammetric analysis. Finally, platinum levels of the samples were multiplied by 1.54 to obtain cisplatin levels.Total platinum (Pt) concentrations in wipe samples were determined as a marker for cisplatin. For analysis, the wipe samples were extracted with 2% hydrochloric acid for 1\u00a0h and analyzed by voltammetry as described previously . Blank values were subtracted from the results. Afterwards, statistical analysis was performed using IBM SPSS Statistics, Version 26 . All data are presented as median and interquartile range (IQR). Mean platinum levels were tested against guidance values reported in the literature using a one-sample The results for each sampling location are given in Table 2) followed by the gloves of the perfusionist (0.69\u00a0pg Cis-Pt/cm2). However, the results showed a high variance. In five samples (50%) cisplatin levels on the surgeon's gloves showed a moderate contamination and four (40%) were severely contaminated (>\u20096\u00a0pg Cis-Pt/cm2). The gloves of the perfusionist showed significantly lower contamination. In fact, only three samples (30%) were moderately contaminated. In the one-sample t test the surface contamination of the perfusionist\u2019s glove lies significantly below the reference value of a severe contamination were found on the surgeon\u2019s gloves and one severe (10%) contaminations were observed at this location in the cleaning sample already. After HITOC, there were only three (30%) samples found in moderate contamination ranges. Comparing the values of the display before and after HITOC, no clear trend is apparent since the individual value of the sample before perfusion was sometimes even higher than after HITOC Fig.\u00a0. The mea0% and on2 in 90% of cases. In one case (10%) the cisplatin level next to the perfusion device increased from 0.24\u00a0pg/cm2 pre-HITOC to 13.62\u00a0pg/cm2 post-HITOC which represented a severe contamination. One sample (10%) of the opposite floor surface was classified as moderate contamination. The data revealed no clear trend between cleaning, pre-surgery and post-surgery samples .In the floor area next to the perfusion device as well as on the opposite side, platinum could be detected both before and after the HITOC. At both locations, however, observed values were low after HITOC and below the lower reference range of 0.9\u00a0pg Cis-Pt/cm2). Therefore, the differences in the observed cisplatin concentration before and after HITOC consisted of minimal changes in the absolute values. A conversion to a surface concentration would disproportionately represent these minimal deviations, so that the absolute values are shown. Absolute levels of cisplatin (median: 0.15\u00a0ng Cis-Pt) in the endobronchial tube were relatively low, but higher than the blank sample (0.05\u00a0ng Cis-Pt).After extubation, the endobronchial tube was wiped endoluminally. The tube thus contained condensed breathing air from the patient. Compared to the other investigated locations, the wiped area in the endobronchial tube was very small and Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC), can result in substantial contamination of OR surfaces with the applied cytostatic drugs. For both surgical methods, surface contaminations were observed on the perfusion device, the floor and the gloves of surgeons and perfusionists. However, the studies showed that surgeries with overall low contaminations and occupational exposure are possible if adequate occupational safety and cleaning standards are ensured during and after these surgical procedures on the reservoir of the perfusion device and 75th (4\u00a0pg Pt/cm2 \u2259 6\u00a0pg cisplatin/cm2) percentiles of the platinum analysis in 1008 wipe samples as guidance values to compare the results between different locations , all other localizations were below it. Since they are disposed of immediately after HITOC, the risk of exposure and cross-contamination in clinical practice can be presumed to be low if the gloves are disposed properly. Besides a perfusion on the closed chest performed in this study, perfusion with an open chest is also performed in some clinics internationally (Ried and Hofmann 2) and the floor next to the perfusion device (13.62 vs 0.24\u00a0pg/cm2) were clearly higher compared than in samples taken at the start of the procedure. This may be explained by an accidental spillage of the cisplatin solution during the procedure and subsequent cross-contamination. In fact, cisplatin levels of the perfusionist\u2019s glove (2.00\u00a0pg/cm2) from the same day were relatively high, too. These pre-surgery and post-surgery samples in our samples should be addressed by a thorough cleaning procedure of the perfusion device after HITOC. We recommend cleaning of critical surfaces using a two-step procedure by first using an aqueous cleaning agent followed by an alcoholic solution (e.g. 70% isopropanol) directly after finishing HITOC.In our study, the wipe samples of the surgeon\u2019s gloves showed the highest level of contamination. This localization was also the only one with a median contamination level above the reference value of 0.9\u00a0pg Cis-Pt/cm2 after the procedure. Again, this may be caused by an accidental spillage during the perfusion or undetected leakage of surgical wounds.The floor areas showed similar values in median after HITOC both close to the perfusion device and further away, but the variability of contamination close to the perfusion device was considerably higher. This can be explained by the fact that the cytostatics were introduced into the circulation directly above this test area, thus posing a risk of unintentional spillage of the substances. In one HITOC, cisplatin levels on the floor next to the surgeon increased from 0.10 at the start to 1.14\u00a0pg/cmWe also investigated the potential contamination of the breathing tube as a potential source of airborne exposure to cisplatin. Therefore, we wiped the endobronchial tube endoluminally and thus collected condensed exhaled breath of the patient. The endobronchial tube showed cisplatin levels above the blank value, although in very small amounts. Since the measurement was taken in a distance from the distal end of the tube (>\u200910\u00a0cm) it can, therefore, be assumed that patients' breathing air contained cisplatin during/after HITOC. Due to its high molecular weight, cisplatin's vapor pressure is low, and so is its tendency to form aerosols (Connor et al. Hyperthermic intrathoracic chemotherapy is generally safe to perform, but there are critical areas to consider. After each use, the perfusion device should be thoroughly cleaned in a two-step procedure by first using an aqueous cleaning agent followed by an alcoholic solution. The risks of direct contact with the cytostatic drug can be minimized very well by a correct use of personal protective equipment and careful handling. During and after HITOC, the protective equipment should include gloves, reinforced gowns, and safety glasses to prevent dermal uptake. Gloves should be worn for any patient contact or contact with near-patient surfaces and disposed of immediately afterwards.In this study, only selected sites were monitored that the authors felt to be especially relevant for direct contact to cisplatin or as an origin of cross-contamination. No systematic ambient air study has been conducted to evaluate airborne transmission of cisplatin. The examination of the endobronchial tube in this regard represents only indirect evidence of aerosol-bound transmission. Although our results suggest that the risk of an accidental exposure of the OR personnel to cisplatin can be considered to be very low, biomonitoring would have been necessary to ultimately exclude any uptake of cisplatin."} +{"text": "N = 1,129) in German and English. Confirmatory factor analyses supported the four-factor structure, as well as scalar invariance, of the different language versions. Moreover, the subscales showed convergent and divergent validity with related constructs such as requirements for problem solving or autonomy. The criterion validity for emotional exhaustion, engagement, positive work rumination, negative work rumination, and problem-solving pondering suggested that cognitive demands of flexible work can be construed as challenge demands. However, relationships with emotional exhaustion were not significant. Overall, the CODE scale was shown to be a reliable and valid instrument to measure cognitive demands of flexible work.With globalization, digitalization, and the spread of information and communication technologies, rules regulating work have been softened or completely abolished. Consequently, employees face additional cognitive demands to plan, structure, and coordinate their work. To capture these demands of contemporary work, we constructed and initially validated the Cognitive Demands of Flexible Work (CODE) scale. The scale comprises four subscales . We initially validated the scale in three independent studies (overall The organization of work and the associated working conditions change according to societal and technological developments scale. To validate the scale, we will assess construct validity and reliabilities and show that the factor structure of the CODE subscales is the same across German and English versions. We will further test convergent and divergent validity with other job-related factors, as well as criterion validity with work outcomes. Finally, we will investigate whether the means of the CODE subscales differ between groups of employees that should be exposed differently to flexible work environments.The last decades have brought significant growth in globalization and global operations , we will focus in the following on the aspects of flexible work that reflect deregulated working conditions in terms of how work is conducted. Many employees in today's companies and organizations are exposed to different forms of flexible work, such as flexible working hours, telework , there has been a shift from standard operating procedures to goal-oriented performance, where work activities and coordination with others are also no longer determined only by the organization.Such deregulation of work does not necessarily mean that control in organizations has become irrelevant. Rather, employees, instead of management, are required to take control. Thus, control has become increasingly indirect by being transferred to the employees . All items were positively worded to avoid artificial factor solutions, which could occur when including negatively worded items alongside positively worded items within the same scale . Information processing at work refers to requirements to monitor and process data or other information in general, whereas problem solving is characterized by more specific requirements to actively process information, come up with ideas and solutions to solve non-routine problems, and deal with errors . Both decision-making autonomy and work methods autonomy can be described as facets of general job autonomy that reflect how much freedom, independence, and discretion employees have at work . Work scheduling autonomy is also considered an aspect of general job autonomy . Both working from home and telework from other locations can be considered aspects of flexplace that describe alterations in the physical boundaries around work . Interdependence can be described as the extent to which a job is connected with that of others. It is oftentimes differentiated into initiated interdependence, i.e., the extent to which others depend on one to finish one's work, and received interdependence, i.e., the extent to which one's work depends on others to finish their work . Our protocol fully complied with the ethical principles of psychologists and the code of conduct of the American Psychological Association . Particiwww.respondi.com). This panel recruits its participants via offline and online methods and ensures high quality through minimizing participation frequency and focusing on intrinsic motivation instead of financial dependency.The sample consisted of employees from Germany working at least 30 contractual hours per week that were accessed via an ISO 26362-certified online panel (SD = 11.11); they worked on average 41.70 h per week (SD = 6.09) and had worked for an average of 11.37 years (SD = 10.11) for their current company. About half (49.2%) of the participants held an academic degree.In total, 303 employees participated in study 1. About half (51.5%) of the sample was female. The average age of the participants was 43.13 years .Participants responded to the initial set of 24 items generated for the CODE scale. They were asked to indicate on a 5-point scale how the statements of the generated items applied to their work in general . The English version of the items can be found in the Appendix of Morgeson and Humphrey on a 7-point rating scale . As both items were highly correlated (r = 0.85), we combined them to create a score for the availability of flextime.Availability of working from home and availability of telework from other locations were each measured using a single self-created item. Participants were asked to indicate whether their employer allowed them to work from home or other locations outside their employer's premises. If they indicated that they were allowed to do so, they were also asked to indicate for how many hours per week they were allowed to work from home or other locations outside their employer's premises. We divided these numbers by the contractual working hours to get percentage scores that indicated how much of the participants' working time was available to work from home or telework from other locations.We conducted exploratory factor analyses in Mplus 8.3 techniques based on maximum likelihood estimation in Mplus 8.3 , we conducted a series of confirmatory factor analyses compare . For eacThe goal of the second study was to translate the newly created scale for measuring cognitive demands of flexible work from German to English and check for invariance between the two versions. Further, this study aimed to gather information on the criterion validity of the newly developed scale.Drawing on challenge\u2014hindrance stressor literature . Our protocol fully complied with the ethical principles of psychologists and the code of conduct of the American Psychological Association . ParticiThe sample consisted of employees from the United Kingdom working at least 30 contractual hours per week and was recruited via the same certified online panel that had been used in the first study.SD = 10.64); they worked on average 37.53 h per week (SD = 3.46), and they had worked on average 7.41 years (SD = 6.86) for their current company. About half (47.4%) of the participants had a leadership position, while slightly more than half (58.4%) of the participants held an academic degree.In total, 274 employees participated in study 2. About half (49.1%) of the sample was female. The average age of the participants was 41.22 years .The items were first translated from German to English by native speakers in both languages using a back-translation method. Participants responded to the translated version of the CODE scale in English. They were asked to indicate on a 5-point scale how the statements of the generated items applied to their work in general . A sample item is, \u201cI feel emotionally drained from my work.\u201dEmotional exhaustion was measured with 3 items that we selected from the Maslach Burnout Inventory . A sample item is, \u201cAt my work, I feel bursting with energy.\u201dPositive work rumination and negative work rumination were assessed by adapting a measure that Frone (does not apply to 5 = fully applies). We adapted the measure by Frone . We adapted the measure by Cropley et al. . Our protocol fully complied with the ethical principles of psychologists and the code of conduct of the American Psychological Association . ParticiParticipants of study 3 were employees working at the headquarters of an Austrian company, where an activity-based flexible office system had been introduced recently. Activity-based flexible office systems are characterized by their offering of different work locations that match the requirements of different tasks and work activities. They are usually open-office environments complemented by open, half-open, and enclosed working locations, without assigned workstations, that provide space for concentrated work, as well as for collaboration and conversation in different areas ; they worked on average 42.03 h per week (SD = 8.27) and had worked 14.09 years (SD = 13.44) for their current company. About a quarter (24.7%) of the participants had a leadership position, while about half (49.5%) of the participants held an academic degree.In total, 552 employees participated in study 3. About half (42.3%) of the sample was female. The average age of the participants was 43.63 years .Participants responded to the CODE scale in German. They were asked to indicate on a 5-point scale how the statements of the generated items applied to their work during the past 3 months , and we measured the use of flextime and use of flexplace using a self-created item for each on a 5-point rating scale .Information on whether participants had a To check for internal consistency in the organizational sample, we calculated Cronbach's alphas structuring of work tasks, (2) planning of working times, (3) planning of working places, and (4) coordinating with others. Based on data from samples of employees in Germany, the United Kingdom, and Austria, the CODE scale proved to be a reliable and valid measure.Results from confirmatory factor analyses supported the four-factor structure in three separate studies, and measurement invariance tests suggested that even scores can be compared across the English and German versions of the scale. Convergent and divergent validity with information processing and problem solving, as well as with other closely matched constructs, were quite satisfactory overall. Tests for criterion validity indicated that the CODE scale was not significantly associated with emotional exhaustion, although analyses regarding associations with work engagement, positive work rumination, negative work rumination, and problem-solving pondering showed satisfactory results overall.Because the cognitive demands of flexible work did not show a significant positive relationship with emotional exhaustion in our study, the question arises whether the CODE scale can really be considered a measure of job demands or whether\u2014although we made sure that items were worded in terms of the requirements placed on employees\u2014it represents a measure of job resources. As a reviewer noted, it also seems possible that the CODE scale captures job autonomy more neutrally than established scales that tend to have a rather positive appraisal of job autonomy. Although providing a more neutral measure of job autonomy might in itself be a relevant contribution to the literature, we think that the CODE scale represents a measure of job demands rather than job resources.The main difference between job demands and job resources lies in their relationships with indicators of work strain , there are also some limitations that need to be considered. In our efforts to initially validate the scale, we relied on cross-sectional data that did not allow us to infer causality from the observed relationships between the constructs. For example, it could be that cognitive demands of flexible work do not promote work engagement but rather that organizations tend to grant highly engaged employees more autonomy and flexibility P29408-G29.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": "We report a 15\u2010year\u2010old male with hypoplastic left heart syndrome (HLHS) after Fontan operation with recurrent, drug\u2010resistant atrial tachycardia. With the use of electro\u2010anatomical mapping system (EnSite) an atrial flutter (AFl) with reentry activation around the tricuspid valve was diagnosed. Successful radiofrequency catheter ablation (RFCA) was performed. Since its inception, the procedure has undergone a lot of modifications to improve hemodynamics, which resulted in longer life expectancy of these patients. Despite the improvement in surgical and interventional techniques, many patients after Fontan operation develop atrial tachycardia (AT) with increasing frequency of arrhythmia episodes during the follow\u2010up that poorly respond to pharmacological treatment. Although radiofrequency catheter ablation (RFCA) could be an optimal invasive treatment for supraventricular tachyarrhythmias, the limited access because of multiple surgical interventions in patients with complex congenital heart diseases may be a challenge.We report a case of a child with lateral tunnel Fontan and an epicardial pacemaker who underwent RFCA for drug resistant atrial flutter (AFl).2A 15\u2010year\u2010old child with complex congenital heart disease, after multiple hospitalizations because of persistent, drug resistant supraventricular tachyarrhythmia was admitted to a high\u2010volume tertiary care university center presenting atrial tachycardia with ventricle response of about 130 bpm. He had been diagnosed as a newborn with a hypoplastic left heart syndrome, mitral stenosis, and aorta atresia, initially palliated with a modified Norwood operation in 2003, followed by Rashkind operation and balloon angioplasty of coarctation of the aorta 3 months later. Then he underwent hemi\u2010Fontan and Fontan operation followed by tricuspid valve and left pulmonary artery plasty. Because of a Fontan operation, he developed sick sinus syndrome which was treated by epicardiac dual chamber pacemaker implantation in September 2018.2 saturation of 93%. In the ECG the atrial tachycardia with a ventricular rate of 130 bpm was observed was cut off and connected to the pulmonary artery\u2014the new vascular canal was also connected with right atrium (RA) via fenestrations. Because of the atresia of femoral veins, a hydrophilic leader and a long vascular sheet were used to reach the vascular canal. With the use of Lasso catheter, the canal was reconstructed (on the EnSite Cardiac Mapping System) and atrial reference signals were recorded. Subsequently, with the use of transseptal sheet, the RA (via fenestrations) was reached to create an activation and potential map of RA. A reentrant tachycardia (AFl with the cycle of 300 ms) around the tricuspid valve, with the isthmus between the tricuspid valve and the scar that was created by the excision of the SVC and creation of the vascular canal during the second Fontan procedure was found Figure . The appDuring 20 months of follow\u2010up the patient was in general good condition, without any syncope and remained free of arrhythmia.3Atrial arrhythmias following Fontan\u2010type surgery are very common and increase in the postoperative time reaching about 50% of patients during 20 years of follow\u2010up and are associated with significant morbidity and mortality. Furthermore, almost half (47%) of these tachyarrhythmias are resistant to antiarrhythmic drugs.Several isthmuses of tachycardia could be developed by anatomical sectors made by the orifices of vena cave, coronary sinus, suture lines of atriopulmonary anastomosis, or lateral tunnel repair. Catheter ablation in post\u2010Fontan patients is challenging because of distorted anatomy of the heart after multiple surgical interventions consequently barriers to access the native atrial tissue, limited access to the heart or possible hemodynamic instability. It is even more challenging in patients with lateral tunnel or extracardiac conduit because the caval veins are not directly connected to the heart and the access to the heart is possible only through the fenestration if present.Furthermore, the acute and long\u2010term results of RFCA in Fontan patients are significantly worse than in other congenital heart diseases with a high risk of recurrent tachycardia, which reaches 40%. For these reasons, RFCA in these group of patients is not very common. There is a case report of a 7\u2010year\u2010old boy with HLHS after Fontan procedure who required RFCA because of drug resistant reentry atrial tachycardia. The ablation electrode could not have been inserted through small fenestration. After dilatation with the use of angioplasty balloon, the RFCA was successfully performed. Other case report of a 24\u2010year\u2010old female after Fontan operation has shown efficacy of RFCA for paroxysmal AF. However, pulmonary vein stenosis was developed as a consequence of the ablation procedure. Moore et al. observed that AT of extracardiac total cavopulmonary connection (E\u2010TCPC) in 36 patients was treated with success via catheter ablation in 83% of the cases.4Radiofrequency catheter ablation with the use of electro\u2010anatomical mapping system could be effective and feasible treatment of drug resistant atrial flutter in pediatric patients with hypoplastic left heart syndrome after Fontan operation.Ewa J\u0119drzejczyk\u2010Patej and Rados\u0142aw Lenarczyk\u2014consultant fees from Medtronic, Biotronik, Abbott, and Boston Scientific. Beata \u015aredniawa\u2014consultant: Medtronic, Zoll, Bayer, lectures fee for: Boehringer\u2010Ingelheim, Bayer, Pfizer."} +{"text": "The carcinogenic effect of NUP37 has been reported recently in a variety of tumors, but its research in the field of glioma has not been paid attention. The main purpose of this study is to reveal the relationship between NUP37 and prognosis or clinical characteristics of glioma patients.First, as a retrospective study, this study included thousands of tissue samples based on a variety of public databases and clinicopathological tissues. Second, a series of bioinformatics analysis methods were used to analyze the NUP37 and glioma samples from multiple databases such as the CGGA, TCGA, GEO, HPA, and GEPIA. Third, to analyze the relationship between the expression level of NUP37 in tumor tissues and cells and a variety of clinical prognostic molecular characteristics, whether it can be an independent risk factor leading to poor prognosis in glioma and whether it has clinical diagnostic value; GSEA was used to analyze the cancer\u2010related signaling pathways that may be activated by high expression of NUP37. Fifth, CMap was used to analyze small molecule drugs that may inhibit NUP37 expression. Finally, the meta\u2010analysis of thousands of tissue samples from seven datasets and cell proliferation and migration experiments confirmed that NUP37 has a malignant effect on glioma.NUP37 is highly expressed in glioma patient tissues and glioma cells, significantly correlates with reduced overall survival, and may serve as an independent prognostic factor with some diagnostic value. Silencing NUP37 suppresses malignant biological behaviors of glioma cells. 4 small molecule drugs that had potential targeting inhibitory effects on NUP37 overexpression.This study demonstrates for the first time a malignant role of NUP37 in glioma and provides a vision to unravel the complex pathological mechanisms of glioma and a potentially valuable biomarker for implementing individualized diagnosis and treatment of glioma. The present study used a series of analytical methods and confirmed that the NUP37 expression level was elevated in gliomas and led to a poor prognosis of patients. We partially revealed the mechanism of NUP37 in glioma and provided a novel potential diagnostic and therapeutic target.\u200b The median survival after standardized treatment for adult glioblastoma multiforme (GBM) is only 14.2\u00a0months.Many biological targets are used in the diagnosis and treatment of glioma. For example, EGFR amplification has a high incidence in gliomas, which may be used as a reference index to determine the pathological grades of the tumor.Chromosomal aneuploidy, which is an abnormal chromosome segregation during mitosis, is a common feature and possible cause of cancer. The NUP family, also known as the nuclear pore complex (NPC), occupies a significant position in cell mitosis and kinetochore\u2013microtubule interactions,Therefore, the present study examined the relationship between the expression of NUP37 and the clinical features of gliomas through the joint analysis of a large sample size in multiple databases. First, it is reported for the first time that the high expression of NUP37 as an oncogene is obviously associated with the prognosis of glioma especially grades III. Second, by gene set enrichment analysis (GSEA), we identified the possible oncogenic pathway of NUP37. Third, we identified four small molecule compounds that may inhibit the expression of NUP37 using the CMap database. Finally, we verified the effect of NUP37 on the behavior of glioma cell line by traditional experimental methods. Therefore, we suggest that NUP37 is a valuable potential biomarker and molecular target for the diagnosis and treatment of glioma.22.1http://www.cgga.org.cn/) is a genomics database that contains 2000 Chinese glioma specimens. The mRNA\u2010seq data of 1018 gliomas and microarray data of 301 glioma specimens were obtained from this database, and matching clinical information was acquired. Specimens that lacked information, such as survival time and molecular characteristics, were removed. Finally, 748 cases of mRNA\u2010seq data and 268 cases of microarray data were retained for subsequent analysis and processing. The basic clinical characteristics of the sample are shown in Tables\u00a0The Chinese Glioma Genome Atlas is the most widely used public tumor database, and it contains RNA\u2010seq, miRNA\u2010seq, methylation data, and matching clinical information for a variety of tumors. The present study obtained 698 cases of glioma mRNA\u2010seq data from the database. After excluding the specimens that lacked relevant clinical information, 653 specimens were included for follow\u2010up research. The basic clinical characteristics of the included specimens are shown in Table\u00a0The Cancer Genome Atlas database is an open biological database that contains high\u2010throughput gene expression data submitted by research institutions around the world. We retrieved two datasets from the database: GSE50161 and GSE116520. GSE50161 contained 34 glioma and 13 normal brain tissue specimens, and the GSE116520 contained 34 glioma and 8 normal brain tissue specimens.Gene expression omnibus is an online data analysis and visualization tool that contains the RNA sequencing data of various human tumors and matching normal tissues.Gene Expression Profiling Interactive Analysis . Three normal brain tissues, two low\u2010grade gliomas and one high\u2010grade gliomas were randomly downloaded to detect the protein expression of NUP37 in gliomas and normal brain tissues at all levels were obtained from this database.The Human Protein Atlas (HPA) uses transcriptomic and proteomic techniques to study the protein expression in different human tissues and organs at protein levels.2.2p\u00a0<\u00a00.05 and FDR <0.25 were considered significantly enriched.Gene set enrichment analysis (GSEA) is a bioinformatics analysis tool that is widely used to annotate and predict gene functions. After normalization of the above three sets of data , the specimens were split into high and low expression groups according to the expression level of NUP37. To reveal how NUP37 participated in the pathological process of glioma, we performed enrichment analysis using GSEA 4.0.2 jar software. The number of permutations was set to 1000, and the genome database was set to the KEGG cell signaling pathway. Normal 2.3CMap, https://portals.broadinstitute.org/cmap/) is an online drug analysis tool that discovers and predicts potential therapeutic drugs for certain diseases based on genome expression profiles.p\u00a0<\u00a00.01 and enrichment < \u22120.7) were identified as potential inhibitors of NUP37. In addition, we also obtained the 3D chemical structure of these small molecule drugs using PubChem (https://pubchem.ncbi.nlm.nih.gov/).Connectivity Map (2.42. The short interfering (si)RNA to NUP37 was purchased from GenePharma. The siRNA sequence was as follows: sense, 5\u2032\u2010GCAUGUGGUAGAAUUUAAUTT\u20103\u2032 and antisense, 5\u2032\u2010AUUAAAUUCUACCACAUGCTT\u20103\u2032. The negative control (NC) siRNA sequence was sense, 5\u2032\u2010UUCUCCGAACGUGUCACGUTT\u20103\u2032 and antisense, 5\u2032\u2010ACGUGACACGUUCGGAGAATT\u20103\u2032. Cells were transfected using Lipofectamine 2000 reagent following the manufacturer's protocol. After 24\u00a0h of transfection, the knockdown efficiency was detected by RT\u2010qPCR technology.Glioma cells and Human astrocytes cells (HA cells) were obtained from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM medium with 10% FBS (GIBCO), 100\u00a0U/ml penicillin, and 100\u00a0mg/ml streptomycin (Invitrogen) at 37\u00b0C with 5% CO2.5Glioma tissues and normal brain tissues that underwent surgical treatment for glioma or primary epilepsy were obtained from the operating room. A total of 49 glioma tissues and 12 normal brain tissues were obtained. These tissue samples were further embedded in paraffin to detect the protein expression of NUP37 by immunohistochemistry . In addition, the other 8 patients with epilepsy and 23 patients with glioblastoma were collected and stored in liquid nitrogen, and the mRNA level of NUP37 was detected by RT\u2010qPCR. The diagnosis of glioma was pathologically confirmed. All patients provided written informed consent. The study was approved by the ethics committee.2.6\u2212\u0394\u0394CT.Total RNA from cells was extracted using the Total RNA kit I (Omega Biotek). RNA concentration was detected using Nanodrop (Thermo Fisher Scientific). RNA was reverse\u2010transcribed into cDNA by NovoScript Plus All\u2010in\u2010one 1st Strand cDNA Synthesis SuperMix (Novoprotein). Finally, RT\u2010qPCR was performed to detect the expression level of NUP37 using Novostart SYBR qPCR SuperMix Plus Kit (Novoprotein). Primers were manifested as follows: GAPDH forward 5\u2032\u2010CAAGGTCATCCATGACAACTTTG\u20103\u2032 and GAPDH reverse 5\u2032\u2010GTCCACCAC CCTGTTGCTGTAG\u20103\u2032; NUP37 forward 5\u2032\u2010TAGGACACCCTCAGCCCATC\u20103\u2032 and NUP37 reverse 5\u2032\u2010TTCAGTCACCCAAAACAACA\u20103\u2032. The relative NUP37 expression levels were determined using the 22.75/well and incubated in an incubator at 37\u2103 with 5% CO2. After the cells had grown to confluence, a linear scratch in the cell monolayer was made with a 200\u00a0\u00b5l sterile pipette, the microscope was immediately applied for photograph, and the migrated distance was observed after 24\u00a0h. The distance migrated by cells in each group was measured using ImageJ software .A172 cells to make single\u2010cell suspension, then cells were seeded in 6\u2010well plate at 2\u00a0\u00d7\u00a0102.8A172 cells were prepared as a single\u2010cell suspension and then seeded in 6\u2010well plate at 500\u00a0cells/well. After 14\u00a0days of culture, the cells were fixed using paraformaldehyde for 20\u00a0min and stained with Crystal violet solution (Solarbio). Colony numbers were counted using ImageJ software .2.9The 4 normal brain samples, 7 low\u2010grade gliomas samples, and 19 high\u2010grade gliomas samples were collected from the operating room for NUP37 immunochemical stabilization. For immunohistochemical (IHC) staining, tissue sections of 2\u00a0\u00b5m thickness were placed in xylene and graded alcohols for deparaffinization and hydration. Antigen retrieval was performed using a microwave oven in EDTA (pH 8.0) buffer for 15\u00a0min. Blocking was performed with 10% goat serum. The appropriate amount of primary antibody NUP37 working solution was then dropped on the sections and incubated overnight at 4\u2103. The staining results were observed under light microscope and photographed. IHC results were analyzed using Image\u2010Pro Plus software (version 6.0).2.10The treated cells were fixed with 4% paraformaldehyde for 30\u00a0min and then perforated with 0.1% Triton X\u2010100 for 10\u00a0min at room temperature. Primary antibody Ki67 was added and incubated overnight at 4\u2103. Then, incubated with DyLight 594\u2010conjugated AffiniPure goat anti\u2010rabbit IgG in a humidified atmosphere at room temperature in the dark for 1\u00a0h. Finally, the staining results were observed and photographed under a fluorescence microscope.2.11GSE43378:50 patients; GSE4412:85 patients; GSE74187:60 patients; GSE83300:50 patients) including 1959 glioma samples. HR value and 95% CI were considered as an important indicator to evaluate the analysis results. The heterogeneity between multiple datasets was analyzed by the Q test (I2 statistics). A fixed effects model or a random effects model was selected for meta\u2010analysis based on the cut\u2010off criterion was (I2\u00a0=\u00a050%) in R 3.4 software.In order to find more evidence to confirm the effect of NUP37 on the prognosis of glioma, we used meta\u2010analysis to test that NUP37 is a risk factor for the prognosis of glioma patients. First of all, we searched several databases to find the research about NUP37 in gliomas. Unfortunately, up to now, there is no literature about the relationship between NUP37 and gliomas. Therefore, this study can only use the existing data in the database for meta\u2010analysis and evaluation of NUP37 on the overall survival of gliomas patients. In this study, we collected seven sets of data . NUP37 expression in glioma and normal brain tissues was detected using the Wilcoxon method. The overall survival of the NUP37 high expression and low expression groups was determined using Cox regression and Kaplan\u2013Meier analyses. To analyze the main factors affecting the prognosis of patients, univariate and multivariate Cox analyses were used. The ROC method was used to detect whether NUP37 may be used as an independent prognostic factor for glioma. The relationship between NUP37 expression and the clinical features of the specimens was tested using the Wilcoxon or Kruskal\u2013Wallis test. Finally, the cor.test was used to analyze the correlation between NUP37 and other genes.33.1Using the GEPIA database, we obtained the expression of NUP37 in different tumors and corresponding normal tissues. As shown in Figure\u00a03.2To reveal the role of NUP37 in the pathological process of glioma, we performed a multilevel analysis from glioma data derived from multiple database sources . The above data were divided into two groups according to the expression level of NUP37: high expression and low expression. Survival analysis was performed using the Kaplan\u2013Meier analyses, and the results are shown in Figure\u00a03.3p\u00a0<\u00a00.05, [HR] >1). In addition, PRS type, histology, and chemotherapy in databases other than TCGA RNA\u2010seq also suggest risk for the prognosis of glioma . Besides, in the CGGA RNA\u2010seq and CGGA microarray datasets, IDH mutation status was a protective factor for the prognosis of glioma . Finally, in the CGGA RNA\u2010seq datasets, 1p19q codeletion status was a low\u2010risk factor for the prognosis of glioma patients ).First, we employed univariate analysis of data from the three datasets to interpret the impact of NUP37 and each clinical feature of the patients on the prognosis of glioma Figure\u00a0.We foundp\u00a0<\u00a00.05, [HR] >1). In addition, there are also some clinical characteristics of patients that was also an independent risk factor for the prognosis of glioma such as: tumor grade, PRS type, histology, and age in some datasets . Finally, in some datasets such as CGGA RNA\u2010seq, IDH mutation status, 1p19q codeletion status, and chemotherapy was an independent protective factor for glioma prognosis . We do not make excessive interpretations of the remaining data, and details are shown in Figure\u00a0Second, through univariate analysis, we learned that NUP37 or some patient's clinical characteristics were a risk factor for glioma prognosis, but to clarify whether such a result was an independent factor, we adopted multivariate analysis to explain Figure\u00a0. We founThe results of the univariate and multivariate analyses suggest that NUP37 may be used as a prognostic and diagnostic factor for glioma patients, but its diagnostic value must be further examined.3.4ROC curves were used to determine the diagnostic value of high NUP37 expression for glioma prognosis based on the three datasets. As shown in Figure\u00a03.5p\u00a0<\u00a00.001). As shown in Figure\u00a0p\u00a0<\u00a00.001). As shown in Figure\u00a0p\u00a0<\u00a00.001). The expression level of NUP37 was positively correlated with the age of glioma patients , we could only choose a random effects model to present the results in Figure\u00a0We retrieved multiple well\u2010known databases and found no reports about NUP37 in glioma, so we could only collect the extant data in the database to complete the meta\u2010analysis to further confirm the effect of NUP37 on the prognosis of glioma patients. A total of 7 datasets containing 1959 glioma samples were included in this analysis, and the results of the meta\u2010analysis found that each individual dataset revealed NUP37 as a risk factor for glioma prognosis ([HR] >1). The results of the seven datasets as a whole showed that NUP37 was also a risk factor for glioma prognosis ([HR]\u00a0=\u00a01.98 (95% CI [1.42\u20132.75])). Because the heterogeneity among the seven datasets was greater than the cut\u2010off criterion was . The formulation of this cut\u2010off criterion was based on the rationale of CMap analysis and the enrichment index was set up from \u22121 to 1, which indicates the strength of inhibition of the target gene by the drug.It is of great value to reveal the mechanism of oncogenes in tumors for the formulation of clinical treatment strategies. Recognizing that some tumors contain abnormal expression of coding proteins can be targeted for the development of small molecule inhibitors. Based on this rationale, we employed CMap analysis to obtain small molecule drugs with inhibitory effects on NUP37, and finally we obtained four drugs according to the cut\u2010off criterion . The use of patient samples conformed to the declaration of Helsinki. All patients provided informed written consent.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file."} +{"text": "China set the goal of expanding early childhood education (ECE) in 2018, by encouraging the development of public interest kindergartens (PIK) to provide high-quality, low-cost preschool services to the general public. This is in response to the challenges of accessibility, affordability, and accountability besetting China\u2019s current ECE system. However, the transition toward PIK has been slow due to various complex problems, including the lackluster willingness of ECE providers to become PIK. To better understand the challenges leading to low participation, this study explores the external pressures affecting ECE providers and evaluates the external factors that influence their level of social responsibility. A stratified-random sampling questionnaire survey solicited responses from 832 ECE personnel representing 261 kindergartens from across China. Our findings suggest that institutional pressure has a positive effect on social responsibility and inclusive participation. We also found that institution visibility positively regulates the relationship between institutional pressure and social responsibility. At the same time, the level of environmental perception positively governs the relationship between social responsibility and participation willingness. Kindergartens should have certain social values, including assuming certain behaviors and participating in social activities in the spirit of social service and ensure multiple subjects\u2019 synergetic governance. Early childhood development and education (ECDE) has been recognized for its irreplaceable role in childhood development and care by the educational development community. In the United Nations Sustainable Development Goals, one of the targets is to ensure that all children have access to quality early childhood education (ECE), development, and care that prepare them for primary education . Early cTo ensure that all children have equitable access to programs and services, inclusive early childhood education has become a significant emphasis of national governments, education systems, and institutions . ECDE caIn China, total ECE providers are 281,174 in 2019, while 76.96% of them are public interest kindergartens (PIK) and located in urban areas . AlthougThe huge disparity between urban and rural (71.79% in number and 59.42%) student\u2019s access to ECE is caused by several factors, such as high costs and difficulties entering kindergarten . AlthougStudies on public interest kindergartens have mainly focused on the connotation characteristics , status This study focuses on the whole social responsibility consciousness of the industry in which preschool education institutions are located. The findings of this study can help to make a strategy for the supervision and construction of corporate social responsibility, improving the service quality, and creating a positive and harmonious atmosphere of social responsibility. Policy-makers can use the results of this study is proposing new strategies for social responsibility management of preschool education. This study can also help ECE providers and local government units to guide them in preschool education reform.In China, early childhood education is provided for children 3\u20136\u2009years old. Since its beginning in 1949, ECE in China has undergone significant changes. From 1949 to 1992, ECE focused on women\u2019s employment and family support. At this stage, school choices were limited, and the allocation of educational resources mainly depended not on market forces but on government policies and planning . From 19Factors that hinder the transformation of kindergartens to PIK are complex and multifaceted . PIK\u2019s pThough the issues are important, only a few studies have closely examined the social responsibility of kindergartens and the various institutional pressures and forces affecting the system within China. In recent years, the mainstream research concepts of \u201cvoluntariness principle\u201d and \u201cutilitarianism principle\u201d have shifted toward macroscopic notions of \u201ccompulsory responsibility\u201d and \u201cvoluntarism principle\u201d . As a quThe new institutionalism theory gradually clarified the internal mechanism of external institutional pressure and organizational behavior . The insInstitutional theorists propose that institutional pressure causes organizations to conform to social conventions to accept and recognize legitimacy . This viIn China, institutional pressures on kindergartens mainly come from regulatory pressure, social norms, and cognitive pressure on organizations to promote social responsibility . Figure Does institutional pressure affect the social responsibility of ECE providers in China?Is the participation of ECE providers in social responsibility intermediary?Does the level of visibility among ECE providers affect institutional pressure and social responsibility?Does the environmental perception of ECE providers affect social responsibility and inclusive participation?The conceptual model diagram is constructed, as shown in H1: Institutional pressure positively influences social responsibility.H1a:\u2002Regulatory pressure positively influences social responsibility.H1b:\u2002Normative pressure positively influences social responsibility.H1c:\u2002Cognitive pressure positively influences social responsibility.According to institutional theory, the organizational choice is limited by various external pressures because the environment is collective and interrelated . InstituH2:Institutional pressure positively influences inclusive participation.H2a:\u2002Regulatory pressure positively influences inclusive participation.H2b:\u2002Normative pressure positively influences inclusive participation.H2c:\u2002Cognitive pressure positively influences inclusive participation.H3: Social responsibility positively influences inclusive participation.H4: Social responsibility plays a mediating role between institutional pressure and inclusive participation.Visibility refers to the degree to which an enterprise attracts the attention of various stakeholders, such as the public, regulators, and the media, and is an important feature of strategic corporate social responsibility . The visH5:Institution visibility positively affects the relationship between institutional pressure and social responsibility.H5a:\u2002Institution visibility positively affects the relationship between regulatory pressure and social responsibility.H5b:\u2002Institution visibility positively affects the relationship between normative pressure and social responsibility.H5c:\u2002Institution visibility positively affects the relationship between cognitive pressure and social responsibility.Environmental perception means that an enterprise can fully recognize the changes and development trends of the environment . PrahalaH6:Environmental perception positively affects the relationship between social responsibility and inclusive participation.H6a:\u2002Environmental perception positively affects the relationship between responsibility management and inclusive participation.H6b:\u2002Environmental perception positively affects the relationship between customer responsibility and inclusive participation.H6c:\u2002Environmental perception positively affects the relationship between staff responsibility and inclusive participation.H6d: Environmental perception positively affects the relationship between social services and inclusive participation.H6e: Environmental perception positively affects the relationship between organizational responsibility and inclusive participation.The primary research object in this study is Chinese ECE providers. With the rapid development of China\u2019s economy and society, preschool education has received significant attention from the government and the people. Along with this heightened interest, China\u2019s current \u201c3A\u201d problems in ECE providers have become highly pronounced and require considerable strengthening of the country\u2019s PIK. Preschool education in most developed countries has also long been oriented to serve the public interests. In other countries, such as China, ECE requires significant policy overhaul and long-term development strategies. PIK development is a key focus in the United Nations Sustainable Development Goals on Quality Education. It aims to ensure that children have access to quality early childhood development, care, and preschool education by 2030 to prepare them for primary education.Institutional pressure was measured by measuring regulatory, normative, and cognitive pressure. Similar methodology was used by By following convenience sampling approach, a total of 1,050 questionnaires were distributed to 400 ECE providers in China\u2019s 31 provincial-level administrative divisions, of which 1,050 questionnaires were recovered, and 832 were valid. The valid samples cover 261 kindergartens, which come from 27 provinces and 103 cities. Among the valid samples, 46.1% are from economically developed regions, where kindergartens are more representative and typical. To ensure the quality and accuracy of results, the samples were taken from people top the management level according to inclusion criteria. Each questionnaire takes around 30\u2009min to answer all questions. The descriptive statistics of the valid samples are summarized in After collecting the questionnaires, the data were recorded according to the requirements of social statistical analysis. SPSS22.0 and Amos24.0 software were used to analyze the sample data. Before conducting hypothesis testing, Harman\u2019s single-factor detection method was used to test for the common method variation (CMV). This CMV approach is simple and easy to use. Since the scales used in this study are all recognized, exploratory factor analysis and confirmatory factor analysis were conducted to determine whether the indexes adopted have structural validity.All ethical guidelines and standards of the research were followed in the study. Ethical approval was obtained from the ethics review committee of Sichuan University, Chengdu, China. Besides, verbal approval was obtained from each interviewee before an interview. This study also maintained the confidentiality of the interviewee.Using Harman\u2019s single-factor detection test, the existence of CMV was examined in the survey data. If the variance explained by the first common factor reaches 50% or more, it would suggest that there is significant CMV present in the dataset. The exploratory factor analysis found that the first principal component variance interpretation rate was 27.33% in this study. Since the calculated value is less than 50%, this indicates that the dataset does not contain serious CMV and that the accuracy of the results is not considerably affected .p =\u20090.000). Confirmatory factor analysis was then conducted to evaluate the suitability of the model. The best model can be determined by comparing multi-factor models. As shown in X2/DF\u2009=\u20091.200, CFI\u2009=\u20090.991, NFI\u2009=\u20090.950, TLI\u2009=\u20090.990, IFI\u2009=\u20090.991, and RMSEA\u2009=\u20090.022. The five-factor model passes the confirmative factor analysis , normative pressure , and cognitive pressure are all positively affected by social responsibility. Model 3 is based on Model 2 that incorporates regulatory variables. The regression results from Model 3 show that institution visibility , institution visibility*regulatory pressure , institution visibility*normative pressure , and institution visibility*cognitive pressure have significant positive correlation with social responsibility. The results also indicate that the positive impact of regulatory, normative, and cognitive pressure on social responsibility performance will be strengthened, and the moderating effect is established in the case of high organizational visibility.To ensure the accuracy of the research results, the age, size, and nature of the ECE provider were included in the hierarchical regression as control variables and whether the ECE provider is a PIK. For Model 2, additional independent variables were added to Model 1, and the summary of results is presented in \u03b2\u2009=\u20090.234, p\u2009<\u20090.001), normative pressure , and cognitive pressure positively influence inclusive participation. In Model 6, the mediating variable social responsibility was added to the model and used as an independent variable. The results show that social responsibility is positively affected by inclusive participation. Model 7 is based on Model 6 and includes regulatory variables. The regression results from Model 7 show environmental perception , environmental perception*social responsibility , environmental perception*responsibility management , environmental perception*customer responsibility , and environmental perception*social services positively affect inclusive participation.Additional independent variables were added to Model 5. The results show regulatory pressure and environmental perception*organizational responsibility did not have significant effect on inclusive participation. This suggests that the positive influence of social responsibility, responsibility management, customer responsibility, and social service on inclusive participation should be strengthened with acute environmental perception ability data to analyze and explore the causal relationship between these research variables. Second, this study developed a structural equation model that statistically analyzed the role path of inclusive participation. However, certain details and variables may have been overlooked, which could affect the findings. Future studies can go deeper into the subject matter.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YL have designed the research plan, collected the data, analyzed the data, and wrote the manuscript. YL, CM, MW, XL, and XH analyzed the data and revised the manuscript. All authors have checked the final version of the manuscript and approved for publication.The article is funded by the National Social Science Fund of China Education Science Western Region Project entitled \u201cThe Behavior Logic and Realization Mechanism of Multiple Subjects\u2019 Synergetic Governance on Vocational Education.\u201dThe 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": "Zolpidem is indicated in cases of severe insomnia in adults and, as for BDZs, its assumption should be limited to short periods under close medical supervision. Since several drugs cause corrected QT interval (QTc) elongation, the authors investigated whether high daily doses of Zolpidem could cause QTc elongation. The study was conducted in the Addiction Medicine Unit of the G.B. Rossi University Hospital in Verona. The data were collected from hospitalizations carried out between January 2015 and February 2020 and refer to a total of 74 patients, 38 males and 36 females, who were treated for detoxification from high doses of Zolpidem with the \u201cVerona Detox Approach With Flumazenil.\u201d One patient out of 74 had QTc elongation (479 ms). The patient was male and took a daily dose of 50 mg of Zolpidem; he did not take concomitant therapies that could cause QTc lengthening. He had no electrolyte alterations, no contemporary or previous intake of barbiturates, heroin, cocaine, THC, alcohol, NMDA or nicotine which could cause an elongation of the QTc interval. The present study highlights the low risk of QTc elongation due to high dosages of Zolpidem; however, if, on one hand, we can affirm that Zolpidem is a safe drug, on the other, the widespread use of high dosages of this drug for prolonged periods of time is problematic and worrying. Benzodiazepines (BDZs) and Z-drugs are among the most prescribed drugs in Western countries and, while there has been a downward trend in annual BDZ prescriptions, Z-drug prescriptions have instead increased exponentially. This is probably due to a widespread opinion among doctors that the latter don\u2019t involve the problems associated with chronic use of BDZs, while, in fact, Z-Drug (ZD) use requires the same safeguards that apply to BDZs \u20134.The use of ZDs is indicated in cases of severe insomnia in adults and, as for BDZs, it should be limited to short periods under close medical supervision. The pharmacokinetic differences between the molecules allow for some important considerations with respect to the selection criteria of these drugs. Zolpidem and Zopiclone, being eliminated more slowly, are more suitable for the treatment of insomnia with central awakenings, unlike Zaleplon, which has a rapid elimination and is thus best suited for the management of initial insomnia . The maiZDs, like BDZs, are characterized by high manageability, and starting from their introduction on the market it was initially thought that they could offer advantages in terms of efficacy and safety compared to classic BDZs. However, over time it was shown that even this class of drugs can lead to important side effects, such as cognitive impairment, worsening of psychomotor performance, and increasing the risk of falls and fractures, especially if taken at higher doses or in association with other psychoactive substances such as alcohol. \u20139.Zolpidem and Zopiclone abuse are primarily described in patients with a history of drug addiction, alcoholism or psychiatric disorders and, minimally, also in patients not belonging to these categories. Their use should, therefore, reflect careful indications concerning posology , 11.Zolpidem is an imidazopyrimidine and, although it is not structurally connected to BDZs, it has a similar mechanism of action: it enhances the inhibitory effect of GABA on nerve transmission, binding the related receptor with consequent increases in permeability to chlorine ions.Although early reports highlighted a profile of low abuse risk for Zolpidem, in recent years an important increase in Zolpidem dependence has been detected; for this reason, Zolpidem was transferred to Schedule IV of the 1971 Convention in 2001 \u201314.High-dose Zolpidem use (600-2000 mg/die) has been associated with psychostimulant effects, such as feelings of well-being, euphoria (\u201chigh\u201d), energy, alertness, sociability, talkativeness, delusions and psychotic experiences, sleepwalking, falling asleep while driving, sleep-related eating disorders or engaging in other activities while not fully awake \u201317.It is also interesting to note that, in a large number of hypnotic drug abusers, a selective preference for Zolpidem was reported by subjects that were positive at screening tests for adult Attention Deficit/Hyperactivity Disorder , 19.Finally, intense craving, inability to stop use and withdrawal were associated with long-term high dose Zolpidem consumption. Through the analysis of adverse reaction data provided by the European Medicines Agency and after assessing the potential for abuse and dependence of ZDs, it has been shown that Zolpidem is more frequently involved in both abuse and withdrawal problems.The QT interval begins with the beginning of the QRS complex and ends with the end of the T wave and has an inverse relationship with heart rate (HR). A rate-related (or corrected) QT interval (QTc) according to Bazett\u2019s formula can be cThe upper limit of a normal QTc interval is 470 ms in males and 480 ms in females. As for the lower limits of the QTc, they have not been well established, but sometimes values between 330 and 360 ms are mentioned. .Long QT syndrome (LQTS) is a myocardial repolarization disorder characterized by a prolonged QT interval on the electrocardiogram (ECG). The main symptoms in patients presenting LQTS include palpitations, syncope, seizures and sudden cardiac death. This syndrome can be congenital or acquired. The acquired form is related to drug therapy and the presence of hypokalemia and hypomagnesaemia may accentuate the risk of drug-induced LQTS development. One of the main risks of LQTS is that it generates polymorphic ventricular tachycardia, i.e., a ventricular rhythm greater than 100 bpm with frequent changes in the QRS axis, its morphology, or both , 23.Torsades de pointes (TdP) is a form of polymorphic ventricular tachycardia which derives from either acquired or congenital QT interval prolongation, and manifests with a heart rate between 160 and 250 bpm. These variations take the form of a progressive, sinusoidal and cyclical evolution of the QRS axis; the peaks of the QRS complexes appear to \u201ctwist\u201d around the isoelectric line of the recording. This condition tends to spontaneously regress; however, multiple episodes can occur in rapid succession and may degenerate into ventricular fibrillation and sudden cardiac death. Determining the absolute and comparative risk of many drugs associated with QT interval prolongation is difficult, as most of the available data comes from case reports or small series of observations. Furthermore, the incidence of QT prolongation without torsades de pointes (Tdp) is much higher than the incidence of TdP itself.The pathophysiological mechanism underlying drug-induced TdP is the development of abnormal depolarizations of the cell membrane in the final part of the action potential, defined as early post-depolarization (EAD), or during diastolic repolarization, termed late post-depolarization (DAD).Almost all drugs that cause LQTS cause blockage of the potassium channel, thereby inhibiting the rapid outward flow of potassium ions and, therefore, cellular repolarization , 25.Furthermore, lower heart rates result in a smaller potassium output from the cell during repolarization, as there are fewer repolarization events; also, the reduction of extracellular potassium increases the degree of inhibition induced by the drug on the rapid potassium current, consequently increasing the QT interval. .Most patients with drug-induced LQT have one or more risk factors for the condition.In a review of the literature that included 249 patients with non-cardiac drug-associated QT prolongation, 97% had at least one risk factor and 71% had at least two. These included: female sex in 71%, history of heart disease in 41%, concomitant use of another QT prolonging drug in 39%, hypokalemia in 28%, a high dose of the drug in 19%, and a previous history of LQTS in 18%.The most common risk factor for drug-induced LQTS is being female. In a review of the literature of 332 patients with cardiovascular drug-associated Torsades de pointes (Tdp), 70% were women. Compared to males, females have a longer QTc and a greater response to drugs that block the rapid potassium channel, favoring Tdp, possibly due to the effect of sex steroids on ion channel expression. Estrogen potentiates the prolongation of the QT interval induced by bradycardia and the development of arrhythmia. Conversely, androgens reduce the QT interval and make it less sensitive to drugs. \u201328.In scientific literature, benzodiazepines and Z-Drugs are considered safe concerning LQTS ; to dateSince several drugs cause QTc elongation, with this case series we want to analyze if QTc lengthening occurs on a total of 74 patients admitted to the Addiction Medicine Unit in Verona, Italy for daily use of high doses of Zolpidem.The study was conducted in the Addiction Medicine Unit of the G.B. Rossi University Hospital in Verona. The data were collected from hospitalizations carried out between January 2015 and February 2020 and refer to a total of 74 patients, of which 38 were males and 36 were females, that were being treated for detoxification from high doses of Zolpidem with \u201cVerona Detox Approach With Flumazenil\u201d , 31.The criterion of dependence on high doses of ZDs was defined on the criteria established by the DSM IV-TR, which provide for the presence of continuous use for a period greater than 6 months, a daily intake of Zolpidem greater than at least five times the maximum recommended daily dose and problematic use of ZDs, such as mixing various molecules, increasing dosages, using for pleasure, obtaining them through illegal means or deriving negative social consequences. The dosage assumed was obtained from a drug use history assessment performed by the staff doctors.The variables examined were demographic ones, that considered age and sex, and clinical ones, that considered the following: type of BDZ used; DDDE (mg); heart rate ; QY (ms); QTc (ms); Na+; K +; Cl-; additional therapy other than the BDZ.Upon admission, blood samples were taken to assess electrolyte concentration and an electrocardiogram was performed in order to obtain HR and the QT interval. QT was corrected with HR using Bazett\u2019s formula : QTc = QT/Vrr.A total of 74 patients including 38 males and 36 females that werThe average age of these patients was 44 years with a minimum of 23 and a maximum of 74 years; 38 were workers, 25 were unemployed and 11 were students .The mean daily dose of Zolpidem was 402.36 mg/day (SD \u00b1 368.88) , with a Assuming that the threshold value of QTc is 470 ms in males and 480 in females, we recorded a single case exceeding these threshold values . The patIt should also be noted that the QTc value of one of the 74 patients was not recorded.Zolpidem is a non-benzodiazepine receptor modulator used in short-term treatment of insomnia aimed at patients with difficulty starting sleep; it improves measures of sleep latency, sleep duration, and reduces the number of awakenings in patients with transient insomnia. It also improves sleep quality in patients with chronic insomnia and can act as a minor muscle relaxant. \u201335.The main thing to consider when starting patients on Zolpidem is the possibility of addiction and the development of withdrawal symptoms following drug dismission if the drug has been taken for a long time. An interprofessional team approach comprising healthcare professionals such as a pharmacist, a therapist, a nurse, and a clinician is critical; patients should also be educated on possible withdrawal effects resulting from the medication. Because Zolpidem has the potential to cause dependence, it should not be prescribed for long periods of time.Moreover, long term use of Z-drugs causes cognitive impairments: several case control studies report that benzodiazepine or Z-drug use approximately doubles the risk of being involved in a motor vehicle accident , 36. OthThere is a mistaken belief that Z-drugs have advantages both in terms of efficacy and safety compared to BDZs, and, for this reason, many countries have seen a lower prescription of BDZs in favor of an increase in prescriptions of Z-drugs, especially Zolpidem. However, even this class of drugs can have important side effects, such as reduced functionality of cognitive abilities and worsening of psychomotor performances , 6, 8, 9Inadequate prescribing and poor overall assessment of the risks associated with their prescribing carries many potential risks to patients, including the risk of developing addiction.Several drugs have been proven or suspected to cause QT prolongation; many of these medications are frequently used in the intensive care unit (ICU), such as different types of anesthetics, sedatives, antibiotics, antimycotics, antidepressants and antipsychotics. The mechanism behind drug-induced QT prolongation is primarily the blockade of potassium ion channels (Ikr) in myocytes, causing prolonged cardiac repolarization. A secondary mechanism is the blockade of hepatic drug degradation because of inhibition of the cytochrome P450 enzyme CYP3A4. Awareness on the many medications known to cause drug-induced LQTS is imperative, especially when the drugs are combined in the same patient , 40.In literature there is no study that correlates the use of high doses of Z-drugs, in particular Zolpidem, with the analysis of QTc on the ECG. With this study we analyzed whether there was a correlation between the intake of high doses of Zolpidem and QTc prolongation.The data collected showed that 1.35% of Zolpidem abusers developed QTc elongation, which corresponds to only one subject of the 74 total (with the exception of the single patients for whom we didn\u2019t record QTc data). We could deduce that Zolpidem does not represent a significant cause of this side effect, but this is the only study in literature that treats this problem with very selective criteria. Regarding the population that was taken into consideration, we can affirm that QTc lengthening can be explained by the simultaneous intake of other drugs that present this problem among their side effects , 42.With the information we have, we are unable to formulate a hypothesis that explains this phenomenon. Perhaps other studies focusing on gender differences of patients taking high doses of BDZs or Z-drugs might be more insightful.This study presents several limitations: it\u2019s a retrospective study, Zolpidem dosage was reported by subjects, no follow-up was included and the sample size is small.The present study shows a low risk of QTc elongation caused by high daily doses of Zolpidem; however, it must be considered that this is the only study currently present in literature and it could be useful to expand the data by analyzing a larger sample. Z-drugs and Zolpidem, proved to be safe drugs considering the risk of QTc lengthening, however the problem of abuse and dependence of this drug represents a growing phenomenon which is often underestimated by healthcare professionals.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of the Verona University Hospital . The patients/participants provided their written informed consent to participate in this study.SC, LZ, RC, and FL were responsible for the study concept and design. SC, FF, and LZ drafted the manuscript. LZ was responsible for the study methodology. All authors critically reviewed the content and approved the final version of the manuscript for publication."} +{"text": "Adrenal rest tumors are rare collections of aberrantly located adrenocortical tissue. They are most commonly found in the kidneys, and hepatic involvement is rare with few published case reports. When located in the liver, imaging findings are frequently indistinguishable from hepatocellular carcinoma (HCC), but when resected, histologic examination shows adrenocortical tissue. Here, we present a patient with a history of nonalcoholic steatohepatitis with advanced fibrosis who was identified as having HCC by cross-sectional imaging but was found to have a hepatic adrenal rest tumor (HART) after resection. HARTs can share imaging characteristics with HCC, and this alternative diagnosis should be considered, especially for hepatic segment VII lesions. Adrenal rest tumors are defined as collections of aberrantly located adrenal tissue ,2. The k2. Physical examination revealed a soft abdomen without tenderness or hepatomegaly. No stigmata of chronic liver disease were noted.A 72-year-old woman was referred to our hepatology clinic at Saint Louis University Hospital for the management of newly diagnosed HCC. She had a history of nonalcoholic steatohepatitis with stage 3 fibrosis, obesity, gastroesophageal reflux disease, obstructive sleep apnea, and hypertension. There was no history of tobacco, alcohol, or illicit drug use. She had no gastrointestinal symptoms. Vital signs were within normal limits. BMI was 33.8 kg/mComplete blood count and comprehensive metabolic panel were notable for elevated alanine aminotransferase of 113 U/L and aspartate aminotransferase of 65 U/L. Alkaline phosphatase, total bilirubin, hemoglobin, platelet count, and international normalized ratio (INR) were within reference ranges. Serum alpha-fetoprotein was 2.89 ng/ml. A right upper quadrant ultrasound, performed for screening for HCC\u00a0revealed a 26-mm right hepatic lobe lesion. Dynamic MRI and CT imaging of the abdomen showed a 23-mm segment VII lesion that demonstrated arterial enhancement with washout on delayed phase, consistent with HCC as per the Liver Imaging Reporting and Data System (LI-RADS) criteria (Figure She was evaluated for possible surgical resection with a transjugular liver biopsy showing steatohepatitis with stage 3 fibrosis and a hepatic vein pressure gradient measurement of 4 mmHg. As the patient was not a candidate for liver transplantation, given her age and morbid obesity, she elected to undergo partial hepatectomy of the segment VII lesion with ablation of the margins. The liver was noted to be nodular intraoperatively. Her postoperative course was unremarkable, and she was discharged home on day 3. Histologic examination of the resected lesion revealed benign adrenocortical tissue with clear margins Figure . ImmunosAdrenal rest, also referred to as adrenal rest \u201ctumor,\u201d is a term used to describe ectopic adrenal tissue. The kidneys are most commonly involved followed by genital structures, and hepatic involvement is rare\u00a0. They arWe conducted a search of the English literature using the MEDLINE database for published cases of HARTs. We identified a total of 11 published case reports describing 11 patients\u00a0,4-12. TaThe finding of HART in the setting of chronic liver disease has been described in only one case report earlier . Dalla VHART should be considered in the differential diagnosis of HCC in patients with chronic liver disease, especially in patients with normal alpha-fetoprotein and lesions involving hepatic segment VII."} +{"text": "Clostridioides difficile (C. difficile) is an opportunistic pathogen that leads to antibiotic-associated diarrhoea and is a leading cause of morbidity and mortality worldwide. Antibiotic usage is the main risk factor leading to C. difficile infection (CDI), as a dysbiotic gut environment allows colonisation and eventual pathology manifested by toxin production. Although colonisation resistance is mediated by the action of secondary bile acids inhibiting vegetative outgrowth, nutrient competition also plays a role in preventing CDI as the gut microbiota compete for nutrient niches inhibiting C. difficile growth. C. difficile is able to metabolise carbon dioxide, the amino acids proline, hydroxyproline, and ornithine, the cell membrane constituent ethanolamine, and the carbohydrates trehalose, cellobiose, sorbitol, and mucin degradation products as carbon and energy sources through multiple pathways. Zinc sequestration by the host response mediates metabolic adaptation of C. difficile by perhaps signalling an inflamed gut allowing it to acquire abundant nutrients. Persistence within the gut environment is also mediated by the by-products of metabolism through the production of p-cresol, which inhibit gut commensal species growth promoting dysbiosis. This review aims to explore and describe the various metabolic pathways of C. difficile, which facilitate its survival and pathogenesis within the colonised host gut. Clostridioides difficile (C. difficile) infection (CDI) is a global health threat being the leading cause of antibiotic-associated diarrhoea. CDI is mediated by the toxins TcdA and TcdB, which act to damage the gut epithelium leading to a breakdown in the gut barrier integrity. In addition, CDI possesses a high recurrence rate, owing to the ability of C. difficile to produce highly resilient endospores allowing it to persist following antibiotic treatment regimes. The ability of a pathogen to successfully colonise the gut relies on its ability to acquire and metabolise nutrients from its environment. C. difficile is proposed to act as a bacterial generalist, capitalising on the broad range of available nutrients present within a dysbiotic gut environment. This review describes the metabolic capabilities of C. difficile within the current literature to provide an overview of the genetic and molecular basis in which its metabolism operates following gut colonisation and how this contributes towards pathogenicity. Clostridioides (formerly Clostridium) difficile is a spore forming, gram-positive bacterium that is the major cause of antibiotic-associated diarrhoea worldwide and whose pathogenic lifestyle relies on an ability to produce the toxins TcdA and TcdB [C. difficile occurs via the conversion of primary bile acids to secondary bile acids, thus preventing the germination of its spores [C. difficile in vivo [C. difficile colonisation [Colonisation resistance is the ability of the gut microbial community to inhibit and outcompete pathogenic organisms preventing them from colonising the gut environment . Clostriand TcdB . Gut mics spores . Additio in vivo . Howevernisation .C. difficile colonisation. Wilson and Sheagren (1983), for example, reported that precolonisation by a nontoxigenic strain of C. difficile in a mouse model protected against colonisation by a toxigenic strain [C. difficile by depleting carbohydrates in a gut model environment [C. difficile prevented colonisation by toxigenic strains, although surprisingly, this protection could not be provided by other Clostridium species [Paraclostridium bifermentans has also been shown to prevent C. difficile colonisation in vivo by consuming glycine, a cogerminant for spores [C. difficile is occupied, then the pathogen is outcompeted and unable to colonise.The ability of the gut microbiota to occupy nutrient niches and restrict the use of nutrients therein is thought to play a key role in preventing c strain while thironment . Similar species \u20139. Paracagren 198, for exar spores . Taken tC. difficile possesses a large and mosaic genome, capable of metabolising a diverse range of nutrients for growth [C. difficile, are enriched in the gut metabolome following antibiotic disruption of the colonic microbiota [C. difficile to capture these \u201cfree\u201d nutrients vital for its outgrowth post-spore germination using a specific subset of metabolic pathways, regardless of the gut community structure in a post-antibiotic environment [C. difficile also has an intimate relationship with nutrient availability as, upon nutrient limitation in the gut environment, C. difficile responds by producing toxins that liberate host-derived nutrients through cytotoxic activity and creation of a proinflammatory environment [+ ratio) inhibit toxin expression when their cognate nutrient is present in excess [C. difficile\u2019s production of toxins can then be thought of as a means to maintain a favourable nutrient environment to sustain growth during infection. The purpose of this review is to highlight recent advances in our understanding of how the interplay between C. difficile, its host and the gut microbiota\u2014with respect to the availability and metabolism of certain nutrients\u2014is implicated in the pathogenesis of this important gut pathobiont , and glycine reduced to acetyl phosphate by glycine reductase (GR) [prd) operon is induced by the presence of its substrate through the regulatory protein, PrdR, which also leads to the repression of the glycine reductase (grd) operon [Clostridium sporogenes has been shown to be coupled to the production of a proton motive force allowing ATP generation, this has yet to be characterised in C. difficile [C. difficile, accompanied by its reduction to 5-aminovalerate [grd operon in excess proline is thought to shuttle all available selenium towards PR as this is the main means of NAD+ regeneration in C. difficile metabolism [C. difficile is then thought to manage Stickland metabolism in a hierarchical manner, preferentially utilising specific amino acids as substrates to maximise energy intake during growth.Early studies defining growth requirements in glycine . Prolinease (GR) , with boase (GR) . The pro) operon . While pifficile . Prolinevalerate ,44,45, wtabolism . C. diffC. difficile colonisation with free proline shown to increase in a dysbiotic state [prdB mutant [Clostridium leptum, Clostridium scindens, or Clostridium hiranonis depleted proline, protecting against CDI [C. difficile metabolic needs, specifically amino acid metabolism through the release of proline and hydroxyproline from collagen (see below) [C. difficile has been shown to be of importance for successful colonisation of the gut once dysbiosis occurs [In vivo mice model studies have also demonstrated the importance of proline for ic state . In humaB mutant , decreasinst CDI . Furthere below) . Unsurprs occurs .hypD) and a pyrroline-5-carboxylate reductase (proC), with the former essential for hydroxyproline-dependent growth in C. difficile [prdF), with the resultant D-proline being the substrate for PR [C. difficile toxin activity, which induces the production of host matrix metalloproteases in vivo to degrade collagen, liberating hydroxyproline (and proline) to be metabolised [hypD gene and thus may exclude a competitor that could compete with C. difficile for amino acids [C. difficile Stickland metabolism and maintain redox balance. Of note, C. difficile strains show differing abilities to metabolise hydroxyproline with R20291 and VPI 10463 strains having hypD expression induced 10-fold in its presence, whereas the 630 strain showed no expressional change [C. difficile in the gut may differ depending on the colonising strain, and only a subset of strains may benefit by its release from degraded collagen.Hydroxyproline, a host-modified form of proline and a major constituent of collagen, can be converted to L-proline by the glycyl radical enzyme, 4-hydroxyproline reductase functions to reduce COate NAD+ ,54. C. d strains ,55. The genesis\u201d . By coupneration . This wa glucose . The WLPC. difficile is thought to occur widely in populations with up to 90% of infants and 15% of adults carrying toxigenic strains [C. difficile for infection of others [C. difficile metabolism in the gut under dysbiotic conditions [Asymptomatic infection of the human gut by strains . Asymptof others . While tnditions ,27,47,52C. difficile during asymptomatic colonisation of mice [in C. difficile led to a competitive disadvantage against wild-type strains during in vitro or in vivo competitive coculture [To date, ornithine has received most attention as a substrate of importance for the maintenance of of mice . Ornithi of mice . Alterna of mice ,24. The Clostridium sardiniese enriched ornithine availability, leading to a significant increase in expression of putative C. difficile ornithine degradation genes allowing its import and metabolism as a Stickland substrate [C. sardiniese can significantly increase a putative C. difficile ornithine cyclodeaminase gene, which possibly converts it to proline for its own Stickland metabolism [P. bifermentans, its generation of ornithine acts as a substrate for its own metabolism to produce proline, depriving C. difficile of an important Stickland substrate [C. difficile growth.Further, in an in vivo coinfection mouse model, ubstrate . Furthertabolism . Conversubstrate . ClearlyNos2), representative of a type 1 inflammatory response [Arg1), which is representative of a type 2 inflammatory response [Mammalian macrophages act as one of the key drivers in regulating inflammation, generating either a proinflammatory, cytotoxic, type 1 inflammatory response involved in pathogen clearance, or an anti-inflammatory, tissue-repairing, type 2 inflammatory response involved in maintaining tissue integrity . Ornithiresponse . Alternaresponse .\u2212/\u2212) mouse infected with wild-type C. difficile shows increased ornithine concentrations in faeces, as well as an up-regulation of the oraE gene, suggesting its enrichment in a noninflammatory environment [\u2212/\u2212 knock-out mice infected with the wild-type and an oraSE mutant, where the former showed a competitive advantage due to the availability of ornithine from ablated iNOS activity [C. difficile colonisation under noninflammatory conditions further highlighting its adaptive nature. Contrarily, CDI may then reduce ornithine levels within the gut due to the promotion of a type 1 inflammatory response, which uses arginine for iNOS production [C. difficile with the potential to allow for the development of a targeted therapeutic to diminish its persistence.During CDI, iNOS consumes arginine shunting it away from its conversion to ornithine by arginase . A host ironment . This waactivity . Host anoduction . The benC. difficile are relieved, leading to toxin production and acquisition of alternative nutrients [Once nutrients become depleted within the gut environment, the repressive effects of CcpA and CodY in utrients ,18,61. Tutrients . Phosphautrients .C. difficile can metabolise ethanolamine as a carbon and nitrogen source and occurs within a macromolecular bacterial microcompartment (BMC) [nt (BMC) . This sent (BMC) ,65. The nt (BMC) . The relnt (BMC) .eut) utilisation cluster within the genome of C. difficile is induced by the presence of ethanolamine in vitro, as well as in a mouse model of CDI [eutG-eutW and eutA-eutQ, with the only form of regulation thought to be mediated by the early-sporulation sigma factor, SigF, via repression by an indirect means [eut gene clusters through the EutR regulatory protein has been reported in Citrobacter rodentium, enterohaemorrhagic Escherichia coli, and Salmonella typhirmurium; however, eutR cannot be found in the C. difficile genome [Enterococcus faecalis also lacks a EutR regulator, instead using the EutV-EutW two-component system to activate the eut operon [C. difficile [eut genes has not been demonstrated experimentally.The 19-gene ethanolamine , nc_085, repressing t system and proving sRNA .C. difficile eutA mutant, onset of pathogenesis occurred earlier than the wild type. However, ethanolamine has been shown to have no regulatory effect on known virulence factors, including toxin production, sporulation, and motility within C. difficile [C. difficile, it was proposed that it is not necessary for in vivo growth but does delay the onset of pathogenesis during CDI [In a ifficile . This obifficile ,75,76. Aring CDI . Follow-C. difficile is evidenced by its possession of numerous genes associated with carbohydrate metabolism genes and also a raft of phosphoenolpyruvate-dependent phosphotransferase transport system (PTS) genes [C. difficile colonisation, these carbohydrate metabolism genes increase in expression with concomitant decreases in carbohydrate abundance due to their utilisation by the bacterium in vivo [C. difficile to colonise the intestine relies on a dysbiotic microbial community lacking competitor species that would normally utilise carbohydrate sources more readily [Carbohydrates, such as nondigestible polysaccharides in the host\u2019s diet, are an important energy source for microorganisms residing within the large intestine. Their significance for S) genes ,14. Duri in vivo . This im readily .C. difficile metabolises a number of carbohydrates including, glucose, fructose, mannose, mannitol, melezitose, sorbitol, cellobiose, trehalose, and derivatives of mucin [+ for further metabolism and ATP . Despit mannose ,30\u201332,78C. difficile is encoded and regulated by a transcriptional antiterminator (srlR), a transcriptional activator (srlM), sorbitol-specific PTS components , and a sorbitol-6-phosphate dehydrogenase (srlD) [srlD mutant unable to grow in the presence of the substrate [C. difficile mutant had lower expression of its sorbitol metabolism genes in vivo in comparison to the wild type, suggesting the metabolism of sorbitol following toxin-mediated inflammation [C. difficile toxin production in vitro and in vivo, while a srlD mutant increases its production in vivo [C. difficile [C. difficile [C. difficile.Sorbitol metabolism in e (srlD) . In vitrubstrate . Sorbitoubstrate ,55. Sepaammation . Sorbitoammation . Additio in vivo . This su in vivo . Subsequifficile . Finallyifficile . AltogetC. difficile has a heterogeneous arrangement of the typical trehalose operon, where strains belonging to ribotypes (RT) 012, 027, 017, and 078 possess the canonical treR and treA genes encoding a trehalose regulatory protein and phosphotrehalase, respectively, with no equivalent of a treP [treA2 and treR2, the putative trehalase gene treX (which is truncated in RT023 strains), and the trehalose-specific PTS ptsT [Trehalose is a disaccharide consisting of two glucose molecules bonded together by an \u03b1 1,1 glycosidic bond, which can be used by microorganisms not only as a carbon and energy source but also as an osmoprotectant ,80. Comphe cell) ,29,81. IPTS ptsT ,29.C. difficile isolates (50 mM) [treR gene, they were also capable of utilising trehalose when present at 10 mM [treA gene [treA in low concentrations of trehalose [Recently, strains from RT027 and RT078 were shown to be capable of growth in vitro on trehalose (10 mM) at concentrations much lower than required by other (50 mM) . It has (50 mM) . To suppat 10 mM . It is preA gene Fig 1)..C. diffiptsT was shown to be essential for this phenotype supplied trehalose precolonisation of C. difficile in a single feed [C. difficile spore germination while preventing toxin production [Intriguingly, a trehalose-enriched diet is linked to increased disease severity in mice, seemingly associated with elevated levels of TcdB, when infected with the hypervirulent R20291 strain (RT027) . In addited mice . Taken tdB loads . Importagle feed ,82. Subsoduction .C. difficile clade 2 and 4 isolates found its occurrence to be more frequent than previously thought and was actually clade specific rather than ribotype specific [C. difficile clades, and, importantly, its presence lacked any association with increased mortality in CDI cases [C. difficile species, these genotypes were established long before the widespread introduction of synthetic trehalose into the human diet since 2000. Indeed, the hypothesis that C. difficile exhibited increased virulence due to this increase in trehalose in the human diet is disputed, as it is not supported by CDI patient data nor human gut models.Interest in the distribution of the variant TreR across specific . AdditioDI cases ,85. It cC. difficile by an operon containing a cellobiose-specific PTS (celABC), a 6-phospho-beta glucosidase (celF) and a repressor regulatory protein (celR) [Cellulose, a complex carbohydrate found in plant-based foods in the human diet, acts as a major nutrient source for the colonic microbiota . Cellulon (celR) .celA to be essential for growth on cellobiose [celA [cel operon under control of carbon catabolite repression (CCR) [Knock-out experiments have shown llobiose . CelR wase [celA . CelR alon (CCR) .C. difficile and CelR, in particular, was found to have an impact on sporulation and toxin production [C. difficile, is involved in negatively regulating toxin production and sporulation [celA mutant showed lowered levels of colonisation, as well as the inability to cause recurrent infection [C. difficile pathogenesis by affecting the rate of sporulation and toxin production. Altogether, cellobiose utilisation and its associated regulatory processes are shown to play an important part in C. difficile pathogenesis. Nutrient-specific metabolic regulators in C. difficile then may have broader roles in its physiology by indirectly modulating aspects of virulence. This then should encourage the identification of nutrient-specific regulator target genes outside of their canonical metabolic operons.Interestingly, uptake of cellobiose in oduction . It was rulation \u201389. Therrulation . Finallynfection . These fC. difficile colonisation during infection [Bacteroides thetaiotaomicron liberates free sialic acid from mucin to support the growth of C. difficile [C. difficile lacks the genes required to cleave sialic acid from host mucin, it does possess the nan operon encoding the catabolic pathway for sialic acid [B. thetaiotaomicron and C. difficile in gnotobiotic mice [C. difficile in vivo was further highlighted in a conventional mouse model following antibiotic use, which showed increased sialic acid levels with an associated up-regulation of the C. difficile nan operon postinfection [C. difficile to exploit the now readily available nutrients that would otherwise be efficiently consumed by other bacterial species in an unperturbed environment. Similar phenomena have been observed during Salmonella typhimurium infection, whereby gut microbiota metabolites, including sialic acid, influence pathogen colonisation [Mucous found on the gut lining of the large intestine consists of mucin, a glycosylated protein that contains ribose, N-acetylglucosamine, fucose, mannose, and sialic acid . These cnfection ,55. In aifficile . While Clic acid , with intic mice . Sialic nfection . This sunisation ,75,93.Akkermansia muciniphila, Ruminococcus torques, and again B. thetaiotaomicron, have been shown to facilitate the growth of C. difficile by cross feeding when mucin is the sole carbon source during coculture and from cell-free supernatant filtrates [C. difficile, which supports increased growth relative to glucose [C. difficile ribotypes, where its use as a sole carbon source facilitates robust growth in several C. difficile strains [C. difficile species and, thus, their importance in its lifestyle. Similar findings of cross feeding from mucin to facilitate pathogen growth have also been shown during the coculture of vancomycin-resistant Enterococcus and R. torques, with the former unable to grow in mucin alone [C. difficile pathogenesis, whereby the release of monosaccharide by-products is thought to facilitate colonisation of the intestinal mucous layer by chemotaxis while also providing substrates for growth. It is then plausible there is a level of synergy between C. difficile and mucin-degrading commensal microorganisms in the perturbed gut environment; therefore, deeper insight into whether other gut community members support CDI progression are warranted to fully understand how this pathogen colonises the gut.In a similar study, the mucin-degrading commensal microorganisms, iltrates . Specifi glucose . Mannose strains ,94,95. Tin alone . Mucin dMicronutrients play an essential role in many cellular processes in microorganisms and are considered to be under constant limiting amounts within the gut environment. Pathogens can acquire micronutrients through the release of metal chelating molecules, the use of high-affinity metal transporters, or the exploitation of the metal scavenging mechanisms of other microorganisms to acquire divalent metal ions such as iron, zinc, manganese, and cobalt .C. difficile, Zn limitation caused by the metal-chelating protein calprotectin in vitro leads to the expression of the zupT gene, predicted to have a role as a transporter of a diverse range of metal ions [C. difficile growth, colonisation, and recurrence in an in vivo mouse model of CDI [prdA gene during Zn sequestration; however, during this, in vitro, the PrdB subunit of PR requires selenium for its catalytic activity [selD, which encodes the selenophosphate synthetase, is thought to lead to a shift in metabolism from Stickland fermentation of proline to mannitol fermentation, as mannitol utilisation genes are induced within this selD mutant [C. difficile colonisation, it decreases in abundance over time [C. difficile, and it can be used as a primary nutrient source in minimal medium [C. difficile may then adapt to this limitation by utilising mannitol to potentially regenerate NAD+ [C. difficile metabolic response to calprotectin may then be influenced by the dynamic nutrient landscape found within the host, which drives its growth, specifically regulating proline reduction in combination with the abundance of selenium, which is required for this process [Zinc (Zn) is an important micronutrient that plays an essential role in the structural and functional activity of many proteins . In C. dtal ions ,46. ZupTl of CDI . In addiactivity ,46. SeleD mutant . Mannitover time ,55. In al medium ,13. Giveate NAD+ to susta process .C. difficile toxin activity [eut genes by calprotectin, involved in ethanolamine metabolism, which are increased during toxin-mediated inflammation as described above [C. difficile as it inadvertently acts as one signal to indicate an inflamed gut, leading to metabolic adaptation so it can capitalise on those nutrients now available.Lopez and colleagues (2019) then proposed that the induction of proline reductase by Zn limitation was part of a broader metabolic adaptation in which the effects of calprotectin indicate an immune response and, in turn, inflammation mediated by activity . As mentactivity ,23. Thised above ,46. TherC. difficile can metabolise tyrosine to an intermediate, p-hydroxyphenylactate (p-HPA), and, subsequently, to p-cresol [Escherichia coli, Klebsiella oxytoca, and Proteus mirabilis [p-Cresol is thought to increase membrane permeability and hence the loss of small molecular compounds such as phosphate from other bacteria [C. difficile has a tolerance towards p-cresol, although this varies from strain to strain and its importance in colonisation and/or pathogenesis remains unclear [p-cresol , a bacteirabilis . p-Cresobacteria . Importa unclear .hpdBCA operon, found across all 5 toxigenic C. difficile clades, is required for the conversion of p-HPA to p-cresol [C. difficile as observed during coculture experiments with gut commensals in vitro [p-cresol for sustaining infection was further exemplified by the fitness defect of a C. difficile hpdC mutant in an in vivo mouse relapse model of CDI [hpdBCA operon is induced by p-HPA rather than tyrosine [p-HPA within the gut in addition to C. difficile\u2019s own biosynthetic capability are thought to include being derived from host cells, and other gut commensals including Klebsiella and other Clostridium species [HpdBCA decarboxylase, encoded by the p-cresol ,101,102.in vitro . The impl of CDI . The hpdtyrosine , and, in species .hpdBCA operon, a SigA consensus sequence, has been found, which facilitates induction in the presence of p-HPA even if provided exogenously [p-HPA uptake, has yet to be identified [p-cresol, all strains so far examined show similar levels of efficiency of p-HPA conversion to p-cresol, with production being 30-fold higher in the presence of p-HPA than compared to tyrosine supplementation [hpdBCA operon, CodY, the nutrient-sensing global regulator, has been shown to be indirectly involved in its regulation [C. difficile R20291 strain were shown to contain CodY consensus binding sequences in their promoter regions [p-HPA in excess amounts was shown to be inhibitory to C. difficile growth; in turn, HpdBCA activity provides a means to lower p-HPA concentrations in the gut environment to a tolerable level [p-cresol as a by-product of p-HPA, and possibly tyrosine, metabolism governs C. difficile\u2019s success within the gut environment by diminishing the numbers of gut commensal species that establish colonisation resistance towards it.Upstream of the genously . Howeverentified . Interesentation . Despitegulation . This ma regions ,105. Finle level . Altogetp-Cresol production is one factor that prevents the reestablishment of antagonistic gut microbiota species to sustain dysbiosis and, thus, C. difficile colonisation; however, as a whole, these mechanisms are poorly understood. Understanding the processes C. difficile uses to suppress growth of the gut microbiota is critical in order to fully understand its pathogenesis. Therefore, given the importance of the gut microbiota in preventing C. difficile colonisation, inhibiting these processes could act as an effective treatment to restore antagonistic species towards C. difficile, in turn reestablishing gut homeostasis.C. difficile\u2019s ability to capitalise on a wide array of nutrients classifies it as a bacterial generalist for nutrient acquisition, possessing high genetic flexibility to metabolise a range of nutrients from its environment. The ability to effectively metabolise nutrients available in the gut environment is the key to the success of a pathogen colonising and establishing infection, where in C. difficile, a number of nutrient sources are shown to be important for survival and persistence (+ regeneration to drive catabolic flux in metabolism, being highlighted several times as important nutrients during C. difficile colonisation [C. difficile may utilise the WLP as an adaptive response to maintain redox balance, thus sustaining the catabolic flux of carbohydrate fermentation [C. difficile metabolism under homeostatic conditions, specifically the production of ornithine by immunometabolism, which is thought to contribute to persistence within its host [C. difficile, rewarding it with an appropriate metabolic adaptation to thrive on metabolites abundant in this environment such as proline, hydroxyproline, and ethanolamine [p-cresol is an important metabolic trait in C. difficile, as it provides a competitive advantage over other gut commensal species paving the way for continued dysbiosis [sistence . Host annisation ,20,23,47entation . The useits host . Carbohyits host ,30\u201332,78nolamine ,46. Prodysbiosis ,100,102.C. difficile metabolism studies are their reliance on the ability to produce mutant strains to provide evidence of a gene\u2019s function. Further work using the ClosTron mutagenesis [C. difficile\u2019s metabolic capabilities in the context of pathogenesis. The understanding of aspects of C. difficile\u2019s metabolism, which aid in its success as a pathogen, is slowly being realised, and continued research will further elucidate the adaptive mechanisms that contribute towards C. difficile\u2019s success during infection.A hallmark of agenesis , allelicagenesis , and CRIagenesis genetic C. difficile also has the potential to provide an insight into those gut microbiota species that may compete or efficiently consume its preferred metabolites in the gut. The use of gut competitor species as probiotic therapeutics then represents a novel strategy for the treatment of C. difficile infection; however, whether these can achieve clinical efficacy remains unanswered. Theoretically, probiotic therapeutics could reestablish colonisation resistance in the gut through the exclusion and consumption of those nutrient sources that are integral to C. difficile success and pathogenesis. Much is still to be learned about the metabolic aspects of CDI and how this shapes the pathogenesis of this important gut pathogen.Understanding the metabolic needs of"} +{"text": "Clostridioides difficile is a spore-forming anaerobic bacterium that causes gut infections in both human and non-human animals. In the gut, C. difficile colonization and pathogenicity are affected by nutrients, chemicals , other bacteria and inflammation. Investigations of interactions with microbes and immune cells are important for effective disease treatment, and prevention and control of bacterial transmission. New strategies based on re-shaping patient gut microbiota, such as through probiotics or fecal microbiota transplantation, are effective but have biologically complex mechanisms yet to be comprehensively understood.C. difficile interactions with gut microbiota, intestinal cells and metabolites. Horvat et al. examined C. difficile cell-cell and supernatant-cell interactions with gut microbiota derived from healthy children through batch culture. They found both C. difficile cells and cell-free conditioned medium affected diversity of bacterial communities and abundance of metabolites, indicating direct and indirect ways in which C. difficile can influence its environment. Peroxisome proliferator-activated receptor-\u03b3 (PPAR-\u03b3), normally expressed in adipose tissues and colonic epithelial cells, was shown for the first time to be involved in C. difficile infection (CDI) by Lai et al. Using mouse models, the authors showed PPAR-\u03b3 downregulation in CDI led to intestinal permeability. An agonist of PPAR-\u03b3, pioglitazone, limited disease symptoms in C. difficile-infected mice. This provides a new strategy for restoring intestinal integrity during CDI. Another strategy for reshaping patient gut microbiota could be through probiotics. Wu et al. found that Akkermansia muciniphila, a commensal in the healthy human gut, protected against CDI in a mouse model by limiting intestinal tissue damage. A. muciniphila maintained microbial diversity and metabolite levels in the presence of C. difficile at levels similar to a healthy gut. The overall effect was colonization resistance against CDI. Currently prescribed probiotics to prevent recurrent CDI are Saccharomyces boulardii, Lactobacillus acidophilus and L. casei, however, these did not prevent primary CDI produced as metabolites of C. difficile when grown in minimal medium, 28 of which were new. The authors proposed a biosynthetic pathway involving cysteine and methionine for VOC production, some of which have known effects on prokaryotic and eukaryotic cells. This study forms a basis for targeted investigations of C. difficile biochemical pathways corresponding to nutrient availability. In the gut, nutrients are dynamic and C. difficile gene transcription must be regulated in response to such changing external factors in large part through sigma factors. A previously predicted alternative sigma factor, SigL, was shown by Clark et al. to control metabolism, virulence and sporulation in C. difficile. Through insertional mutation of sigL in two isolates representing ribotypes 027 and 078, the authors revealed pleiotropic, strain-specific and growth phase-specific gene regulation in part corresponding to genetic differences of the two ribotypes. Although not examined in this study, mobile genetic elements in these strains could have contributed to strain-specific variation and would be a logical next target for further investigations.Metabolites of C. difficile and a disrupted gut microbiome, unsurprisingly, CDI is associated with inflammatory bowel disease (IBD). Morbidity and mortality are higher in IBD patients with CDI and, with a global increasing incidence of both IBD and community-associated CDI, it is a significant problem. Mahnic et al. determined gut bacterial communities in non-IBD and IBD patients with CDI. Only in non-IBD patients, CDI was associated with lower diversity of gut bacteria compared to uninfected patients. In IBD patients, reduced gut bacterial diversity was found regardless of CDI. Interestingly, 14 differentially represented operational taxonomic units (OTUs) were common to both CDI and IBD, and four OTUs were significantly decreased in IBD patients with CDI, suggesting a role in disease severity in this patient group.Due to the consequences of the combination of C. difficile strains can behave very differently to the same environmental stimuli, and chemicals they release have as great a role to play in modulating other bacteria and host cells as C. difficile toxins. Colonization is dependent on a disrupted gut microbiota and agents capable of restoring microbiome richness are effective in limiting CDI. While much C. difficile research has been focused on virulence and sporulation, it is equally important to study C. difficile in the presence of other gut constituents for a more holistic understanding of its pathogenicity.The main messages from these papers are: SG drafted the manuscript. TR and PM edited it. All authors approved the final version for submission."} +{"text": "Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhea, and is responsible for a spectrum of diseases characterized by high levels of recurrence, morbidity, and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to the classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to the formation of robust endospores, and an array of adhesins promote gut colonization. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrheal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen. Clostridioides difficile is a gram-positive obligate anaerobe, capable of causing disease through the fecal-oral transmission of robust endospores. These metabolically dormant spores are able to persist in a range of environments, being resistant to oxygen, heat, and many common disinfectants \u2013 contributing to both the organism\u2019s success as a pathogen, and the associated healthcare costs and difficulty of treating infection . He. He65]. C. difficile, owing to the significant immune response to TcdA, but not TcdB, observed in animal infection models [C. difficile clinical isolates producing only TcdB led to a re-evaluation of the roles of each toxin in disease [C. difficile virulence factor, due to its involvement in invoking both local and systemic host damage, and activation of the host inflammatory response [The relative contributions of TcdA and TcdB to virulence have commonly been disputed \u2013 historically, TcdA was accepted as the major virulence factor in n models . However disease . Isogeni disease . However disease . Despiteresponse ; 103. Inresponse .tcdC at the end of the PaLoc, its divergent transcription and its inverse expression profile compared with the rest of the PaLoc genes has led to its common association with PaLoc repression [in vivo \u2013 mutations truncating TcdC are widespread in hypervirulent clinical isolates, and are commonly acknowledged to contribute to the high mortality of ribotype 027 strains [C. difficile ribotype 027 strains revealed that mutation of tcdC led to hypervirulence, and complementation reduced virulence in hamster models [tcdA promoter. The mechanism of this inhibition is unclear \u2013 TcdC may inhibit interaction of the TcdR sigma factor with RNAP, or prevent recognition of the tcdA promoter [The location of pression ,105. The strains . Howeverr models . Despitepromoter . More repromoter .tcdC mutation leading to increased PaLoc expression, and possession of a further toxin \u2013 C. difficile binary toxin (CDT) [C. difficile adherence to host cells, both in vitro and in mouse models [Characterization of the hypervirulent ribotype 027 epidemic strain, first reported at the start of the millennium, showed a combination of factors putatively involved in increased virulence: high-level fluoroquinolone resistance, in (CDT) . CDT is in (CDT) ; 110. CDin (CDT) . This biin (CDT) ,112. In in (CDT) . CDTa thin (CDT) ; 115. Hoin (CDT) . CDT hije models . Detailse models .cdtA and cdtB are located on a separate 6.2 kb chromosomal region of the genome, known as CdtLoc. CdtLoc contains the two genes encoding CDT, and cdtR \u2013 a LytTR family orphan response regulator. CdtR is a positive regulator of both CDT and the PaLoc in hypervirulent strains [C. difficile strains contain either a truncated version of the CdtLoc, or a 68 bp insertion sequence at this site [The regulation of CDT is distinct from, but entwined with, the PaLoc; since strains . Non-CDThis site .C. difficile pathogenesis has become increasingly apparent through both clinical and experimental works. Several cases of patients with C. difficile infections, with the unusual toxinotype of TcdA and TcdB negative/CDT positive have been reported [C. difficile strain, expressing only CDT, caused disease phenotypes in hamster models [C. difficile. CDT also induces inflammation via the Toll-like receptor 2-dependent pathway, and suppresses the protective host eosinophil response [C. difficile pathogenesis, particularly in hypervirulent strains.The substantial contribution of CDT to reported . Althougr models . Howeverr models . This alresponse . Recent response . CollectC.difficile surface proteins are a group of important virulence factors which support C. difficile colonization through adherence to the gut epithelium, activation of the host immune response, and other aspects of pathogenesis. The S-layer is an evolutionary conserved paracrystalline array of protein which envelops the cell, and is ubiquitous among C. difficile strains [ strains ; 124. It strains . These s strains ; 127. AcslpA mutants, has impeded the complete functional characterization of the S-layer. However, isolation and analysis of a spontaneous S-layer null strain has implicated the S-layer in sporulation, and resistance to innate immune effectors including lysozyme and LL-37 [in vivo and in vitro, toxin release of the S-layer null strain was markedly reduced compared to wildtype, associating the S-layer with toxin production, albeit via an unknown mechanism [C. difficile strains \u2013 collectively suggesting an important role in colonization and disease establishment [C. difficile ecology in the gut that we do not fully understand.The essentiality of SlpA, and thus the lack of available isogenic nd LL-37 . Collectechanism . Strong echanism . Bindinglishment . The appslpA, and related genes, and adjacent to a cluster of genes thought to be involved in the synthesis of the cell wall polysaccharide PS-II [The 28 members of the CWP family are defined by the presence of three tandem copies of the cell wall-binding 2 domain (PF04122), with many also having additional individual domains conferring function . Twelve de PS-II . The remde PS-II .cwp2 knockout displayed no defects in growth, sporulation, or virulence in hamster models [cwp84 mutants were fully virulent in hamster models [C. difficile classified adhesin, since antibodies to Cwp66 reduced cellular adherence [cwp66 mutant implicated this surface protein in adhesion, motility and stress tolerance [cwp66 suggested a wider cellular involvement of the protein in antimicrobial resistance and metabolism \u2013 signifying a multifactorial role in pathogenesis [cwp22 led to reduced toxin production, and increased cell permeability and autolysis, as well as reduced cellular adherence. Further, cwp22 mutants displayed reduced fitness compared to WT in mice, collectively suggesting that this protein plays important roles in cell envelope integrity and pathogenesis [C. difficile toxins, Cwp19, has been identified as a transglycosylase, contributing to C. difficile pathogenesis through autolysis, resulting in toxin release in specific environmental conditions [C. difficile strains, with five distinct repeat types identified to date [in vivo [Many CWPs are associated with pathogenesis, and are often highly immunogenic. Antibodies to a range of CWPs, most prominently Cwp2 and Cwp84, were found in convalescent patient sera . Such CWr models . Howeverr models . Cwp66 idherence . Recentlolerance . Furtherogenesis . Cwp22 iogenesis ; 139. Munditions . CwpV isnditions . As withnditions . The Cwp to date . Functio[in vivo .C. difficile. CD2831 is a collagen binding protein, which further promotes adhesion and biofilm formation [C. difficile produces an additional collagen-binding surface protein, CbpA. Despite the cpbA knockout being indistinguishable from its respective WT during immobilized collagen V binding assays, this protein enhances collagen interaction and extracellular matrix adherence, demonstrating the large redundancy involved in host interaction and pathogenesis [As with all pathogenic bacteria, multiple complex mechanisms allow fine-tuned host interactions and immune evasion. One such mechanism, binding to the host extracellular matrix, has recently been described in ormation . CD2831 ormation . Similarogenesis .C. difficile is highly resistant to cell wall hydrolysis via lysozyme, due to a combination of important virulence factors. The C. difficile S-layer clearly provides some protection against lysozyme, since an S-layer null mutant becomes sensitive to physiological concentrations of the enzyme [C. difficile susceptible to lysozyme, suggesting that a steric barrier function contributes to S-layer-mediated resistance to innate immune effectors [C. difficile also has a further inducible resistance system that is controlled by the extracytoplasmic sigma factor \u03c3V [Lysozyme is a ubiquitous and highly conserved antimicrobial protein involved in the innate immune response. This antimicrobial targets the bacterial cell wall, cleaving the \u03b2(1\u20134) bond between N-acetylglucosamine and N-acetylmuramic acid of peptidoglycan . C. diffe enzyme . Deletioe enzyme increaseffectors . In addiactor \u03c3V . \u03c3V is aactor \u03c3V . Classicactor \u03c3V . Deacetyactor \u03c3V . Moreoveactor \u03c3V .C. difficile biofilms. C. difficile forms part of the healthy, multi-species biofilm during asymptomatic carriage [C. difficile. Of note, the sporulation master-regulator Spo0A is associated with regulation of biofilm formation, with mutants exhibiting significantly reduced biofilm [C. difficile biofilm formation [cwp84 mutants displayed a severe defect in biofilm formation, as did mutants lacking the quorum sensing regulator LuxS [dnaK result in stronger biofilms [lexA mutants showing reduced sporulation, motility, and increased biofilm formation [C. difficile biofilm regulation can be found here [The clear contribution of biofilms to both virulence and antimicrobial resistance, along with improved tools and novel study methods, has led to the emergence of biofilms as a \u201chot topic\u201d in microbiology over the last decade. Despite this, little is known about the formation, regulation and maintenance of carriage . Howevercarriage . Despite biofilm . The secormation . Increasormation ; 155. Mutor LuxS . The biobiofilms . Likewisormation . The comund here .C. difficile-host interactions has been explored through confocal laser scanning microscopy in mouse models. In a mono-associated mouse model, which simplifies analysis of pathogen\u2013host interactions without competition from the microbiota, C. difficile was found to produce a 3D biofilm associated with the mucus layer [C. difficile biofilm in relation to the host gut, 16S rRNA analysis identified C. difficile as a minor part of the complex multispecies host biofilm, composed of Bacteroidetes and Firmicutes [C. difficile biofilms may be important for virulence, since they enhance survival through improved resistance to antibiotics and oxygen stress [in vivo biofilms are needed to fully understand the contribution of this lifestyle to pathogenesis.More broadly, the contribution of biofilm to us layer . Cells wrmicutes . C. diffn stress . HoweverC. difficile pathogenesis is antibiotic resistance family of antibiotics, encompassing erythromycin and clindamycin, is achieved through ribosomal methylation, and is gained via acquisition of transposons, such as Tn5398, containing erm genes [erm encodes a 23S rRNA methylase, which modifies the 23S rRNA of the 50S ribosomal subunit, reducing drug binding affinity [C. difficile erythromycin-resistant strains have been identified which lack erm genes \u2013 suggesting the presence of yet uncharacterized alternative resistance mechanisms [C. difficile, however conjugative transposons have allowed transfer of tetM to certain strains, providing a mechanism of ribosome protection against tetracycline [C. difficile 027 clinical isolates found fluoroquinolone resistance did not lead fitness costs in vitro, suggesting that this property will persist in the species even in the context of improved fluoroquinolone stewardship [The success of osporins ; 176. Thrm genes ,177. ermaffinity . Howeverchanisms . Tetracyacycline . Perhapsacycline . Howeverwardship .C. difficile as an urgent threat; and (ii) such treatment options are likely to be further limited through the high degree of adaptation and flexibility in the C. difficile genome. Until recently, three antibiotics were commonplace for the treatment of CDI. Metronidazole was typically the antibiotic of choice for mild-to-moderate CDI in first instance of infection, while vancomycin was reserved for severe and severe-complicated disease. Fidaxomicin \u2013 a narrow-spectrum antibiotic, effective against gram-positive anaerobes \u2013 was often overlooked due to higher cost, being significantly more expensive than metronidazole [Of course, being resistant to a wealth of antibiotics poses two challenges: (i) the extensive resistance displayed greatly reduces treatment options for CDI, warranting the status of nidazole ; 183. Innidazole . This moC. difficile was previously thought to be transient, however a recent explosion in research focussed on characterizing metronidazole resistance has led to the discovery of multiple heritable pathways to reduced susceptibility , phage therapy, and narrow-spectrum antimicrobials.The increasing threat of antimicrobial resistance, coupled with the diminishing number of available treatments has driven interest in both novel antimicrobials and alternative therapeutics for the treatment of CDI. Since the root of the problem lies with broad-spectrum antibiotics, new approaches aim to shift the archetypal FMT involves administration of faeces from a healthy individual (heterologous), or from one\u2019s own previously healthy microbiome (autologous) to restore the natural gut flora. This has gained popularity as a treatment for CDI over the last decade, however the procedure has yet to be standardized, and there have been reports of adverse events post-transplantation . TypicalDespite the clear effectiveness of FMT, its unconventional nature has limited public acceptance, and the lack of process standardization poses a worry to clinicians. Further, upon progression to pseudomembranous colitis, FMT has reduced efficacy and often requires repeat treatment . There iC. difficile have been identified, and increasing evidence suggests at least some of these are viable therapeutic agents [C. difficile growth and toxin levels in a batch fermentation model of CDI [C. difficile phage have been identified, and all of the characterized temperate phage are double-strand DNA viruses, either contractile myoviruses or non-contractile siphoviruses [C. difficile cell surface receptors have been identified, however the S-layer seems to be a common target [C. difficile lysis in vitro, and to reduce disease symptoms and bacterial colonization in hamster models, suggesting targeted phage cocktails may be feasible treatment options [C. difficile eradication in fermentation vessels [Phage therapy involves the use of naturally-occurring bacteriophages to infect and lyse pathogenic bacteria . This hac agents , for exal of CDI . To dateoviruses . Addition target ; 215. Gin target , it is l options . More re vessels .Mycobacterium abscessus infection [in vitro and in vivo, by engineered reduction of lysogeny and redirection of the endogenous type 1-B CRISPR Cas system to target the host\u2019s own genome [C. difficile phage could be engineered to be lytic, albeit not completely, and showed that cargo DNA could be added to the phage genome with no apparent impact on the efficiency of infection or formation of progeny phage. As our understanding of both phage infection and host resistance improves it seems likely that engineered optimized phage will play an important role in the treatment of bacterial infections, particularly those such as CDI, where species-specificity is paramount.R-type bacteriocins, phage tail-like particles that are structurally similar to the contractile myoviruses, have also been explored as potential therapeutic agents ,218. Thenfection . Similarnfection and enhan genome . This lain vivo models, suggesting the possibility of adverse reactions which may worsen disease [C. difficile therapeutic.As with all novel therapeutics, phage therapy does not come without limitations. While phage therapy is generally regarded as safe, and small human trials have not reported adverse effects, the possibility of harm to the patient cannot be ruled out . Host in disease . One pos disease , with ph disease . OverallC. difficile specific therapeutics have come to market in recent years. Fidaxomicin was approved by the FDA in 2011 and the human monoclonal antibody bezlotoxumab followed in 2016. Bezlotoxumab binds to two highly similar sites within the TcdB CROPs domain, thereby blocking binding of the toxin to carbohydrate receptors [C. difficile toxins [C. difficile . Whilstiewed in . Recentlnilazole . TogetheIn addition to the novel compounds that are currently in clinical development, there has also been significant interest in repurposing existing licensed drugs. Among these are the antirheumatic agent auranofin and antiC. difficile as an opportunistic pathogen. Over the last decade, work on C. difficile has exploded, owing greatly to the ever-expanding array of genetic tools available. Despite such victories, there are still many research gaps to address. Further understanding of virulence factors, resistance mechanisms and host interactions will no doubt aid development of novel therapeutics, and exploring alternative therapeutic avenues may also prove fruitful. It should not be forgotten, however, that the success of C. difficile as a pathogen is owed largely to its remarkable genome plasticity \u2013 allowing the acquisition of virulence factors and an array of resistance mechanisms. With this in mind, it is clear that the road to combatting this pathogen is far from complete [The increasing disease incidence, coupled with growing reports of community-acquired CDI and the threat of antimicrobial resistance has focussed efforts on characterization of complete ,260,261."} +{"text": "Panax ginseng C. A. Meyer) has a long history of medicinal use worldwide. The quality of ginseng is governed by a variety of internal and external factors. Nuclear factor Y (NF-Y), an important transcription factor in eukaryotes, plays a crucial role in the plant response to abiotic stresses by binding to a specific promoter, the CCAAT box. However, the NF-Y gene family has not been reported in Panax ginseng. In this study, 115 PgNF-Y transcripts with 40 gene IDs were identified from the Jilin ginseng transcriptome database. These genes were classified into the PgNF-YA (13), PgNF-YB (14), and PgNF-YC (13) subgroups according to their subunit types, and their nucleotide sequence lengths, structural domain information, and amino acid sequence lengths were analyzed. The phylogenetic analysis showed that the 79 PgNF-Y transcripts with complete ORFs were divided into three subfamilies, NF-YA, NF-YB, and NF-YC. PgNF-Y was annotated to eight subclasses under three major functions by GO annotation, indicating that these transcripts perform different functions in ginseng growth and development. Expression pattern analysis of the roots of 42 farm cultivars, 14 different tissues of 4-year-old ginseng plants, and the roots of 4 different-ages of ginseng plants showed that PgNF-Y gene expression differed across lineages and had spatiotemporal specificity. Coexpression network analysis showed that PgNF-Ys acted synergistically with each other in Jilin ginseng. In addition, the analysis of the response of PgNF-YB09, PgNF-YC02, and PgNF-YC07-04 genes to salt stress treatment was investigated by fluorescence quantitative PCR. The expression of these genes increased after salt stress treatment, indicating that they may be involved in the regulation of the response to salt stresses in ginseng. These results provide important functional genetic resources for the improvement and gene breeding of ginseng in the future.Jilin ginseng (Conclusions: This study fills a knowledge gap regarding the NF-Y gene family in ginseng, provides systematic theoretical support for subsequent research on PgNF-Y genes, and provides data resources for resistance to salt stress in ginseng.The online version contains supplementary material available at 10.1186/s12870-022-03687-6. NF-Y gene family have been identified in a variety of plants, including Arabidopsis thaliana\u00a0[Oryza sativa\u00a0[Prunus persica\u00a0[Populus\u00a0[NF-Y gene family influences flowering in plants [Arabidopsis\u00a0[ZmNF-YB16 functions by regulating the expression of photosynthesis-related genes to improve the antioxidant capacity of cells and thus achieve drought resistance [Lycopersicon esculentum, SlNFYA10 negatively regulates the AsA (ascorbic acid) biosynthetic pathway by binding to the CCAAT box in response to oxidative stress [GmNFYA5 gene in soybean enhances drought resistance [NF-Y gene family has been extensively studied in other species, it has not been reported in ginseng.Nuclear factor Y (NF-Y) family transcription factors are important regulators of plant development and physiology . Nuclearthaliana\u00a0, Oryza sa sativa\u00a0, Prunus persica\u00a0, and Pop[Populus\u00a0. Studiesn plants , improven plants , regulatbidopsis\u00a0, and assbidopsis\u00a0\u201312. NF-Ysistance . In Lycoe stress . Overexpsistance . AlthougPanax ginseng C.A. Meyer) is a perennial herb in the Araliaceae family that has a cultivation history of at least 4,000\u00a0years. The Chinese \"Sheng Nong\u2019s Herbal Classic\" records in detail that ginseng tastes sweet, can be used as a tonic for the five organs, calms the spirit, fixes the soul, stops panic and palpitations, removes evil spirits, brightens the eyes, makes the mind happy, educates the mind, lightens the body and prolongs life when taken for a long time [NF-Y gene family members in response to salt stress treatment by screening the adventitious roots of ginseng, which will provide genetic resources for the subsequent improvement of salt stress resistance in ginseng cultivars.Jilin ginseng . Subsequently, we analyzed the evolutionary relationships of the PgNF-Y gene family, and conserved motifs and annotated them with GO functions. In addition, expression pattern analysis and coexpression network analysis were performed based on the PgNF-Y gene expression data. Finally, the response of PgNF-Y family members to different concentrations of salt was explored. The results of this experiment provide important theoretical information and experimental data on the NF-Y gene family for the subsequent study of functional genes in ginseng.In this study, 40 NF-Y gene family in the Jilin ginseng database containing 248,993 transcripts [http://pfam.xfam.org/), and using PFAM IDs as the interrogated sequences, tBlastn was performed in the Jilin ginseng transcriptome. Second, the coding and protein sequences of NF-YA, NF-YB, and NF-YC were downloaded from the Korean Ginseng Genome website (http://ginsengdb.snu.ac.kr/pathway.php); these sequences were used as the interrogation sequences for Blastn and tBlast of the Jilin ginseng transcriptome database at an e-value of 1\u2009\u00d7\u200910\u20136. Finally, 10 protein sequences from the NF-Y family with verified functions were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/) and used as interrogation sequences for tBlastn of the transcriptome of Jilin ginseng. These 10 sequences were from Arabidopsis thaliana\u00a0[Triticum aestivum\u00a0[Oryza sativa\u00a0[Glycine max\u00a0[Solanum tuberosum\u00a0[Nicotiana tabacum [Zea mays [http://itak.feilab.net/cgi-bin/itak/index.cgi) for online analysis while maintaining the sequence information regarding NF-Y structural domains. Based on the results of iTAK, all the obtained NF-Y transcripts were named PgNF-Ys. Finally, the obtained transcripts were verified one by one for structural domains by the SMART online tool (http://smart.embl-heidelberg.de/).To maximize data integrity and reliability, we adopted three different approaches to screen members of the nscripts . First, thaliana\u00a0, Triticuaestivum\u00a0, Oryza sa sativa\u00a0, Glycinecine max\u00a0, Solanum tabacum , and ZeaZea mays . SubsequPgNF-Y gene transcripts based on ORF Finder in NCBI . Based on these results, 12 nucleic acid sequences in the NF-YA, NF-YB, and NF-YC subgroups from Lycopersicon esculentum, Arabidopsis thaliana, Oryza sativa, and Helianthus annuus were downloaded from the NCBI database as outgroups for the phylogenetic tree. These nucleic acid sequences were translated into protein sequences. In MEGA-X software [PgNF-Y genes with 1,000 bootstrap replicates to further illustrate the evolutionary relationships between the PgNF-Y genes in different species. Finally, the evolutionary tree was optimized by Evolview (https://www.evolgenius.info/evolview/#/treeview). To determine the conserved sequence patterns in the NF-Y gene family members in Jilin ginseng, we analyzed the motifs of PgNF-Y transcription factors by the MEME online tool (https://meme-suite.org/meme/doc/cite.html?man_type=web). Finally, the obtained results were visualized by TBtools [We identified 79 transcript sequences with complete open reading frames from 115 software , the max TBtools .Chi-square test at Level 2. The functions of 109,781 transcripts that were annotated by previous authors were used as reference information [We used Blast2GO version 6.0.3 to annotormation .PgNF-Y genes in Jilin ginseng in time and space was determined. To further investigate the interrelationship between PgNF-Y gene expression in 42 farm cultivars, Spearman's correlation coefficients of PgNF-Y gene expression were calculated using R, and BioLayout Express 3D version 3.3 software was used to form a visual network of the obtained results.Since gene expression is subject to a variety of conditions, we obtained data on the expression of PgNF-Ys in 42 farm cultivars, 14 different tissues of 4-year-old ginseng, and 4-year-old ginseng roots, and we plotted the heatmap in R. Thus, the expression pattern of the Panax ginseng C.A. Meyer) were treated with different concentrations of salt in B5 medium , and the treated adventitious roots were incubated under dark conditions at 22\u00a0\u00b0C for 30\u00a0days.In this experiment, the adventitious roots of Jilin ginseng treated with different salt concentrations was weighed separately, and total RNA was extracted from ginseng adventitious root tissue using TRIzol . The HiFiScript gDNA Removal cDNA Synthesis Kit was used to reverse transcribe the extracted RNA into cDNA. Actin 1 was selected as the internal reference gene, and according to the instructions for the UltraSYBR One-step RT-qPCR Kit (Low ROX) , a 7500 Real Time PCR System was used to perform the reaction. The total reaction system was 10 \u00b5L, which included UltraSYBR Mixture (Low ROX) at 5 \u03bcL, upstream and downstream primers at 0.2 \u03bcL each, template at 1 \u03bcL, and ddHNF-Y gene family were obtained. We collected basic information on the PgNF-Y gene family members of Jilin ginseng, including transcript ID, gene ID, mRNA sequence information, the number of transcripts with a length ranging from 240\u20132,624, and the number of amino acids with complete open reading frames (ORFs) ranging from 70\u2013336 . We explored the evolutionary relationships of the PgNF-Y gene family from the perspectives of closely related species, model plants, monocotyledons, and dicotyledons. We selected Solanum lycopersicum, Arabidopsis thaliana, Oryza sativa, and Helianthus annuus as outgroups for phylogenetic analysis via the maximum likelihood method (Table SPgNF-YC04 and PgNF-YC10), and the NF-Y members that were distributed in the same subclass had functional similarity. Based on the evolutionary relationship between outgroups and PgNF-Y transcripts, NF-Y members in these species have an evolutionary origin from a common ancestor . At Level 2, 4 sublevels were enriched in BP, 2 sublevels were enriched in CC, and 2 sublevels (transcription regulator activity and binding) were enriched in MF. The results of 109,781 previously annotated transcripts from the Jilin ginseng transcriptome data were used as a reference, and 115 PgNF-Y transcripts were interrogated. The obtained results were subjected to the chi-square test in SPSS version 23.0 software, and the results showed that all eight sublevels obtained from interrogated gene enrichment were significantly different from those for reference genes (P\u2009\u2264\u20090.05). The cellular anatomical entity and the protein-containing complex were not annotated in the reference genes and 14 different tissues , as well as four ginseng root samples of various ages of PgNF-Y gene expression data (Table SPgNF-Y transcripts (85%) were expressed in at least one cultivar and 36 transcripts (31%) were expressed in all 42 cultivars were expressed in at least one tissue, and 41 transcripts (36%) were expressed in all 14 tissues appeared in at least one age stage and 48 PgNF-Y transcripts (42%) were expressed in all four age stages . The coexpression network results showed that at P\u2009\u2264\u20095.0E-02, the 96 transcripts formed a coexpression network containing 95 nodes with 554 edges to construct the regulatory network and randomly selected another 65 transcripts as the negative control from 115 PgNF-Y gene transcripts. The results of fluorescence quantitative PCR showed that all three PgNF-Y genes were upregulated in ginseng adventitious roots under different salt stress treatments is a common phenomenon in angiosperms [The NF-Y gene family is prevalent in plants. Therefore, the NF-Y transcription factor family has been extensively studied in plants, including thaliana\u00a0, Glycinecine max\u00a0, Triticuaestivum\u00a0, and Lotaponicus\u00a0. The funiosperms , and theArabidopsis by binding to the CCAAT box [PgNF-Y transcripts were annotated to molecular functions (binding and transcription regulator activity); therefore, these genes are likely to bind the CCAAT box of other genes to regulate the growth and development of Jilin ginseng. Nine PgNF-Y transcripts were annotated to biological processes, and 91 PgNF-Y transcripts were annotated to cellular components, suggesting that PgNF-Y genes act not only as a functional gene but also as structural genes in Jilin ginseng. In summary, the PgNF-Y transcripts are functionally diverse in Jilin ginseng.Genome sequencing has shown that most of the genes specifying core biological functions are common to all eukaryotes . MultiplCAAT box . AccordiPgNF-Y transcripts is helpful for exploring the function of these transcripts. From the analysis of the expression levels of PgNF-Y transcripts in the 42 farm cultivars, the relatively close expression patterns of 115 PgNF-Y transcripts suggest that they are widely expressed in Jilin ginseng. The PgNF-YB13-02 and PgNF-YC08-06 genes had high expression levels in the 42 farm cultivars; therefore, these two genes may be housekeeping genes in Jilin ginseng and thus maintain the basic life activities of the plant. The expression of PgNF-Y genes in 14 different tissues of 4-year-old ginseng showed that their expression was tissue specific, and PgNF-YA12, PgNF-YA13, PgNF-YB01, PgNF-YB08-15, PgNF-YC10, and PgNF-YC12 were expressed only in the fruit pedicel, suggesting that these five PgNF-Y transcripts may be involved in the development of ginseng fruit pedicels. The PgNF-YC13 gene was expressed only in leg roots and may be related to their development in ginseng. The median expression levels of the PgNF-Y gene in the leaf blade and fruit pedicel were higher than that in other tissues, indicating that PgNF-Ys are likely to be involved in ginseng fruit and flower development. In ginseng roots of plants at different ages, approximately 42% of the PgNF-Y transcripts were expressed at different times, and approximately 13% of the PgNF-Y transcripts were expressed only in a specific year. The median expression levels of PgNF-Y genes across the four ages was fairly consistent, indicating that the expression levels of the PgNF-Y gene family members did not increase with growth year. The specific expression of PgNF-Y transcripts at four different ages suggested that not all PgNF-Y transcripts were constitutively expressed in Jilin ginseng, and some transcripts might be induced to be expressed in response to changes in external conditions such as soil and climate.Clarifying the expression pattern of P\u2009\u2264\u20095.0E-02, suggesting that such heterologous trimers may also be formed in Jilin ginseng. However, this result needs to be verified using yeast two-hybrid and yeast three-hybrid techniques.The NF-Y transcription factor is a heterotrimer formed by evolutionarily conserved subunits: NF-YA, NF-YB, and NF-YC . NF-YB aNF-YC13, was found in indica rice [TaNF-YA10-1 gene was isolated from the salt-tolerant wheat variety SR3; and overexpression of the NF-YA10 gene in Arabidopsis thaliana regulates the response of Arabidopsis to salt stress [PgNF-YB02, PgNF-YC09, and PgNF-YC07-04, in ginseng responded to salt stress. This result also indicates that the NF-Y gene family is prevalent in plants responding to salt stress.The cultivation time for both garden and forest ginseng is usually 5\u20137\u00a0years. During the long cultivation cycle, the soil environment is one of the key factors controlling the quality of ginseng . A recenica rice ; the TaNt stress . In thisPgNF-Y genes from Jilin ginseng and analyzed their evolution, structure, function, expression pattern and coexpression network to verify the response of PgNF-Y genes to salt stress treatment. The results of the above analysis showed that PgNF-Y gene family members are functionally diverse and exhibit tissue specificity, time specificity, and interspecies differences in their expression patterns. PgNF-Y gene family members act synergistically with each other in the plant. Moreover, the PgNF-Y gene family plays an important role in the plant response to salt stress. This study further demonstrates the role of the NF-Y gene family in the response to salt stress and provides a theoretical basis for the genetic breeding of ginseng.In this study, we screened 40 Additional file 1: Table S1. Basic information of the PgNF-Y gene familyAdditional file 2: Table S2. The identified genes used as evolutionary controls for the PgNF-Y gene phylogenetic analysis.Additional file 3: Table S3. The classification, annotation and GO functional categorization of the PgNF-Y gene transcriptsAdditional file 4: Table S4. The expressions of the PgNF-Y gene transcripts in 14 tissues, 42 farm cultivar roots and 4 ages roots (TPM)."} +{"text": "In 2020, over 1,089,000 new cases of GC and approximately 769,000 GC-associated new deaths were estimated worldwide ranks 5orldwide . Notablyorldwide .via individualized routes.This Research Topic, which has collected six high-quality original researches, aimed to highlight some of the novel emerging advancements pertaining to chemotherapy for GC, as well as the ways that physician scientists help patients with GC receiving chemotherapy by streamlining the treatment, and by delivering the drugs Chen et\u00a0al. explored the risk factors and prognostic impacts of delayed (>60 days after resection) or omitted adjuvant chemotherapy, which may be unacceptable, in resected TNM stage II-III GC by retrospectively analyzing data of 1520 patients undergoing radial gastrectomy, and demonstrated that delayed or omitted postoperative chemotherapy was significantly associated with inferior overall survival (OS) and disease-free survival (DFS). Various factors associated with delayed or omitted adjuvant chemotherapy were further revealed, including female sex, old age, history of intraabdominal surgery, serious postoperative complications, etc. These data call for further standardization of GC care.Adjuvant chemotherapy may need to be administered timely in patients with resected GC upon recovery of performance statuses ; Chen etTao et\u00a0al. analyzed the associations between PNI and efficacy of fluoropyrimidine (FU)-based adjuvant chemotherapy in patients with radically resected stage IB-III GC from two independent retrospective cohorts. Both univariable and multivariable analyses showed that adjuvant chemotherapy was significantly associated with both enhanced OS and DFS only in PNI-positive cases irrespective of cancer stages, but not in PNI-negative ones. For the underlying mechanisms, the authors chemotherapy and the related adverse events were associated with the risk of postsurgical complications. The authors found that preoperative comorbidities, clinical T4b stage, and more cycles (5-6 vs 3-4) of preoperative chemotherapy were independent risk factors for more frequent postoperative complications, while the presence of preoperative SOX chemotherapy, neoadjuvant chemotherapy-associated adverse events, or their severity was not significantly associated with the occurrence of postsurgical complications.In patients with resectable advanced GC undergoing resection, adverse events associated with neoadjuvant chemotherapy may have an impact on postsurgical complications; during exploration of the safety of neoadjuvant chemotherapy for GC, Feldbr\u00fcgge et al. for the first time retrospectively evaluated the safety of systemic chemotherapy (with or without the VEGFR2 antagonist ramucirumab) combined with pressurized intraperitoneal aerosol chemotherapy (PIPAC), a local chemotherapy method using the laparoscopic technique, for GC with peritoneal metastasis. Ramucirumab may cause wound healing problems, while the authors found that the addition of ramucirumab to systemic chemotherapy before PIPAC did not increase the risk of postoperative adverse events, regardless of the length of the treatment-free interval before PIPAC , and was thus a safe alternative for the management of GC with peritoneal metastasis , and demonstrated the safety, feasibility, and efficacy of AIC for NGAS after esophagectomy. Further prospective and randomized evidence is needed to confirm the findings.After removal of esophageal cancer, neoplastic gastroesophageal anastomotic stricture (NGAS) is a highly clinically challenging condition. Together, this Research Topic has included interesting and important publications \u201318 addreIn this era of precision and targeted medicine, future researches may need to focus more on forefront aspects including the application of advanced techniques using artificial intelligence for more personalized prediction of net benefits and noteworthy side effects associated with chemotherapy, novel treatment modalities including immune cell therapies and treatment based on innovative antibody drug conjugates , utilizaConception or design: LH and YS. Acquisition, analysis, or interpretation of data: LH and YS. Drafting of the manuscript: LH. Critical revision of the manuscript for important intellectual content: LH and YS. Administrative, technical, or material support: LH and YS. Both authors have approved the current version of the manuscript for submission and publication.Our study was supported by Shanghai Pujiang Program (21PJ1409700), and the Start-up Fund for the Introduction of High Level Talents by Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. The funders had no role in study design; in the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.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": "The disease profile of low- to middle-income countries is moving towards one seen in Westernised countries, where deaths are mainly attributed to chronic diseases. Children develop risk factors at a young age predisposing them to noncommunicable diseases in adulthood. Most of the risk factors are preventable through healthy lifestyles. Results from South Africa (SA) show that many children, particularly from marginalized communities, do not achieve the minimal requirements of physical activity (PA). Thus, more emphasis needs to be placed on primary prevention strategies, such as incorporating health promotion interventions within established educational and workplace structures. Primary schools present unique opportunities for holistic prevention interventions.Using an ecosystem approach, an interprofessional team of PA researchers, public health specialists and digital innovators, together with partners from the ministry of education and ministry of health in SA, was set up to map and tackle the role of physical education (PE) in the SA school system. Experts identified actionable changes at the school, teacher and policy levels. First, a comprehensive health intervention was developed and implemented in primary schools in low resourced settings in the Eastern Cape of SA. The intervention was followed to learn and adapt. Finally, changes in the educational system will be scaled-up and sustained through governmental institutionalization.In 1994 PE lost its stand-alone subject status and became part of Life Orientation. Ever since, non-specialist teachers lack the confidence and understanding to adequately teach the subject. The interdisciplinary team developed \u2018the KaziBantu model \u2019, to promote PA and healthy lifestyles in public primary schools through two complementary programs: KaziKidz, a PE toolkit for schoolchildren, and KaziHealth, a workplace health intervention program for teachers. Furthermore, Short Learning Programs have been developed for continued professional development of life orientation teachers, thereby introducing lasting changes within the educational system.PE and health literacy are oftentimes neglected in the SA curriculum, especially in marginalized areas. System-wide changes initiated and sustained through local ownership are critical to ensure long-lasting impact. Our multilateral intervention aimed to achieve this to offer children and teachers a quality education."} +{"text": "Recent inconsistent empirical findings on the impact of context on foreign language development (FLD) are related to some conflicting context views, which hinders healthy FLD. Given this, an ecocontextualized approach/perspective is presented as a \u201drecipe\u201d, holding that inter-intrastratally interactive context-oriented learning starts with the alignment of implicit sound-meaning mapping (phonic listening and speaking only) with the low cognitive levels of early starters and physical objects/visual realia in the proximate context, and only when the learners\u2019 cognitive levels develop several years later can it turn to explicit formal learning through abstract written language and contents. Based on this view, a sound-meaning mapping prioritizing (SMMP) route to healthy/sustainable FLD is proposed and testified via questionnaires and an interview/oral test. Results showed: (1) SMMP early starters surpassed the non-SMMP (NSMMP) early starters in oral proficiency at the late stage despite their homogeneity at the early stage; (2) oral, especially listening abilities, could not be well developed at the late stage by NSMMP learners; (3) written proficiency could be developed later by both types. These findings reveal the SMMP route to healthy/sustainable FLD in the Chinese context. The role of environment/context in language learning has attracted increasing interest ,2,3. HowSuch differences in either contextual concepts or empirical findings have been at large for ages in SLA, lacking a consideration of contextual order parameters across timescales and ranges within a dynamic and holistic contextual framework. Given such a research lacuna, this study aims to present an ecocontextualized approach/perspective with a focus on the interactions between language(s) and environments/contexts, viz. an ecointeractive context-oriented perspective that coordinates external , linguistic , and internal parameters through sequential inter-intrastratal interactions. From this perspective, it will then investigate a route for healthy/sustainable FLD regarding early learning effects that will be verified by structured questionnaire surveys and interviews while focusing on the listening bottleneck in context-poor FLD. Prior context models and underlying views tend to focus on parts rather than a whole. One of the views in the Socio-linguistic School is Language Socialization, holding that language learners are in a communicative setting in which social, cultural, and even political factors play a dominating role. In such settings, learners and the language they learn are supposed to be socialized with the help of parents/teachers, viz. socializing agents. Ochs (p. 408)Another socio-linguistic view is more language-based, represented by Halliday and Hasan . This moHalliday\u2019s context view is more explicit, starting with linguistic context, and extending it to situational and cultural contexts. Regarding the relationship between context and language as realizational, Halliday (p. 49) In sum, the Socio-linguistic School focuses on the relatedness of language to sociality. However, some limitations to the Socio-linguistic School may lie in its underestimation of cognitive and psychological factors in language learning and the difficulties in its applications to FLL in \u2018socializing agents\u2019-poor settings. Halliday\u2019s model can tell us quite a bit about \u2018input\u2019 and the \u2018target language\u2019 but much less about \u2018intake\u2019 and \u2018online processing\u2019 as pointed out by , ignorinThe Cognitive School as the mainstream school in SLA, in particular, traditional cognitivists, sees the mind/brain as the locus of human thought and learning, and thought and learning of this type as an information-processing form. Cognitivism concentrates on aggregates of individuals and then construes processed results as representations of \u201caverage human being[s]\u201d (p. 97).Relatedly, being influenced by the cognitive decontextualization view and cognitive psychology, cognitive linguists then view context as a psychological construct, known as Cognitive Context . They seThe Sociocognitive School is mostly influenced by the Sociocultural Theory which focuses on the role of interaction in cognitive and language development and endeRegarding SLA, Ref. pinpointThe sociocognitive context view proposed by van Dijk after crGiven the above conflicting context views, L2 studies have come up with different understandings and inconsistent empirical findings on learning context in the field. In a broad sense, studies show that the effect of learning L2 in the target language countries is greater than that of FLL in classroom instruction at home, especially in terms of communicative strategies , narratiIn a narrow sense, however, the role of external/physical context on interaction is still controversial in SLA. Unlike some researchers\u2019 ,19 claimUnder the above-reviewed conditions, this article, from a healthy/sustainable development perspective, holds that FLD needs ecocontextualization as a \u201cprescription\u201d to treat its \u201cdisease\u201d. Dialectically, things change through mutual interactions. The same is true of language learning. Language learning is an ecocontextualized process in which meaning is made via interstratal and intrastratal interactions . Similar to the well-known semantic triangle , the universally accepted three categories of meanings, i.e., conceptual, textual, interpersonal/associative, cf. ,41, may Ecologically, language and context are symbiotic and the rationality of a learning context depends on its holistic construction through the interaction between language(s) and environments. The co-occurrence of cotextual and contextual variables facilitates meaning-making and understanding of spoken interaction . The sitThe external world is stratified as indicated in the hierarchical society and animal world. The same is true of the external context which can be tristratified: the physical, the situational, and the cultural. \u201c[T]he actual physical setting in which a text might unfold\u201d is viewed as the material setting p. 108)081,45,46The situational stratum is the one that points up to the cultural and down to the physical. Halliday identifiThe cultural stratum is a broader background against which a text is interpreted. Halliday relates The view that language has content and expression planes exists in prior linguistic studies ,50,51. TUnlike the systemicians\u2019 onion-stratified language-context relation, cf. , the linThe internal context also has three strata: information input, formal schema, and content schema. The latter two are cognitive schema . The lowThe cognitive context model views context as a mental construct concerning the listener\u2019s subsets of assumptions about the world . The stuThe relationship between strata within each context circle is intrastratal. Such intrastrata consist of different features of language(s) or signs with different representations. They are the intra-systems of self-organizations that interact with one another in a circulating triangle cf. . Thus, tThe internal context connects both external and linguistic contexts interactively. The interstratal relationship among the three contextual circles is interdependent and ecologically symbiotic. Mental representations link and ground themselves on the outside world. The latter, including the collectively formed ideological and natural world, shapes an individual\u2019s mental representations. Language can be viewed as an expression of meaning as well as a reflection of the external world. In this sense, well-educated adult individuals\u2019 mental representations are shared reflections of collectively formed linguistic, cultural/ideological, and natural knowledge. The relationship between the internal context and the external context is reciprocal, and so is the one between the internal context and the linguistic context. Thus, the relationship between context and language is realizational and symbiotic, and the linguistic system (language) can be instantiated by text (languaging).Interaction arises between at least two types of context variables . In spoken interaction, rapid relevance of phonic utterances to meaning via internal processing can be more dynamic (as with object-meaning processing), whereas slow relevance in the interaction with/of written texts with sociocultural meaning at the slow timescales, can be less dynamic. Let alone the evolution of organic life forms and the interaction with other humans or creatures in outer space at galactic timescales of the physical universe. In sum, characterized by the dynamic continuum , the comThe ECM presents a clearer picture of how L1 and L2/FL contextual variables interact with one another in their interactional processes. In the process of contextualization, L1 and L2/FLL learning contexts differ in the way they are situated in social interactions and based on what functions they have in their respective contexts. L1 is more socially contextualized and has more social functions than L2/FL. FL learners learn FL in their L1 and culture-dominated external environment. As a result, they learn FL mostly through interacting with intuitively written target texts in textbooks rather than with real native speakers or their authentic texts in immediate contexts. Many learners still have listening comprehension bottleneck even after learning FL for over a decade. Under such circumstances, how to work out a route to healthy/sustainable FLD is a major objective herein.Based on the ECM, the tripartite contextual interaction processing enables forms and functions to be mapped via the abstraction of symbolic units from examples in the FL context. Such mapping aligns interstratally and intrastratally . Interstratal interaction means the incorporation of internal, linguistic, and external contextual variables and intrastratal interaction means the ascending order of language learning from lower strata to higher strata, ranging from implicit/phonic/physical/early stage to explicit/graphic/abstract/late stage. In this developmental process, the phonic expression of meaning is mainly implicit whereas the graphic expression of meaning is chiefly explicit. Such differences can be found between L1 and FL learning. Regarding explicit and implicit learning, L1 learning can be classified into three stages in its socializing process: (1) the unawareness (phonic) stage of learning language, (2) the awareness (graphic) stage of learning about language, and (3) the unawareness (automatic) stage of learning through language see .At the first unawareness stage, children are exposed to the phonic input in L1 without any conscious learning of the language form. At this phonic stage, they try to understand meanings by constructing links of sounds with meanings through verbal/nonverbal interpersonal interaction with adults/parents. They are being illiterate or \u2018cognitively underdeveloped\u2019 primeval roles (baby/child/peer) as they cannot read or write, which is the sound-meaning mapping or the intuitively contextualized physical-phonic stage. At the second stage, learners gradually notice and generalize \u201crules\u201d such as plural nouns, with the help of increasing awareness as they become more socially experienced and cognitively developed. They start to learn how to read and write, which is subject to the interaction with written language/genre in a linguistic context, though the interpersonal phonic interaction still continues. Learners\u2019 conscious/analytical learning of the written language dominates at this stage. They learn how to spell words, make sentences, and compose articles of different genres from sample texts which mostly take place with the assistance of teachers at school. Noticing facilitates language learning ,63. that are more concrete are prone to learning, needing less cognition. Thus, the unawareness of FLL is removed from the early stage in the FLL context. This is probably one of the decisive factors for the unsuccessful FLD of most learners. A functional-semantic interpretation for this is that nonverbal meaning-exchanging and functions take place much earlier than verbal ones. Similarly, the meaning-exchanging and functions in phonic L1 take place much earlier than the ones in graphic L1. That Hasan distinguIn compliance with the above objectives and contentions, three hypotheses are made below to testify whether there is a sound-meaning mapping prioritizing route to sustainable FLD from the ecocontextualized perspective, i.e., the learning trajectory from physical-phonic stratum (unconscious learning FL) up to abstract-formal/graphic (conscious learning about FL) and the automatic (unconscious learning through FL) content-based stratum.Hypothesis\u00a01.There is no homogeneity between NSMMP and SMMP learners of both senior high schools and universities in terms of their oral and written English proficiencies at the early stage.Hypothesis\u00a02.There are no significant differences between the NSMMP and SMMP learners at both senior and tertiary levels in terms of oral and written English proficiencies.Hypothesis\u00a03.If the above two hypotheses are true, there are no significant differences between NSMMP and SMMP learners in their scores in entrance examinations, interview/oral tests, and multiple choices.Considering the hypotheses and the time span of the order parameters discussed above, this study adopted a cross-sectional design and a structured questionnaire for surveys, followed by an interview as immediate experiments or longitudinal and quantitative studies are unlikely to be carried out in different schools or universities over a decade. Furthermore, based on the ecocontextualized view and the SMMP route to FLD, the study design implemented a cross-sectional comparison among juniors, seniors, and tertiary participants via questionnaire and interview/oral test due to the potentially limited population of SMMP learners. It is hoped, thus, that in so doing, learners\u2019 different age, cognitive levels, and contextual knowledge of lower/higher intra/interstrata will be highlighted and thus their effects on SMMP vs. NSMMP FLD will be displayed more clearly.The questionnaire collection sites were intact parallel classes in Guangzhou city, China. 281 students in total were recruited on a voluntary basis after being given a full explanation regarding the purpose of the study by the research assistants of the project. Among them, there were 99 middle school students . 182 university students filled out the questionnaire forms online via WeChat. While most FL learners began with letters, i.e., reading, speaking, listening, and writing simultaneously, students from an experimental high school in Guangzhou, one of the national key middle schools, could be an exception, which helped with a potential collection of the data required herein. According to the students\u2019 answers to demographic questions, they were divided into NSMMP and SMMP early starters. The inclusion criteria were as follows: All the students were able to communicate in Mandarin and complete the questionnaire written in Chinese. The exclusion criterion, as explained by the research assistants to the participants, was that students who moved from English-speaking countries to Guangzhou with their parents were excluded due to their L2 or L1 acquisition environment during childhood.The study\u2019s questionnaire consists of 6 sections. The interview was carried out in a foreign language school. 30 juniors were recruited from a grade, among whom fifteen were randomly selected from those who reported that they were SMMP early starters whilst the other fifteen, NSMMP early starters. Before the interview, they were kept in a separate room. Each was separately called into the interview room and was asked in turn the same four questions. To examine how fluently and correctly the students answered, two testers, while recording response-reaction time, assessed the testees\u2019 responses and gave marks according to the evaluation criteria. Average marks of the two testers were calculated for the participants whose SMMP or NSMMP types were unknown during the test. The interview was designed to compensate for the self-reported questionnaire items.p values of the proficiencies at early and late stages were calculated so as to analyze potential significant differences between the NSMMP and the SMMP learners of both secondary school and universities.This study adopted the SPSS for data analysis. First, descriptive statistics were adopted to display basic demographic information. Next, Independent Sample Analysis was used to explore the mean values and t values for the early and late oral and written proficiencies of the four cohorts. These values were calculated for listening and speaking as well as reading and writing (written proficiency). All the entrance examination scores the participants filled in the forms were converted into percentages as some examinations have different total scores. For some reason, only 100 students filled the questionnaire examination items correctly . Finally, the p (t) = 0.34 (\u22121.06) and written proficiency, p (t) = 0.48 (\u22120.77). There were no significant differences between university NSMMP and SMMP learners in either the oral proficiency, p (t) = \u22120.71 (0.66), or written proficiency, p (t) = 0.77 (\u22120.33), indicating that all the participants of different levels agreed that they had homogeneity in either oral or written FLL at the early stage though they had different time spans and routes of FLL.p (t) = 0.00 (\u22125.11), and written proficiency, p (t) = 0.00 (\u22125.77). Similar gains were also found between university NSMMP and SMMP learners in both the oral proficiency, p (t) = 0.00 (\u22125.49); and written proficiency, p (t) = 0.00 (\u22124.69). These results may indicate that they can be subjective based on the questionnaire items and thus more convincing results need to be found from other data collections such as tests in what follows.p (t) = 0.009 (\u22122.22), whereas there were no significant differences in their oral accuracy, p (t) = 0.418 (\u22120.82). As for the tertiary learners\u2019 multiple choices of translation and grammar, no significant differences were found between the two types, p (t) = 0.063 (1.89); p (t) = 0.97 (1.68) though the NSMMP early starters were better than the SMMP ones. These results corroborate the SMMP route to FLD, i.e., SMMP learning can be more sustainable than NSMMP learning as sound-meaning can be well mapped by early starters if they solely begin with sound-meaning mapping rather than simultaneous reading, speaking, listening, and writing.p (t) = 0.034 (\u22122.167), and University Entrance Oral Examination scores, p (t) = 0.032 (\u22122.187), except University Entrance Written Examination scores, p (t) = 0.577 (\u22120.561). These results indicate that NSMMP learners turn out to have the same learning achievements in written language as SMMP learners whereas they cannot achieve so high a proficiency as the latter in terms of oral English. Another indication is that scores can be more reliable than elicited intuitive judgments as shown in the tertiary written results from questionnaires . Generally, language learning through interaction in rich interactive contexts facilitates implicit learning of implicit knowledge as seen in context-rich au pair or international community FLL settings ,17. ConvAddressing Hypothesis 2, our finding indicates significant differences between NSMMP and SMMP learners of either the senior high school or the universities in terms of both oral and written English proficiencies. In contrast to Baumert et al.\u2019s findingsCognitively, young starters, due to their low cognitive level, are unlikely to process complex information with the four skills simultaneously involved in learning. If forced, they will be unable to comprehend and produce the target language automatically at the third stage. Interacting with the intuitively produced graphic/written language during the onset stage belongs to the less dynamic end of the dynamic context continuum rather than the more dynamic phonic one, viz. sound-meaning mapping, which makes a huge difference in processing. Thus, this finding may have provided empirical evidence for the acquisition hypothesis that hasGiven the FLD route, a good beginning presupposes an early age and prioritizes the phonic learning stage. One possible reason is that the material/physical objects are always situated in the early starters\u2019 learning, particularly the phonic elements, i.e., the material base of language. One point to note is: if the SMMP early starters had more gains in oral proficiency, why did the results also show more gains in written proficiency than the NSMMP early starters? Generally, written language learning requires more analytical abilities at which the SMMP starters are assumed not to excel. Our interview may prove it. The junior SMMP students had two advantages that the NSMMP junior students did not possess. One is that their fluency was better, and the other is their reaction time was quicker in conversations, indicating that they had better oral proficiencies. However, their accuracy was not as good as their counterparts\u2019. This finding is even hidden in their score on Senior High School Entrance Examination cf. . Thus, tAddressing Hypothesis 3, our result reveals significant differences between the NSMMP learners and the SMMP learners in their oral English scores rather than written language scores for the juniors\u2019 interview (accuracy test results), tertiary entrance written examinations, and translation and grammar (discrete) multiple choices. However, despite their strength in oral proficiency, the SMMP learners could learn well the written language, especially grammatical knowledge explicitly as the NSMMP learners did at the late stage, as there were no intergroup significant differences in the written language results. This finding indicates that listening and speaking can be better learned implicitly without reading and writing involved at the initial stage. Hence, such an SMMP route to FLD has been proven more sustainable at the late stage. The results herein provide evidence for the route to FLD and support the over ten-year case study finding \u2014an FL leReturning to the above irresponsive question, the SMMP starters\u2019 written English proficiencies were significantly different from the NSSMP starters\u2019 based on the self-reported questionnaire data in Socially, young learners map sound and meaning in immediate contexts in which simple utterances, physical objects, simple social roles, and more are aligned implicitly with one another via their socializing experiences. Thus, the older they are, the more sociolinguistically appropriate their language(s) will be. Given this, language teaching should take into consideration the three stages for the FLD route in which the order parameters change. Learners of different ages and cognitive levels are bound to have different roles and surroundings to align with at the three stages. On the one hand, FLD contexts are short of native speakers of the target language, especially their social roles in society. Thus, pseudo roles and their speech extended cognitively in linguistic contexts are essential in the FLL context. On the other, social roles and role relations are enacted by the target language to which one is exposed. The roles one plays range from baby, child, peer, roommate, classmate to worker or boss and more, which demonstrates a bottom-up trajectory of development from the low strata of contexts to the upper ones , a continuum in fact. Such roles are closely realized by their corresponding language at the different stages. Furthermore, social roles embody their thoughts, emotions, values (cognitive schema), and responsibilities . Thus, tThis article has presented an ecocontextualized approach/perspective by integrating the concepts of existing context studies from the perspective of healthy/sustainable FLD to clarify the controversial issues on the role of context and inconsistent underlying learning theories in L2 studies. To understand the differences in the interactional L1 and L2/FL processes, this perspective has been further analyzed and discussed in terms of sequential inter-intra-stratified contextualized learning and a sound-meaning mapping prioritized route for sustainable FLD. Empirical research was conducted to verify the perspective and the route. Findings show that SMMP learners excelled over the NSMMP learners, especially in oral proficiency, and that oral, especially listening abilities, could not be developed well at the late stage by NSMMP learners, whereas written proficiency could be developed later by both. Thus, following the sound-meaning mapping prioritizing route will lead to healthy/sustainable FLD.Implications of this perspective and route are significant as traditional approaches and teaching methods could not solve the listening bottleneck problem of most FLL students. The application of this route will promote the FLD of worldwide learners and their international communication. Thus, it is envisaged that more SMMP early starters in future research will help further corroborate the findings of the present study, and overcome the shortcoming of the limited number of participants in this study due to the restricted availability of SMMP learners at present. Furthermore, further research can be conducted on the effects of SMMP FLL early starters from different L1 backgrounds. Considering the limitations in the research methods, more sound methods such as triangulation methods can also be utilized to verify the ecocontextualized approach and the SMMP route for FLD."} +{"text": "This study evaluated the susceptibility to groundwater pollution using a modified DRASTIC model. A novel hybrid multi-criteria decision-making (MCDM) model integrating Interval Rough Numbers (IRN), Decision Making Trial and Evaluation Laboratory (DEMATEL), and Analytical Network Process (ANP) was used to investigate the interrelationships between critical hydrogeologic factors (and determine their relative weights) via a novel vulnerability index based on the DRASTIC model. The flexibility of GIS in handling spatial data was employed to delineate thematic map layers of the hydrogeologic factors and to improve the DRASTIC model. The hybrid MCDM model results show that net recharge (a key hydrogeologic factor) had the highest priority with a weight of 0.1986. In contrast, the topography factor had the least priority, with a weight of 0.0497. A case study validated the hybrid model using Anambra State, Nigeria. The resultant vulnerability map shows that 12.98% of the study area falls into a very high vulnerability class, 31.90% falls into a high vulnerability, 23.52% falls into the average vulnerability, 21.75% falls into a low vulnerability, and 9.85% falls into very low vulnerability classes, respectively. In addition, nitrate concentration was used to evaluate the degree of groundwater pollution. Based on observed nitrate concentration, the modified DRASTIC model was validated and compared to the traditional DRASTIC model; interestingly, the spatial model of the modified DRASTIC model performed better. This study is thus critical for environmental monitoring and implementing appropriate management interventions to protect groundwater resources against indiscriminate sources of pollution.The online version contains supplementary material available at 10.1007/s11356-023-25447-1. The importance of groundwater resources to all life forms cannot be over-emphasized, particularly as valuable water resources for irrigation and livestock maintenance Skevas . GloballSeveral studies have shown that groundwater pollution is a significant cause of environmental and health concerns in most developing countries due to poor regulation of anthropogenic activities from industries, agriculture, mining, and waste disposal is subjective, requiring expert opinion or engagement using MCDM models , net recharge (R), aquifer media (A), soil media (S), topography (T), vadose zone impact (I), and hydraulic conductivity (C) were developed. Primary and secondary data were obtained for this study. A geophysical survey using a VES system was employed for primary data collection to determine the resistivity of various soil layers. The data from the geo-electric survey was used to delineate the impact of the vadose zone, the depth-to-water table, hydraulic conductivity, and aquifer media thematic map layers in ArcGIS. Secondary data obtained for this study include rainfall data from the Worldbank\u2019s Climate Database used to delineate net recharge factor, soil data from the Harmonized World Soil Database used to delineate soil media factor, and the Digital Elevation Model (DEM) from the United States Geological Survey used to delineate the topography factor. The weights and ratings of the factors of the DRASTIC model were obtained using a hybrid MCDM model (IRN-DEMATEL-ANP). The model was also used to assess the degree of impact of the factors. Expert opinion obtained in the form of a matrix was used as data input for the model. The IRN was first employed to treat uncertainty and imprecision contained in the expert opinion. The DEMATEL method was then employed to evaluate the significance of the factors and create a relationship network between them. Finally, the ANP method was employed to determine the relative weights of the factors. Based on the determined weights and thematic maps of the factors, the DRASTIC model was employed to present a spatial distribution of the susceptibility to groundwater pollution in the area.The study area (Anambra State) is located in the eastern part of Nigeria and lies between latitudes 5\u00b0 40\u02b9 and 6\u00b0 48\u02b9 north and longitudes 6\u00b0 35\u02b9 and 7\u00b0 50\u02b9 east. The state is located in the tropical rain-forest zone of West Africa Fig.\u00a0 with an A geophysical survey was conducted to determine the resistivity of various soil layers in the study area. The survey was aimed at determining the variations in resistivity with soil depth. A geo-electrical method that utilizes a vertical electrical sounding (VES) system was employed for the survey. The VES was conducted by deploying an ABEM Terrameter Self-Averaging System (SAS) 1000C device. The instrument can display apparent resistivity values calculated from Ohm\u2019s law digitally. It also automatically records the voltage and current, piles up the results, and determines the resistance in real-time with an instant digital read-out , the aforementioned depth-to-water table values, resistivity values, and the calculated hydraulic conductivity values were imported into the GIS software. The IDW was subsequently applied for interpolation to delineate the impact of the vadose zone, the depth-to-water table, hydraulic conductivity, and aquifer media thematic map layers.The soil data were derived from the Food Agriculture Organization (FAO) database, precisely the Harmonized World Soil Database (HWSD). After geo-referencing and digitization, the soil data was converted to a raster format to delineate the thematic map layer for soil media. The soil media factor describes the influence of the uppermost part of the soil layer, the topsoil. The satellite imagery of the Shuttle Radar Topography Mission (SRTM) was sourced from the United States Geological Survey (USGS) website, and the data was used to define the slope using the surface tool in ArcGIS software. The slope represents the topography as a factor in the DRASTIC model. The mean monthly precipitation data were obtained from World Bank\u2019s climate database for 26\u00a0years (1991\u20132016). The net recharge, a factor that describes the amount of water per unit area that penetrates the ground surface and percolates down to the water table, was calculated as 12% of the average annual precipitation , c denotes the number of criteria (hydrogeologic factors).The next step in the IRN method is aggregating the rough sequence of all the decision-makers, which is done by applying Eqs. and 3)..3).2\\docThe last step of this method is the transformation of interval rough numbers into crisp numbers to obtain our initial decision matrix. Hence, the matrix ing Eqs. and 6) \\document}D (Eq.\u00a0).5\\documD obtained using the IRN method is used as the input data for the DEMATEL method. The first step of this method involves the normalization of the elements of the initial decision matrix The second phase of the hybrid MCDM model involves the application of the DEMATEL method to analyze the structure and the strength of the relationships between the factors. The initial decision matrix n in Eq.\u00a0.8\\documeThe elements ing Eqs. .910T illustrates the direct/indirect relationships of the hydrogeologic factors and is defined using Eqs. \\documenti has on other conditions; this is obtained by calculating the sum of the i-th row of the matrix j criterion, and this is obtained by calculating the sum of the j-th column of the matrix After determining the total relationship between the conditioning factors, values of (see Eq.\u00a0). \\docum(see Eq.\u00a0).13\\docui criterion that has on the remaining criteria and designates its position in the problem. i-th criterion is effective and falls into the category of \u201ccauses.\u201d Also, a negative value of i-th criterion will be under the influence of others and fall into the category of \u201ceffects.\u201d Criteria with a high value of The values T obtained in the model\u2019s previous phase is used as the input data for this phase. The first step in this phase is the creation of an unweighted supermatrix from the total relationship matrix total-relation matrix The third and final phase of the hybrid MCDM model involves applying the ANP method to determine the relative weights of the hydrogeologic factors. The total relationship matrix n in Eq.\u00a0. If \\docThe matrix k denotes any number. After determining the individual weights of the hydrogeologic factors, the DRASTIC model was applied to aggregate them and produce a groundwater vulnerability map of the study area.The final step of this method involves the determination of the weight of the hydrogeologic factors. Here, a limit supermatrix is obtained by multiplying the weighted supermatrix by itself multiple times. The weighted supermatrix can be raised to the limiting powers until the supermatrix has converged and become a long-term stable supermatrix to obtain global priority vectors, called IRN-DEMATEL-ANP influence weights, such as surface region,introduction of contaminant to the ground precipitation is responsible for the transportation of pollutants to groundwater,the contaminants have enough water mobility to reach the water table, andthe area under study is 100 acres or more significantly affects water table net recharge determines the propensity of pollutants to surface runoff or retention toward the water table impacts the speed of pollutant migration into an aquifer ..12). EquThe elements of the matrix 22) and 2. The resx-axis contains values of the y-axis contains values of the Each row of the matrix by Eq.\u00a02 to obtaisee Fig.\u00a0.Table 4VThe interpretation of the result obtained for the The individual weights of hydrogeologic factors were determined using the ANP method, and the total relationship matrix was employed as input data. Expert opinion was relied upon to arrive at an optimum \u03b1 threshold value of 1.08. This was used to filter out minor influences from the matrix 2, with the resultant groundwater vulnerability map showing that 12.98% (592.21 km2) of that land mass falls into the very low vulnerability class, 31.90% (1455.78 km2) falls into low vulnerability class, 23.52% (1073.20 km2) falls into average vulnerability class, 21.75% (992.42 km2) falls into high vulnerability class, and 9.85% (449.40 km2) falls into very high vulnerability class. Anambra West and East LGA were classified as highly vulnerable areas; this agrees with previous studies on flooding, indicating that the study area\u2019s western region is prone to flooding , the vulnerability map based on our hybrid MCDM model classified six under the high vulnerability class and four under the average vulnerability class. Out of 12 boreholes with a lower concentration of nitrate (>\u20094\u00a0mg. L\u22121), ten were classified under low vulnerability, and the remaining two were under average vulnerability class. In comparison, the traditional DRASTIC model ranked 4 out of 10 higher concentrations under high vulnerability, four under average vulnerability and two under low vulnerability classification. For the lower concentrations, the traditional DRASTIC model classified 6 out of 12 under low vulnerability, two under average vulnerability and four under high vulnerability. See Supplementary information \u22121 was under the average classification of the hybrid model. At the same time, it was classified in the traditional model within the low vulnerability zone. Generally, the hybrid model shows better agreement with the nitrate concentration, an indication of improvement in vulnerability assessment, which is critical in groundwater resource management.Out of 10 boreholes with a higher concentration of nitrate Below is the link to the electronic supplementary material."} +{"text": "Emergency psychological responding professionals are recruited to help deal with psychological issues as the Corona Virus Disease 2019 (COVID-19) continues. We aimed to study the neural correlates of psychological states in these emergency psychological responding professionals after exposure to COVID-19 related trauma at baseline and after 1-year self-adjustment.t-tests. The brain functional network correlates of psychological symptoms were explored.Resting-state functional MRI (rs-fMRI) and multiscale network approaches were utilized to evaluate the functional brain activities in emergency psychological professionals after trauma. Temporal (baseline vs. follow-up) and cross-sectional differences were studied using appropriate At either time-point, significant changes in the ventral attention (VEN) and the default mode network (DMN) were associated with psychological symptoms in emergency psychological professionals. In addition, the emergency psychological professionals whose mental states improved after 1\u2009year demonstrated altered intermodular connectivity strength between several modules in the functional network, mainly linking the DMN, VEN, limbic, and frontoparietal control modules.Brain functional network alterations and their longitudinal changes varied across groups of EPRT with distinctive clinical features. Exposure to emergent trauma does cause psychological professionals to produce DMN and VEN network changes related to psychological symptoms. About 65% of them will gradually adjust mental states, and the network tends to be rebalanced after a year. In December 2019, an epidemic named COVID-19 occurred in Wuhan, Hubei Province, China . BesidesThese healthcare workers in emergency care settings are always exposed to stressful environment and trauma, so they are particularly at risk for post-traumatic stress disorder (PTSD) , 9. HowePrevious studies have demonstrated that resting-state fMRI (rs-fMRI) could be considered a promising imaging modality to diagnose and evaluate many mental diseases . ThroughThis work reports the results of a longitudinal project in emergency psychological responding professionals assessed by clinical assessments and rs-fMRI data after supporting Wuhan. The aims were to: (i) investigate functional network alterations at baseline in EPRT; (ii) analyze longitudinal progression trends of brain functional network between EPRT subtypes based on rs-fMRI data; and (iii) explore the impacts of trauma experience and its underpinning neural basis in emergency psychological responding professionals. We hypothesized that alterations of multiscale functional networks in EPRT would be correlated with changes of psychological symptoms, and subsequent improvement of psychological symptoms would be underpinned by the dynamic alterations of brain functional network.Our initial sample included 85 participants\u201446 EPRT members, and 39 healthy controls (HC). All participants were aged between 18 and 55, right-handed, and had an education of at least 15\u2009years. All participants were screened with Mini International Neuropsychiatric Interview (M.I.N.I.) and free of current and past psychiatry diseases. Other exclusion criterions included: substance abuse or dependency within 6\u2009months, contraindication to MRI scan, pregnancy, history of neurological disorders, and family history of mental illness. Three EPRT members was excluded for incomplete MRI or movement artifacts. One subject was identified as outlier of the Posttraumatic Stress Disorder Checklist for DSM-5 (PCL-5) total score and excluded from our present study. Besides, the local psychiatrists from Shanghai were enrolled as HC and matched the EPRT members with age and gender. All HCs were never exposed to COVID19 patients or highly suspected ones. Seven subjects were excluded either for the lack of clinical assessment or the poor image quality. All study procedures were approved by the Institutional Review Board of Shanghai Mental Health Center, which was in accordance with the Declaration of Helsinki. Written informed consents were provided by all the participants.Due to the loss during follow-up, a longitudinal imaging sample of 31 EPRT members and 25 HCs was included. Among EPRT members, six had an increase in PCL-5 score, 20 had a decrease in PCL, and five had no change in PCL during the 1-year follow-up, so EPRT members were divided into EPRT+ group and EPRT\u2212 group according to the changes in PCL-5 scores. Participant flow through the study is presented in Six psychological scales were applied to assess the clinical features among participants. Patient Health Questionnaire (PHQ9) is a reliable and valid measure of depression severity . General2, and number of slices 70 and 220 time points. Structural images were acquired with magnetization-prepared rapid acquisition with gradient echo (MPRAGE) sequence, with the following parameters: acquisition time 221\u2009s, repetition time 1,800\u2009ms, echo time 2.28\u2009ms, flip angle 8\u00b0, voxel size 1*1*1\u2009mm, field of view 256*256\u2009mm2, and number of slices 160.MRI data were obtained with 3\u2009T Magnetom Scanner at the Radiology Department of Renji Hospital. Eyes opened resting-state fMRI images were acquired using the echo-planar imaging (EPI) sequence. The EPI sequence parameters were listed as follows: acquisition time 452\u2009s, repetition time 2,000\u2009ms, echo time 30\u2009ms, flip angle 90\u00b0, voxel size 3.3*3.6*2.4\u2009mm, field of view 230*230\u2009mm3. Next, the normalized images were smoothed using a Gaussian filter with 6-mm full-width at half maximum, and its linear trend was removed by temporal linear regression. Also, a set of noise signals were regressed, including the average signals of white matter, average signals of cerebrospinal fluid and the Friston 24-parameter of head motion. At last, rs-fMRI data were bandpass filtered (0.01\u20130.08\u2009Hz) to reduce physiological artifacts. For each dataset, motion correction was checked to ensure that the maximum absolute shift did not exceed 2\u2009mm, and the maximum absolute rotation did not exceed 2\u00b0. There were no significant group differences in head motion . We further evaluated frame-wise displacement (FD), which was defined as the sum of the absolute derivative values of realignment parameters. The maximum FD was <0.5\u2009mm with a mean\u2009\u00b1\u2009SD across all participants of 0.118\u2009\u00b1\u20090.09. So, motion scrubbing was not performed to remove the spike volume.All rs-fMRI data were preprocessed using DPARSF software . The firThe whole cerebral cortex was parcellated into 300 functionally homogenous regions based on the Schaefer-300 template. Each brain region was classified as one of seven network: the VIS, SOM, dorsal attention (DOR), ventral attention (VEN), limbic (LIM), and frontoparietal control modules (FCP), and the default mode network (DMN), according to the canonical seven-module parcellation given by Yeo et al. Figure . The regThe brain functional network was constructed by a weighted adjacency matrix where nodes corresponded to brain regions and edges corresponded to functional connectivity between network nodes. Intramodular connectivity represented the connection strength within the module, and the intermodular connectivity between two modules represented the strength connecting both modules. Intra- and inter-modular connectivity provide information about the organization and integration of brain networks, which can help us understand how the brain processes information and supports cognition. Besides, the absolute weighted functional network for each subject was used to calculate the following topological properties: betweenness centrality, degree centrality, clustering coefficient, nodal efficiency, local efficiency, assortativity, hierarchy, local network efficiency, global network efficiency, and small-world. Nodal metrics, which have one value for every region in each subject, include betweenness centrality (BC), degree centrality (DC), clustering coefficient (CC), nodal efficiency (NE), and local efficiency (NLE). Global metrics which have one value for each subject, are assortativity, hierarchy, local network efficiency (Eloc), global network efficiency (Eglob), and small-world properties. In order to investigate the complete connectivity patterns related to psychological symptoms, we calculated the area under curve (AUC) for topological properties using a range of sparsity from 0.10 to 0.40 for each subject. This AUC index for each topological property is calculated to provide a scalar independent of a specific threshold to reduce the influence of different imaging devices and their parameters , and is t-test, and categorical data were analyzed by chi-square test. Since the ages, gender, and years of education were no significant difference in participants, we compared the network properties between EPRT members and HC at baseline. In addition, because the EPRT members was divided into EPRT+ group and EPRT\u2212 group according to the changes in PCL-5 scores during follow-up, the paired t-test was used to test specific within-group (EPRT+ and EPRT\u2212) time effects, and the two sample t-test was used to test between-group baseline and follow-up differences. In order to further understand the reasons for the changes in psychological symptoms in the EPRT group after 1\u2009year, we also compared the brain functional activities of the EPRT- and non-EPRT groups, as well as the EPRT- and HC groups. We also used paired t-tests to compare the relevant measures at baseline and follow-up for HC group. The multiple comparison corrections for p values used the false discovery rate (FDR) method (q\u2009<\u20090.05), and uncorrected p\u2009<\u20090.05 was considered a trend. All statistical analyses were performed by MATLAB R2019b. Additionally, associations between cross-sectional and longitudinal network metrics that showed significant differences and clinical measures were explored using Pearson correlation coefficients. The significant level was set to p <\u20090.05, two-sided.Continuous variables were analyzed by Student\u2019s p\u2009=\u20090.045). Moreover, substantially more subjects in the EPRT group felt affectively fragile and were stuck in a constant state of sadness, especially exposed to COVID-19 affairs. For the scale of IRI, there were no significant differences between groups.The clinical and demographic features of our recruited sample were illustrated in No significant difference in the intramodular and intermodular connectivity strength of the functional network was found between EPRT vs. HC groups. In contrast, graph analysis showed significantly reduced NLE of frontal operculum insula_7 (FrOperIns_7) and parietal operculum_3 (ParOper_3) areas involving VEN and increased DC and NE of temporal_4 (Temp_4) and temporal_6 (Temp_6) areas involving DMN in EPRT groups relative to controls see .No difference was found in global and nodal functional topological properties as well as in intramodular and intermodular connectivity strength between EPRT+ and EPRT\u2212 groups at baseline.Significant difference in the connectivity strength of the DMN and LIM intermodular functional network was found between EPRT\u2212 vs. non-EPRT\u2212 groups. Graph analysis showed increased DC of temporal pole_2 (TempPole_2) and CC of orbital frontal cortex_1 (OFC_1) area involving LIM in EPRT\u2212 group relative to non-EPRT\u2212 group.r =\u20090.2316, p =\u20090.033) and NE of temporal_4 (Temp_4), and PCL-5 was correlated with NE of temporal_6 (Temp_6) involving DMN in participants. Besides, SRQ was inversely correlated with frontal operculum insula_7 involving VEN. However, these correlations did not persist after FDR correction. Following the Pearson correlation analysis, we found that the SRQ was correlated with DC . This is in keeping with the known role of the DMN module reflecting the neural basis of the self and thinking about others in the resting state scan is a potential limitation of our study. Finally, our sample is relatively small, especially after 1\u2009year follow-up, which will limit the generalization of the result.In conclusion, capitalizing on the comprehensive clinical mental evaluations and the rs-fMRI scans, we identified the impacts of psychological trauma and the brain functional activity alteration in emergency psychological responding professionals. Approximately 65% of EPRT may improve the psychological condition mainly through the significant self-adjustment of DMN and VEN after trauma.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Renji Hospital, School of Medicine, Shanghai Jiao Tong University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.YH, ZW, and YZho: study design. HH, YS, and YW: data collection. YH, YS, and HH: analysis and interpretation. YH: drafting of the manuscript. YZha, XH, SS, KZ, ZW, YZho, HH, and YS: critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by the National Natural Science Foundation of China (grant no.82171885 and 82001457), Medical Engineering Cross Research Foundation of Shanghai Jiao Tong University (no. YG2022QN037), Shanghai Science and Technology Committee Project , and Shanghai Rising Stars of Medical Talent Youth Development Program, Youth Medical Talents-Medical Imaging Practitioner Program (grant no. SHWRS(2020)_087).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": "As water\u2010saturated polymer networks, hydrogels are a growing family of soft materials that have recently become promising candidates for flexible electronics application. However, it remains still difficult for hydrogel\u2010based strain sensors to achieve the organic unity of mechanical properties, electrical conductivity, and water retention. To address this challenge, based on the template, the excellent properties of MXene nanoflakes are fully utilized in this study to prepare the P(AA\u2010co\u2010AM)/MXene@PDADMAC semi\u2010interpenetrating network (semi\u2010IPN) hydrogel. The proposed hydrogel continues to exhibit excellent strain response and flexibility after 30 days of storage at room temperature, and its performance do not decrease after 1100 cycles. Considering these characteristics, a hydrogel\u2010based device for converting sign language into Chinese characters is successfully developed and optimized using machine learning. Therefore, this study provides novel insight and application directions for hydrogel families. MXene semi\u2010interpenetrating network hydrogels take full advantage of the characteristics of MXene nanoflakes and the interaction between components. The strain sensor based on it has high stability, high sensitivity, and fast response recovery time. And the portable device can convert sign language into Chinese characters. Therefore, flexible electronic devices have attracted increasing attention in recent years. The excellent flexibility of electronic materials is key to ensuring their mechanical and electrical properties. Flexible electronic include conductive polymers , carbon\u2010based nanomaterials , liquid metals , metal nanowires (such as silver and gold nanowires), and semiconductor materials (such as silicon and germanium). Compared with other flexible materials, composite conductive polymers have greater potential because two parts are included: a polymer matrix and conductive fillers. Therefore, they can be considered functional materials for flexible electronic devices.With the rapid development of science and technology, traditional rigid electronic devices can no longer meet the developmental needs of emerging fields such as electronic skins (E\u2010skins),1 pers Moreover, P\u2010hydrogels do not generally retain water. After losing water, both conductivity and also inherent characteristics, such as flexibility are lost. This significantly limits practical applications for P\u2010hydrogels. Therefore, it is necessary to prepare composite hydrogels (C\u2010hydrogels) with more harmonious and uniform properties, and to improve conductivity, it is necessary to identify suitable conductive filler materials as carriers. To improve stability, the interaction between the internal structure and water molecules must be strengthened to improve water retention, and to reduce hydrogel toxicity the cross\u2010linking agent must be improved. Therefore, suitable conductive fillers, cross\u2010linkers, and water retainers are critical to the preparation of effective C\u2010hydrogels with excellent performance.As a type of water\u2010saturated cross\u2010linked polymer network, pristine hydrogels are a type of composite conductive polymer\u2010based material that has been widely studied in recent years. Compared to other base materials, P\u2010hydrogels obtain unique properties that common polymers do not possess. This is achieved by changing the types of monomer materials and polymerization conditions, including, albeit not limited to, adhesion, self\u2010healing, and network structure. Therefore, various P\u2010hydrogels can be prepared according to practical requirements. However, P\u2010hydrogels lack charge carriers and/or mobile charges, and their conductivity predominantly depends on water absorption, which reduces their conductivity.8 More MXenes have been widely used to improve hydrogel performance. However, most existing studies have solely used MXenes as conductive fillers without fully exploiting their hydrophilicity, high specific surface area, multiple active sites, and abundant surface functional groups. Few studies have been published using MXenes as cross\u2010linking agents, however, only single network hydrogels were constructed, resulting in a relatively single performance and low tunability compared with semi\u2010IPN (semi\u2010interpenetrating network) or IPN hydrogel. Therefore, constructing a semi\u2010IPN or IPN hydrogel that fully utilizes the characteristics of MXenes is a feasible solution for preparing C\u2010hydrogels with excellent performance. In this study, we used MXene@PDADMAC ) as a template, selected common AA (acrylic acid) and AM (acrylamide) as polymer monomers, and prepared a P(AA\u2010co\u2010AM)/MXene@PDADMAC semi\u2010IPN hydrogel (referred to hereafter as MXene\u2010gel). Because of the characteristics of MXene nanoflakes , hydrogen bonding, and electrostatic interactions between the components, hydrogels exhibit excellent mechanical properties, high conductivity, and water retention. The flexible strain sensor based on the hydrogel had a sensitivity that reached 0.98, and no obvious performance degradation was observed over the 30\u2010day continuous and 1100 cycle tests. Finally, in light of these advantages, a machine for converting sign language into Chinese characters was developed and optimally trained using an artificial neural network (ANN), thus demonstrating the significant potential of flexible electronic material applications in a broad range of fields.As emerging two\u2010dimensional transition metal carbons and/or nitrides,10 MXe22.1Figure\u00a03AlC2 MAX precursor to prepare colloidal solutions of delaminated Ti3C2Tx MXene nanoflakes , the mixed solution was finally in a stable state, in which the two monomers (AM and AA) were uniformly arranged on the MXene@PDADMAC composite linear template. Owing to the electronegativity and abundant surface functional groups of MXene, the MXene@PDADMAC composite linear template and an orderly arrangement of monomer molecules were formed, which was conducive to the formation of longer polymer chains and more stable uniform hydrogels.The MXene\u2010gel network structure is shown in y etched.16 SubFinally, after adding APS and increasing the temperature to 55 \u00b0C, the monomers were polymerized in an orderly manner. After cooling to room temperature, a flexible and stable MXene\u2010gel is formed. In addition to replacing the MXene colloidal solution with the traditional cross\u2010linking agent, MBAM powder, the preparation conditions of the control group P(AA\u2010co\u2010AM)@PDADMAC hydrogel (referred to as MBAM\u2010gel) were identical to those of the MXene\u2010gel. Comparing the SEM images of the MXene\u2010gel in different states, in the normal hydrated state is significantly smaller than that of MBAM\u2010gel (17.76%), indicating that it has higher stability and durability. This is very important for the application of hydrogels. The successive three times compression cycle tests to (0012) could also be observed. The presence of these characteristic peaks indicates the presence of delaminated Ti3C2Tx MXene rather than oxides in the hydrogel, which ensures MXene\u2010gel conductivity. By comparing the XRD patterns of different hydrogels, it was identified that the broad peak at \u22482\u03b8 = 20\u00b0 is a characteristic peak associated with polymers. Moreover, compared to the MBAM\u2010gel, the MXene\u2010gel solely consisted of additional MXene nanoflakes without other materials. However, because of the low crystallinity of the polymers, XRD cannot fully distinguish the specific polymer species contained in the hydrogel.A series of relevant physical characterizations were performed to verify the MXene\u2010gel water retention. By comparing the XRD patterns of pristine Til Figure\u00a02a, the of PAA. The featured band \u22481677 cm\u22121 is ascribed to the stretching vibrations of C\u2550O bonds from PAM. The characteristic absorption band at 1647 and 1461 cm\u22121 correspond to the stretching vibrations of \u2500NR4+ and \u2500CH3 groups from PDADMAC, respectively. Absorption bands at 1427 and 1276 cm\u22121 are ascribed to C\u2500N stretching and \u2500NH2 rocking for primary amide (\u2500CONH2), respectively, which are related to PAM. The FTIR spectra of the hydrogel in the original hydrated state are similar to those in the lyophilized state in the spectrum of MXene\u2010gel. This is because XPS mainly analyzes the surface of the material, while the Ti3C2Tx MXene nanoflakes are mainly coated inside the MXene\u2010gel, which is also consistent with SEM and TEM images.To thoroughly analyze the hydrogel polymer components, the lyophilized MXene\u2010gel was tested using FTIR spectroscopy Figure\u00a0. Sharp a) of PAA.24 The\u22121. However, the formation of hydrogen bonds (O\u2500H\u2026O) increases the bond length (the bond strength decreases) of the original hydroxyl groups, which means that the characteristic peaks of the hydroxyl groups will be red\u2010shifted. Therefore, the stretching vibration of the hydroxyl groups at the lower wavenumber of 3100\u20133500 cm\u22121 means the existence of hydrogen bonding interactions. As shown in Figure\u00a0\u22121, which means that the MXene\u2010gel contains a large number of hydrogen bonds. The hydrogen bonds of lyophilized MXene\u2010gel mainly come from the surface functional groups of MXene and each component, which fully enhances the mechanical properties of the MXene\u2010gel. As shown in Figure\u00a0 This helps to fully enhance the water retention of the MXene\u2010gel.In addition to identifying the kinds of polymers, FTIR spectra can also be used to analyze hydrogen bonds, which has an important contribution to the water retention of hydrogels. It is well known that for free hydroxyl groups (\u2500OH), the sharp absorption peak is located at 3500\u20133700 cmractions.29 As \u22121 can be used to estimate the ratio of strong intermolecular hydrogen bonds to weak intramolecular hydrogen bonds. As shown in Figure\u00a03287/I3446 ratio of MXene\u2010gel is higher than that of MBAM\u2010gel, indicating that a relatively large number of strong hydrogen bonds were formed after MXene was selected as the cross\u2010linking agent. This is because the surface functional groups of MXene nanoflakes form a large number of intermolecular hydrogen bonds, which also weaken the intramolecular hydrogen bonds. These intermolecular hydrogen bonds mean that more water molecules in the hydrogels are converted from free water to bound water, thus fundamentally improving the water retention of the hydrogel.In addition to FTIR spectra, Raman spectra are also used to further analyze hydrogen bonds in hydrogels. The vibration bands corresponding to hydrogen bonds are centered at 3287 and 3446 cm1 Figure\u00a0. These a2. This important parameter can reveal the different mobility and distributions of water components in the aqueous system. Due to the chemical exchange between water protons and labile protons on the polymer and the diffusion exchange of water in different microenvironments, T2 of water protons in polymer solution is usually smaller than T2 of neat water. Therefore, a shorter T2 means that the water is more tightly bound to the material. As shown in Figure\u00a02 relaxation time decreases, which means that the water molecules in the hydrogels are restricted by the polymers. According to the peak area of T2 distribution curve, the relative ratio of bound water and free water in the hydrogels was calculated to further evaluate the binding ability between the structure of the hydrogel and water molecules. As shown in Figure\u00a0To measure the binding state of water in hydrogels, LF\u2010NMR relaxometry, a powerful tool for studying the mobility of water molecules, was used to analyze different hydrogels. LF\u2010NMR relaxometry can be employed to elucidate proton mobility that reflects structural heterogeneity and interactions by measuring its spin\u2013spin relaxation time, Tmaterial.32 As 2.3Figure\u00a0 Therefore, the current change in the strain sensor during subsequent tests was caused by the resistance change in the MXene\u2010gel. And it can be seen from Figure To quantify the MXene\u2010gel response to external strain, a strain sensor consisting of MXene\u2010gel as the sensing layer and Cu wires as the electrode was fabricated, as shown in Figure\u00a033 TheI = |I \u2212 I0|, I and I0 represent the current with and without strain respectively and s represents the applied strain. Based on the above standards, our sensors show different sensitivity in three different strain ranges , the MXene nanoflakes are still in close contact with each other, and the resistance change of semi\u2010IPN hydrogel is mainly caused by the reduction of the contact area between the polymer and the MXene nanoflakes caused by the stretching of the polymer network. As the stretching increases (40\u2212100%), the MXene nanoflakes also begin to separate slowly. And, the sensitivity of the strain sensor also changes due to the change of the conductive path. Finally, when the MXene nanoflakes are almost completely separated from each other, the conductive path also becomes the final form, so the sensitivity gradually approaches zero. And the work still has some advantages over the hydrogel sensors that have been reported, especially in terms of water retention, response time, and recovery time /MXene@PDADMAC semi\u2010IPN hydrogel was synthesized using the template copolymerization of AA and AM in the presence of MXene@PDADMAC. In MXene\u2010gels, hydrogen bonds and electrostatic interactions between the components produce excellent mechanical properties, high conductivity, and water retention in hydrogels. As a cross\u2010linking agent, MXene nanoflakes not only act as conductive fillers to increase hydrogel conductivity but also increase hydrogel moisture retention owing to their hydrophilicity and functional groups. A series of physical characterizations and complementary experiments proved that the P(AA\u2010co\u2010AM)/MXene@PDADMAC semi\u2010IPN hydrogel has a unique structure (honeycomb\u2010like chamber structure), high strain performance (high sensitivity and stability), and excellent moisture retention (not dried for 40 d). In addition, a machine\u2010learning\u2010based hydrogel device was developed for converting sign language into Chinese characters, facilitating daily life communication between the hearing\u2010impaired and general population.43C2Tx MXene colloidal solutions could be obtained in two steps, including the preparation of the multilayer Ti3C2Tx powder and the centrifugation of the aqueous dispersion of the powder. Wet etching was used to remove the Al\u2010element layers in the middle of Ti3AlC2 MAX to prepare multilayer Ti3C2Tx MXene powder. Specifically, 3\u00a0g Ti3AlC2 MAX powder was first added to a pre\u2010mixed etchant solution (4.8\u00a0g LiF and 60\u00a0ml of 9\u00a0m HCl) at room temperature. Then the above solution was stirred for 48\u00a0h under the condition of 35 \u00b0C oil bath. After the etching, the above solution was centrifuged at 3500\u00a0rpm for 5\u00a0min, and the obtained precipitate was stirred in 60\u00a0mL of 2\u00a0m H2SO4 for 30\u00a0min to remove the remaining etchant. Afterward, the precipitate was continuously washed with deionized (DI) water and centrifuged until the supernatant is close to neutral. At this time, the precipitate was the required multilayer Ti3C2Tx MXene powder. The Ti3C2Tx MXene powder was dissolved in 75\u00a0ml of DI water, centrifuged at 3500\u00a0rpm for 1\u00a0h, and the supernatant was the colloidal solution of delaminated Ti3C2Tx MXene flakes, which was referred to as the Ti3C2Tx MXene colloidal solution.The Ti3C2Tx MXene colloidal solution (\u22488.5 mg\u00b7mL\u22121), 1\u00a0g of AM powder, 4\u00a0mL of AA solution, 0.1\u00a0g of APS powder, and 10\u00a0mL of DI water to the 2\u00a0mL of PDADMAC solution in sequence. Then, the uniformly mixed solution was transferred to the mold, wrapped in tin foil, and then placed in an oven at 55 \u00b0C for heating for 12\u00a0h. After naturally cooling to room temperature, the hydrogel was peeled from the mold for subsequent experiments. Other materials and conditions remained unchanged, 5\u00a0mg of traditional cross\u2010linking agent MBAM powder was used to replace MXene colloidal solution to prepare P(AA\u2010co\u2010AM)@PDADMAC hydrogel (referred to as MBAM\u2010gel) as the control group. After that, the MXene\u2010gel and Cu wires are encapsulated by polydimethylsiloxane (PDMS) to make a strain sensor.The MXene\u2010gel was produced by heating and copolymerization. Add 5\u00a0mL of TiadsE) was calculated as:First\u2010principles calculations were implemented in Vienna Ab\u2010Initio Simulation Package (VASP) using density functional theory (DFT) with generalized gradient approximation (GGA) of the form proposed by Perdew\u2010Burke\u2010Ernzerhof (PBE). The valence electronic states were expanded on the basis of plane waves. The projector augmented plane wave (PAW) method with a cutoff of 550\u00a0eV was used to express the core\u2010valence interaction. In order to obtain high accuracy through geometry optimization, the Brillouin zone integration was sampled with 4 \u00d7 4 \u00d7 1 k\u2010grid mesh. The adsorption energies of different hydrogels for water molecules were calculated by DFT calculations. The adsorption energy . Scanning electron microscopy (SEM) images were obtained using Magellan 400 to obtain high\u2010magnification images of the treated powders. For the testing of hydrated hydrogels, the water on the surface of the hydrogel needs to be completely removed first, and the entire testing process needs to be completed as quickly as possible. Transmission electron microscopy (TEM) images in this work were obtained using JEOL JSM\u20132010F. The tensile\u2010sensing properties were tested using an A11719 Iviumstat electrochemical interface and CHI 760D electrochemical workstation. At the same time, in order to measure the stability of the strain sensor, cyclic tensile tests were carried out using an electric horizontal test bench . Tensile and compression tests were performed using a high\u2010precision micromechanical testing system . The water retention of hydrogels was evaluated by exposing them to the environment with the relative humidity (RH) of 40% and the temperature of 25 \u00b0C. Fourier transform infrared spectroscopy (FTIR) was performed on a Nicolet IS10 spectrometer to investigate the surface bonding of the composites. The components of the composites were analyzed using X\u2010ray photoelectron spectroscopy (XPS) . Raman spectroscopy of all MXene materials and composites was performed by using a 532\u2010nm laser . Each hydrogel was subjected to low\u2010field nuclear magnetic resonance (LF\u2010NMR) analyses . The transversal relaxation time (T2) were measured at 32 \u00b0C with 90\u00b0 pulse of 12 \u00b5s, 180\u00b0 pulse of 24 \u00b5s, scans of 8, echoes of 18000 and echo time of 0.6\u00a0ms.X\u2010ray diffraction (XRD) patterns were obtained with a powder diffractometer (DX\u20102700B) using Cu K\u03b1 radiation of wavelength The authors declare no conflict of interest.Supporting InformationClick here for additional data file."} +{"text": "The cerebellum is involved in learning of fine motor skills, yet whether presynaptic plasticity contributes to such learning remains elusive. Here, we report that the EPAC-PKC\u03b5 module has a critical role in a presynaptic form of long-term potentiation in the cerebellum and motor behavior in mice. Presynaptic cAMP\u2212EPAC\u2212PKC\u03b5 signaling cascade induces a previously unidentified threonine phosphorylation of RIM1\u03b1, and thereby initiates the assembly of the Rab3A\u2212RIM1\u03b1\u2212Munc13-1 tripartite complex that facilitates docking and release of synaptic vesicles. Granule cell-specific blocking of EPAC\u2212PKC\u03b5 signaling abolishes presynaptic long-term potentiation at the parallel fiber to Purkinje cell synapses and impairs basic performance and learning of cerebellar motor behavior. These results unveil a functional relevance of presynaptic plasticity that is regulated through a novel signaling cascade, thereby enriching the spectrum of cerebellar learning mechanisms. The cerebellum has historically been viewed as a motor coordination center . Recent In particular, the function of cAMP-dependent protein kinase A (PKA) on transmission release has been the subject of debate. In this study, we identified a new presynaptic signaling module that comprises EPAC (exchange protein directly activated by cAMP) and PKC\u03b5 (epsilon isozyme of protein kinase C). This signaling module controls threonine phosphorylation of RIM1\u03b1, initiates the assembly of a Rab3A-RIM1\u03b1-Munc13-1 tripartite complex, and thereby facilitates docking and release of synaptic vesicles at parallel fiber (PF) to Purkinje cell (PC) synapses, which is in line with previous work showing +EAAT4+) and climbing fiber (CF) synapses (vGluT2+EAAT4+) constituted 88.8% and 7.5% of the total, respectively mice. The reason for this strategy is that EPAC1 and EPAC2 share highly conserved cAMP-binding domains, and have significant cross-talk and redundant roles in many physiological processes , whereas phosphorylation at PKC\u03b1-S657 or PKC\u03b1-T638 was unchanged (Prkce (the gene coding for PKC\u03b5) deletion specifically in cerebellar granule cells (Prkce-cKO) by crossing Atoh1Cre to WT synaptosomes. The addition of \u03b5V1-2 to the synaptosomes strongly attenuated RIM1 p-Thr induced by 8-pCPT . In cont changed . OverallOur finding that the EPAC-PKC\u03b5 module regulates RIM1 activity through phosphorylation leads to an interesting question: whether the EPAC-PKC\u03b5 module functions on synaptic formation and function through acting on RIM1, which is known to be critical to organization of the presynaptic active zone and neurotransmitter release .Rapgef3/4-dKO (n=4) or Prkce-cKO (n=4) mice, compared to corresponding WT (n=4) and Prkcef/f (n=4) mice . Similarly, the specific deletion of PKC\u03b5 in granule cells also decreased the number of vesicles in the docked vesicle pool . These data demonstrate that both EPAC and PKC\u03b5 regulate the docking of presynaptic vesicles.To address this question, we first visualized PF-PC synapses using transmission electron microscopy (EM), in which PF boutons were identified by their presence of synaptic vesicles as well as their asymmetric synaptic contacts with PC spines . No appa=4) mice . However<0.0001) . This di<0.0001) . MeanwhiAtoh1Cre;Rapgef3f/f;Rapgef4f/f and Prkce-cKO mice, the former of which caused specific deletion of Rapgef3 and Rapgef4 in granule cells revealed significant reductions in Pr in Rapgef3;Rapgef4-cKO (Prkce-cKO mice (We next examined the effect of the ablation of EPAC or PKC\u03b5 on synaptic transmission. Miniature excitatory synaptic currents (mEPSCs) at PF-PC synapses were recorded in cerebellar slices from le cells , while Aenotypes . Similarf/f mice . A decref/f mice . Comparegef4-cKO and PrkccKO mice . FurthercKO mice . These rcKO mice , indicatRapgef3/4-dKO , indicating that presynaptic LTP at PF-PC synapses occurs upon a rise in the cellular level of cAMP.To test this hypothesis, presynaptic LTP at PF-PC synapses was induced by a tetanus stimulation (8 Hz for 5 min) at voltage-clamp mode (\u201370 mV) . The potRapgef3/4-dKO and Rapgef3;Rapgef4-cKO mice. We made whole-cell recordings from PCs and found that 8 Hz stimulation failed to induce potentiation of EPSCs in Rapgef3/4-dKO mice (and D). This finding was confirmed in recordings from slices of Rapgef3;Rapgef4-cKO mice, in which EPAC is deleted from the granule cells innervating the PCs, showing that presynaptic PF-PC LTP was also blocked and D. Tp=0.059) . In contPrkcef/f and Prkce-cKO mice. Similar to WT and Atoh1Cre mice, the potentiation of PF-EPSCs evoked by 8 Hz stimulation reached 120 \u00b1 3% of baseline in control Prkcef/f mice . In cont p=0.76) . Similarp<0.001) , but not p=0.34) .Atoh1Cre, Rapgef3;Rapgef4-cKO, Prkcef/f, and Prkce-cKO mice (P21). We found that presynaptic LTP was induced in Atoh1Cre and Prkcef/f mice and Prkce-cKO mice in cerebellar slices from p<0.001) , but not92) mice .2+ concentration in the aCSF from 2 mM to 0.5 mM, and then recorded presynaptic LTP in cerebellar slices from Atoh1Cre, Rapgef3;Rapgef4-cKO, Prkcef/f, and Prkce-cKO mice (P21). Again, presynaptic LTP was induced in Atoh1Cre and Prkcef/f mice (and C), but not in Rapgef3;Rapgef4-cKO and Prkce-cKO mice and C, b53) mice . These Therefore, on the basis of our experiments in PCs from mice with presynaptic specific deletion of EPAC and PKC\u03b5, we conclude that the presynaptic EPAC-PKC\u03b5 module is critical for presynaptic PF-PC LTP in the cerebellum.cAMP is also required for presynaptic LTP induced by electrical stimulation , and itsAtoh1Cre, Rapgef3;Rapgef4-cKO and Prkce-cKO mice. In Atoh1Cre control mice, external application of forskolin produced a long-lasting elevation in PF-EPSC amplitude and again examined forskolin-induced EPSC potentiation. In this case, we found that combined blockade of EPAC and PKA completely eliminated the action of forskolin on EPSC potentiation , we induced postsynaptic LTP by stimulating PFs at 1 Hz for 5 min in current-clamp mode . In WT m p=0.26) . While t p=0.26) , we had of mice , while P of mice , suggestd to PFs with unad to PFs , confirmRapgef3/4-dKO PCs showed robust PF-PC LTD and VOR (Basic performance parameters included amplitude (gain) and timing (phase) of the optokinetic response (OKR), vestibulo-ocular reflex (VOR), and visually enhanced VOR (VVOR) . We founrements) . In contrements) .Rapgef3;Rapgef4-cKO and Prkce-cKO mice as well as their littermate controls. Basic eye movement performance was also affected in Rapgef3;Rapgef4-cKO mice in that their OKR gains were significantly lower than those of Atoh1Cre littermates , that threments) , and tharements) . No signctively) . Moreoverements) , whereasrements) . Meanwhirements) . No signctively) . AltogetAtoh1Cre and Prkcef/f) exhibited gain reductions similar to previous work . WT , they were so during sessions on days 2\u20135 . Therefore, we conclude that Rapgef3/4-dKO, Rapgef3;Rapgef4-cKO and Prkce-cKO mice had prominent deficits in phase-reversal learning of their VOR.Next, we tested the VOR phase-reversal protocol, which is considered the type of motor learning sensitive to the perturbation to the vestibulo-cerebellum . VOR phaous work . Gain ref3/4-dKO , Rapgef3gef4-cKO , as wellrkce-cKO mice, bu.15). WT , Atoh1CrAtoh1Cre as well Prkcef/f mice shoPrkcef/f . WhereasIn the current study we demonstrate that triggering EPAC induces PKC\u03b5 activation and threonine phosphorylation of RIM1\u03b1, which in turn facilitates the assembly of the Rab3A-RIM1\u03b1-Munc13-1 tripartite complex and thereby docking and release of synaptic vesicles at active zones of PF-PC synapses . The forOur finding that the EPAC-PKC\u03b5 module can phosphorylate RIM1\u03b1 raises a simple but fascinating mechanistic concept that the phosphorylation level of RIM1\u03b1 determines presynaptic release. RIM1\u03b1 specifically interacts with a number of presynaptic proteins, such as Munc13-1, liprin-\u03b1 and ELKS, so as to form a scaffold complex regulating homeostatic release of synaptic vesicles . RIM1\u03b1 ccAMP-mediated signaling pathways that are mediated by EPAC and PKA regulate a multitude of physiological and pathological processes . EPAC shRapgef3/4-dKO and Prkce-cKO mutants is reduced, whereas the general structure of PF-PC synapses is unchanged. As the ablation of either EPAC or PKC\u03b5 attenuated protein interactions in the Rab3A-RIM1\u03b1-Munc13-1 complex, which is required for the docking and priming of presynaptic vesicles , which are involved in cerebellar ataxias and Wei Mo , respectively. Rapgef3f/f, Rapgef4f/f and Prkcef/f mice were made by us with the assistance of GemPharmatech and Nanjing Biomedical Research Institute . The resulting offspring were genotyped using PCR of genomic DNA. Mice were kept at the Experimental Animal Center of Zhejiang University under temperature-controlled condition on a 12:12 hr light/dark cycle. All experiments were performed blind to genotypes in age-matched littermates of either sex.Original breeding pairs of AB_2238250 and Cat# 140023, RRID:AB_2177807), Rab3 and Munc13-1 were from Synaptic Systems . Antibodies against phosphor-threonine , EPAC1 and EPAC2 were from Cell Signaling . The antibody to phosphor-serine was from Millipore . Antibodies against HA , Flag and His were from Abmart . Antibody against PKC\u03b1 was from sigma . Antibodies against PKC\u03b1-pS657 , PKC\u03b1-pT638 , PKC\u03b5-pSer729 , EPAC1 , EPAC2 , anti-mouse IgG for IP (HRP) and VeriBlot for IP Detection Reagent (HRP) were from Abcam . Antibody against \u03b2-tubulin was from Santa Cruz . Antibody against PKC\u03b5 , Goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated , Goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated were from Thermo Fisher Scientific . Anti-vGluT1 antibody was a gift from Dr. Masahiko Watanabe . The antibody against PKC\u03b5 was from Proteintech . Mouse IgG and rabbit IgG were from Beyotime . Protease inhibitor cocktail (04693132001) was from Roche . G\u00f66976 (2253), 8-pCPT (4853) and FR236924 (3091) were from Tocris . Dulbecco\u2019s modified Eagle\u2019s medium , Penicillin-Streptomycin (15140\u2013122), Sodium Pyruvate (11360\u2013070), Fetal Bovine Serum , lipofectamine 2000 (11668\u2013019), OPTI-MEM (31985\u2013062), and Alexa Fluor-conjugated secondary antibodies were from Invitrogen . GammaBind Plus Sepharose (17-0886-01) was from GE healthcare. Other chemicals were from Sigma unless stated otherwise.Antibodies against RIM1 , rat Rapgef3 gene (GenBank# NM_021690.1), rat Rapgef4 gene (GenBank# XM_017592164.1), and rat Prkce gene (GenBank# NM_017171.1), respectively. All constructs were verified by DNA sequencing.The construction of plasmids was performed according to previous work . HA-RIM1Rapgef3, F: 5\u2019- GCT TGT TGA GGC TAT GGC-3\u2019; R: 5\u2019- ACA CAG TTC CTG CCT TGC-3\u2019. Rapgef4, F: 5\u2019- CAT TCT CTC TCG AGC TCC-3\u2019; R: 5\u2019 TGG TTG AGG ACA CCA TCT-3\u2019. Prkce, F: 5\u2019- ATT GAC CTG GAG CCA GAA \u20133\u2019; R: 5\u2019- CTT GTG GCC ATT GAC CTG-3\u2019. Gapdh, F: 5\u2019-GGT GAA GGT CGG TGT GAA CG-3\u2019; R: 5\u2019-CTC GCT CCT GGA AGA TGG TG-3\u2019.RT-PCR was used to determine the mRNA level of EPAC1, EPAC2 and PKC\u03b5 in granule cells. The contents of individual granule cells (P21) were harvested as described in previous work . The har2/5% CO2; 37 \u00b0C). The plasmids were transfected to HEK cells in OPTI-MEM using lipofectamine 2000 (Invitrogen) at 70\u201380% confluency.HEK cells were cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 10 \u03bcg/ml streptomycin and stored in an incubator (95% Og (4 \u00b0C for 2 min) and the supernatant was spun again at 9500\u00d7g (4 \u00b0C for 12 min). The compacted white layer containing the majority of synaptosomes was gently resuspended in sucrose (320 mM) supplemented with protease inhibitors, and an aliquot of synaptosomal suspension (2 ml) was placed onto a 3 ml Percoll discontinuous gradient containing (in mM) 320 sucrose, 1 EDTA, 0.25 DL-dithiothreitol, and 3, 10, or 23% Percoll. After centrifugation at 25,000\u00d7g (4 \u00b0C for 10 min), synaptosomes were recovered from between 10% and 23% bands and diluted in a medium (in mM) supplemented with protease inhibitors. The synaptosomes good for experiments were harvested from the pellet after the final centrifugation at 22,000\u00d7g (4 \u00b0C for 10 min).Synaptosomes were purified according to previous work . CerebelFor immunocytochemistry, synaptosomes were added to a medium containing 0.32 M sucrose (pH 7.4), allowed to attach to polylysine-coated coverslips for 1 hr, and fixed for 10 min in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at room temperature. Following several washes with PB (pH 7.4), synaptosomes were incubated for 1 hr in 10% normal goat serum diluted in PBS (pH 7.4) containing 0.2% Triton X-100. Subsequently, they were incubated for 24 hr with primary antiserum for EPAC1 (1:500), EPAC2 (1:500), PKC\u03b5 (1:500) and vGluT1 (1:500). After washing in PBS, synaptosomes were incubated with secondary antibodies for 2 hr. Coverslips were mounted with Prolong Antifade Kit (Molecular Probes) and synaptosomes were viewed using a confocal microscope (Nikon A1R) with a\u00d7100 objective.After measuring protein concentration using the BCA assay, a tenth of lysis supernatant derived from synaptosomes or cultured cells was used for input and the remainder were incubated with anti-RIM1 or anti-HA antibody, which was precoupled to GammaBind Plus Sepharose at 5\u201310 \u03bcg antibody/1 ml beads for 3 hr. Proteins on the beads were extracted with 2\u00d7SDS sample buffer plus protease inhibitors and boiled for 5 min before western blot.The protein concentration was determined using BCA protein assay. Equal quantities of proteins were loaded and fractionated on SDS-PAGE, transferred to PVDF membrane , immunoblotted with antibodies, and visualized by enhanced chemiluminescence (Thermo Fisher). The dilutions of primary antibodies were 1:1,000 for RIM1, Munc13-1, PKC\u03b1-pS657, EPAC1, EPAC2, p-Thr, p-Ser, \u03b2-tubulin, and PKC\u03b5-pSer729; 1:2,000 for Rab3A and PKC\u03b5; 1:5,000 for PKC\u03b1-pT638; 1:10,000 for HA, His, Flag, GAPDH, and PKC\u03b1. Secondary antibodies were goat anti-rabbit , goat anti-mouse , anti-mouse IgG for IP (HRP) , VeriBlot for IP Detection Reagent (HRP) . Film signals were digitally scanned and quantified using ImageJ 1.42q .After anesthetic mice (P21) were transcardially perfused with saline and ice-cold fixative, brains were removed and stored at 4 \u00b0C for 2.5 hr in fixative. Sagittal slices of vermis (200 \u03bcm) were prepared and rectangular molecular layer sections from lobules IV-V were dissected. The samples were dehydrated and embedded in an epoxy resin. Ultrathin sections (90 nm) were cut using an ultra-microtome (Leica), stained with 2% uranyl acetate and lead solution, and mounted on grids. EM images were captured at \u00d730,000 magnification using a Tecnai transmission electron microscope . PF-PC synapses were identified by asymmetrical and short contacts, which were distinct from GABAergic or climbing fiber synapses . ImageJ Golgi staining was performed using Rapid Golgi Stain Kit according to the manufactory\u2019s instruction. PCs at the apical region were imaged using a bright field microscope . ImageJ was used to count the spine number and dendrites length of PCs with manual assistant.2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3 and 25 D-glucose, bubbled with 95% O2/5% CO2. When low Ca2+ (0.5 mM) was used, Mg2+ concentration was increased to 2.5 mM. After recovery for 30 min at 37 \u00b0C, slices were placed in a submerged chamber that was perfused at 2 ml/min with aCSF supplemented with GABAzine (10 \u03bcM) during recordings.Sagittal slices of cerebellar vermis (250 \u03bcm) were prepared from anesthetic mice (P21) using a vibrating tissue slicer (Leica VT1000S) and ice-cold standard artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2ATP, and 0.4 Na3GTP . PCs were held at \u201370 mV to prevent spontaneous spikes that might escape clamp. For PF stimulation, standard patch pipettes were filled with aCSF and placed in middle third of molecular layer. Presynaptic PF-PC LTP was induced by stimulating PF input 120 times at 8 Hz equipped with a 40\u00d7water-immersion objective and infrared differential interference contrast enhancement. Whole-cell recordings were made on PCs from lobules IV-V with a MultiClamp 700B amplifier (Molecular Devices). Currents were digitized at 10 kHz and filtered at 3 kHz. Patch electrodes (3\u20135 M\u03a9) were filled with an intracellular solution containing (in mM) 135 Cs-methanesulfonate, 10 CsCl, 10 HEPES, 0.2 EGTA, 4 Na at 8 Hz . Postsyn at 8 Hz . PF-LTD at 8 Hz . mEPSCs at 8 Hz . To esti at 8 Hz . A tempeMice (P60) were surgically prepared for head-restrained recordings of compensatory eye movements. A pedestal was attached to the skull after shaving and opening the skin overlaying it, using Optibond primer and adhesive and under isoflurane anesthesia in O2 (induction with 4% and maintained at 1.5% concentration). Mice were administered xylocaine and an injection with bupivacaine hydrochloride to locally block sensation. The pedestal consisted of a brass holder (7\u00d74 mm base plate) with a neodymium magnet (4\u00d74 \u00d7 2 mm) and a screw hole for fixation. After a recovery period of at least 3 days, mice were placed in a mouse holder, using the magnet and a screw to fix the pedestal to a custom-made restrainer, and the mouse was placed with the head in the center on a turntable (diameter 60 cm) in the experimental setup. A drum (diameter 63 cm) surrounded the mouse during the experiment. The recording camera was calibrated by moving the camera left\u2013right by 20\u00b0 peak to peak at different light levels. Compensatory eye movement performance was examined by recording the OKR, VOR, and VVOR using a sinusoidal rotation of the drum in light (OKR), rotation of the table in the dark (VOR), or rotation of the table (VVOR) in the light. These reflexes were evoked by rotating the table and/or drum at 0.1\u20131 Hz with a fixed 5\u00b0 amplitude. In order to evaluate motor learning, a mismatch between visual and vestibular input was used to adapt the VOR. The ability to perform VOR phase reversal was tested using a 5 day paradigm, consisting of six 5 minute training sessions every day with VOR recordings before, between, and after the training sessions. On the first day during training, the visual and vestibular stimuli rotated in phase at 0.6 Hz and at the same amplitude, inducing a decrease of gain. On the subsequent days, the drum amplitude was increased relative to the table and induced the phase reversal of the VOR, resulting in a compensatory eye movement in the same direction as the head rotation instead of the normal compensatory opposite direction . Between recording sessions, mice were kept in the dark to avoid unlearning of the adapted responses.Eye movements were recorded with a video-based eye-tracking system . Recordings were always taken from the left eye. The eye was illuminated during the experiments using two table-fixed infrared emitters and a third emitter that was mounted to the camera, aligned horizontally with the optical axis of the camera, which produced the tracked corneal reflection. Pupil size and corrected vertical and horizontal pupil positions were determined by the ISCAN system, filtered , digitized and stored for offline analysis. All eye movement signals were calibrated, differentiated to obtain velocity signals, and high-pass\u2013filtered to eliminate fast phases, and then cycles were averaged. Gain\u2014the ratio of eye movement amplitude to stimulus amplitude\u2014and phase values\u2014time difference between eye and stimulus expressed in degrees\u2014of eye movements were calculated using custom-made MATLAB routines .t test for two-group comparison, or one-way ANOVA followed by Tukey\u2019s post hoc test for multiple comparisons, or repeated measures ANOVA for repeated measures. The accepted level of significance was p<0.05. \u2018n\u2019 represents the number of preparations or cells. Data in the text and figures are presented as mean \u00b1 SEM.Experimenters who performed experiments and analyses were blinded to the genotypes until all data were integrated. Data were analyzed using Igor Pro 6.0 , Graphpad Prism , SPSS 16.0 , and MATLAB. No statistical methods were used to pre-determine sample sizes, which were based on our previous studies. All data sets were tested for the assumptions of normality of distribution. No data were excluded except electrophysiological recordings with \u226515% variance in series resistance, input resistance, or holding current. Standard deviations for control were calculated from the average of all control data. Statistical differences were determined using unpaired or paired two-sided Student\u2019s The cerebellum plays a critical role in motor learning, but exactly which forms of synaptic plasticity contribute to learning and the underlying molecular mechanisms remain poorly understood. In this study, Wang and colleagues show that presynaptic long-term potentiation at the parallel fiber to Purkinje cell synapse is required for one form of motor learning, and involves a previously-unknown signaling cascade, where EPAC activation leads to PKC\u03b5-dependent threonine phosphorylation of RIM1\u03b1. The evidence is compelling and convincing. This study provides fundamental and new insights into the underlying mechanisms and functional consequences of presynaptic LTP. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Lu Chen as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article \"cAMP-EPAC-PKC\u03b5-RIM1\u03b1 signaling regulates presynaptic long-term potentiation and motor learning\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) 1. The stimulation protocol for estimating the RRP is not sufficient \u2013 The authors need to provide additional experimental data.2) Please conduct additional control experiments \u2013 apply KT5720 on control animals to look at the effect.3) The cumulative EPSC analysis in its present form is confusing. Please plot RRP size instead of commutative EPSC values.4) Substantial revision of the test is recommended \u2013 Please follow the reviewers' detailed suggestions below, such as tuning down the conclusion, providing a better rationale in the introduction, and adding a discussion on the difference in experimental conditions between ex vivo and in vivo experiments, and lastly provide more citations of the prior publications.Reviewer #1 (Recommendations for the authors):1. My primary concern is the manner in which the authors choose to refer to previous studies, which seem designed to highlight the novelty of their work rather than provide context and information for the reader. For example, Line 44 \"However, the molecular underpinnings of presynaptic plasticity in the cerebellar cortex are merely starting to be explored \" is a baffling statement, given that the molecules involved in presynaptic LTP at this synapse have been described since the 1990s in studies like Salin et al., 1996 (cited by the authors), and Storm et al. 1998 (uncited).Line 45: \"whether presynaptic plasticity plays a role in cerebellar motor learning remains to be elucidated\". Again, Storm et al. 1998 showed impaired rotarod performance and learning in AC1 knockout mice. The genetic manipulations in the current study are far cleaner than Storm et al., and the physiology and behavior are more convincing, but they should not minimize the impact of past studies.The introduction and beginning of the results provide no context for the authors' decision to study EPAC. I can only assume that the results of Martin et al. 2020 provided a compelling rationale for this study, and think it should be included in the introduction.2. Figure 1C: The statement that ENACs bind to RIM would be more convincing if the authors provide a negative control showing that the assay does not detect another presynaptic protein that does not interact with RIM does not appear in the pulldown. That said, interactions between ENAC and RIM have been reported before, and those studies could be cited.3. Figure 2E, F: I have multiple concerns with this data. First, the RRP is calculated from EPSC trains collected evoked by only 40 stimuli. 40 stimuli are insufficient to reach a steady state at PF-PC synapses, which is evident in the example EPSC traces. Without reaching a steady state, it is impossible to estimate the resupply rate. Thus the RRP measurements are not accurate. Second: Without an accurate measure of the RRP, the initial release probability cannot be determined. Finally, although Line 166 states that the RRP was significantly reduced in the mutant mice, that data is not presented in the text of the figure. Instead, the figure shows the \"cumulative EPSC\", which is highly dependent on the number of presynaptic terminals that were stimulated. The average RRP sizes, measured by back extrapolation, actually appear to be very similar across genotypes. The authors should not make claims about release probability or RRP unless they repeat these experiments with longer stimulus trains that reach steady state amplitudes, and the RRP is normalized to the size of the initial EPSC in each experiment.4. Line 240: \"However, it remains unclear which downstream effector, EPAC or PKA , is responsible for cAMP-induced potentiation.\" Martin et al. 2020 present compelling evidence that presynaptic LTP is driven by EPAC at the PF-PC synapse. This study is cited obliquely, and should not be ignored to enhance the novelty of the current study. For example, Line 433 understates the relevance of Martin to the current study: \"EPAC1 and EPAC2 have been shown to be involved in synaptic release in the hippocampus and cerebellum\".5. The rationale for proceeding immediately to EPAC1/2 DKOs was not clear, leaving the reader to guess the reason? Are EPAC1/2 redundant in the EPAC-Rim1 pathway, or does one isoform dominate in potentiating this synapse? Could the authors make it clear why all experiments were done with DKOs?6. Figure 4: What concentration of KT5720 was used to inhibit PKA? The authors note on Line 441 that previous studies used 10 microM, a concentration that produces off-target effects, but I cannot find the concentration the authors used. From their experiments, the authors conclude that the EPAC-PKC module is dominant in driving forskolin-induced potentiation. However, an obvious experiment would have been to apply KT to the control animals (Math1-Cre). This would rule out the possibility that PKA is activated somewhere within the EPAC-PKC, and plays a major role in potentiation.7. The authors hypothesize that PLC is involved in this pathway . They might use the discussion to highlight which aspects of their signaling pathway remain to be experimentally verified in presynaptic LTP.Reviewer #2 (Recommendations for the authors):The introduction part should elaborate more on the rationale behind the selection of EPAC to conduct the study of presynaptic plasticity.The activities of EPAC and PKC\u03b5 required for presynaptic LTP are shown. However, the change of the basal synaptic properties due to the knockdown of two proteins is not shown, such as the input-output relation of evoked EPSC in the PF-PC synapse. Therefore, studying the protein during the normal condition may be of interest.The visual diagram of the authors' hypothesis on a process describing EPAC-mediated phosphorylation of RIM1 is a bit difficult to follow. Arrows may be indicated with more comprehensible signs/ words.Following the compensatory eye movement training, the deficit is more prominent in phase than gain. Mutants catch up the gain at the end of 5 days of training but not the phase. The authors may explain how the presynaptic LTP is more essential to phase in the discussion. In addition, the reason to choose VOR phase reversal learning other than OKR learning or VOR gain-up learning may be related to this discussion.In the discussion part, some more thoughts and scientific predictions of the limited role of EPAC and PKC\u03b5 on postsynaptic plasticity should be stated.Overall, specific claims made by the authors were straightforward and supported by an ample amount of experimental results. This study has widened the spectrum of understanding of the molecular complexity of cerebellar motor learning by demonstrating the active contribution of presynaptic plasticity and its related signaling cascade.Reviewer #3 (Recommendations for the authors):The conclusions often overstate the findings of the data. The language can be toned-down a without diminishing the importance of these findings.In the present form, I don't think the cumulative EPSC analysis is informative. The authors should repeat the analysis using the Elmqvist and Quastel (EQ) method . This method is more reliable at low Pr and can provide (when combined with the Train method) a high and low bracket of possible Pr and RRP values. Ideally, the authors would redo these experiments with a higher stimulus frequency. It would also be helpful to show a simple PPR value for each genotype. This can be done without additional experiments.2+ concentration) do not match in vivo conditions. These differences and potential changes in synaptic plasticity should be discussed.Slice conditions in LTP/LTD experiments based on the relative number of each synapse? This would help the interpretation of the synaptosome data. Essential revisions:1) 1. The stimulation protocol for estimating the RRP is not sufficient \u2013 The authors need to provide additional experimental data.We have included new data using a new 100-Hz train protocol to estimate RRP and Pr, according to the reviewers\u2019 suggestions. These results have been added in the updated text and figure legends , and the corresponding methods have also been updated.2) Please conduct additional control experiments \u2013 apply KT5720 on control animals to look at the effect.We have conducted new experiments to investigate the effect of KT5720 on forskolin-induced potentiation. The results have been presented in Figure 4\u2014figure supplement 1. We found that KT5720 (3 \u03bcM) inhibited the forskolin-induced potentiation by 15%, which is significant but much smaller than EPAC effect.3) The cumulative EPSC analysis in its present form is confusing. Please plot RRP size instead of commutative EPSC values.Math1-Cre: 861 \u00b1 113 vs cKO;Epac2cKOEpac1 790 \u00b1 101, p = 0.31; Prkcef/f 764 \u00b1 100 vs PrkcecKO 728 \u00b1 106, p = 0.40). These results have been included in new Figure 2E and 2F.The related sentences have been rewritten in text and figure legend. We have measured RRP and Pr using an additional protocol as the reviewers suggested. Under this condition, the RRPs of control and cKO mice were similar (4) Substantial revision of the test is recommended \u2013 Please follow the reviewers' detailed suggestions below, such as tuning down the conclusion, providing a better rationale in the introduction, and adding a discussion on the difference in experimental conditions between ex vivo and in vivo experiments, and lastly provide more citations of the prior publications.We have tuned down the language, provided the rationale in the introduction, revised the discussion, and added more citations as suggested. Please see our detailed answers to each comment.Reviewer #1 (Recommendations for the authors):1. My primary concern is the manner in which the authors choose to refer to previous studies, which seem designed to highlight the novelty of their work rather than provide context and information for the reader. For example, Line 44 \"However, the molecular underpinnings of presynaptic plasticity in the cerebellar cortex are merely starting to be explored \" is a baffling statement, given that the molecules involved in presynaptic LTP at this synapse have been described since the 1990s in studies like Salin et al., 1996 (cited by the authors), and Storm et al. 1998 (uncited).We apologize for the confusions. In the original manuscript, we intended to make the introduction more concise. This choice resulted in a simple, short introduction that lacked relevant contextual information. We have re-written the introduction to present current status of presynaptic LTP at PF-PC synapses. In particular, the sentences and references that the reviewer indicated have been modified and added, respectively. \u201cRelatively speaking, the molecular underpinnings of presynaptic plasticity in the cerebellar cortex are less understood , though early studies have shown that presynaptic Ca influx, Ca-sensitive adenylate cyclase, and cyclic adenosine monophosphate (cAMP) production are required for presynaptic LTP \u201d (Line 45).Line 45: \"whether presynaptic plasticity plays a role in cerebellar motor learning remains to be elucidated\". Again, Storm et al. 1998 showed impaired rotarod performance and learning in AC1 knockout mice. The genetic manipulations in the current study are far cleaner than Storm et al., and the physiology and behavior are more convincing, but they should not minimize the impact of past studies.We have re-written the introduction to describe the prior knowledge regarding the relationship between presynaptic plasticity and cerebellar motor learning in more details. \u201cMoreover, the function of presynaptic plasticity on cerebellar motor learning remains to be elucidated , though it was suggested that adenylyl cyclase-dependent LTP participates in rotarod learning \u201d (Line 49).The introduction and beginning of the results provide no context for the authors' decision to study EPAC. I can only assume that the results of Martin et al. 2020 provided a compelling rationale for this study, and think it should be included in the introduction.This is an excellent comment. We actually started this project in 2014, and finished part of current data and submitted the manuscript entitled \u201cEPAC-PKC\u03b5 Module Induces Threonine Phosphorylation of RIM1 and Presynaptic Long-Term Potentiation\u201d to Neuron on August 2018. In that cover letter of Neuron submission, we wrote \u201cTo our knowledge, the present work provides compelling evidence to answer two major neuroscientific questions: (1) Compared to postsynaptic plasticity, the molecular underpinnings for the plastic changes at presynaptic side are poorly understood. It is shown that presynaptic LTP requires cAMP and scaffold protein RIM1\u03b1. However, to what extent these two processes are related and through which binding partners and/or enzymatic processes remains to be elucidated ; (2) Lonart et al. found that RIM1\u03b1-Ser413 is phosphorylated by PKA, which is required for presynaptic LTP. However, mice with a Ser413 mutation exhibit normal presynaptic LTP in the cerebellum and the hippocampus , questioning the central roles of RIM1\u03b1-Ser413 and PKA in presynaptic LTP and how phosphorylation of RIM1\u03b1 comes about\u2026 Here, we show that, upon cAMP activation, EPAC (a cAMP target protein) induces PKC\u03b5-dependent threonine phosphorylation of RIM1\u03b1 and that presynaptic parallel fiber-Purkinje cell LTP depends on an EPAC-PKC\u03b5 module.\u201d Therefore to be frank, our decision to study EPAC in PCs was based on the prevailing scientific questions.Epac1-cKO, Epac2-cKO, and Prcke2-cKO) during the very hard pandemic time. Furthermore, we built VOR setup and re-conducted all electrophysiological and behavioral studies.Although three reviewers agreed to the conceptual advance and found it interesting, they finally rejected the submission with two major reasons: (1) the knockout was not cell specific (EPAC1 and EPAC2 double knockout was used in that submission); (2) there was no behavioral study. To answer these critiques, we endeavored to create 3 conditioned knockout mice . Mart\u00edn et al. (2020) paper was also added (Line 63) \u201cwhich is in line with previous work showing \u03b2-adrenergic receptors/EPAC signaling modulates PF release using EPAC2 knockout mice\u201d.2. Figure 1C: The statement that ENACs bind to RIM would be more convincing if the authors provide a negative control showing that the assay does not detect another presynaptic protein that does not interact with RIM does not appear in the pulldown. That said, interactions between ENAC and RIM have been reported before, and those studies could be cited.Thank you. We have cited previous work showing the interactions betweenEPAC and RIM (Lines 483). Our preliminary experiments have detected the interaction of EPACs with other several important transmitter release-related proteins, such as MUNC13, MUNC18, and synaptophysin. However, the immunoprecipitation assay revealed no interaction between EPACs and these proteinssee .3. Figure 2E, F: I have multiple concerns with this data. First, the RRP is calculated from EPSC trains collected evoked by only 40 stimuli. 40 stimuli are insufficient to reach a steady state at PF-PC synapses, which is evident in the example EPSC traces. Without reaching a steady state, it is impossible to estimate the resupply rate. Thus the RRP measurements are not accurate. Second: Without an accurate measure of the RRP, the initial release probability cannot be determined. Finally, although Line 166 states that the RRP was significantly reduced in the mutant mice, that data is not presented in the text of the figure. Instead, the figure shows the \"cumulative EPSC\", which is highly dependent on the number of presynaptic terminals that were stimulated. The average RRP sizes, measured by back extrapolation, actually appear to be very similar across genotypes. The authors should not make claims about release probability or RRP unless they repeat these experiments with longer stimulus trains that reach steady state amplitudes, and the RRP is normalized to the size of the initial EPSC in each experiment.We thank the reviewer for this valuable feedback. Combined with the suggestion of the reviewer#3, we have redone the RRP and Pr experiments using a 100-Hz train (instead of 20-Hz which was used in the previous submission). This method has allowed us to have a much better estimation on RRP and Pr at PF-PC synapses, according to Thanawala and Regher (2016). The new results have been presented in new Figure 2E and 2F.Question #1: in new experiments, we have paid particular attention to ensure that recorded EPSCs reached a steady-state level by giving 50 stimuli, so that the regression can be conducted using at least 20 data points for the back extrapolation.Questions #2 and #3, we have created a linear fit from normalized steady-stateEpac1cKO;Epac2cKO 790 \u00b1 101, p = 0.31; PKC\u03b5f/f 764 \u00b1 100 vs PKC\u03b5cKO 728 \u00b1 106, p = 0.40). Actually, there was also no difference in RRP between control and cKO mice in our previous submission, but we incorrectly wrote \u201crepeated stimulation (20 Hz) revealed significant reductions in both RRP and Pr in Epac1cKO;Epac2cKO and PrkcecKO mice \u201d. Here, \u201cRRP\u201d was added incorrectly, we meant that only \u201cPr\u201d was reduced. In the revised figure, we add the statistics of RRP in Figure 2E and 2F.EPSCs and back-extrapolated the curve to the y-axis to obtain the RRP. Our results showed that RRP was similar between control and cKO mice . These results have been added in the text and figure legend , and corresponding methods have also been updated.Finally, we have found that the conditional knockout of either EPACs or PKC\u03b5 produces significant decrease on Pr , is responsible for cAMP-induced potentiation.\" Martin et al. 2020 present compelling evidence that presynaptic LTP is driven by EPAC at the PF-PC synapse. This study is cited obliquely, and should not be ignored to enhance the novelty of the current study. For example, Line 433 understates the relevance of Martin to the current study: \"EPAC1 and EPAC2 have been shown to be involved in synaptic release in the hippocampus and cerebellum\".We are sorry for these confusions. We have modified the paragraph in Line 283 to \u201cNext, we wondered which downstream effector, EPAC or PKA , is responsible for cAMP-induced potentiation. The role of PKA in presynaptic LTP has been contradicted by the studies showing that presynaptic LTP is intact when serine phosphorylation of RIM1 by PKA is interrupted . Moreover, Mart\u00edn et al. (2020) showed that EPAC2 regulates synaptic release at PF synapses and is required for presynaptic PF-PC LTP. These findings inspired us to investigate whether perhaps the EPAC-PKC\u03b5 module mediates cAMP-triggered EPSC potentiation\u201d.Although early studies have shown that EPAC1 and EPAC2 are involved in synaptic release in the hippocampus and the cerebellum , which was further strengthened by Mart\u00edn et al., (2020), our finding that PKC\u03b5 acts\u2026\u201d (Line 486).Both EPAC1 and EPAC2 are the effectors of cAMP, and widely present in the brain. In earlier studies, the lab of Prof. Youmin Lu has shown the roles of EPAC1 and EPAC2 in synaptic release in the hippocampus using double and single EPAC knockout mice . Considering the reviewer\u2019s suggestion, we have modified the sentence to \u201c5. The rationale for proceeding immediately to EPAC1/2 DKOs was not clear, leaving the reader to guess the reason? Are EPAC1/2 redundant in the EPAC-Rim1 pathway, or does one isoform dominate in potentiating this synapse? Could the authors make it clear why all experiments were done with DKOs?EPAC1 and EPAC2 share highly conserved cAMP-binding domains, and have significant cross-talk and redundant roles in many physiological processes . Thus, we simultaneously deleted EPAC1 and EPAC2 in the granule cells in the present work, leaving the question open whether one might dominate the modulation of Rim1\u03b1 phosphorylation\u201d (Line 449).EPAC1 and EPAC2 are both homologous receptors of cAMP and share highly conserved cAMP-binding domains . They both act on the downstream Rap1 signaling, and have significant cross-talk and redundant roles in many physiological processes . Thus, we cannot exclude the possibility that they both work on Rim1 at PF synapses, plus previous studies in the hippocampus using EPAC1 or EPAC2 knockout mice . It remains unclear whether EPAC1 or EPAC2 alone is sufficient and which one may dominate modulation of Rim1 phosphorylation. We thereby have added a paragraph in the discussion to explain the rationale for using EPAC1/EPAC double cKO mice \u201c6. Figure 4: What concentration of KT5720 was used to inhibit PKA? The authors note on Line 441 that previous studies used 10 microM, a concentration that produces off-target effects, but I cannot find the concentration the authors used. From their experiments, the authors conclude that the EPAC-PKC module is dominant in driving forskolin-induced potentiation. However, an obvious experiment would have been to apply KT to the control animals (Math1-Cre). This would rule out the possibility that PKA is activated somewhere within the EPAC-PKC, and plays a major role in potentiation.Here are our responses.1) We apologize for the missing information. The concentration of KT5720 used was 3 \u03bcM, which is close to the IC50 of PKA. According to Murray (2008), this concentration should minimize KT5720 effects on irrelevant signaling molecules, including EPAC, but still act on PKA.2) We showed that KT5720 further decreased the potentiation by forskolin in conditional EPAC1/2 or PKC\u03b5 knockout mice, albeit its effect was rather limited . This result was also depicted in our summary Figure 7\u2014figure supplement 2 (dashed line). Therefore, we believe that the impact of the EPAC-PKC module is much more significant than that of PKA in cAMP signaling pathway, at least at PF-PC synapses.3) Following the reviewer\u2019s comment, we have conducted new experiments to investigate the effect of KT5720 on the forskolin-induced potentiation in Math1-Cre mice. We found that KT5720 inhibited the forskolin-induced potentiation around 15%, significantly different but much smaller than the effect of EPAC or PKC\u03b5 ablation. The result has been presented in Figure 4\u2014figure supplement 1 and text (Line 304).Science Signaling 1:re4.Reference: Murray AJ. 2008. Pharmacological PKA inhibition: all may not be what it seems. 7. The authors hypothesize that PLC is involved in this pathway . They might use the discussion to highlight which aspects of their signaling pathway remain to be experimentally verified in presynaptic LTP.Great suggestion. We have added the point in the discussion \u201cSimilarly, it would be of interest to investigate the role of Rap1, the presynaptically expressed substrate of EPAC a , on RIM1\u03b1 phosphorylation\u201d (Line 452).Reviewer #2 (Recommendations for the authors):The introduction part should elaborate more on the rationale behind the selection of EPAC to conduct the study of presynaptic plasticity.Thank you very much for this suggestion. We actually initiated this study based on previous controversy, regarding whether PKA is involved in the cerebellar plasticity. We then found EPAC, another downstream molecule of cAMP, and considered it a good candidate to test.In the revised manuscript, we have added our rationale \u201cIn particular, the function of cAMP-dependent protein kinase A (PKA) on transmission release has been the subject of debate. Lonart et al. (2003) found that RIM1\u03b1-Ser413 is phosphorylated by PKA, which is required for presynaptic LTP. However, the mice with dysfunctional RIM1\u03b1-Ser413 mutation exhibit normal presynaptic LTP in the cerebellum and the hippocampus , questioning the role of RIM1\u03b1-Ser413 and PKA in presynaptic LTP. Thus, how RIM1\u03b1 is activated during presynaptic plasticity needs to be revisited\u201d (Line 52).The activities of EPAC and PKC\u03b5 required for presynaptic LTP are shown. However, the change of the basal synaptic properties due to the knockdown of two proteins is not shown, such as the input-output relation of evoked EPSC in the PF-PC synapse. Therefore, studying the protein during the normal condition may be of interest.In the new experiment, we have conducted input-output relationship of evoked EPSCs. Our results showed that the amplitudes of evoked EPSCs with various stimulation intensities were reduced by presynaptic deletion of EPAC or PKC\u03b5. The data have been shown in Figure 2\u2014figure supplement 1 and in the text \u201cFurthermore, we examined the evoked PF-PC EPSCs with different stimulation intensities (3-15 \u03bcA) in control and mutant mice. Our results showed that presynaptic deletion of either Epac1/Epac2 or Prkce significantly decreased evoked EPSCs in response to all stimuli \u201d (Line 181).The visual diagram of the authors' hypothesis on a process describing EPAC-mediated phosphorylation of RIM1 is a bit difficult to follow. Arrows may be indicated with more comprehensible signs/ words.We are sorry for the confusion. We have updated the Figure 1G and legend to make the point clear.Following the compensatory eye movement training, the deficit is more prominent in phase than gain. Mutants catch up the gain at the end of 5 days of training but not the phase. The authors may explain how the presynaptic LTP is more essential to phase in the discussion. In addition, the reason to choose VOR phase reversal learning other than OKR learning or VOR gain-up learning may be related to this discussion.The reviewer is correct in that the phase difference is the only difference present at the end of our VOR training. However, we found that the gain was also different in multiple days in both EPAC-cKO and PKC\u03b5-cKO mice compared to their controls. VOR phase reversal training subject mice to multiple days of changing training stimuli, unlike VOR or OKR gain increase, and thereby tests different aspects of adaptation. It should be noted that the aim of VOR phase reversal training is to first decrease the gain, followed by an increase in phase. If the training is continued long enough, the gain will increase again, usually when the phase reaches the levels higher than 120 degrees. Therefore, the initial decrease of gain followed by the late-stage increase presumably underlies the absence of significant differences in gain between control and mutant groups in days 4 and 5. This does not imply that presynaptic LTP is more essential for the VOR phase than VOR gain, as the VOR gain decrease is affected during the first days of training.This explanation has been included in the end of the manuscript \u201cAlthough we observed only a difference in phase at the end of VOR phase reversal training, it should be noted that the gain was different on multiple days in both Rapgef3;Rapgef4-cKO and Prkce-cKO mice compared to their controls. VOR phase reversal training subjects to multiple days of changing training stimuli to test different aspects of adaptation. The first aim is to decrease the gain, followed by an increase in phase. Once the phase has increased above 120o, the gain will increase again . Therefore, the initial decrease of gain followed by the late-stage increase presumably underlies the absence of differences in gain between control and mutant groups in days 4 and 5. This does not imply that presynaptic LTP is more essential for the phase than the gain, as VOR gain decrease is affected during the first day of training\u201d.In the discussion part, some more thoughts and scientific predictions of the limited role of EPAC and PKC\u03b5 on postsynaptic plasticity should be stated.We appreciate this comment. In our work, we used the mouse models with presynaptic deletion of EPAC or PKC\u03b5, which affects PF transmitter release but not postsynaptic plasticity.2\u03b1/COX2-PKC\u03b1 and CaMKII activation, GluA2 trafficking and its regulators, and CB1 receptor activation and high NO production; the induction of postsynaptic PF-LTP requires low Ca influx, cPLA2\u03b1 activation, low NO production, phosphatase activation, and GluA2 trafficking . Based on current studies, we speculate that EPAC or PKC\u03b5 might be unrelated to these molecular processes.In the classical theory, induction of postsynaptic PF-LTD requires massive Ca influx, mGluR1 activation, cPLAInterestingly, Gutierrez-Castellanos et al. (2017) showed that EPAC might regulate GluA3 conductance and is essential to postsynaptic LTP. Since an inhibitor of EPAC was used in that study, the genetic ablation of EPAC in PCs would be needed to repeat the results. However, this study gives us a hint that postsynaptic EPAC or PKC\u03b5 may regulate the conductance of AMPA subunits, and then regulate postsynaptic LTP and LTD.We have added a paragraph in Line 519 \u201cIn addition, Gutierrez-Castellanos et al. (2017) showed that EPAC may regulate GluA3 conductance in PCs, suggesting that postsynaptic EPAC or PKC\u03b5 may regulate the conductance of AMPA receptor subunits, and thereby postsynaptic LTP or LTD at PF-PC synapses\u201d.Overall, specific claims made by the authors were straightforward and supported by an ample amount of experimental results. This study has widened the spectrum of understanding of the molecular complexity of cerebellar motor learning by demonstrating the active contribution of presynaptic plasticity and its related signaling cascade.Thank you for these positive comments.Reviewer #3 (Recommendations for the authors):The conclusions often overstate the findings of the data. The language can be toned-down a without diminishing the importance of these findings.We thank you for this important comment. We have modified our conclusions throughout the manuscript, also based on the comment of the other reviewer.In the present form, I don't think the cumulative EPSC analysis is informative. The authors should repeat the analysis using the Elmqvist and Quastel (EQ) method . This method is more reliable at low Pr and can provide (when combined with the Train method) a high and low bracket of possible Pr and RRP values. Ideally, the authors would redo these experiments with a higher stimulus frequency. It would also be helpful to show a simple PPR value for each genotype. This can be done without additional experiments.p to be constant throughout a stimulus train . Although p may be constant for the calyx of Held synapses they studied, it cannot be case for PF-PC synapses. Therefore, we decided to redo the estimations of RRP and Pr using 100-Hz train (previously 20-Hz train).Thanks for the very insightful comment. In the previous experiments, we measured RRP and Pr based on parameter taken from the work in the hippocampal CA1 neurons , which, in our opinion, is similar to PF-PC synapses concerning low release probability. We have carefully read Thanawala and Regher (2016) paper and compared different methods. While the performance of the EQ method is in general more reliable to estimate small RRP and low Pr, it relies on p and allows us to have a better estimation on RRP and Pr at PF-PC synapses .This method does not require constant 0 and follow-up EPSCs were smaller in cKO mice, indicating that both the initial release and the replenishment are reduced by the conditional knockout o EPACs or PKC\u03b5. Compared to 20-Hz train, the 100-Hz train resulted in steady-state EPSCs brought EPSCs into steady state faster. We created linear fit from normalized steady-state EPSCs and back-extrapolated the curve to the y-axis to calculate Pr. Indeed, we found that the Pr value estimated from the 100-Hz train stimulus was significantly larger than that from the 20-Hz train, showing 0.17 (Math1-cre) and 0.19 (PKC\u03b5f/f) with 100-Hz, but 0.07 (Math1-cre) and 0.08 (PKC\u03b5f/f) in previous submission. This result was similar to Thanawala and Regher (2016), in which they claimed that the accuracy of estimation from a 100-Hz train is about three times of that from a 20-Hz train. Moreover, we found that the conditional knockout of either EPACs or PKC\u03b5 produced significant decrease on Pr . These results have been added in the text and figure legend , and corresponding methods have also been updated.The new results have been presented in new Figure 2E and 2F. The PF-PC synapses were stimulated at the frequency of 100 Hz, and the artifacts were truncated and the EPSCs were aligned . Note that the aim of this experiment was to investigate whether there is difference between control and cKO mice. Indeed, we found that the amplitudes of both EPSC2+ concentration) do not match in vivo conditions. These differences and potential changes in synaptic plasticity should be discussed.Slice conditions in LTP/LTD experiments . Our new data show that, under these conditions, EPACs and PKC\u03b5 are still needed for the induction of presynaptic PC-LTP .To date, almost all PC plasticity in published work were recorded in young adult mice (< 1 month) and at room temperature, and most behavioral experiments were conducted around 2-3 months of age. To better answer the reviewer\u2019s comment, we tried our best to redo the LTP experiments under the requested, alternative conditions .Is it possible to provide an estimate of what percent of synaptosomes arise from parallel fibers versus mossy fibers based on the relative number of each synapse? This would help the interpretation of the synaptosome data.+EAAT4+) or CF-PC (vGluT2+EAAT4+) synapses. Our staining results showed that PF-PC synapses covered 88.8% of the total and CF-PC synapses covered 7.5% of the total. Thus, we estimated the number of mossy fiber synapses to be less than 3.7%, which would not affect our conclusion. These results have been presented in Figure 1\u2014figure supplement 1.We have performed synaptosome staining vGluT1/vGluT2, EAAT4 and bassoon to identify PF-PC synapses (vGluT1"} +{"text": "Objective: This meta-analysis aimed to determine the efficacy of curcumin in preventing myocardial ischemia/reperfusion (I/R) injury in animal models.Methods: Studies published from inception to January 2023 were systematically searched in databases including PubMed, Web of Science, Embase, China\u2019s National Knowledge Infrastructure (CNKI), Wan-Fang database, and VIP database (VIP). The SYRCLE\u2019s RoB tool was used to determine methodological quality. Sensitivity analysis and subgroup analysis were performed when there was high heterogeneity. Publication bias was assessed using a funnel plot.Results: Thirty-seven studies involving 771 animals were included in this meta-analysis with methodology quality scores ranging from 4 to 7. The results indicated that curcumin treatment significantly improved myocardial infarction size standard mean difference (SMD) = \u22125.65; 95% confidence interval (CI): 6.94, \u22124.36; p < 0.01; I2 = 90%). The sensitivity analysis for infarct size showed that the results were stable and reliable. However, the funnel plot was asymmetric. The subgroup analysis included species, animal model, dose, administration, and duration. The results showed that the subgroup dose was statistically significant between subgroups. In addition, curcumin treatment improved cardiac function, myocardial injury enzymes, and oxidative stress levels in animal models of myocardial I/R injury. The funnel plot revealed that there is publication bias for creatine kinase and lactate dehydrogenase. Finally, we performed a meta-analysis of inflammatory cytokines and apoptosis index. The results showed that curcumin treatment downregulated serum inflammatory cytokine levels and myocardial apoptosis index.Conclusion: This meta-analysis suggests that curcumin has excellent potential for the treatment of myocardial I/R injury in animal models. However, this conclusion needs to be further discussed and verified in large animal models and human clinical trials.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/, identifier CRD42022383901. The patte death . Therefote death .The inherent health benefits and hypotonicity of natural products have attracted growing interest worldwide, leading to a remarkable increase in the use of nutraceuticals and dietary supplements. Turmeric, a curry spice originating in India, has attracted increasing attention in recent years for its medicinal properties. Curcumin, an extract of turmeric, is a lipophilic polyphenol with antioxidant, anti-inflammatory, and anti-fibrotic properties . CurcumiRecent studies have shown that curcumin protects cardiomyocytes from myocardial I/R injury through multiple and diverse mechanisms . CurcumiThis meta-analysis has been registered in PROSPERO (ID: CRD42022383901). This study was conducted in Covidence from literature selection to data extraction.Six databases, including Web of Science, PubMed, Embase, China\u2019s National Knowledge Infrastructure (CNKI), the Wan-Fang database, and the VIP database (VIP), were searched systematically between database inception and January 2023 without language restrictions. The keywords mainly used were \u201cmyocardial infarction\u201d, \u201cmyocardial ischemia\u201d, \u201cmyocardial I/R\u2033, \u201cmyocardial I/R injury\u201d, \u201cmyocardial ischemia-reperfusion injury\u201d, \u201cmyocardial ischemia-reperfusion\u201d, and \u201ccurcumin\u201d.The included studies met the following criteria: : myocardin vitro studies, . Subgroup analysis and sensitivity analysis were conducted to find the source of heterogeneity. Publication bias was evaluated, when more than 10 studies were included, by funnel plot. p < 0.05 indicated that the difference was statistically significant.The summary statistics were considered by standard mean difference (SMD) and 95% confidence interval (CI). The Cochran Q test and the ISeven hundred and ninety-one articles were involved after the primary retrieval. One hundred and twenty-eight duplicate articles were excluded by Covidence. After carefully reading the abstracts and titles, 579 articles were excluded. Forty-seven studies were excluded after full-text articles were assessed due to articles with unclear data, irrelevant to our topic, the original text being unavailable, and duplicate data. Finally, a total of 37 studies involving 755 animals were included in the present meta-analysis . The PRIIn terms of species, 21 studies used Sprague-Dawley rats, 10 studies used Wistar rats, and 6 studies used C57BL/6 mice. In terms of gender, only 3 studies used female animals and all the others were male. The methods used to establish animal models of myocardial I/R injury included LAD ligation in 26 studies, ISO injection in 10 studies, and coronary microembolization in 1studies.Curcumin was administered by multiple routines including oral in 20 studies, intraperitoneal in 7 studies, intravenous in 4 studies, and perfused in 6 studies. Additionally, the duration of curcumin treatment varied from 1\u00a0min to 12\u00a0weeks. The publication year of the included studies varied between 1996 and 2022. Twenty-four of 37 studies were conducted in China, 4 studies in India, 3 studies in Korea, 2 studies each in Iran or Romania, and 1 study each in France or the United States. All the basic information about the included studies in this meta-analysis is shown in The studies included in this meta-analysis scored between 4 and 7 on the quality assessment. The details of the methodological quality of the included literature are shown in p < 0.01; I2 = 90%) in the animal model of myocardial I/R injury , suggesting that 100\u00a0mg/kg may be the most well-documented effective dose for the treatment of myocardial I/R injury.In conclusion, our results demonstrated a significant cardioprotective effect of curcumin at multiple levels in animal models of myocardial I/R injury. This result provided a solid theoretical basis for subsequent large animal studies and human clinical trials.The basic pathological processes of myocardial I/R injury include in inflammation, oxidation stress, cardiomyocyte apoptosis, etc . CurcumiCurcumin reduces myocardial I/R injury through multiple molecular mechanisms, including reducing NF-\u03baB activation , activat+-dependent histone deacetylase, regulates cell stress, energy metabolism, and cell apoptosis through the deacetylation of specific substrate proteins such as NF-\u03baB, FOXO1, TP53, and AMPK, and thus plays a cardiovascular protective role (Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD)ive role . The uprive role . Myocardive role . Curcumiive role .Myocardial fibroblasts and macrophages play vital roles in cardiac repair and remodeling after myocardial infarction and affects the prognosis of patients . Pro-infOxidative stress is involved in the entire process of myocardial I/R injury . ExcessiAnimal researches are limited by design variations and methodological flaws. In addition, publication bias also limits the clinical translation of animal studies because positive experimental results are easier to publish . StudiesThe results of this study showed that curcumin significantly reduces myocardial IS, improves cardiac function, downregulates myocardial enzyme levels, inhibits oxidative stress, decreased serum inflammatory cytokines, and myocardial apoptosis index to play a cardioprotective role in animal models of myocardial I/R injury. However, the results of this study only used the target animal model and did not include animal models with multiple coexisting diseases, which may differ from the pathology of patients in the actual clinical setting. Although high heterogeneity exists, our results still suggest that curcumin is effective in myocardial I/R injury, and this study can provide a solid theoretical basis for further clinical trials.Firstly, only small animals were used in the included studies. There is a lack of evidence in larger animals. According to the criteria of IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) , large aThis meta-analysis suggests that curcumin has excellent potential for the treatment of myocardial I/R injury in animal models. However, this conclusion needs to be further discussed and verified in large animal models and human clinical trials."} +{"text": "Objective: Myocardial ischemia-reperfusion (I/R) injury is a complex clinical problem that often leads to further myocardial injury. Curcumin is the main component of turmeric, which has been proved to have many cardioprotective effects. However, the cardioprotective potential of curcumin remains unclear. The present systematic review and meta-analysis aimed to evaluate the clinical and preclinical evidence regarding the effect of curcumin on myocardial I/R injury.Methods: Eight databases and three register systems were searched from inception to 1 November 2022. Data extraction, study quality assessment, data analyses were carried out strictly. Then a fixed or random-effects model was applied to analyze the outcomes. SYRCLE\u2019s-RoB tool and RoB-2 tool was used to assess the methodological quality of the included studies. RevMan 5.4 software and stata 15.1 software were used for statistical analysis.Results: 24 animal studies, with a total of 503 animals, and four human studies, with a total of 435 patients, were included in this study. The meta-analysis of animal studies demonstrated that compared with the control group, curcumin significantly reduced myocardial infarction size (p < 0.00001), and improved the cardiac function indexes (p < 0.01). In addition, the indexes of myocardial injury markers, myocardial oxidation, myocardial apoptosis, inflammation, and other mechanism indicators also showed the beneficial effect of curcumin (p < 0.05). In terms of clinical studies, curcumin reduced the incidence of cardiac dysfunction, myocardial infarction in the hospital and MACE in the short term, which might be related to its anti-inflammatory and anti-oxidative property. Dose-response meta-analysis predicted, 200\u00a0mg/kg/d bodyweight was the optimal dose of curcumin in the range of 10\u2013200\u00a0mg/kg/d, which was safe and non-toxic according to the existing publications.Conclusion: Our study is the first meta-analysis that includes both preclinical and clinical researches. We suggested that curcumin might play a cardioprotective role in acute myocardial infarction in animal studies, mainly through anti-oxidative, anti-inflammatory, anti-apoptosis, and anti-fibrosis effects. In addition, from the clinical studies, we found that curcumin might need a longer course of treatment and a larger dose to protect the myocardium, and its efficacy is mainly reflected on reducing the incidence of myocardial infarction and MACE. Our finding provides some meaningful advice for the further research. Cardiovascular disease (CVD), especially acute myocardial infarction (AMI), has become one of the main risk factors threatening human health . ThromboTurmeric is a commonly used herb as a traditional medicine in Asian countries . According to the records of ancient Chinese medical books, turmeric is capable of relieving pain through promoting qi and blood circulation. Therefore, it is frequently used in the treatment of chest tightness, chest pain, shoulder and back pain, dysmenorrhea, et al. Curcumin , Wanfang database, China Science and Technology Journal Database (VIP), and China Biology Medicine disc (CBM), were searched systematically from inception to 1 November 2022. Moreover, references of eligible studies were also manually searched to identify additional eligible studies. The search language was restricted to English and Chinese, and the search strategy was as follows. The detailed search strategies in eight databases and retrieved results were provided in Inclusion criteria were as follows: 1) The I/R experimental model were established by ligating the left coronary artery (LCA) such as left anterior descending (LAD) or other proper methods, or ischemic heart disease patients receiving PCI or CABG in the hospital; 2) The treatment group received any dose of curcumin as monotherapy, and the control group received the same amount of non-functional fluid or no treatment at all; 3) The primary outcome of animal studies were myocardial infarction (MI) size and echocardiogram indicators. The secondary outcomes of animal studies were biomarkers of myocardial injury and mechanisms of curcumin in the treatment of myocardial I/R injury; 4) The human studies were RCT study. Exclusion criteria were set as follows: 1) not a myocardial I/R model or patients who beyond the relevant clinical diagnostic criteria; 2) combined with other drugs; 3) no control group; 4) duplicate publication; 5) not an animal model or not RCT study .Two independent reviewers extracted the following details from the included studies: 1) first author name and year of publication; 2) Specific information about the animals and patients in each study, including species, number, sex, and body weight; 3) Myocardial I/R model and the anesthetic method used to prepare the model; 4) Information about curcumin treatment, including dose, method of administration, course of treatment; as well as corresponding information in the control group; 5) The mean and standard deviation (SD) of the results. Suppose there were many different time point results, only the last time point results would be recorded. Since some of the data is only presented in graphical format, we tried to contact the authors for detailed information. If we did not receive any response, the digital ruler software would be used to measure the value of the graph.The quality of the included animal studies was assessed using Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk of bias (RoB) tool 10-item scale as folloThe quality of the included human studies was assessed using RoB-2 tool as folloThe problem of curcumin divided into different dose subgroups in the original study was handled following the strategy recommended by Cochrane Handbook for Systematic Reviews of Interventions (CHSRI) . We mergp < 0.05 was considered statistically significant) and I2-statistical test. According to the Cochrane handbook on heterogeneity analysis, I2 value ranges from 0% to 40% means heterogeneity might not be important; 30%\u201360% may represent moderate heterogeneity; 50%\u201390% may represent substantial heterogeneity; 75%\u2013100% means considerable heterogeneity. A fixed-effects model was adopted if I2 < 50%; otherwise, a random-effects model would be applied. All outcomes were continuous variables, so we used weighted mean difference (WMD) with 95% confidence intervals (CIs) to present, for outcomes reported in various measurement methods or different scales of measurement, we calculated with a standard mean difference (SMD). p < 0.05 was considered statistically significant.RevMan software (version 5.4) was used for statistical analysis. Heterogeneity was determined by the Q-statistical test (2 > 50%) and the preset subgroups failed to find the source of heterogeneity.We preset four subgroups: 1) method of model establishment, 2) administration method, 3) genus of animals, 4) intervention moment. We conducted a subgroup analysis on the primary outcomes to evaluate the impact of variables and to explore the source of heterogeneity. A meta-regression analysis was further performed using Stata 15.1 software once the heterogeneity in the study was significant was modeled using STATA 15.1 software. According to the characteristics of point distribution, linear, exponential, logarithmic, quadratic, and cubic regression equations can be selected to try to fit. The generalized least squares method (GLS) was used to calculate each of the parameters while R2 represents the degree of fitting (R2 closer to one indicates a better fit of the prediction model).The relationship between the standard dosage of curcumin and the ratio of means . Then the same two reviewers individually perused the remaining 60 full-texts, and finally 24 animal studies publisheTwenty studies were pubAll human studies are EnglWe evaluated the quality of all included studies. The score of study quality ranged from two to six with a total of ten points. One study got two RoB-2 tool was used to evaluate the methodological quality (deviation risk) of the four included human studies. Two of the studies were judged as low risk, while the other two were judged as some concerns. The details are shown in p < 0.00001, I2 = 97%) size was calculated by infarct area/left ventricular area. A meta-analysis of 16 animal studies demonstrated that curcumin significantly decreased MI size compared with the control group , 2 = 97%) . Becauseexperimental/meanscontrol). Then, scatter plot was drawn for curcumin dose and RR (Left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular end-diastolic dimension (LVDD), left ventricular end-systolic dimension (LVSD) were examined to demonstrate improvement in cardiac function induced by curcumin. A total of four animal studies reportedI2 = 0%) . ResultsI2 = 0%) showed c2 = 47%) . Results2 = 47%) showed c2 = 35%) . Results2 = 35%) showed cI2 = 0%) .p = 0.04, I2 = 98%) (p < 0.05), and another one (p < 0.05).Compared with the control group, meta-analysis of three animal studies showed c2 = 98%) . A meta-2 = 98%) showed c2 = 93%) . Through2 = 93%) on CK di2 = 99%) . Moreove2 = 99%) showed cther one showed cp = 0.008, I2 = 95%) . A meta-2 = 95%) showed t2 = 89%) . Three a2 = 89%) showed t2 = 90%) . A meta-2 = 90%) showed t2 = 91%) . A meta-2 = 91%) indicate2 = 79%) . Results2 = 79%) showed c2 = 89%) . Results2 = 89%) showed c2 = 87%) .p-value indicates comparison with the control group). These indicators included Bcl-2 (p < 0.05), Caspase-3 (p < 0.01), MMP-9 (p < 0.05), MPO (p < 0.05), IL-6 mRNA (p < 0.05). The included animal studies also reported the regulation of curcumin on other proteins. However, because the frequency of occurrence was <2, they would not be described in detail , other mechanism indicators reflecting the cardioprotective function of curcumin only are described as follows. Unless otherwise specified, these reports indicated positive effects of the curcumin group on the mechanism of anti-myocardial I/R injury (ed Bcl-2 (p < 0.0aspase-3 (p < 0.0), MMP-9 (p < 0.005), MPO (p < 0.0L-6 mRNA (p < 0.0p < 0.05) (p < 0.05), while the outcome of between ischemia and reperfusion group was inconsistent with the overall results (p > 0.05). Moreover, I2 did not decrease, suggesting that the preset subgroups did not explain the source of heterogeneity of MI size.We performed the subgroup analysis on primary outcomes according to preset subgroups. The LVEF, LVFS, LVDD, LVSD in the primary outcome indicators were not included in the subgroup analysis because of not enough animal studies, so we only carried out the subgroup analysis on MI size. Analysis of the three preset subgroups indicated outcomes that were consistent with the overall rusults ( < 0.05) \u20139. Some < 0.05) . The subTo further explore the source of heterogeneity, we conducted a meta-regression analysis. Eight characteristics were selected for meta-regression, including: 1) Region , 2) year of publication , 3) whether the original study set the dose subgroup, 4) In vivo or ex-vivo experiments, 5) myocardial ischemia duration , 6) times of administration (single-dose or multiple-dose), 7) No. of sample size (<20 or \u226520), 8) publication language (English or Chinese). However, meta-regression analysis did not p = 0.92 > 0.05).A funnel plot was adopted to assess the publication bias regarding myocardial infarction size . FurtherOral curcumin nanomicelle was used in four human studies. Two studies used a sp = 0.912), cTnI , CK-MB . One study (p = 0.02).cTnI, cTnT and CK-MB have been used clinically for a long time to evaluate the myocardial injury in patients. Three studies detectedne study showed tp = 0.87), but the results of study (p = 0.031) and MDA .Serum CRP and MDA were used to evaluate the degree of inflammation and oxidative stress injury. The results of study showed tof study showed tp = 0.021), and NT-proBNP . The results of two studies (p = 0.02).For patients with ischemic heart disease, in-hospital MI and MACE after revascularization are the most important clinical outcome, and cardiac function is the most important factor affecting the above outcome. The study showed t studies showed t studies showed tThis is the first systematic review and meta-analysis consisting of preclinical and clinical evidence to investigate the cardioprotective effects of curcumin on myocardial I/R injury. Twenty-four studies with 503 animals and four human studies with 435 patients were included, and the overall methodological quality of the included studies was moderate. The results of animal studies meta-analysis suggested that curcumin might diminish myocardial infarction size, improve heart function, suppress biomarkers concentration for myocardial infarction, as well as ameliorate cardiomyocyte inflammation, oxidation, apoptosis, and myocardial fibrosis in animal studies.p = 0.75). Although p > 0.05, both indicators showed descending trend. Besides, it should be noted that the protocol of curcumin treatment in these two studies is often accompanied by a variety of conditions, such as aging, hypertension, diabetes, etc. Therefore, more complex animal models such as aging animal models, should be used to study the pharmacological effects and mechanisms of curcumin in future researches.The rationality and standardization of in vivo researches directly affect the further transformation from pre-clinical studies to clinical applications. According to SYRCLE 2.0 tool, the methodology quality of included animal studies is moderate. Therefore, when designing experimental protocols, researchers should strictly follow the animal experiment guidelines, such as random allocation, blinding investigators, random outcome assessment, etc., and record this information in the paper.Many animal studies have shown that curcumin has acceptable security, and no significant toxicity was observed even when rats or mice were given a high dose (5\u00a0g/kg BW per day). According to the dose-response model of animal studies, the cardioprotective effect of curcumin may be stronger with the further increase of the dosage. Further, researchers developed a Polylactic-co-glycolic acid (PLGA) and Plga-polyethylene (PEG) (Plga-peg) nanoparticle containing curcumin, and it was observed that the nanoparticles increased the mean half-life of curcumin in about 6\u00a0h, enhanced the maximum blood concentration of curcumin by 7.4 times, and improved the bioavailability of curcumin by 55.4 times compared with curcumin aqueous suspension . TherefoConducting more clinical trials should be encouraged because animal experiments have shown excellent effectiveness. In the existing clinical trials, the results showed that curcumin significantly reduced the incidence of MACE in patients after PCI or CABG in the short term. In addition, curcumin is widely consumed as a food material in Southeast Asia, and a large number of studies supported its high biological safety.In clinical research, researchers should not only focus on the incidence of MACE events caused by MIR in the short term after PCI or CABG, but also focus on the incidence of MACE and cardiac function within 1 year after discharge by increasing follow-up time.Patients with myocardial injury and reperfusion often need to take antiplatelet drugs. Some studies claimed that curcumin plays an antiplatelet role by inhibiting platelet aggregation , inhibitFirst of all, we only retrieved English and Chinese studies, which may lead to selection bias. Secondly, positive results are no doubt more likely to be published, so the dominance of positive studies may lead to an overestimation of the efficacy of curcumin, although the funnel plot and Egger\u2019s regression test did not show significant publication bias. Thirdly, the overall quality of the animal studies was moderate, ranging from two to six points out of 10, for example, many animal studies ignored the need for randomization. In addition, myocardial infarction usually occurs in patients with cardiovascular risk factors, such as aging, diabetes, hypertension, and hyperlipidemia . HoweverOur study is the first study that includes both preclinical and clinical evidence in terms of curcumin in treatment of MIR, and our results suggest that curcumin might play a cardioprotective role in acute myocardial infarction, mainly through its anti-oxidative, anti-inflammatory, anti-apoptosis, and anti-fibrosis effects. In addition, we found that curcumin might need a longer course of treatment and a larger dosage to exert cardioprotection in the clinical studies, and its efficacy is mainly reflected in reducing the incidence of myocardial infarction and MACE in short term. Finally, our study summarized the main defects of the existing research and suggested how such primary studies need to be improved." \ No newline at end of file