Background: Coronary stenosis severity determines ischemic symptoms and adverse outcomes. The metabolomic analysis of human fluids can provide an insight into the pathogenesis of complex disease. Thus, this study aims to investigate the metabolomic and lipidomic biomarkers of coronary artery disease (CAD) severity and to develop diagnostic models for distinguishing individuals at an increased risk of atherosclerotic burden and plaque instability.Methods: Widely targeted metabolomic and lipidomic analyses of plasma in 1,435 CAD patients from three independent centers were performed. These patients were classified as stable coronary artery disease (SCAD), unstable angina (UA), and myocardial infarction (MI). Associations between CAD stages and metabolic conditions were assessed by multivariable-adjusted logistic regression. Furthermore, the least absolute shrinkage and selection operator logistic-based classifiers were used to identify biomarkers and to develop prediagnostic models for discriminating the diverse CAD stages.Results: On the basis of weighted correlation network analysis, 10 co-clustering metabolite modules significantly (p < 0.05) changed at different CAD stages and showed apparent correlation with CAD severity indicators. Moreover, cross-comparisons within CAD patients characterized that a total of 72 and 88 metabolites/lipid species significantly associated with UA (vs. SCAD) and MI (vs. UA), respectively. The disturbed pathways included glycerophospholipid metabolism, and cysteine and methionine metabolism. Furthermore, models incorporating metabolic and lipidomic profiles with traditional risk factors were constructed. The combined model that incorporated 11 metabolites/lipid species and four traditional risk factors represented better discrimination of UA and MI  compared with the model involving risk factors alone . The combined model was successfully used in discriminating UA and MI patients (p < 0.001) in a three-center validation cohort.Conclusion: Differences in metabolic profiles of diverse CAD subtypes provided a new approach for the risk stratification of unstable plaque and the pathogenesis decipherment of CAD progression. Coronary artery disease (CAD) refers to underlying coronary artery atherosclerotic lesions that cause vascular lumen stenosis or occlusion and insufficient blood supply and result in myocardial ischemia, hypoxia, or necrosis . DespiteEmerging metabolomics is a powerful tool to systematically investigate the functional small molecule in biological fluid samples. An abnormal metabolome can reportedly characterize CAD, further providing clues for physiological and pathological explorations . ElevateThe goals of the present study were to comprehensively investigate the plasma metabolomic and lipidomic signatures associated with increased CAD severity and to evaluate the significantly differential metabolites and lipid species for their use in discriminating the subgroups of CAD, thereby providing an enhanced understanding of disease progression. Herein, we performed a widely targeted metabolomic and lipidomic evaluation in plasma of patients with SCAD, UA, and MI and identified specific features of metabolite profiles that are associated with increase in CAD severity and can be used to differentiate these three subgroups. The subsequent pathway analysis revealed that glycerophospholipid metabolism was the most significantly altered metabolic pathway. Disease diagnostic classifiers for discriminating between different CAD subgroups were constructed and validated based on novel metabolic markers and traditional risk factors.N = 942), we evaluated the association of plasma metabolome and lipidome with CAD using consecutively enrolled samples with clinical and demographic information obtained from Guangdong Provincial People’s Hospital (N = 493), we enrolled multi-center patients with CAD from three centers  from 2017 to 2018.An overview of the workflow is depicted in Hospital  in 2010–All subjects were 18–80 years and met the diagnostic criteria of CAD. They were further stratified into three subgroups  on the basis of a detailed diagnosis performed by cardiologists, their symptoms, ischemic changes in electrocardiogram, laboratory measurements, and coronary angiographic results. The specific diagnostic criteria of CAD subtypes are summarized under The study fully complies with the guidance of the Helsinki Declaration. The Medical Ethical Review Committee of Guangdong Provincial People’s Hospital granted ethics approval (GDREC2010137 and GDREC2017071H). Written informed consent was obtained from all subjects.g, 10 min, 4°C) within 2 h and refrigerated at −80°C until analysis.Each eligible subject fasted for at least 8 h to minimize the influence of nutrition on metabolite levels. The subjects’ venous blood samples were collected in ethylene diamine tetraacetic acid (EDTA) vacutainer tubes in the morning (between 9 AM and 12 PM) after overnight fasting and cooled in a freezer (4°C) immediately. Plasma was separated by centrifugation (2095 Coronary angiography (CAG) was performed to define the extent and severity of CAD in patients with suspected symptoms whose clinical characteristics and results of noninvasive testing indicated a high likelihood of CAD and who are amenable to, and candidates for coronary revascularization . CAG wasThe hydrophilic and hydrophobic compounds were extracted from each plasma sample and detected via ultra-performance liquid chromatography and electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) system in the positive and negative ionization modes in Metware Biotechnology . Details for the sample preparation and UPLC-MS/MS experiment parameters are provided in the In total, 202 metabolites  and 667 lipid species  were identified and quantified.Quality control (QC) samples were utilized for the normalization of the data. A QC sample was created via pooling aliquots from all samples and was injected every 10 samples throughout the run to assess the instrument’s stability. Highly stable QC data showed that the run had great repeatability and reliability .For metabolomic and lipidomic analyses, raw signals with more than half of the missing rate in the QC samples (those with zero ion intensity) were removed. Missing metabolomic data were imputed by replacing the missing value with a minimum value of the metabolite quantified. To adjust signal drift, we applied the Quality Control–based Robust LOESS  Signal Correction (QC–RLSC) algorithm for analytical batch effect correction , which iβ = 4 and min module size = 5 were chosen for the analysis. Spearman correlation between metabolite modules and clinical parameters was calculated using R. The Benjamini–Hochberg method was used to control the false discovery rate (FDR). Hub metabolites indicated a high degree of connectivity in biological interaction networks and ,thus, they were considered biologically important. Clusters of co-abundant plasma metabolites were identified using the “WGCNA” package in R.A metabolic network was constructed by the weighted correlation network analysis (WCNA), which used metabolites’ pairwise correlations to identify modules of highly correlated metabolites . An unsip < 0.05.Among the baseline characteristics of the study population, continuous variables were described using medians (interquartile ranges) and were compared using Mann–Whitney U tests . Categorical variables were presented as counts (percentages) and were compared with Chi-squared tests. Statistical significance was determined as The linear regression analysis, adjusted for traditional Framingham risk factors, including age, sex, hypertension, diabetes mellitus, smoking, low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), and triglycerides (TG) , was appp-value of <0.05. Open database sources, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (http://www.genome.jp/kegg/) and the MetaboAnalyst  (version 4.0), were used to identify the highly enriched metabolic pathways based on the significantly differential levels of metabolites and lipid species.To assess the association of individual metabolomic and lipidomic signatures against the different stages of CAD, we performed adjusted logistic regression of metabolomic and lipidomic profiles against SCAD vs. UA and UA vs. MI to estimate the odds ratios (ORs) and 95% confidence intervals (CIs). To avoid potential confounders, traditional risk factors, including age, sex, hypertension, diabetes mellitus, smoking, LDLC, HDLC, and TG, were used as covariates for adjustment. Subjects with missing covariates were omitted. Statistical significance was determined as a p < 0.05) associated with UA (vs. SCAD) and MI (vs. UA) in the adjusted logistic regression analysis were included into least absolute shrinkage and selection operator (LASSO) penalized models (“glmnet” package) to further reduce the number of markers and select the most powerful predictive features. In the LASSO selection analysis, the optimal value for the tuning parameter λ was determined via 5-fold cross-validation (200 iterations). We adopted the largest value of lambda, such that the error was within one standard error of the minimum, known as “1-se” lambda. The relative contribution of features to classification assignment (UA vs. SCAD and MI vs. UA) was determined by the occurrence frequency in our multivariate model training. The feature was selected with an occurrence frequency of more than 100 times.In the development of diagnostic models to classify CAD subgroups, firstly, the following were added to develop the traditional risk factor-based model in a stepwise regression (forward and backward) with the aim to minimize the Akaike information criterion (AIC): age, sex, hypertension, diabetes mellitus, smoking, LDLC, HDLC, and TG ; APOA and Lp(a) (risk lipid traits); and left ventricular ejection fraction . This procedure was performed within 10 iterations of a 5-fold cross-validation framework . Subsequently, metabolites and lipid species that were nominally significantly  = 1/1 + exp [-(intercept + coefficient1 (biomarker1) + coefficient2 (biomarker2) … + coefficient n (biomarker n))]. The area under the curve  of the receiver operating characteristic (ROC) was applied to calculate the proportions of concordant pairs among all pairs of observation with 1.0 indicating perfect prediction accuracy. Moreover, the continuous net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were calculated in assessing the models. The 95% confidence intervals (CIs) were estimated for each parameter. The difference of combined diagnostic scores between CAD subgroups in the validation cohort was examined by the Wilcoxon rank sum test.http://www.R-project.org/).All the above analyses were conducted on the R platform , UA (N = 368), and MI (N = 264). The baseline characteristics and laboratory data of each group are shown in p < 0.001). The systemic atherosclerotic burden of CAD was determined using SYNTAX score system, and the median scores of each group were as follows: SCAD, 13.0 ; UA, 14.0 ; and MI, 19.0 . The SYNTAX score showed a significant difference between SCAD vs. MI (p < 0.001) and UA vs. MI (p < 0.001). The MI group exhibited a higher proportion of three-stenosed vessels (37.56%), a larger left ventricular mass index (LVMI), and a lower LVEF. The median levels of cTnI, an indicator of myocardial infarction, were 0.01 , 0.02 , and 0.3  μg/ml in the SCAD, UA, and MI groups, respectively. Significant differences in cTnI levels were found with SCAD vs. UA (p = 0.025) and UA vs. MI (p < 0.001).A total of 1,435 CAD patients were included from three independent centers in China . The disThe validation cohort from three centers included 493 participants. Their baseline characteristics are summarized in In the WCNA, 756 of the metabolites and lipid species in the discovery set were parsed into 35 co-abundance modules, whereas the gray module comprised unassigned metabolites and lipids due to weak correlation with others. However, each metabolite and lipid were further analyzed individually.p < 0.05 between CAD stages  correlated with four severity indicators  of 0.082 ± 0.040, p = 0.042 , cTnI , and hs-CRP (inflammatory state) was conducted by adjusting for traditional risk factors, including age, sex, hypertension, diabetes mellitus, smoking, LDLC, HDLC, and TG. Numerous metabolites and lipid species showed strong association for one or more severity indicators . Three ldicators . We also = 0.042 .p < 0.05) associated with UA (p < 0.05) associated with MI  and valine, leucine, and isoleucine biosynthesis . Furthermore, the pathway analysis revealed that cysteine and methionine metabolism  and glycerophospholipid metabolism  were the main perturbed pathways for UA vs. MI. The glycerophospholipid metabolism was the most significantly altered pathway among all paired comparisons.We focused on SCAD vs. UA for transition from coronary stability to instability and UA vs. MI for cardiac events. The logistic regression analysis of the metabolic and lipidomic profiles against UA (vs. SCAD) adjusting for traditional risk factors, identified 72 metabolites/lipid species that were significantly ( with UA . The reg with MI . The enrp < 0.05) associated with MI (vs. UA) were considered as input variables. The model was obtained by the LASSO logistic analysis  and IDI of −0.0178 , and an IDI of 0.105 , 11 conventional CAD risk factors, including age, sex, hypertension, diabetes mellitus, smoking, TG, LDLC, HDLC, APOA, Lp(a), and LVEF, were considered in the stepwise variable selection modeling. Finally five variables, namely, age, hypertension, TG, HDLC, and LVEF were retained in the model with minimal AIC and had an AUC value of 0.758 ; Table 2p < 0.05) selected from adjusted logistic regression, LASSO logistic analyses were further applied to identify the most predictive biomarkers. The optimized model consisting of 17 features exhibited a considerable performance with an AUC of 0.688 (p < 0.0001), and a IDI of 0.105 ; UA (N = 184); and MI (N = 157). We used the established optimal LASSO models to further demonstrate the potential ability of subgroup discrimination. Consistently, the combined diagnostic score could help differentiate UA vs. MI patients  and MI (vs. UA), respectively. Moreover, the pathway analysis of these potential biomarkers indicated that glycerophospholipid metabolism exhibited the most significantly altered metabolic pathway in all paired comparisons. Lastly, the newly developed combined diagnostic models improved stratification performance of CAD subtypes compared with the traditional risk model, offering further evidence of dysbiotic metabolome and lipidome and highlighting its potential to distinguish various stages of CAD.Specifically, the co-clustering modules within lipid classes including phosphatidylcholine (PC), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylethanolamine (PE(P)), and alkylphosphatidylcholine (PC(O)) tended to decrease with plaque instability and were inversely correlated with CAD severity and myocardial markers. Moreover, modules containing five PCs were positively correlated with HDLC  and primarily decreased in the MI group. Different PC species showed diverse effects on CAD progression. We observed that PCs with longer and more unsaturated acyl chain had an inverse association with UA (vs. SCAD). PCs are the most abundant membrane lipids in mammals  and are PC in lipoproteins or from cell membrane can be further hydrolyzed on the sn-2 position fatty acid to generate LPC and free fatty acid by the phospholipase A2 enzyme . AlthougOne of the prominent features observed was that a number of PE(P) species with polyunsaturated fatty acids displayed a significant inverse association with MI compared with UA patients . AlkylphHowever, the module containing ceramides was elevated with disease shifting and was positively associated with CAD severity, myocardial markers, and inflammatory state. Notably, hexosylceramide species played an important role in the development of CAD. Specific hexosylceramide species  were related to the enhanced coronary atherosclerosis burden. Both mono- and dihexosylceramide have a direct association with the risk of future cardiovascular events in patients with type 2 diabetes, which is a potential atherogenesis-contributing factor .Some plasma ceramide levels were observed to be up-regulated with the disease shifting direction as SCAD, UA, and MI and positively correlated with atherosclerosis burden quantified by the SYNTAX score and SYNTAX score Ⅱ and the evidence of subclinical myonecrosis quantified by cTnI. This result corroborates the finding that elevated plasma ceramide levels are independent biomarkers of MACE . Cer d1. As the In addition to the use of certain lipid species of sphingolipids and glycerophospholipids as predictors of CAD progression, the downregulation of deoxycholic acid in MI, which plays key roles in bile acid and cholesterol metabolism, indicated that the metabolism of cholesterol and phospholipids might be inhibited . MoreoveOur study had some limitations that needed be considered. First, due to the upgrading of analytical platform and technical issues with the mass spectrometry, the metabolites and lipid species detected were not in accordance, thereby resulting in the lack of four independent predictors for UA (vs. SCAD) model and one for MI (vs. UA) model which affected the model estimation in the verification cohort. Second, site-to-site and observer-to-observer variations in the evaluation of coronary stenosis may exist, leading to diagnostic bias. Third, the improvement in AUC for a model is often very minor, yet the category-free NRI may overstate the incremental value of a biomarker. Last, our study population tended to consist of middle-aged to elderly Chinese patients. Thus, other ethnicities within Asia and other races, such as Caucasians and Africans, should be included in future studies.Multiple plasma metabolites and lipid species differed between CAD subgroups, and the alterations were correlated with CAD severity. The metabolites involved in glycerophospholipid metabolism appeared to be a predominant alteration in CAD progression. A small number of these biomarkers significantly improved the diagnostic value for differentiating patients between CAD types. These findings may help to predict disease progression and clinical outcome and indicate the potential for novel intervention strategies to attenuate disease progression.


Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (ΔOCTgfbr2) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice  by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 ( Retinal degeneration is among the leading causes of blindness worldwide ,2. IntriTGFβ signaling controls a plethora of cellular responses such as proliferation, differentiation, tissue homeostasis, morphogenesis and regeneration . In addiTgfbr2 was notably upregulated following photoreceptor degeneration , w, wMapk7,ignaling .Ngf is dependent upon TGFβ2 treatment in vitro and is significantly enhanced in the juvenile retina of a mouse model with increased TGFβ signaling activity .The number of degenerating photoreceptor cells was determined using TUNEL . This method was performed on retinae of one-month-old animals, as described previously ,50. TUNEEyes of three-month-old mice were carefully enucleated and fixed for 24 h in Ito’s fixative . The eyet-test in Excel . One-way ANOVA analyses were performed in SPSS  if more than two individual groups were compared (post-hoc test: Bonferroni). p values ≤ 0.05 were considered to be statistically significant.Data are expressed as mean ± SEM. Statistical comparative analyses between the mean variables of two individual test populations were performed using a two-tailed Student’s


Giardia duodenalis is an important parasite with veterinary and public health significance worldwide. The presence and zoonotic assemblages of G. duodenalis have previously been reported in rabbits. In this study, to understand the infection status of G. duodenalis in rabbits from Shaanxi province, a total of 537 fecal samples were collected from two breeds of rabbits in four age groups  from four geographical origins . The presence of G. duodenalis in these samples was assessed using molecular assays based on beta-giardin (bg). The glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) loci were then amplified in the bg-positive samples for multi-locus genotype (MLG) analysis. The total prevalence of G. duodenalis in these rabbits was 3.54% (19/537). Giardia duodenalis infection was found in both breeds of rabbits, and in all farms and age groups, but with no statistically significant differences related to these factors (p > 0.05). Two assemblages, including B and E, were identified, with the former the predominant assemblage detected in both breeds, and in all age groups and farms. Sequence analysis revealed 2 (named as rbg1-2), 1 (named as rtpi1), and 2 (named as rgdh1-2) haplotypes at the gene loci of bg, tpi, and gdh, respectively, forming a multilocus genotype (MLG) of assemblage B . These findings reveal the significant zoonotic potential and genetic diversity of G. duodenalis in rabbits in Shaanxi Province, PR China. Giardia duodenalis is an important intestinal parasite of humans and more than 40 animal species, making it the 11th most important foodborne parasite globally according to the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) ..bg gene Giardia duodenalis has been widely reported in animals worldwide, with prevalence of 1.6–91.33% [G. duodenalis infection by nested-PCR targeting the bg gene, for a total prevalence of 3.54% (19/537). This is in the range of prevalence for G. duodenalis infection, but lower than most previous studies, e.g. 8.40% in Henan [G. duodenalis prevalence in rabbits in different studies may reflect the different examination methods used. For example, a study in European countries used coproantigen ELISA to investigate the prevalence [G. duodenalis in rabbits from Henan (3.35%) [G. duodenalis. A study from Nigeria [G. duodenalis infection. A previous study in Shandong used the same target gene as this study to detect prevalence, with higher prevalence (11.2%) than that detected here [6–91.33% , 25, 68.in Henan , 7.41% iin Henan , 7.60% iin Henan , 13.79% in Henan , 11.20% in Henan  and Xinjin Henan . The difevalence , while pevalence , 56, 70 evalence , 56, 57. (3.35%)  by PCR wG. duodenalis in Shaanxi province may not be affected by these factors. A previous study conducted in Henan province, China assessed differences in prevalence among rabbits from different farms and age groups and found no significant differences  among rabbits from nine farms or in different age groups  [No significant differences in prevalence were detected among rabbits from different farms and different age groups in our study, suggesting that the infection of  > 0.05) . Future G. duodenalis assemblages, A, B and E, were previously reported in rabbits [G. duodenalis and its clinical appearance is a controversial topic. Some reports showed that asymptomatic infection was associated with infection in children [G. duodenalis related to assemblage A [G. duodenalis assemblage B, including malodorous and poorly formed feces and severe weight loss [G. duodenalis assemblage B in both humans and animals. There is also evidence that assemblage E, a previously hoofed animal-specific assemblage, has zoonotic potential, since assemblage E has been detected in humans from some areas with poor conditions, such as Egypt and Brazil [G. duodenalis from rabbits to humans and other animals in Shaanxi province. Future work should increase testing by rabbit farmers to confirm the prevalence and genotypes of G. duodenalis.Three  rabbits , 70, 71. rabbits , 70, 71. rabbits , 62. Thechildren , 29, butmblage A , 36. Themblage A , 49, 62.mblage A , and sevght loss . These fd Brazil . These rG. duodenalis isolates from humans and animals [bg gene locus was identical to that in assemblage B isolates from rabbits, Lemur catta, and humans [G. duodenalis isolates from rabbits in Shaanxi Province, eight and five samples were amplified at the gene loci of tpi and gdh, respectively. The haplotype rtpi1 was identified as assemblage B-IV identical to the isolates from rabbits in China [gdh haplotype (rgdh1) was identical to G. duodenalis assemblage B isolates from humans in Zambia, Canada, Brazil, Poland, and water in Canada [Genetic variability has been detected within  animals , 27. Assd humans , 65. Hapd humans , 34, 65.d humans . To furtin China , 71. Then Canada , 61. Hapn Canada . InteresG. duodenalis infection in rabbits from Shaanxi province, with a total prevalence of 3.54%. Giardia duodenalis was detected in both Rex and IRA rabbits, and detected on all farms and in all age groups (G. duodenalis from rabbits in Shaanxi province, and provided the basis to implement control strategies of G. duodenalis in this province as well as other regions of the world.This study is the first report of e groups . Assemble groups . GeneticWe declare that we do not have any commercial or associative interests that represent a conflict of interest in connection with the work submitted.


While survival after hematological malignancies in adolescent and young adult patients is improving, patients report poor oncofertility care. This population-based, retrospective, cohort study used data from the Ontario Cancer Registry and billing codes to identify fertility consultations for lymphoma patients between 2000 and 2018. Consultation trends across time and different patient and physician characteristics were analyzed. We identified 2088 patients and a consultation rate of 3.4% (increasing from 1% in 2000–2006 to 8% in 2014–2018). Patient parity and regional deprivation scores decreased rates. Despite mild improvement, there is ample missed opportunity for fertility discussions. Greaves et al. demonstrated that HM survivors were more likely to remain childless than the general population.2 In Hodgkin's lymphoma (HL), the most common HM in the AYA population, premature ovarian failure occurs in up to 37% of patients.3 To address these concerns, the discipline of oncofertility emerged with the hopes to accelerate the inclusion of fertility in oncology care.4The adolescent and young adult (AYA) age group, 15–39 years of age, presents unique challenges in oncology. Hematological malignancies (HMs) make up 21%–34% of cancer diagnoses among this age group. Since the 5-year survival rate for HMs diagnosed before the age of 39 ranges from 50% to 95%, research foci are shifting to improve quality of life (QOL) after cancer.5–7 However, oncology patients continue to report dissatisfaction.8 Specifically, in AYA patients with HM, rates of infertility discussions vary from 17% to 83%.2 Factors that affect these rates include patient demographics, physician characteristics, and complex societal factors.9The American Society of Clinical Oncology (ASCO) has released guidelines necessitating fertility discussions in oncology care for AYA patients.10 This article investigates a similar trend in lymphoma patients by capturing the province of Ontario. We determined patient, physician, and socioeconomic factors associated with fertility consultation rates, highlighting areas for improvement.While the need for fertility consultations led to the ASCO guideline updates, the impact of these guidelines is unknown. Similarly, patient surveys cannot capture the scope of care in a population. Our group published a population-based analysis of referral rates for AYA, breast cancer patients showing a modest improvement after the 2013 guideline.11 Patients with a history of infertility, sterilization procedures, or previous cancer and patients ineligible for health insurance were excluded.This population-based cohort study of AYAs diagnosed with lymphoma from January 2000 to March 2018 identified AYAs residing in Ontario, Canada's largest province by population (13.2 million) using the Ontario Cancer Registry (>98% of cancer incidents).www.ices.on.ca). The primary outcome, gynecology consultation about fertility, between the diagnosis of HM and commencement of chemotherapy, was identified as a gynecology consult billed as an International Classification of Diseases, Ninth Revision (ICD-9) code 628 (infertility diagnosis).Datasets were linked by encoded identifiers and analyzed at ICES . Chemotherapy billing codes identified treatment commencement. Community deprivation and income quartile scores measured socioeconomic factors.p < 0.05 for significance. Data were analyzed using SAS version 9.4 . This study was approved by the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board.Individual characteristics were analyzed using chi-square analyses for categorical variables, one-way analysis of variance (ANOVA) for continuous variables, and Kruskal–Wallis tests for medians. Logistic regression examined how factors influenced fertility consults, adjusting for confounding variables through a backward selection modeling. All statistical tests were two sided with 10 There was a significant increase in fertility consultation rates across the study period (p for trend <0.001).This population-based study assessed fertility consultation rates in female AYAs with lymphoma in Ontario, Canada. A total of 2088 female AYAs diagnosed with lymphoma were identified. 2 However, fertility consultation in that study was publicly funded. In contrast, universal funding for fertility treatment began in Ontario in 2015. Referral rate in a private, American system for HMs, breast and gastrointestinal malignancy combined, was 5% between 1993 and 2007, similar to our study.12A survey-based study investigating HM patients between 1957 and 2006 in Britain showed a slightly higher rate of 12% with improvement after 2000.Notably, we used fertility billing codes, which preclude referrals that were suggested, but not completed. While this code, ICD-9 code 628, is the only one for infertility and required for billing insured services in Ontario, its use as a substitute for fertility consultations has not been validated. Furthermore, it does not represent discussions had by other health care professionals, including oncologists, patient flow coordinators, and nursing staff. Together, these limitations may contribute to the low rates observed.p < 0.001). This modest improvement is consistent with the literature that demonstrates knowledge gaps even after guidelines are released.13 Campbell et al. identified that 35% of pediatric oncologists surveyed read the 2013 guideline.14 In addition to guidelines, other mechanisms to promote oncofertility include the following: referral pathways, multidisciplinary teams, and provider education.9 One Canadian initiative is the establishment of the Canadian National Task Force on Adolescents and Young adults with Cancer, in 2008, which identified improving fertility outcomes in cancer survivors as a priority issue.15Fertility consults increased after the 2013 ASCO update .10Patient and socioeconomic factors may affect care. In this study, fertility consultations were not altered by patient age or cancer type. Several studies demonstrate that pediatric and older-AYA (30–39 year old) patients have reduced odds of referral than patients in their 20s.16 In Ontario, public funding of one cycle of in vitro fertilization for a female patient is covered under the Ontario Fertility Program, introduced in December 2015. Prior to this program, the consultation was publicly funded, but treatment was not. Thus, some patients may not have followed through with a consultation if they knew they could not afford fertility preservation.Since economic factors alter referral patterns, in Ontario, future research into the impacts of public funding is warranted.9 as well as heterosexuality and being Caucasian. This suggests social determinants of health influence oncofertility care.17Likewise, income quintile did not alter fertility consultation odds; however, more complex socioeconomic factors  captured by a higher deprivation score decreased odds of consultation . Being educated was similarly associated with increased referral trends in a review by Loren and Senapati,p = 0.02). Notably, we did not measure cancer stage, and therefore, cannot comment if urgency affects odds of referral. One possibility is that a less severe stage allows for more time for a referral. The literature and guidelines suggest early referrals and referral pathways reduce delays in treatment.13 Furthermore, more recent ovarian stimulation protocols allow for less delay.7Fertility consultation was more likely if the time between diagnosis and chemotherapy was >6 weeks . However, this result does not mean that fertility referrals delay treatment. The median time to chemotherapy was 5 weeks (IQR 3–8) and 6 weeks (IQR 4–9) for patients with and without a fertility consultation, respectively, thus demonstrating a modest delay .18 Of the physicians who made a referral, 55% were older than 45. The most common specialty to refer was hematology (48%), which aligns with the target audience of the guidelines, followed by family physicians (20%) and medical oncology (11%).Several studies demonstrate that physician characteristics, including sex, attitudes, age, and specialty, affect referrals.Fertility consultation improved after the ASCO guidelines for female AYAs with lymphoma. By identifying referral trends, we can tailor future interventions toward demographics in need. Despite the modest improvement demonstrated in this study, oncofertility research demonstrates vast potential for improving care.


Staphylococcus aureus and Staphylococcus epidermidis. Our coatings exhibited several features that made them particularly effective for antibiofilm protection: (i) UA was successfully integrated with the graphene material, (ii) a steady release of UA was documented, (iii) steady UA release ensured strong inhibition of bacterial biofilm formation. Interestingly, even after the initial burst release of UA, the second phase of steady release was sufficient to block bacterial colonization. Based on these results, we propose that graphene coatings loaded with UA can serve as effective antibiofilm protection of biomedical surfaces.Protecting surfaces from bacterial colonization and biofilm development is an important challenge for the medical sector, particularly when it comes to biomedical devices and implants that spend longer periods in contact with the human body. A particularly difficult challenge is ensuring long-term protection, which is usually attempted by ensuring sustained release of antibacterial compounds loaded onto various coatings. Graphene have a considerable potential to reversibly interact water insoluble molecules, which makes them promising cargo systems for sustained release of such compounds. In this study, we developed graphene coatings that act as carriers capable of sustained release of usnic acid (UA), and hence enable long-term protection of surfaces against colonization by bacterial pathogens  The biofilm formation is considered as a key factor for majority (˃ 60%) of bacterial infections such as cystic fibrosis, nonhealing wounds and medical device associated infections in humans worldwide5. The bacterial community inside biofilms represents a protected mode of survival, due to the presence of exopolymeric substances which allows bacteria to thrive in hostile environments9. Bacterial cells in biofilms are found to be 100–1000 times more resistant to antibiotics than bacterial cells in planktonic state, leading to higher risks of treatment failure by developing antibacterial resistance12. Most biomedical devices in contact with the human body are always at risk of bacterial colonization, and this leads to device-associated infections, which often lead to failure of such devices, patient suffering and financial losses14. Hence, there is significant incentive to development of antibacterial coatings to prevent or minimize bacterial attachment to biomedical surfaces.Bacterial growth, surface colonization and subsequent biofilm formation is one of the major challenges for biomedical devices16. For example, antibacterial activity of graphene and its derivatives has been documented extensively21. Similarly, direct antibacterial effects of metalling nanoparticles have been frequently reported. Antimicrobial activity of silver nanoparticle is widely studied and demonstrated to have strong bactericidal effect23. However, the ability of these nanomaterials and nanoparticles to protect surfaces over extended periods of time is questionable. In past decade, combining nanomaterials with antibiotics to enhance the antibacterial efficiency was intensively explored 26. Metallic nanoparticles are not particularly suitable for long-term protection, as they are not chemically inert and release metal ions that may be toxic to mammalian cells28. By contrast, graphene and its derivatives are chemically inert and possess excellent physicochemical properties for long-time protection, such as high thermal stability, biocompatibility, and high mechanical strength. Therefore, they could provide the optimal cargo system for sustained drug release to ensure long-term antibacterial protection of biomedical devices30. There are a few available studies showing the loading of antibiotics and other therapeutics to graphene derivatives, which have resulted in sustained drug release and enhancement in antimicrobial activity32. However, these tests were performed only against planktonic state of bacterial cells, which are highly sensitive to antibiotics in compared to sessile cells.Many nanomaterials are known to exhibit strong antibacterial effects18. To maximize this effect, graphene coatings/flakes need to be oriented perpendicular to the surface. Graphene coatings where flakes are predominantly arranged parallel to the surface and have no exposed edges available for interaction with bacterial cells, are typically not bactericidal20. Such planar or parallel-to-the-surface coatings have a high adsorption capacity, which in fact directly favours the adhesion of bacterial cells and promotes biofilm growth34.It should be mentioned that graphene coatings themselves may be engineered to provide a degree of antibacterial activity, which is based on direct mechanical interaction of bacterial cells with the exposed sharp edges of graphene35. Sea lichens are surface-attached symbiotic communities composed of fungi and algae or bacteria. By producing antibacterial compounds such as UA, lichens protect themselves from colonization of surrounding bacterial communities. Considering the inhibitory activity of UA on bacterial colonization, the potential of UA to prevent surfaces of biomedical devices has already been proposed36. Strong antibacterial activity of UA against various human pathogens was demonstrated by previous studies36. This activity of UA is mainly based on its inhibitory potential on nucleic acid synthesis, which, via inhibiting RNA synthesis also blocks bacterial translation35. UA has been used as an antibacterial additive in medicinal products, toothpaste, mouth wash, wound healing patches and creams. However, poor solubility of UA limits its medical application37. In order to improve delivery and expand the scope of antibacterial applications of UA, this bioactive molecule has been incorporated into liposomes, nano emulsions and polymeric and metallic nanoparticles38. These strategies were effective in delivery of UA for several applications, e.g. wound treatment and targeted delivery into macrophages infected with Mycobacterium tuberculosis38.UA is a secondary metabolites of sea lichen with strong antibacterial activityS. aureus and S. epidermidis. These pathogens often colonise to biomedical implants, forms biofilms, infect surrounding tissues and leads towards the implant failure. We found that our coatings provide a sustained release of UA for up to one week, and we found them to be extremely effective against all forms bacterial that we tested, for periods of up to 96 h. Even after the primary burst release of UA during the initial 24 h, the coatings retained excellent antibiofilm properties. Our results indicate that this combination of graphene coatings and UA could pave the way for more effective antibacterial protection of biomedical devices, leading to diminished risks of device-associated infections.In this study, we attempted to develop graphene coatings loaded with UA, which would be capable of sustained UA release and thereby providing long-term antibiofilm protection. the effectiveness of our coatings was evaluated against planktonic state and early- and late-stage biofilms of opportunistic pathogens S. epidermidis ATCC 35,984 and S. aureus CCUG10778 were used to evaluate the antibacterial and antibiofilm activity. Tryptic soy broth (TSB)/agar was used to grow bacteria.The graphene flakes (powder form) was purchased from Graphitene (UK). The usnic acid (UA) was purchased from Sigma-Aldrich and dissolved in DMSO. The The graphene flakes were dispersed in deionized and double distilled water by ultrasonication (125 W) for 4 h at the concentration of 2 mg/mL as a stock solution. Parallelly, UA was dissolved in di-methyl sulfoxide  at the concentration of 5 mg/mL. By mixing these stock solutions to sterile water, various concentrations of graphene—UA mixture were prepared: total concentrations of 200 µg/mL, 100 µg/mL, 50 µg/mL and 25 µg/mL. All mixtures contained equal concentrations of graphene and UA (1:1 molar ratio) in sterile water with 5% of DMSO. For example, 200 µg/mL of mixture containing final concentration of 200 µg/mL of graphene and 200 µg/mL of UA in sterile water with 5% of DMSO. After mixing, all mixtures were further sonicated for 4 h. After the sonication, graphene flakes loaded with UA were separated by centrifugation at 10,000 rpm for 15 min. The flakes were further washed twice with deionized and double distilled water. The washed flakes were mixed in original volume of sterile deionized water and further sonicated for 2 h to achieve homogeneous dispersion.Round cover glasses (15 mm diameter) were washed with isopropyl alcohol, followed by 70% ethanol and dried by air drier. Clean and dried glass surfaces were coated with various concentrations of graphene flakes loaded with UA  and non-loaded graphene flakes (100 µg/mL) as control, by using a drop casting method. Specifically, 400 µL of each batch of dispersed graphene flake solution was placed on a glass surface and left overnight for complete drying at 37 °C. The dried surfaces were further cured at 60 °C for 12 h. The coated surfaces were subsequently washed with sterile deionized water to remove the flakes that were not attached to the glass surface dried at 60 °C.−1 and at a resolution of 2 cm−1. Raman spectra were measured by a Dilor LabRAM confocal micro‐Raman spectrometer equipped with a 50 × objective and a HeNe laser (632.8 nm). Each spectrum was recorded in the range 500–3000 cm-1 with 10 min accumulation time and approximately 4 cm−1 resolution.The morphology of coatings was examined by scanning electron microscopy (JEOL JSM 6301F). The graphene coatings were further coated with gold (5 nm) prior to SEM imaging. Infrared spectra of the samples were recorded using an attenuated total reflection (ATR) Alpha FT-IR spectrometer from Bruker, with a diamond crystal as refractive element, in the range of 400–3000 cmTo examine the UA release pattern from the loaded graphene flakes in the coatings, a coated cover glass was transferred to a 15 ml of falcon tube containing 1 ml of PBS buffer (pH 7.4) and kept at 37 °C. After 24 h, the buffer with released UA was collected from the tube and replaced with fresh buffer. The procedure was repeated every 24 h, for a total of 7 days. All collected samples were freeze dried and resuspended in 100 µl of milli-Q water. The amount of released UA was quantified by using HPLC (Dionex UHPLC-PDA-FLD) with the column AAA C-18 5 mm (150 mm × 4.6 mm) . A mixture of methanol—water—acetic acid (80:15:5 v/v) was used as the mobile phase with a flow rate of 1 mL/min. 10 µl of all the resuspended released samples and standard solutions of UA (from 1.56 to 100 µg/ml) were injected to HPLC quantified by comparison of peaks to a standard curve.S. aureus and S. epidermidis. To prepare bacterial inoculum, single colony of S. aureus and S. epidermidis was inoculated to 5 mL of growth medium (TSB) and incubated overnight at 37 °C with continuous agitation. For this assay, 20 µl of inoculum  from overnight grown bacterial cultures were inoculated into fresh TSB medium containing sterile deionized water (control), 12.5, 25, 50, 100 and 200 µg/mL of UA loaded graphene. All samples were incubated at 37 °C with continuous agitation for 24 h. A fraction of culture (100 µL) from each sample was taken for determination of bacterial viability at time points of 0, 2, 4, 6, 8 and 24 h. The collected samples were serially diluted in 0.89% of saline. From the diluted samples, 100 µL was plated onto TSB agar plates, incubated at 37 °C for 48 h and the number of colonies was counted.The bactericidal efficiency of UA loaded on graphene was tested against the planktonic cells of S. aureus and S. epidermidis. The graphene samples loaded with UA and control glass surfaces were sterilized by UV-light exposure for 10 min. The overnight culture of respective bacteria was diluted in fresh TSB broth to obtain the final inoculum of 2–5 × 106 CFU/mL and 400 µL inoculum was seeded on the pre-sterilized coated and non-coated surfaces. Samples with bacterial inoculum were incubated at 37 °C for 24 h without agitation to allow for formation of biofilms. After 24 h of growth, samples were washed twice with sterile deionized water to remove the loosely adhered or free bacterial cells and collected in 5 mL of 0.89% of sodium chloride solution. The biofilms were detached from the surface and homogenized by sonication . The homogenized biofilm suspensions were serially diluted into 0.89% of sodium chloride solution and plated onto TSB agar plates. Agar plates were incubated at 37 °C for 48 h and the number of colonies was counted. The density of biofilms and morphology of bacterial cells in biofilms were examined by using SEM. For this, biofilms of S. aureus and S. epidermidis formed on coated and non-coated surfaces were washed twice and fixed with 3% of glutaraldehyde for 2 h. The fixed biofilms were dehydrated by using graded series of ethanol concentrations  for 15 min each, and with absolute ethanol for 20 min. The dehydrated biofilms were dried overnight at room temperature and sputter coated with gold (5 nm) prior to SEM imaging. The SEM images were acquired by using JEOL JSM 6301F . To visualize the density of live and dead bacterial cells in biofilms grown on the coated and noncoated surfaces, biofilms were stained with the mixture of 6.0 μM SYTO 9 and 30 μM potassium iodide from Live/Dead BacLight Viability kit L13152, . Fluorescence microscopic images of the stained biofilms were acquired by using a Zeiss fluorescence microscope .The biofilm inhibitory efficiency of coatings was evaluated against 6 CFU/mL of respective bacterial inoculum was added onto the surfaces and incubated at 37 °C for 24 h. The number of viable bacterial cells in biofilms and visualization of live and dead bacterial cells were determined by using the methods described above.To evaluate the long-term anti-biofilm potential of coatings, biofilms were grown on the coated and non-coated surfaces for 96 h. Briefly, overnight grown bacterial suspension was inoculated on the surfaces and incubated at 37 °C for 96 h. The culture medium was replaced in the interval of 24 h . After 96 h, the biofilms were collected and homogenized by means of sonication and plated on agar plates after the serial dilution to count the colonies. In addition, antibiofilm activity of coatings after the primary phase of drug release was also evaluated. In order to test this, the sterile TSB medium was placed onto the pre-sterilize coated and noncoated surfaces and incubated at 37 °C for 24 h, to allow for initial release of UA. After this incubation, the medium was removed, and surfaces were washed twice with sterile deionized water to remove any traces of the medium with released UA from the coatings. Thereafter, fresh TSB medium containing 2–5 × 10P-value was < 0.05.All data are presented as the mean ± standard deviation from at least three different biological replicates. Intergroup differences were estimated by one-way analysis of variance (ANOVA), followed by a post hoc multiple comparison (Tukey) test to compare the multiple means. Differences between values were considered statistically significant when the −1) related to defects (edges) and G peak (~ 1585 cm−1) from the sp2 C–C bond denoted the degree of graphitization of material39. The intensity of the D band was observed to be higher compared to the G band, indicating a considerable amount of defects and roughness of the coating surfaces. For both graphene and UA integrated samples, the minor 2D peak was identified, demonstrating the abundance of multilayer graphene in the surface  analysis were used to characterize the structure and morphology of the coatings and to assess the success of UA loading, as presented in Fig. D/IG) was observed to be slightly higher in the graphene loaded with UA than in pure graphene . This ratio is known to be inversely proportional to the crystallite size of nanographite. This suggests the presence of shorter graphene sheets due to some changes in the amount of defects and hinting to sites where UA attaches.The calculated ratio between the intensities of the D and the G bands , in the presence of different concentrations of graphene-UA flakes. The viability of planktonic bacteria was followed for 24 h of growth , and their bactericidal effects continued for the entire duration of the experiment  within a 24-h period.Previous reports of UA toxicity against 42. One promising strategy to reduce the adhesion of bacterial cells and prevent biofilm formation is to protect the surface of biomedical devices by various coatings43. The coating strategies reported so far include coatings containing antibiotics, containing nanoparticles with antibacterial effects, containing carbon-based materials, containing antibacterial polymers and finally, coatings made of hybrid nanomaterials loaded with antibacterial molecules43. So far, the major risk associated with coatings containing antibiotics/antibacterial agents is the rapid drug release, which precludes these coatings from delivering long-term antibacterial effects, and increases the risk of resistance development 45. To address this drawback, different strategies have been proposed, such as incorporation of antibiotics into a polymeric matrix, integration into nanomaterials, loading onto nanofibers followed by layer-by-layer deposition for sequential and sustained release of antibiotics/antibacterial45. Using these approaches, several classical antibiotics, such as gentamycin, vancomycin and rifampicin, have been used for implant coatings as a prophylactic measure against implant-associated infections45. An ideal coating system should be able to ensure sustained release of the drug following a two-phase profile: a first stage of burst release, followed by sustained release capable of maintain a local drug concentration above the minimum inhibitory concentration47.Adhesion of bacterial cells and subsequent biofilm formation is one of the great challenges for biomedical devicesS. aureus and S. epidermidis. Bacterial biofilms were grown on coated and non-coated control surfaces for 24 h. Thereafter, viability of bacteria in biofilms was evaluated by CFU counts and live/dead staining  at all tested concentrations (25–200 µg/mL), except for S. aureus at 25 µg/mL  decrease at 24 h, after 96 h the decrease was closer to 100,000-fold (5 log10 units). This observation can be corelated to sustained release of UA , which was similar to inhibitory effects obtained during primary phase of UA release (Fig. 36. Therefore, antibiofilm activity observed here is potentially due to bacteriostatic as well as bactericidal effect of UA by disrupting the nucleic acid synthesis pathways.After the primary release, coatings with 25 and 50 µg/mL of graphene flakes loaded with UA were not able to inhibit bacterial growth to the extent that was observed when the primary release was present Fig. . Howeverase Fig. . These rase Fig. c, where ase Fig. a,b and tase Fig. . The obs51. While this makes a direct comparison between in vitro and in vivo data impossible, it should be said that a 100 000-fold reduction of biofilm growth over 96 h, as observed with our coatings in conditions optimal for bacterial attachment (Fig. In this work, we loaded graphene flakes with UA and used those to constitute surface coatings with antibiofilm properties. We demonstrated that the sustained release of UA from the coatings is the key factor in ensuring a strong and long-term antibiofilm effect of our coatings. It is difficult to correlate the findings of such in vitro studies—mimicking bacterial attachment to surfaces of biomedical devices—to real life situation in vivo. In our in vitro setup, large numbers of bacterial cells are loaded onto the surface. This is a rather advantageous situation for bacteria, compared to the in vivo conditions. In the human body, human cells compete with bacteria for attachment to the surface and the host immune system interferes with bacterial attachment


Brazil ranks first in the number of HTLV-1/-2-infected individuals worldwide. The high morbidity and mortality of HTLV-1-associated diseases, especially following infection in infancy, requires strong action to reduce vertical transmission. To facilitate the appraisal of the implementation of the HTLV antenatal screening program by the Brazilian Ministry of Health, we determined the costs in distinct scenarios according to HTLV seroprevalence, specificity of the screening test, and type of confirmatory test. HTLV antenatal screening would cost R$ 55,777,012-R$ 77,082,123/year. Screening assays with high specificity reduce the need and cost of confirmatory assays by up to 25%.  Careful selection of the screening assay is required to optimize the program. In Brazil, the estimated number of people living with HTLV-1 is high ,The diagnosis of HTLV-1/2 infection is based on the detection of anti-HTLV-1/2 antibodies using a screening assay, such as enzyme-linked immunoassay (ELISA) or chemiluminescent immunoassay (CMIA), followed by confirmatory testing of the reactive samples. There is a range of commercially available assay kits for the initial detection of HTLV-1 antibodies, and their performances are diverse with specificities varying from 92.0% to 99.5% in the Brazilian settingThe identification of seropositive individuals is crucial to prevent new infections, but policymakers may consider the cost of testing as a limiting factor. Few studies have focused on reducing the cost of HTLV testing. Here, we analyzed the impact of the performances of HTLV-1 screening tests on healthcare expenditure in the context of a national antenatal screening program in Brazil. Additionally, we determined the economic impact of national antenatal screening in the country, using the perspective of the Brazilian Health Unified System . For this purpose, we considered the need for a diagnostic algorithm comprising one screening test (ELISA/CMIA), followed by confirmatory test (either WB or PCR) on seropositive samples. The following inputs were considered:Number of pregnant women in Brazil in 2018 (extrapolated from the number of live births in Brazil): 2,944,932 HTLV-1 screening test specificities: 92.0% (Murex HTLV-1/2), 98.1% (Architect rHTLV-1/2), 99.0% (Anti-HTLV-1/2 SYM Solution), and 99.5% (Gold ELISA)HTLV-1 screening test sensitivity was 100% for each of the above assaysPrevalence of HTLV-1 infection in pregnant women in Brazil (Minimum 0.1%-Maximum 1.05%)Cost of ELISA: R$ 18.55 according to price list management of SUS Cost of western blot: R$ 85.00 Cost of PCR: R$ 65.00 The Brazilian Ministry of Health will have to spend R$ 54,628,489/year on initial screening for HTLV using ELISA, based on the number of pregnant women in Brazil and the cost of ELISA, as defined by SUS. The number of false positive results was calculated by multiplying the total number of pregnant women with (1 − Specificity) × . Next, the cost of testing to confirm and discriminate HTLV-1 and HTLV-2 infections was determined for both WB and PCR.The confirmation of HTLV-1/2 infection is strongly recommended, and the cost will vary according to the specificity of the screening test, the prevalence of HTLV-1 infection, and the choice of the assay, as detailed in Therefore, using a screening test with higher specificity could result in a reduced cost of up to 25.0% (19.8%-25.0%), saving up to R$ 18,748,909/year . This woAdditionally, the diagnosis of HTLV-1 infection during pregnancy is extremely important because this route of infection is associated with a higher risk of HTLV-1 associated diseases. The identification of infected pregnant women represents a unique opportunity to block the silent transmission of HTLV within families and communities, as it allows the implementation of effective measures to significantly reduce the HTLV-1 mother-to-child transmission. The Brazilian Ministry of Health recommends to avoid breastfeeding for all HTLV-1/2 seropositive mothers and provides formula for feeding,,LIA has superior performance than WB, giving fewer indeterminate results,Our analysis focused on individual sample testing. However, the use of pooled sera for HTLV-1/2 screening is an acceptable strategy to reduce costIn summary, we have demonstrated that, regardless of the cost of the screening assay for the Brazilian Ministry of Health, using an assay with high specificity would allow a significant reduction in overall expenditure. The best performing screening assay would not only result in economic savings but would also prevent distress and uncertainty in up to 220,575 pregnant women per year, who would no longer receive a false-positive result in the screening test . This also reinforces the importance of immediate confirmatory testing for assays with low specificity, as it is unacceptable to misinform up to 80 women regarding HTLV-1/2 seropositivity for every true positive infection detected . We also determined the cost of the implementation of HTLV-1/2 national antenatal screening in Brazil, which will facilitate the appraisal of this important public policy that is necessary in Brazil.


The cell-mediated immune response constitutes a robust host defense mechanism to eliminate pathogens and oncogenic cells. T cells play a central role in such a defense mechanism and creating memories to prevent any potential infection. T cell recognizes foreign antigen by its surface receptors when presented through antigen-presenting cells (APCs) and calibrates its cellular response by a network of intracellular signaling events. Activation of T-cell receptor (TCR) leads to changes in gene expression and metabolic networks regulating cell development, proliferation, and migration. TCR does not possess any catalytic activity, and the signaling initiates with the colocalization of several enzymes and scaffold proteins. Deregulation of T cell signaling is often linked to autoimmune disorders like severe combined immunodeficiency (SCID), rheumatoid arthritis, and multiple sclerosis. The TCR remarkably distinguishes the minor difference between self and non-self antigen through a kinetic proofreading mechanism. The output of TCR signaling is determined by the half-life of the receptor antigen complex and the time taken to recruit and activate the downstream enzymes. A longer half-life of a non-self antigen receptor complex could initiate downstream signaling by activating associated enzymes. Whereas, the short-lived, self-peptide receptor complex disassembles before the downstream enzymes are activated. Activation of TCR rewires the cellular metabolic response to aerobic glycolysis from oxidative phosphorylation. How does the early event in the TCR signaling cross-talk with the cellular metabolism is an open question. In this review, we have discussed the recent developments in understanding the regulation of TCR signaling, and then we reviewed the emerging role of metabolism in regulating T cell function. The cell-mediated immunity is carried out by a repertoire of clonally diverse T lymphocytes that display unique antigen-binding receptors on the cell surface called T-cell receptors (TCRs). The TCR binds to the foreign antigen, presented as peptide-major histocompatibility complex (pMHC) through antigen-presenting cells (APCs) with remarkable selectivity and sensitivity  1–4]. T. T1–4]. Central to the TCR response lies a delicate balance that helps discriminate between ‘self’ versus ‘non-self’ peptides while maintaining high sensitivity against small amounts of non-self antigen (agonist). TCR does not possess intrinsic catalytic activity; it responds to antigen binding by recruiting several enzymes and adaptor proteins following a mechanism that may have evolved 500 million years ago in jawed fish . Early T2+ signaling  that together provide an extracellular ligand-binding domain and intracellular segment for recruiting enzymes and adaptor proteins  7,8,43–. The extignaling . 84,85],852+ sigignaling  86–88]..2+ signaT cells are sensitive to a very low abundance of agonist present in a large amount of self-peptide mixture. Experimentally, 60–200 molecules of the specific pMHC are sufficient for generating T-cell response ,90. T ceTwo theories explaining how a proofreading mechanism may regulate T-cell response to antigen binding were proposed in the mid-nineties ,103,104.2+ release , SH3 and SH2 domain, connected to a kinase domain followed by a short C-terminal tail . The aution loop  [135–140Apart from phosphorylation of Y192, Lck is also down-regulated by the phosphorylation of S59 in the UD domain ,144. S59A large intracellular concentration of Lck could phosphorylate the CD3 chain in the basal state, which may initiate TCR signaling even without binding to pMHC. The basal activity of Lck is regulated by a coordinated function of non-receptor tyrosine kinase Csk and the phosphatases (CD45 and SHP-1) ,149. WitUnder the basal condition, the residual activity of Lck could partially phosphorylate ITAMs, but is not enough for activation of ZAP-70. Complete phosphorylation of the ITAMs are required for TCR engagement, which depends on the half-life of the TCR–pMHC complex ,162. Theholo-tSH2 domain rearranges to a Y-shaped closed conformation exposing the Y315 and Y319 in the interdomain B to be phosphorylated [ZAP-70 is indispensable for propagating downstream TCR signaling. ZAP-70 is a Syk family kinase translated as a single polypeptide chain containing an N-terminal regulatory module connected through a flexible linker, interdomain-B, to the C-terminal catalytic module. The regulatory module comprises tandem repeats of two SH2 (tSH2) domains, N-SH2 and C-SH2, interconnected by a helical linker called interdomain-A . The kinorylated ,172–174.orylated ,175–178.apo- and holo-structure of the tSH2 domain revealed that the N-SH2-binding pocket formed at the interface of the tSH2 domains [Steady-state ligand-binding experiments suggest that the tSH2 domain binds to the doubly phosphorylated ITAM peptides in a biphasic manner . In the  domains ,179,182. domains , uncoupl domains ,184.Although partial phosphorylation of ITAMs allows ZAP-70 to localize on the membrane, it cannot initiate the T-cell signaling, indicating an added layer of proofreading preventing ZAP-70 activation. Mass spectrometry-based phosphoproteomics studies suggest that ZAP-70 does not activate or initiate T-cell signaling without binding to its ligand at TCR ,175,185.The LAT is an essential scaffold that coordinates early TCR signaling to downstream cellular responses in a phosphorylation-dependent manner. At the plasma membrane LAT and its binding partners colocalize into micrometer or submicrometer clusters . EliminaSeveral phosphorylation sites on LAT are vital for kinetic proofreading TCR response to antigen binding . Among tZAP-70 also phosphorylates several distal tyrosine residues on LAT  that facilitate the recruitment for the Grb2 family of proteins, Tec family tyrosine kinase interleukin 2 inducible T-cell kinase (Itk) and SLP-76 . The ada2+ response. At the TCR complex, Lck activates Itk by phosphorylating Y511 residue in the activation loop leading to autophosphorylation of Y180 on the SH3 domain [2+ influx to the cytoplasm. Increased cytoplasmic Ca2+ activates the calcineurin by removing the inhibitory interaction with the calmodulin . Phosphorylation of HK-II by Akt promotes localization of HK-II to the mitochondria leading to increase glycolytic flux and catabolism. Together, these observations suggest that both Erk and phosphatidylinositol 3-kinase (PI3K)-Akt pathways may synergistically regulate hexokinase-dependent glucose metabolism upon TCR activation (2+) is another signaling modulator downstream of TCR that regulates the metabolic network of T cells. For example, a defect in the store-operated Ca2+ entry (SOCE) pathway, which influences Ca2+ flux in T cells, inhibits the phosphorylation of Akt and nutrient sensing through the mTOR pathway [In a recent study, Jones and colleagues investigated the effect of TCR: antigen-binding affinity on the metabolic output in T cells ,219. Stutivation . Calcium pathway .2+ homeostasis, which reprograms glycolysis and suppresses T-cell function. Increased potassium ion down-regulates Akt activation through serine-threonine phosphatase PP2A-dependent manner, affecting the nutrient-sensing mTOR pathway and down-regulating glucose uptake [Elevated potassium ion concentration in the tumor microenvironment is also linked to Cae uptake . The rede uptake . Togethee uptake .Tight regulation of signaling pathways is imperative for an accurate immune response at the right time. The adaptive immune system evolved from a lymphoid cell-based systems in jawless vertebrates to a robust BCR-TCR-MHC immune system in jawed vertebrates ,228–230.Deregulation of crucial signaling modules in TCR signaling pathways  often as


This study examines the impact of negative workplace gossip (NWG) on employee political acts (PA) and the role of ego depletion (ED) as a mediator. We also examined the indirect impact of NWG on PA through ED controlled by emotional intelligence (EI). A three-wave time-lagged study (paper-pencil based) was performed with 277 employees from various private organisations in Islamabad, Pakistan. The current data were gathered in three phases to reduce common method bias. Study results indicate that NWG positively affects employees’ PA. The authors also found ED as a potential mediator in the association between NWG and PA. In addition, the results also indicate the indirect effect of NWG on targets’ PA via ED is reduced by targets’ EI, with the result that this connection is weak when targets’ EI is high. Because this research is limited to a single region of Pakistan, particularly Islamabad, its findings cannot be comprehensive. Future studies should use a larger sample size to accomplish the same study. Future studies may include more organisations  to conduct a comparative analysis of the public and private sectors. This article, based on the affective events theory (AET), argues that EI should be utilised to mitigate the effects of NWG. Along with our significant and relevant theoretical contributions, we provide novel insights into the body of knowledge on how managers may prevent or minimise such PA. The current study results support all direct and indirect hypothesised connections, with important implications for theory and practice. A review of the existing literature indicates that EI may be associated with a reduction in employees’ ED; however, EI has not been used as a moderator in mitigating the influence of NWG, ED, and PA in the past. First, our study expands the literature on proactive behaviour  to counter NWG by drawing on AET  and usinetition” . We may EI, “the ability to carry out accurate reasoning about emotions and the ability to use emotions and emotional knowledge to enhance thought” [thought” .EI is a state of individual capability that describes an emotional resource that may positively reflect and reduce the connection between NWG and ED . A frameThe AET was developed by , who alsAccording to AET, individuals actively want to restore their ego by demonstrating EI in the workplace. EI may be considered a distinctive variable of individual resources to back their ego . EI is aActs of politics in the workplace are purely self-serving activities that increase self-interest via influence over other Organisation members’ thinking, perception, or conduct ,47. EmplAdditionally, AET emphasises that people want to spend their time in political activities to heal from and combat emotional losses . As a reMoreover, prestige and professional ability are important workplace resources that help targets compete for Organisational benefits  and other career possibilities . Second,Hypothesis 1 (H1).Negative workplace gossip positively effect political acts.Affective events theory (AET) describes how feelings and emotions affect the performance of targets in their working life and the other tasks they perform. Employees’ reactions to events that occur at work are described in terms of internal variables, such as personality, emotions, and cognition. In reference , the autNWG tends to induce ED because it tends to devour the feelings and emotions of its targets. This may occur in a variety of ways. First, the targets’ moods and emotions will be disrupted because their time and attention will divert from their normal activities to the wake of examining that they are being targeted . HoweverHypothesis 2 (H2).Emotion depletion mediates the positive relationship between negative workplace gossip and political acts.Previous studies have shown that the target may be able to detect NWG, and how much of an impact it has depends on the target’s emotional state ,24,31. IAffective events theory  asserts Hypothesis 3 (H3).Emotional intelligence moderates the relationship between negative workplace gossip and emotion depletion such that the relationship is weaker for employees with high emotional intelligence than for those with low emotional intelligence.Our study so far has supported a theoretical framework in which ED mediates the effect of NWG on PA, and EI works as a moderator that weakens the impact of NWG on ED. Employees with low EI who are subjected to NWG may become politicized. A moderated mediation hypothesis, in which the EI of the targets mitigates the indirect effect of NWG on PA via ED makes sense as a result ,72. As pHypothesis 4 (H4).The indirect effect of negative workplace gossip on targets’ political acts via emotion depletion is moderated by targets’ emotional intelligence. This relationship is weak when the targets’ emotional intelligence is higher.The data for this research were collected via a three-wave time-lagged study, with a month between each data collection phase, which was completed in three waves. Using this technique, the potential of common method bias was reduced to the absolute minimum ,73. FiveAfter distributing questionnaires to 447 employees, we obtained 393 valid responses, resulting in an 87.91% response rate in the first stage. We followed up with the employees who had previously answered the second questionnaire, and 344 valid responses were received, for an overall response rate of 87.53% for the second round of the survey. The final questionnaire a month later, as part of stage three, employees who had replied to the previous stage. During stage three, we received a total of 277 complete and usable questionnaires, resulting in a response rate of 80.5%. A total of 277 employees responded, with 17.32% being female and 82.67% being male. A total of 4.69% had a high school diploma, 12.27% had an HSSC (higher secondary school certificate), 60.28% had a bachelor’s degree, and the remaining 22.74% with a Master’s degree or above. A total of 73.28% of those had between 1–7 years’ of experience. Only 2.16% of employees were over 25 years of age see .All of the variables were evaluated on a Likert scale from 1 to 5, with 1 being strongly disagree and 5 strongly agree.The three-item scale developed by  was usedWe assessed ED in participants by using five questions from the ,75 measuWe assessed acts using a four-item scale developed by , modifieWe utilised a 16-item version of the Wong and Law EI Scale (WLEIS) to assess EI, which included four dimensions: Self-emotion appraisal (SEA), Others’ emotion appraisal (OEA), use of emotion (UOE), Regulation of emotion (ROE) . A samplThe previous study’s findings  have shoThrough maximum likelihood estimation, CFA was performed to evaluate the discriminant validity of all variables using AMOS V. 23. As shown in p < 0.01) as well as with PA , and ED was significantly associated with PA .Data for all variables are shown in p < 0.001, Model 5). As a result, hypothesis 1 was confirmed. Hypothesis 2 suggests that ED mediates the link between NWG and PA. Based on the results, (1) a high level of NWG was linked to lower levels of ED , (2) depletion of one’s ego was shown to be positively associated with political behaviour , and (3) the positive effect of NWG on PA decreased after ED was placed . Thus, Hypothesis 2 is validated. Additionally, we determined the statistical significance of the indirect impact using [p < 0.05, Model 8). To make the moderating impact of EI more visible, this research computed two kinds of EI mean: one with a standard deviation and the other with a lower standard deviation, as suggested by [p < 0.001) than when it was high ; therefore, Hypothesis 4 is confirmed.Hierarchical multiple regression analysis was used to examine Hypotheses 1 and 2. ct using  PRODCLINested by . Figure ested by  bootstraThere are four distinct ways in which this study adds to the current body of knowledge. First, by emphasising the possibility for self-oriented actions, that is, PA, to evolve as a somewhat proactive reaction aimed at mitigating the adverse consequences of NWG, improves our understanding of negative workplace gossip in the workplace. Although previous research has studied the consequences of targets’ work-related behaviours, it has mainly focused on the influence of targets’ in-role and out-of-role actions in the workplace ,82. MoreOur findings emphasise the need for strong management and organisational reaction to NWG, which affects both individuals and organisations in. NWG has previously been shown to be a source of stress ,24. AccoConsequently, managers should be alert for indications of ED in their employees via frequent communication and monitoring of their actions. They should intervene as soon as possible to assist them in replenishing their resources and recovering from the ED . To incrThird, we conclude that our findings also suggest that the higher an employee’s degree of EI the more insignificant an impact NWG has on ED. Managers might consider adjusting their recruitment and training techniques to reflect this. Our findings indicate that Organisations would be wise to choose employees with high emotional stability and strength levels to minimise the effect of NWG on ED on their staff. While at the same time, managers should offer employees training to improve their EI to minimise ED and the likelihood of their being engaged in political activities.The first issue related to this research is that it relies on self-reporting by the participants, which is problematic since NWG, ED, EI, and PA are all subjective emotions. This measurement technique has the potential to cause CMB ,89. AlthAlmost all employees will be the target of negative workplace gossip at some time in their careers, leading them to suffer unfavourable consequences and pushing them to seek methods to rectify the situation before it becomes irreversible. Using AET, this study aims to show how NWG may drain one’s ego while simultaneously motivating one to participate in PA. Furthermore, by examining the moderating effect of EI, we can better understand the process’s model parameters. Line managers must be aware of NWG, minimise its incidence, and reduce its negative effects. This study contributes to the existing body of knowledge and stimulates new ways of thinking about the subject. To make the study design even better, we also circulated a 1 item questionnaire open-ended “Repeated a rumour or gossip about colleagues or manager at work” to express their experiences about their colleagues and immediate bosses to know hierarchy-wise gossip positions. We found that managers  were more involved in NWG than others 84 (30%) out of 277 see . Many emManagers and above are the most likely source of Gossip initiation. Their primary focus is to sustain their position and to keep themselves aware of their surroundings. Gossips work to assess the situation and develop guidelines for managing the departments and maintaining their jobs. We conclude, based on our results, that bosses should be nice and polite and interested in their employees’ well-being, but the kind of colleague with whom you gossip should be someone outside the workplace . When you breach that threshold, you not only create an uncomfortable atmosphere, but you may also create ED among employees, causing them to lose interest in their jobs and engage in political acts to counter.


Bombyx mori has a homolog of the human gene that encodes the 5′-nucleotidase (5′N) involved in purine metabolism. The B. mori gene, Bm5′N, is located upstream of other genes involved in UA metabolism in the silkworm. Disruption of Bm5′N via the CRISPR/Cas9 system resulted in decreased UA levels in the silkworm epidermis and caused a translucent skin phenotype. When Bm5′N mutant silkworms were fed with the uric acid precursor inosine, the UA levels in the epidermis increased significantly. Furthermore, the metabolomic and transcriptomic analyses of Bm5′N mutants indicated that loss of the Bm5′N affected purine metabolism and the ABC transport pathway. Taken together, these results suggest that the UA pathway is conserved between the silkworm and humans and that the Bm5′N gene plays a crucial role in the uric acid metabolism of the silkworm. Thus, the silkworm may be a suitable model for the study of UA metabolism pathways relevant to human disease.Uric acid (UA) is the end-product in the human purine metabolism pathway. The UA that accumulates in silkworm tissues is excreted as a nitrogen waste product. Here, we first validated that  In human and other higher primates, uric acid (UA) is the purine final degradation product. It is synthesized mainly by the liver and excreted by the kidneys ,2. Elevaurate oxidase (Uro) but also produce allantoin as the end product of purine metabolites, different from purine metabolism in human , as a DsRed selectable marker to identify transgenic plasmids expressing Bm5′N single guide RNAs (sgRNAs) , reported in our previous studies, used the (sgRNAs) . Details(sgRNAs) ,31. A MuBm5′N mutants (G2) with double fluorescence, which were used in subsequent experiments.The transgenic silkworm strain and the nos-Cas9 line were previously reported ,30. We iSgRNAs were designed according to the dual sgRNAs strategy, as previously described . SgRNAs Genomic DNA of mutated animals was extracted at the 5th instar larval stage using standard SDS lysis-phenol treatment after incubation with proteinase K, followed by RNase treatment and ethanol precipitation. Mutation events were detected by PCR amplification using gene-specific primers bound upstream or downstream from each target , and ampUA was extracted from various tissues at the fifth larval instar. The excised samples were washed in PBS buffer. After a 10-h drying process, samples were boiled in deionized water for 30 min. The absorbance of the resulting liquid at 510 nm was then measured with a C012-2-1 Uric Acid Kit (Nanjing Jiancheng Bioengineering Institute) using a multi-mode microplate reader (Varioskan Flash) .2 piece of a mulberry leaf to which 100 μL of 20 mg/mL inosine had been applied to each. Controls were fed with a leaf wiped with the same volume of water. Feeding was started from the first day of the fifth instar and continued for 4 days.The fifth-instar larvae were divided into three groups, each group has fifteen animals. Each larva was fed with a 5 cmv/v/v), and stored extracts at 4 °C until analysis. We controlled sample preparation quality with internal standards mix 1 (IS1) and internal standards mix 2 (IS2). IS1 contained d-, l-methionine , phenylalanine , d-, l-alanine , l-threonine , l-aspartic acid , d-, l-2-aminobutyric acid , l-arginine . IS2 was purchased from Avanti  and contains LPC, LPE, PI, PA, SM, cholesterol, CE, PC, PE, PG, PS, MG, DG, and TG. After homogenizing for 5 min using a TissueLyser (JXFSTPRP), we sonicated samples for 10 min followed by incubation at −20 °C for 1 h, centrifuged at 4 °C for 15 min at 25,000 rpm, and freeze-dried the supernatant. We then resuspended metabolites in 300 μL of 10% methanol and sonicated them at 4 °C for 10 min, followed by centrifugation for 20 min at 25,000 rpm. Supernatants were analyzed on a Waters 2D UPLC interfaced with a Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific). LC-MS experiments were performed by Beijing Genomics Institute, Shenzhen, China as previously described [To investigate metabolic changes, we used mass spectrometry imaging to detect compounds produced by Bm5′N mutants and WT animals . Six replicates of each tissue were analyzed. We used a ratio of 25 mg of each tissue to 800 μL pre-cooled extraction reagents  and mixOmics . To meast-test was used to evaluate the differences between WT and mutant individuals. Asterisks mark statistically significant differences.All experiments in this study were performed with at least three biological replicates, and error bars denote means ± SEM. A two-tailed Student’s Bm5′N was expressed in various tissues and that it was expressed at high levels in malpighian tubules and fat body  mutant in the silkworm [Bm5′N mutants compared to WT which can be seen in Bm5′N gene is essential for UA synthesis in the silkworm.In the silkworm, UA is synthesized in the fat body and stored in the epidermis . It is t animals A,B, indisilkworm . The meaBm5′N had a similar function in the silkworm. To investigate whether Bm5′N participates in UA precursor synthesis, we performed feeding experiments with inosine supplementation using fifth-instar larvae. We collected the epidermis for UA testing. Supplementation caused a significant increase in UA content in the epidermis of mutants  bases on the KEGG database  for the gene-editing experiment (ΔBmABCG5 larvae that had translucent integument (Bm5′N gene is an upstream gene in purine metabolism.The joint analysis showed that the purine metabolites are closely linked to the ABC transporter gene. Moreover, the transporter pathway is the main pathway enriched among DEGs A, and thn tubule B. Intereeviously ,50. To tperiment . As expetegument C. Taken Bm5′N gene is involved in UA metabolism in the silkworm. The 5′N enzyme hydrolyzes nucleoside monophosphates or deoxynucleotide monophosphates to nucleotides in humans [Bm5′N gene is expressed in many silkworm tissues with high levels in malpighian tubules and fat body. The malpighian tubules and fat body in insects are sites of purine metabolism and are considered equivalent to the human kidney and liver. Mutation of the Bm5′N gene caused high transparency of the larval integument, where UA accumulates, indicating that the Bm5′N gene is necessary for UA production. Recently, two silkworm genes, BmMoco and BmXDH, homologous to genes in humans, have been shown to encode proteins necessary for the syntheses of xanthine and hypoxanthine [Bm5′N acts upstream of BmMoco and BmXDH because the adverse effects of the deletion of Bm5′N on UA production are relieved by feeding larvae UA precursors inosine. This is consistent with the function of 5′N in the corresponding human metabolic pathway (In this study, we demonstrated that the n humans . In human humans . Our resxanthine ,52. Our  pathway .op exhibited translucent larval integument, mortality, and male infertility [Bm5′N gene is responsible for the op locus [Bm5′N mutants were lethal at the pupal stage. We thought that the gene-knockdown effects work better in the binary transgenic CRISPR/Cas9 system.The natural mutant ertility . The Bm5op locus . We founBm5′N deletion. The additional metabolites identified in the present study will further enhance our understanding of uric acid metabolism in the silkworm.Alanine, aspartate, glutamate metabolism, citrate cycle metabolism, unsaturated lipid metabolism, and other metabolic pathways were disrupted in hyperuricemia and gout patients ,23. In oBm5′N mutant. Our RNA-seq results indicate that ABC family transporter gene expression is disrupted by the loss of Bm5′N. The ABC family transporters are involved in human urate excretion [w-3, od, oa, and otm silkworm mutants, but the underlying mechanism is unknown [ABG2, ABG3, ABG4, ABG5, ABG8, and Bmwh3 were significantly down-regulated in ΔBm5′N. Among these down regulated genes, we obtained ΔBmABCG5 larval that had translucent integument. Interestingly, Bmwh3 had translucent integument that had been demonstrated previously [Our analyses revealed that the UA synthesis pathway is conserved between humans and silkworms. However, patients with hypouricemia and gout may also have abnormalities in uric acid transport and secretion in kidney and kidney tubules ,55,56. Txcretion ,25. Our  unknown . The expeviously ,50. ThesOur data indicate that UA metabolism and UA excretion systems are similar in humans and silkworms. Nevertheless, for a deeper understanding of the complete function of the UA metabolism, it is vital to investigate gene functions along with the entire UA metabolic network in the silkworm. Such research will shed light on the roles of homologous genes in humans with metabolic conditions as hyperuricemia and gout and on the effects of drugs used to treat these conditions, such as the xanthine dehydrogenase inhibitor drug allopurinol, which is widely used to prevent UA production. Considering its short life cycle, ease of mass rearing, and the availability of a genetic tool kit, further investigation of the UA metabolism genes in the silkworm could provide a powerful model to study UA metabolism and UA-associated disease in humans ,58,59.