Current Status of Fertility Preservation in Pediatric Oncology Patients.

Cancer poses significant emotional challenges for children and adolescents, despite improvements in survival rates due to new therapies. However, there is growing concern about the long-term effects, including fertility issues. This review examines recent advancements and future directions in fertility preservation within a pediatric population subjected to oncological therapies. Worldwide, there is variability in the availability of fertility preservation methods, influenced by factors like development status and governmental support. The decision to pursue preservation depends on the risk of gonadotoxicity, alongside factors such as diagnosis, treatment, clinical status, and prognosis. Currently, options for preserving fertility in prepubertal boys are limited compared to girls, who increasingly have access to ovarian tissue preservation. Adolescents and adults have more options available, but ethical considerations remain complex and diverse.

LA-GEM: imputation of gene expression with incorporation of Local Ancestry.

Gene imputation and TWAS have become a staple in the genomics medicine discovery space; helping to identify genes whose regulation effects may contribute to disease susceptibility. However, the cohorts on which these methods are built are overwhelmingly of European Ancestry. This means that the unique regulatory variation that exist in non-European populations, specifically African Ancestry populations, may not be included in the current models. Moreover, African Americans are an admixed population, with a mix of European and African segments within their genome. No gene imputation model thus far has incorporated the effect of local ancestry (LA) on gene expression imputation. As such, we created LA-GEM which was trained and tested on a cohort of 60 African American hepatocyte primary cultures. Uniquely, LA-GEM include local ancestry inference in its prediction of gene expression. We compared the performance of LA-GEM to PrediXcan trained the same dataset (with no inclusion of local ancestry) We were able to reliably predict the expression of 2559 genes (1326 in LA-GEM and 1236 in PrediXcan). Of these, 546 genes were unique to LA-GEM, including the CYP3A5 gene which is critical to drug metabolism. We conducted TWAS analysis on two African American clinical cohorts with pharmacogenomics phenotypic information to identity novel gene associations. In our IWPC warfarin cohort, we identified 17 transcriptome-wide significant hits. No gene reached are prespecified significance level in the clopidogrel cohort. We did see suggestive association with RAS3A to P2RY12 Reactivity Units (PRU), a clinical measure of response to anti-platelet therapy. This method demonstrated the need for the incorporation of LA into study in admixed populations.

Investigation of genomic and transcriptomic risk factors in clopidogrel response in African Americans.

Clopidogrel, an anti-platelet drug, used to prevent thrombosis after percutaneous coronary intervention. Clopidogrel resistance results in recurring ischemic episodes, with African Americans suffering disproportionately. The aim of this study was to identify biomarkers of clopidogrel resistance in African American patients. We conducted a genome-wide association study, including local ancestry adjustment, in 141 African Americans on clopidogrel to identify associations with high on-treatment platelet reactivity (HTPR). We validated genome-wide and suggestive hits in an independent cohort of African American clopidogrel patients (N = 823) from the Million Veteran's Program (MVP) along with in vitro functional follow up. We performed differential gene expression (DGE) analysis in whole blood with functional follow-up in MEG-01 cells. We identified rs7807369, within thrombospondin 7A (THSD7A), as significantly associated with increasing risk of HTPR (p = 4.56 × 10-9). Higher THSD7A expression was associated with HTPR in an independent gene expression cohort of clopidogrel treated patients (p = 0.004) and supported by increased gene expression on THSD7A in primary human endothelial cells carrying the risk haplotype. Two SNPs (rs1149515 and rs191786) were validated in the MVP cohort. DGE analysis identified an association with decreased LAIR1 expression to HTPR. LAIR1 knockdown in a MEG-01 cells resulted in increased expression of SYK and AKT1, suggesting an inhibitory role of LAIR1 in the Glycoprotein VI pathway. Notably, the CYP2C19 variants showed no association with clopidogrel response in the discovery or MVP cohorts. In summary, these finding suggest that other variants outside of CYP2C19 star alleles play an important role in clopidogrel response in African Americans.

Discovery of Ancestry-specific Variants Associated with Clopidogrel Response among Caribbean Hispanics.

Background: High on-treatment platelet reactivity (HTPR) with clopidogrel is predictive of ischemic events in adults with coronary artery disease. Despite strong data suggesting HTPR varies with ethnicity, including clinical and genetic variables, no genome-wide association study (GWAS) of clopidogrel response has been performed among Caribbean Hispanics. This study aimed to identify genetic predictors of HTPR in a cohort of Caribbean Hispanic cardiovascular patients from Puerto Rico. Methods: Local Ancestry inference (LAI) and traditional GWASs were performed on a cohort of 511 clopidogrel-treated patients, stratified based on their P2Y12 reaction units (PRU) into responders and non-responders (HTPR). Results: The LAI GWAS identified variants within the CYP2C19 region associated with HTPR, predominantly driven by individuals of European ancestry and absent in those with native ancestry. Incorporating local ancestry adjustment notably enhanced our ability to detect associations. While no loci reached traditional GWAS significance, three variants showed suggestive significance at chromosomes 3, 14 and 22 (OSBPL10 rs1376606, DERL3 rs5030613, and RGS6 rs9323567). In addition, a variant in the UNC5C gene on chromosome 4 was associated with an increased risk of HTPR. These findings were not identified in other cohorts, highlighting the unique genetic landscape of Caribbean Hispanics. Conclusion: This is the first GWAS of clopidogrel response in Hispanics, confirming the relevance of the CYP2C19 cluster, particularly among those with European ancestry, and also identifying novel markers in a diverse patient population. Further studies are warranted to replicate our findings in other diverse cohorts and meta-analyses.

Spontaneously ruptured hepatocellular carcinoma on non-cirrhotic liver: A prospective case series.

BACKGROUND AND AIMS: Spontaneous ruptured hepatocellular carcinoma is an uncommon complication, and there are scarce data about non-cirrhotic patients. Tumor treatment is not standardized and the risk of peritoneal dissemination is unclear. AIM: we analyzed the treatment and survival in patients with rHCC on non-cirrhotic liver. METHODS: One hundred and forty-one non-cirrhotic patients with hepatocellular carcinoma diagnosed by histology were included in a multicenter prospective registry (2018-2022). Seven of them (5%) presented with hemoperitoneum due to spontaneous rupture. RESULTS: Liver disease was associated in three patients (42.9%). A single nodule was detected in three cases (42.9%). One patient had vascular invasion and none extrahepatic spread. Initial hemostatic therapy and sequential treatment was individualized. Patients with single nodule were treated: resection (one case) with recurrence at 4 months treated with TACE and sorafenib. TACE/TAE followed by surgery (two cases) one in remission 43 months later, the other had liver recurrence at 18 months and was transplanted. Patients with multiple lesions were treated: TAE/emergency surgery and subsequent systemic therapy (two cases), one received lenvatinib (1-year survival) and the other sorafenib (5-month survival). TAE and surgery with subsequent systemic therapy (one case). Initial hemostatic surgery, dying on admission (one case). No patient developed intraperitoneal metastasis. All patients with multiple lesions died by tumor. The 3-year survival rate was 42.9%. CONCLUSIONS: Initial hemostasis was achieved in all patients by TAE/TACE or surgery. Subsequent treatment was individualized, based on tumor characteristics, regardless of rupture. Long-time remission could be achieved in single nodule patients.

Leveraging drug perturbation to reveal genetic regulators of hepatic gene expression in African Americans.

Expression quantitative locus (eQTL) studies have paved the way in identifying genetic variation impacting gene expression levels. African Americans (AAs) are disproportionately underrepresented in eQTL studies, resulting in a lack of power to identify population-specific regulatory variants especially related to drug response. Specific drugs are known to affect the biosynthesis of drug metabolism enzymes as well as other genes. We used drug perturbation in cultured primary hepatocytes derived from AAs to determine the effect of drug treatment on eQTL mapping and to identify the drug response eQTLs (reQTLs) that show altered effect size following drug treatment. Whole-genome genotyping (Illumina MEGA array) and RNA sequencing were performed on 60 primary hepatocyte cultures after treatment with six drugs (Rifampin, Phenytoin, Carbamazepine, Dexamethasone, Phenobarbital, and Omeprazole) and at baseline (no treatment). eQTLs were mapped by treatment and jointly with Meta-Tissue. We found varying transcriptional changes across different drug treatments and identified Nrf2 as a potential general transcriptional regulator. We jointly mapped eQTLs with gene expression data across all drug treatments and baseline, which increased our power to detect eQTLs by 2.7-fold. We also identified 2,988 reQTLs (eQTLs with altered effect size after drug treatment). reQTLs were more likely to overlap transcription factor binding sites, and we uncovered reQTLs for drug metabolizing genes such as CYP3A5. Our results provide insights into the genetic regulation of gene expression in hepatocytes through drug perturbation and provide insight into SNPs that effect the liver's ability to respond to transcription upregulation.

Genomewide Association Study Identifies Copy Number Variants Associated With Warfarin Dose Response and Risk of Venous Thromboembolism in African Americans.

The anticoagulant warfarin is commonly used to control and prevent thrombotic disorders, such as venous thromboembolism (VTE), which disproportionately afflicts African Americans. Despite the importance of copy number variants (CNVs), few studies have focused on characterizing and understanding their role in drug response and disease risk among African Americans. In this study, we conduct the first genome-wide analysis of CNVs to more comprehensively account for the contribution of genetic variation in warfarin dose requirement and VTE risk among African Americans. We used hidden Markov models to detect CNVs from high-throughput single-nucleotide polymorphism arrays for 340 African American participants in the International Warfarin Pharmacogenetics Consortium. We identified 11,570 CNVs resulting in 2,038 copy number variable regions (CNVRs) and found 3 CNVRs associated with warfarin dose requirement and 3 CNVRs associated with VTE risk in African Americans. CNVRs 1q31.2del and 6q14.1del were associated with increased warfarin dose requirement (ß = 11.18 and 4.94, respectively; Pemp  = < 0.002); CNVR 19p13.31del was associated with decreased warfarin dose requirement (ß = -1.41, Pemp  = 0.0004); CNVRs (2p22.1del and 5q35.1-q35.2del) were found to be associated with increased risk of VTE (odds ratios (ORs) = 1.88 and 14.9, respectively; Pemp ≤0.02); and CNVR 10q26.12del was associated with a decreased risk of VTE (OR = 0.6; Pemp  = 0.05). Modeling of the 10q26.12del in HepG2 cells revealed that this deletion results in decreased fibrinogen gene expression, decreased fibrinogen and WDR11 protein levels, and decreased secretion of fibrinogen into the extracellular matrix. We found robust evidence that CNVRs could contribute to warfarin dose requirement and risk of VTE in African Americans and for 10q26.3del describe a possible pathogenic mechanism.

The Role of Global and Local Ancestry on Clopidogrel Response in African Americans.

Pharmacogenomics has long lacked dedicated studies in African Americans, resulting in a lack of indepth data in this populations. The ACCOuNT consortium has collected a cohort of 167 African American patients on steady state clopidogrel with the goal of discovering population specific variation that may contribute to the response of this anti-platelet agent. Here we analyze the role of both global and local ancestry on the clinical phenotypes of P2Y12 reaction units (PRU) and high on-treatment platelet reactivity (HTPR) in this cohort. We found that local ancestry at the TSS of three genes, IRS-1, ABCB1 and KDR were nominally associated with PRU, and local ancestry-adjusted SNP association identified variants in ITGA2 associated to increased PRU. These finding help to explain the variability in drug response seen in African Americans, especially as few studies on genes outside of CYP2C19 has been conducted in this population.

Genetic association of primary nonresponse to anti-TNFα therapy in patients with inflammatory bowel disease.

OBJECTIVES: Primary nonresponse (PNR) to antitumor necrosis factor-α (TNFα) biologics is a serious concern in patients with inflammatory bowel disease (IBD). We aimed to identify the genetic variants associated with PNR. PATIENTS AND METHODS: Patients were recruited from outpatient GI clinics and PNR was determined using both clinical and endoscopic findings. A case-control genome-wide association study was performed in 589 IBD patients and associations were replicated in an independent cohort of 293 patients. Effect of the associated variant on gene expression and TNFα secretion was assessed by cell-based assays. Pleiotropic effects were investigated by Phenome-wide association study (PheWAS). RESULTS: We identified rs34767465 as associated with PNR to anti-TNFα therapy (odds ratio: 2.07, 95% CI, 1.46-2.94, P = 2.43 × 10-7, [replication odds ratio: 1.8, 95% CI, 1.04-3.16, P = 0.03]). rs34767465 is a multiple-tissue expression quantitative trait loci for FAM114A2. Using RNA-sequencing and protein quantification from HapMap lymphoblastoid cell lines (LCLs), we found a significant decrease in FAM114A2 mRNA and protein expression in both heterozygous and homozygous genotypes when compared to wild type LCLs. TNFα secretion was significantly higher in THP-1 cells [differentiated into macrophages] with FAM114A2 knockdown versus controls. Immunoblotting experiments showed that depletion of FAM114A2 impaired autophagy-related pathway genes suggesting autophagy-mediated TNFα secretion as a potential mechanism. PheWAS showed rs34767465 was associated with comorbid conditions found in IBD patients (derangement of joints [P = 3.7 × 10-4], pigmentary iris degeneration [P = 5.9 × 10-4], diverticulum of esophagus [P = 7 × 10-4]). CONCLUSIONS: We identified a variant rs34767465 associated with PNR to anti-TNFα biologics, which increases TNFα secretion through mechanism related to autophagy. rs34767465 may also explain the comorbidities associated with IBD.

Validez de constructo y fiabilidad de una escala de autopercepción de habilidades en investigación científica y estrategias de aprendizaje autónomo / Construction validity and reliability of a self-perceptionscale of skills in scientific research and self-learning strategies

OBJETIVO. El objetivo del estudio es determinar la validez de constructo y de fiabilidad de una escala para medir autopercepción de habilidades en investigación científica y estrategias de aprendizaje autónomo de estudiantes de la Carrera de Nutrición y Dietética de la Universidad Mayor de San Andrés, La Paz 2021. MATERIAL Y MÉTODO. Se llevó a cabo un estudio cuantitativo, psicométrico, de validación de instrumento documental, en 260 estudiantes universitarios, el tipo de muestreo fue probabilística de forma aleatoria. Se construyó la matriz de correlación y se realizó el análisis de variabilidad de los ítems, para la validez interna del instrumento fue mediante el análisis de fiabilidad, análisis factorial exploratorio y confirmatorio con el programa estadístico SPSS v18 y Excel 2016. RESULTADOS. Se validó un instrumento que mide autopercepción de habilidades en investigación científica y estrategias de aprendizaje autónomo. Consta de 52 ítems y 4 dominios o dimensiones, tiene 13 ítems asignados para cada dominio. La fiabilidad del instrumento ítem ­ total fue de 96% (Alfa de Cronbach = 0.96), la fiabilidad dominio total fue 92% (Alfa de Cronbach = 0.92). La fiabilidad dominio total fue también aceptable 87% (Alfa de Cronbach = 0.872). CONCLUSIONES. Por los resultados obtenidos está disponible un instrumento que cuenta con una adecuada validez de constructo y fiabilidad que justifica su uso y aplicación en el ámbito investigativo.

OBJECTIVE. To determine the construct validity and reliability of a scale to measure self-perception of skills in scientific research and autonomous learning strategies of students of the Nutrition and Dietetics Career at the Mayor de San Andrés University, La Paz 2021. MATERIAL AND METHOD. A quantitative, psychometric, documentary instrument validation study was carried out in 260 university students, the type of sampling was random probabilistic. The correlation matrix was built and the variability analysis of the items was carried out, for the internal validity of the instrument it was through the reliability analysis, exploratory and confirmatory factor analysis with the statistical program SPSS v18 and Excel 2016. RESULTS. An instrument that measures self-perception of skills in scientific research and autonomous learning strategies was validated. It consists of 52 items and 4 domains or dimensions; it has 13 items assigned for each domain. The reliability of the item-total instrument was 96% (Cronbach's Alpha = 0.96), the total domain reliability was 92% (Cronbach's Alpha = 0.92). Total domain reliability was also acceptable 87% (Cronbach's Alpha = 0.872). CONCLUSIONS. Due to the results obtained, an instrument is available that has adequate construct validity and reliability that justifies its use and application in the research field.

Paradigma transcomplejo: aventura intelectual necesaria en la residencia médica de medicina física y rehabilitación / Transcomplex paradigm: necessary intellectual adventure in the medical residence of physical medicine and rehabilitation

OBJETIVO. Reflexionar sobre la importancia del paradigma transcomplejo en la formación de los residentes de la especialidad de Medicina Física y Rehabilitación que permita comprender la realidad compleja y diversa que caracteriza la discapacidad. METODO. Revisión teórica de carácter exploratorio y descriptivo sobre la aplicabilidad del paradigma transcomplejo en la formación de los especialistas en Medicina Física y Rehabilitación. Las estrategias de búsqueda se realizaron de la literatura científica publicado en español e inglés en bases de datos como Medline, Lilacs, Scielo, Cochrane, Pubmed y Google Scholar, Dialnet. DESARROLLO Y DISUSION. La trascendencia de la transcomplejidad evidencia la necesidad de la coexistencia con los paradigmas preexistentes con visión integrada complementaria, favoreciendo así el conocimiento científico y la reflexión filosófica. Las patologías emergentes junto con la revolución de nuestra especie en todos sus ámbitos también llaman a un cambio dentro los paradigmas. CONCLUSIONES. El arte de la transcomplejidad hace que la investigación salte al mundo multidimensional, mismo que es ineludible frente a la necesidad de la complementariedad dentro de los trabajos de investigación y educación de los residentes en Medicina Física y Rehabilitación.

OBJECTIVE. To reflect on the importance of the transcomplex paradigm in the training of residents of the specialty of Physical Medicine and Rehabilitation that allows understanding the complex and diverse reality that characterizes disability. METHOD. Theorretical review of an exploratory and descriptive nature on the applicability of the transcomplex paradigm in the training of specialists in Physical Medicine and Rehabilitation. The search strategies were carried out from the scientific literature published in Spanish and English in databases such as Medline, Lilacs, Scielo, Cochrane, Pubmed, Google Scholar and Dialnet. DEVELOPMENT AND DISCUSSIO. The significance of transcomplexity shows the need for coexistence with preexisting paradigms with a complementary integrated vision, thus favoring scientific knowledge and philosophical reflection. The emerging pathologies together with the revolution of our species in all its fields also calls for a change within the paradigms. CONCLUSIONS. The art of transcomplexity makes research jump into the multidimensional world, which is necessary in view of the need for complementarity within the research and education of residents in Physical Medicine and Rehabilitation.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

Incorporation of DNA methylation into eQTL mapping in African Americans.

Epigenetics is a reversible molecular mechanism that plays a critical role in many developmental, adaptive, and disease processes. DNA methylation has been shown to regulate gene expression and the advent of high throughput technologies has made genome-wide DNA methylation analysis possible. We investigated the effect of DNA methylation on eQTL mapping (methylation-adjusted eQTLs), by incorporating DNA methylation as a SNP-based covariate in eQTL mapping in African American derived hepatocytes. We found that the addition of DNA methylation uncovered new eQTLs and eGenes. Previously discovered eQTLs were significantly altered by the addition of DNA methylation data suggesting that methylation may modulate the association of SNPs to gene expression. We found that methylation-adjusted eQTLs that were less significant compared to PC-adjusted eQTLs were enriched in lipoprotein measurements (FDR=0.0040), immune system disorders (FDR = 0.0042), and liver enzyme measurements (FDR=0.047), suggesting that DNA methylation modulates the genetic regulation of these phenotypes. Our methylation-adjusted eQTL analysis also uncovered novel SNP-gene pairs. For example, we found that the SNP, rs1332018, was associated to GSTM3. GSTM3 expression has been linked to Hepatitis B which African Americans suffer from disproportionately. Our methylation-adjusted method adds new understanding to the genetic basis of complex diseases that disproportionally affect African Americans.

Discovery of novel hepatocyte eQTLs in African Americans.

African Americans (AAs) are disproportionately affected by metabolic diseases and adverse drug events, with limited publicly available genomic and transcriptomic data to advance the knowledge of the molecular underpinnings or genetic associations to these diseases or drug response phenotypes. To fill this gap, we obtained 60 primary hepatocyte cultures from AA liver donors for genome-wide mapping of expression quantitative trait loci (eQTL) using LAMatrix. We identified 277 eGenes and 19,770 eQTLs, of which 67 eGenes and 7,415 eQTLs are not observed in the Genotype-Tissue Expression Project (GTEx) liver eQTL analysis. Of the eGenes found in GTEx only 25 share the same lead eQTL. These AA-specific eQTLs are less correlated to GTEx eQTLs. in effect sizes and have larger Fst values compared to eQTLs found in both cohorts (overlapping eQTLs). We assessed the overlap between GWAS variants and their tagging variants with AA hepatocyte eQTLs and demonstrated that AA hepatocyte eQTLs can decrease the number of potential causal variants at GWAS loci. Additionally, we identified 75,002 exon QTLs of which 48.8% are not eQTLs in AA hepatocytes. Our analysis provides the first comprehensive characterization of AA hepatocyte eQTLs and highlights the unique discoveries that are made possible due to the increased genetic diversity within the African ancestry genome.

Characterization of Signaling Pathways Associated with Pancreatic ß-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice.

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic ß-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, ß-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic ß-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, ß-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the ß-cell's adaptive flexibility and indicates that therapeutic approaches applied to encourage ß-cell rest are capable of restoring endogenous ß-cell function. However, mechanisms that regulate ß-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying ß-cell adaptive flexibility in db/db ß-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with ß-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking-core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of ß-cell function, providing improved insight into disease pathogenesis of T2D.

Proteomic Analysis of Restored Insulin Production and Trafficking in Obese Diabetic Mouse Pancreatic Islets Following Euglycemia.

For the treatment of patients with prediabetes or diabetes, clinical evidence has emerged that ß-cell function can be restored by glucose-lowering therapeutic strategies. However, little is known about the molecular mechanisms underlying this functional adaptive behavior of the pancreatic ß-cell. This study examines the dynamic changes in protein expression and phosphorylation state associated with (pro)insulin production and secretory pathway function mediated by euglycemia to induce ß-cell rest in obese/diabetic db/db islet ß-cells. Unbiased quantitative profiling of the protein expression and phosphorylation events that occur upon ß-cell adaption during the transition from hyperglycemia to euglycemia was assessed in isolated pancreatic islets from obese diabetic db/db and wild-type (WT) mice using quantitative proteomics and phosphoproteomics together with bioinformatics analysis. Dynamic changes in the expression and phosphorylation of proteins associated with pancreatic ß-cell (pro)insulin production and complementary regulated-secretory pathway regulation were observed in obese diabetic db/db islets in a hyperglycemic environment, relative to WT mouse islets in a normal euglycemic environment, that resolved when isolated db/db islets were exposed to euglycemia for 12 h in vitro. By similarly treating WT islets in parallel, the effects of tissue culture could be mostly eliminated and only those changes associated with resolution by euglycemia were assessed. Among such regulated protein phosphorylation-dependent signaling events were those associated with COPII-coated vesicle-dependent ER exit, ER-to-Golgi trafficking, clathrin-coat disassembly, and a particular association for the luminal Golgi protein kinase, FAM20C, in control of distal secretory pathway trafficking, sorting, and granule biogenesis. Protein expression and especially phosphorylation play key roles in the regulation of (pro)insulin production, correlative secretory pathway trafficking, and the restoration of ß-cell secretory capacity in the adaptive functional ß-cell response to metabolic demand, especially that mediated by glucose.

Association of Genetic Variants With Warfarin-Associated Bleeding Among Patients of African Descent.

Importance: Major warfarin-related bleeding occurs more frequently in African Americans than in other populations. Identification of potential genetic factors related to this adverse event may help identify at-risk patients. Objective: To identify genetic factors associated with warfarin-related bleeding in patients of African descent at an international normalized ratio (INR) of less than 4. Design, Setting, and Participants: A case-control genome-wide association study involving patients of African descent taking warfarin was conducted in a discovery cohort (University of Chicago [2009-2011] and the University of Illinois at Chicago [2002-2011]), and associations were confirmed in a replication cohort (University of Chicago [2015-2016]). Potential population stratification was examined in the discovery cohort by principal component analysis. Odds ratios (ORs) and 95% CIs were computed for bleeding risk by logistic regression analysis. Summary statistics from the discovery and the replication cohorts were analyzed with a fixed effects meta-analysis. The potential influence of single-nucleotide polymorphisms (SNPs) on gene expression was studied by luciferase expression assays. Exposures: Single-nucleotide polymorphisms associated with warfarin-related bleeding. Main Outcomes and Measures: Major bleeding-defined as bleeding requiring hospitalization, causing a decrease in hemoglobin level of more than 2 g/dL, requiring blood transfusion, or any combination of the 3-while taking warfarin at an INR of less than 4. Results: The discovery cohort consisted of 31 cases (mean age, 60.1 years [SD, 14.9 years], 26 women [83.9%]) and 184 warfarin-treated controls (mean age, 57.1 years [SD, 15.7 years]) with no documented bleeding. The replication cohort consisted of 40 cases (mean age, 55.6 years [SD, 17.3 years], 27 women [67.5%]), and 148 warfarin-treated controls (mean age, 55.4 years [SD, 17.1 years]; 98 women [66.2%]) with no documented bleeding. In the discovery cohort, 4 SNPs in linkage disequilibrium on chromosome 6 (rs115112393, rs16871327, rs78132896, and rs114504854) were associated with warfarin-related bleeding but did not reach genome-wide significance. The SNP rs78132896 occurred in 11 cases (35.5%) and 9 controls (4.9%) in the discovery cohort (OR, 8.31; 95% CI, 3.2-21.5; P < 6.21 × 10-8), and the association was confirmed in the replication cohort (the SNP was present in 14 cases [35.0%] and 7 controls [4.8%]; OR, 8.24; 95% CI, 3.1-25.3, P = 5.64 × 10-5). Genome-wide significance of this SNP was achieved when the cohorts were combined via meta-analysis (OR, 8.27; 95% CI, 4.18-16.38; P = 2.05 × 10-11). These SNPs are found only in people of African descent. In vitro luciferase expression assays demonstrated that rs16871327 (enhancer SNP) and rs78132896 (promoter SNP) risk alleles together increased EPHA7 gene (Entrez Gene 2045) transcription by a mean of 14.95 (SD, 1.7) compared with wild-type alleles (mean, 9.56 [SD, 0.84]; difference, 5.39; 95% CI, 4.1-6.6; P < .001). Conclusions and Relevance: In this preliminary study involving patients of African descent taking warfarin, 4 single-nucleotide polymorphisms in linkage disequilibrium on chromosome 6 were associated with an increased risk of major bleeding at INR of less than 4. Validation of these findings in an independent prospective cohort is required.

ß-Cell Control of Insulin Production During Starvation-Refeeding in Male Rats.

Mammalian metabolism has evolved to adapt to changes in nutrient status. Insulin, the key anabolic hormone, facilitates intracellular storage of nutrient fuels and plays a pivotal role in the transition away from catabolism upon refeeding. Although circulating insulin relative to nutrient levels has been well characterized during fasting and refeeding, how pancreatic ß-cell biology caters to acute changes in insulin demand has not been sufficiently addressed. Here, we examined the dynamics of (pro)insulin production and associated changes in ß-cell ultrastructure during refeeding after a 72-hour fast in male rats. We found that fasted ß-cells had marked degranulation, which inversely coordinated with the upregulation of autophagolysomal and lysosomal organelles. There was also expanded Golgi that correlated with enhanced (pro)insulin biosynthetic capacity but, conversely, blunted in vivo insulin secretion. Within 4 to 6 hours of refeeding, proinsulin biosynthesis, cellular ultrastructure, in vivo insulin secretion, and glucose tolerance normalized to levels near those of fed control animals, indicating a rapid replenishment of normal insulin secretory capacity. Thus, during a prolonged fast, the ß-cell protects against hypoglycemia by markedly reducing insulin secretory capacity in vivo but is simultaneously poised to efficiently increase (pro)insulin production upon refeeding to effectively return normal insulin secretory capacity within hours.

Pancreatic ß-Cell Adaptive Plasticity in Obesity Increases Insulin Production but Adversely Affects Secretory Function.

Pancreatic ß-cells normally produce adequate insulin to control glucose homeostasis, but in obesity-related diabetes, there is a presumed deficit in insulin production and secretory capacity. In this study, insulin production was assessed directly in obese diabetic mouse models, and proinsulin biosynthesis was found to be contrastingly increased, coupled with a significant expansion of the rough endoplasmic reticulum (without endoplasmic reticulum stress) and Golgi apparatus, increased vesicular trafficking, and a depletion of mature ß-granules. As such, ß-cells have a remarkable capacity to produce substantial quantities of insulin in obesity, which are then made available for immediate secretion to meet increased metabolic demand, but this comes at the price of insulin secretory dysfunction. Notwithstanding, it can be restored. Upon exposing isolated pancreatic islets of obese mice to normal glucose concentrations, ß-cells revert back to their typical morphology with restoration of regulated insulin secretion. These data demonstrate an unrealized dynamic adaptive plasticity of pancreatic ß-cells and underscore the rationale for transient ß-cell rest as a treatment strategy for obesity-linked diabetes.

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets.

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a 'model' monomethyladenine, it was found that 3-MA augmented [cAMP]i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3'K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]i and then potentiate glucose-induced insulin secretion and production in parallel.