Dysregulation of long non-coding RNA gene expression pathways in monocytes of type 2 diabetes patients with cardiovascular disease.

BACKGROUND: Monocytes play a central role in the pathophysiology of cardiovascular complications in type 2 diabetes (T2D) patients through different mechanisms. We investigated diabetes-induced changes in lncRNA genes from T2D patients with cardiovascular disease (CVD), long-duration diabetes, and poor glycemic control. METHODS: We performed paired-end RNA sequencing of monocytes from 37 non-diabetes controls and 120 patients with T2D, of whom 86 had either macro or microvascular disease or both. Monocytes were sorted from peripheral blood using flow cytometry; their RNA was purified and sequenced. Alignments and gene counts were obtained with STAR to reference GRCh38 using Gencode (v41) annotations followed by batch correction with CombatSeq. Differential expression analysis was performed with EdgeR and pathway analysis with IPA software focusing on differentially expressed genes (DEGs) with a p-value < 0.05. Additionally, differential co-expression analysis was done with csdR to identify lncRNAs highly associated with diabetes-related expression networks with network centrality scores computed with Igraph and network visualization with Cytoscape. RESULTS: Comparing T2D vs. non-T2D, we found two significantly upregulated lncRNAs (ENSG00000287255, FDR = 0.017 and ENSG00000289424, FDR = 0.048) and one significantly downregulated lncRNA (ENSG00000276603, FDR = 0.017). Pathway analysis on DEGs revealed networks affecting cellular movement, growth, and development. Co-expression analysis revealed ENSG00000225822 (UBXN7-AS1) as the highest-scoring diabetes network-associated lncRNA. Analysis within T2D patients and CVD revealed one lncRNA upregulated in monocytes from patients with microvascular disease without clinically documented macrovascular disease. (ENSG00000261654, FDR = 0.046). Pathway analysis revealed DEGs involved in networks affecting metabolic and cardiovascular pathologies. Co-expression analysis identified lncRNAs strongly associated with diabetes networks, including ENSG0000028654, ENSG00000261326 (LINC01355), ENSG00000260135 (MMP2-AS1), ENSG00000262097, and ENSG00000241560 (ZBTB20-AS1) when we combined the results from all patients with CVD. Similarly, we identified from co-expression analysis of diabetes patients with a duration ≥ 10 years vs. <10 years two lncRNAs: ENSG00000269019 (HOMER3-AS10) and ENSG00000212719 (LINC02693). The comparison of patients with good vs. poor glycemic control also identified two lncRNAs: ENSG00000245164 (LINC00861) and ENSG00000286313. CONCLUSION: We identified dysregulated diabetes-related genes and pathways in monocytes of diabetes patients with cardiovascular complications, including lncRNA genes of unknown function strongly associated with networks of known diabetes genes.

Protein sectors: evolutionary units of three-dimensional structure.

Proteins display a hierarchy of structural features at primary, secondary, tertiary, and higher-order levels, an organization that guides our current understanding of their biological properties and evolutionary origins. Here, we reveal a structural organization distinct from this traditional hierarchy by statistical analysis of correlated evolution between amino acids. Applied to the S1A serine proteases, the analysis indicates a decomposition of the protein into three quasi-independent groups of correlated amino acids that we term "protein sectors." Each sector is physically connected in the tertiary structure, has a distinct functional role, and constitutes an independent mode of sequence divergence in the protein family. Functionally relevant sectors are evident in other protein families as well, suggesting that they may be general features of proteins. We propose that sectors represent a structural organization of proteins that reflects their evolutionary histories.

Genetic predisposition to cancer across people of different ancestries in Qatar: a population-based, cohort study.

BACKGROUND: Disparities in the genetic risk of cancer among various ancestry groups and populations remain poorly defined. This challenge is even more acute for Middle Eastern populations, where the paucity of genomic data could affect the clinical potential of cancer genetic risk profiling. We used data from the phase 1 cohort of the Qatar Genome Programme to investigate genetic variation in cancer-susceptibility genes in the Qatari population. METHODS: The Qatar Genome Programme generated high-coverage genome sequencing on DNA samples collected from 6142 native Qataris, stratified into six distinct ancestry groups: general Arab, Persian, Arabian Peninsula, Admixture Arab, African, and South Asian. In this population-based, cohort study, we evaluated the performance of polygenic risk scores for the most common cancers in Qatar (breast, prostate, and colorectal cancers). Polygenic risk scores were trained in The Cancer Genome Atlas (TCGA) dataset, and their distributions were subsequently applied to the six different genetic ancestry groups of the Qatari population. Rare deleterious variants within 1218 cancer susceptibility genes were analysed, and their clinical pathogenicity was assessed by ClinVar and the CharGer computational tools. FINDINGS: The cohort included in this study was recruited by the Qatar Biobank between Dec 11, 2012, and June 9, 2016. The initial dataset comprised 6218 cohort participants, and whole genome sequencing quality control filtering led to a final dataset of 6142 samples. Polygenic risk score analyses of the most common cancers in Qatar showed significant differences between the six ancestry groups (p<0·0001). Qataris with Arabian Peninsula ancestry showed the lowest polygenic risk score mean for colorectal cancer (-0·41), and those of African ancestry showed the highest average for prostate cancer (0·85). Cancer-gene rare variant analysis identified 76 Qataris (1·2% of 6142 individuals in the Qatar Genome Programme cohort) carrying ClinVar pathogenic or likely pathogenic variants in clinically actionable cancer genes. Variant analysis using CharGer identified 195 individuals carriers (3·17% of the cohort). Breast cancer pathogenic variants were over-represented in Qataris of Persian origin (22 [56·4%] of 39 BRCA1/BRCA2 variant carriers) and completely absent in those of Arabian Peninsula origin. INTERPRETATION: We observed a high degree of heterogeneity for cancer predisposition genes and polygenic risk scores across ancestries in this population from Qatar. Stratification systems could be considered for the implementation of national cancer preventive medicine programmes. FUNDING: Qatar Foundation.

Discovery of new therapeutic targets in ovarian cancer through identifying significantly non-mutated genes.

BACKGROUND: Mutated and non-mutated genes interact to drive cancer growth and metastasis. While research has focused on understanding the impact of mutated genes on cancer biology, understanding non-mutated genes that are essential to tumor development could lead to new therapeutic strategies. The recent advent of high-throughput whole genome sequencing being applied to many different samples has made it possible to calculate if genes are significantly non-mutated in a specific cancer patient cohort. METHODS: We carried out random mutagenesis simulations of the human genome approximating the regions sequenced in the publicly available Cancer Growth Atlas Project for ovarian cancer (TCGA-OV). Simulated mutations were compared to the observed mutations in the TCGA-OV cohort and genes with the largest deviations from simulation were identified. Pathway analysis was performed on the non-mutated genes to better understand their biological function. We then compared gene expression, methylation and copy number distributions of non-mutated and mutated genes in cell lines and patient data from the TCGA-OV project. To directly test if non-mutated genes can affect cell proliferation, we carried out proof-of-concept RNAi silencing experiments of a panel of nine selected non-mutated genes in three ovarian cancer cell lines and one primary ovarian epithelial cell line. RESULTS: We identified a set of genes that were mutated less than expected (non-mutated genes) and mutated more than expected (mutated genes). Pathway analysis revealed that non-mutated genes interact in cancer associated pathways. We found that non-mutated genes are expressed significantly more than mutated genes while also having lower methylation and higher copy number states indicating that they could be functionally important. RNAi silencing of the panel of non-mutated genes resulted in a greater significant reduction of cell viability in the cancer cell lines than in the non-cancer cell line. Finally, as a test case, silencing ANKLE2, a significantly non-mutated gene, affected the morphology, reduced migration, and increased the chemotherapeutic response of SKOV3 cells. CONCLUSION: We show that we can identify significantly non-mutated genes in a large ovarian cancer cohort that are well-expressed in patient and cell line data and whose RNAi-induced silencing reduces viability in three ovarian cancer cell lines. Targeting non-mutated genes that are important for tumor growth and metastasis is a promising approach to expand cancer therapeutic options.

MicroRNA Changes Up to 24 h following Induced Hypoglycemia in Type 2 Diabetes.

Hypoglycemia, as a complication of type 2 diabetes (T2D), causes increased morbidity and mortality but the physiological response underlying hypoglycemia has not been fully elucidated. Small noncoding microRNA (miRNA) have multiple downstream biological effects. This pilot exploratory study was undertaken to determine if induced miRNA changes would persist and contribute to effects seen 24 h post-hypoglycemia. A parallel, prospective study design was employed, involving T2D (n = 23) and control (n = 23) subjects. The subjects underwent insulin-induced hypoglycemia (2 mmol/L; 36 mg/dL); blood samples were drawn at baseline, upon the induction of hypoglycemia, and 4 h and 24 h post-hypoglycemia, with a quantitative polymerase chain reaction analysis of miRNA undertaken. The baseline miRNAs did not differ. In the controls, 15 miRNAs were downregulated and one was upregulated (FDR < 0.05) from the induction of hypoglycemia to 4 h later while, in T2D, only four miRNAs were altered (downregulated), and these were common to both cohorts (miR-191-5p; miR-143-3p; let-7b-5p; let-7g-5p), correlated with elevated glucagon levels, and all were associated with energy balance. From the induction of hypoglycemia to 24 h, 14 miRNAs were downregulated and 5 were upregulated (FDR < 0.05) in the controls; 7 miRNAs were downregulated and 7 upregulated (FDR < 0.05) in T2D; a total of 6 miRNAs were common between cohorts, 5 were downregulated (miR-93-5p, let-7b-5p, miR-191-5p, miR-185-5p, and miR-652-3p), and 1 was upregulated (miR-369-3p). An ingenuity pathway analysis indicated that many of the altered miRNAs were associated with metabolic and coagulation pathways; however, of the inflammatory proteins expressed, only miR-143-3p at 24 h correlated positively with tumor necrosis factor-α (TNFa; p < 0.05 and r = 0.46) and negatively with toll-like receptor-4 (TLR4; p < 0.05 and r = 0.43). The MiRNA levels altered by hypoglycemia reflected changes in counter-regulatory glucagon and differed between cohorts, and their expression at 24 h suggests miRNAs may potentiate and prolong the physiological response. Trial registration: ClinicalTrials.gov NCT03102801.

A de novo synonymous variant in EFTUD2 disrupts normal splicing and causes mandibulofacial dysostosis with microcephaly: case report.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM) is a rare autosomal dominant genetic disease characterized by intellectual and growth retardations, as well as major microcephaly, induced by missense and splice site variants or microdeletions in the EFTUD2 gene. CASE PRESENTATION: Here, we investigate the case of a young girl with symptoms of MFDM and a normal karyotype. Whole-exome sequencing of the family was performed to identify genetic alterations responsible for this phenotype. We identified a de novo synonymous variant in the EFTUD2 gene. We demonstrated that this synonymous variant disrupts the donor splice-site in intron 9 resulting in the skipping of exon 9 and a frameshift that leads to a premature stop codon. CONCLUSIONS: We present the first case of MFDM caused by a synonymous variant disrupting the donor splice site, leading to exon skipping.

Akt-activated endothelium promotes ovarian cancer proliferation through notch activation.

BACKGROUND: One main challenge in ovarian cancer rests on the presence of a relapse and an important metastatic disease, despite extensive surgical debulking and chemotherapy. The difficulty in containing metastatic cancer is partly due to the heterotypic interaction of tumor and its microenvironment. In this context, evidence suggests that endothelial cells (EC) play an important role in ovarian tumor growth and chemoresistance. Here, we studied the role of tumor endothelium on ovarian cancer cells (OCCs). METHODS: We evaluated the effect of activated endothelial cells on ovarian cancer cell proliferation and resistance to chemotherapy and investigated the survival pathways activated by endothelial co-culture. RESULTS: The co-culture between OCCs and E4+ECs, induced an increase of OCCs proliferation both in vitro and in vivo. This co-culture induced an increase of Notch receptors expression on OCC surface and an increase of Jagged 1 expression on E4+ECs surface and activation of survival pathways leading to chemoresistance by E4+ECs. CONCLUSION: The targeting of aberrant NOTCH signaling could constitute a strategy to disrupt the pro-tumoral endothelial niche.

Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer.

Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies.

Long-term Results After Oncoplastic Surgery for Breast Cancer: A 10-year Follow-up.

OBJECTIVE: The aim of this study was to evaluate the long-term oncologic outcome after oncoplastic surgery (OPS). BACKGROUND: OPS combines wide tumor excision with reduction mammoplasty techniques thus extending breast conserving surgery to large tumors that might else be proposed a mastectomy. Little data are available about the oncologic results for breast conserving surgery of these larger tumors. METHODS: From January 2004 until March 2016, a total of 350 oncoplastic breast reductions were prospectively entered into a database. Patients were included if their breast reshaping included a reduction mammoplasty with skin excision (Level 2 oncoplastic techniques). RESULTS: Histologic subtypes were: invasive ductal carcinoma in 219 cases (62.6%), ductal carcinoma in situ (DCIS) in 88 cases (25.1%), and invasive lobular carcinoma in 43 (12.3%) cases. Seventy-three of the invasive cancers (27.9%) received neoadjuvant chemotherapy. The mean resection weight was 177 grams. The mean pathological tumor size was 26 mm (range 0-180 mm) and varied from 23 mm (4-180 mm) for invasive cancers to 32 mm (0-100 mm) for DCIS. Specimen margins were involved in 12.6% of the cases; 10.5% of invasive ductal, 14.7% of DCIS, and 20.9% of invasive lobular. The overall breast conservation rate was 92% and varied from 87.4% for DCIS to 93.5% for the invasive cancers. Thirty-one patients (8.9%) developed one or more postoperative complications, inducing a delay in postoperative treatments in 4.6% of patients. The median follow up was 55 months. The cumulative 5-year incidences for local, regional, and distant recurrences were 2.2%, 1.1%, and 12.4%, respectively. CONCLUSIONS: Oncoplastic breast reductions allow wide resections with free margins and can be used for large cancers as an alternative to mastectomy.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition.

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Breast cancer cells promote a notch-dependent mesenchymal phenotype in endothelial cells participating to a pro-tumoral niche.

BACKGROUND: Endothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness. METHODS: Xenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition. RESULTS: Our results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECs(Mes)) in vitro. The ECs(Mes) acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECs(Mes) were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECs(Mes) were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFß and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFß down-regulated Smad1/5 phosphorylation and Jag1(KD) tumor cells were unable to initiate the process. CONCLUSIONS: Overall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFß pathways in the initiation of mesenchymal phenotype may propose new stromal targets.

Mesenchymal cell interaction with ovarian cancer cells induces a background dependent pro-metastatic transcriptomic profile.

BACKGROUND: The cross talk between the stroma and cancer cells plays a major role in phenotypic modulation. During peritoneal carcinomatosis ovarian cancer cells interact with mesenchymal stem cells (MSC) resulting in increased metastatic ability. Understanding the transcriptomic changes underlying the phenotypic modulation will allow identification of key genes to target. However in the context of personalized medicine we must consider inter and intra tumoral heterogeneity. In this study we used a pathway-based approach to illustrate the role of cell line background in transcriptomic modification during a cross talk with MSC. METHODS: We used two ovarian cancer cell lines as a surrogate for different ovarian cancer subtypes: OVCAR3 for an epithelial and SKOV3 for a mesenchymal subtype. We co-cultured them with MSCs. Genome wide gene expression was determined after cell sorting. Ingenuity pathway analysis was used to decipher the cell specific transcriptomic changes related to different pro-metastatic traits (Adherence, migration, invasion, proliferation and chemoresistance). RESULTS: We demonstrate that co-culture of ovarian cancer cells in direct cellular contact with MSCs induces broad transcriptomic changes related to enhance metastatic ability. Genes related to cellular adhesion, invasion, migration, proliferation and chemoresistance were enriched under these experimental conditions. Network analysis of differentially expressed genes clearly shows a cell type specific pattern. CONCLUSION: The contact with the mesenchymal niche increase metastatic initiation and expansion through cancer cells' transcriptome modification dependent of the cellular subtype. Personalized medicine strategy might benefit from network analysis revealing the subtype specific nodes to target to disrupt acquired pro-metastatic profile.

An investigation of endometrial vascularity in normally menstruating women and those in other physiological states using 3D ultrasound imaging.

OBJECTIVES: This study aimed to determine the normal vasculature indices of the endometrium and to correlate them with those in various physiological states. METHODS: Women undergoing ultrasound at the Feto-Maternal Center, Qatar in 2020-2021 as part of their gynecologic evaluation were enrolled into the study. They were divided into those with normal menses and no additional pathology, those following spontaneous miscarriage, postpartum and menopausal. Three-dimensional (3D) evaluation of the endometrial vasculature was done and the parameters quantified included vascularization index (VI), flow index (FI), vascularization flow index (VFI), endometrial thickness, endometrial volume and uterine volume. JASP, an open-source statistical analysis software, was used for analysis and an independent t-test to compare the vascularity indices. A multivariate regression analysis was also done to look at the factors affecting the endometrial vascular indices within the luteal phase. RESULTS: A total of 461 women were studied: 122 in the follicular phase, 199 in the luteal phase, 90 after a spontaneous miscarriage, 29 postpartum, and 16 menopausal. The vascularity indices were highest after miscarriage and lowest postnatally. There were no significant effects of age, gravida, para, or abortions on VI and VFI. However, there was a significant positive effect of age on FI (P = 0.019) There was a significant increase in endometrial volume and thickness in the luteal phase as compared to follicular phase (P < 0.01), but there was no difference in the vascularity indices. The uterine and endometrial volume in the postnatal group were nearly double that of the luteal group (P value <0.01 and 0.014, respectively). There was a significant decrease in flow index in the postnatal group compared to the luteal group (P < 0.01), suggesting low flow intensity in the postnatal group. CONCLUSIONS: Endometrial vascular indices measured using 3D Doppler can be used to determine normal vascular indices and vary with physiological states such as after miscarriages, postnatally and in the menopausal states.

Assessing the implications of sentinel lymph node removal in cervical cancer: an immunogenetic perspective - a SENTICOL ancillary study.

BACKGROUND: Cervical cancer's lymphatic spread primarily begins from the sentinel lymph nodes (SLNs), underlining their pivotal role in disease metastasis. However, these nodes' immune gene expression profiles and immunoregulation mechanisms have yet to be explored. METHODS: Our study aimed to elucidate the immune cell populations and their roles in the immune gene expression profile of negative SLNs compared with positive SLNs and non-SLNs using Nanostring RNA seq analysis. We performed a principal component analysis on the log2 normalized expression of 685 endogenous genes in the nCounter PanCancer Immune Profiling Panel, followed by an assessment of the differential expression of genes and immune cell type abundance. RESULTS: We found significant variations in gene expression among the groups, with negative SLNs displaying overexpression of genes related to tumor-infiltrating immune cells, specifically innate cell populations. They also demonstrated the upregulation of genes involved in antigen presentation and T-cell priming. In contrast, positive SLNs were enriched in regulatory networks, suggesting their potential role in immune evasion. A comparison of negative SLNs and non-SLNs revealed increased innate and adaptive immune cell types, underscoring the ongoing T cell response to tumor antigens. CONCLUSION: Our findings underscore a specific immunogenetic phenotype profile in negative SLNs, emphasizing their crucial role in the initial anticancer response, immunosurveillance, and the propagation of immune tolerance from the primary cervical tumor. These results highlight the potential of SLNs as a novel target for immunotherapy strategies and underscore the importance of new imaging methods for accurately identifying SLN status without removal. Future investigations are needed to understand further the immunological interplay within SLNs and their influence on cervical cancer progression.

Adaptation of a commonly used, chemically defined medium for human embryonic stem cells to stable isotope labeling with amino acids in cell culture.

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151.

MiRNA and associated inflammatory changes from baseline to hypoglycemia in type 2 diabetes.

Objective: Hypoglycemia in type 2 diabetes (T2D) increases morbidity and mortality but the underlying physiological response is still not fully understood, though physiological changes are still apparent 24 hours after the event. Small noncoding microRNA (miRNA) have multiple downstream biological effects that may respond rapidly to stress. We hypothesized that hypoglycemia would induce rapid miRNA changes; therefore, this pilot exploratory study was undertaken. Methods: A pilot prospective, parallel study in T2D (n=23) and controls (n=23). Insulin-induced hypoglycemia (2mmol/l: 36mg/dl) was induced and blood sampling performed at baseline and hypoglycemia. Initial profiling of miRNA was undertaken on pooled samples identified 96 miRNA that were differentially regulated, followed by validation on a custom designed 112 miRNA panel. Results: Nine miRNAs differed from baseline to hypoglycemia in control subjects; eight were upregulated: miR-1303, miR-let-7e-5p, miR-1267, miR-30a-5p, miR-571, miR-661, miR-770-5p, miR-892b and one was downregulated: miR-652-3p. None of the miRNAs differed from baseline in T2D subjects. Conclusion: A rapid miRNA response reflecting protective pathways was seen in control subjects that appeared to be lost in T2D, suggesting that mitigating responses to hypoglycemia with blunting of the counter-regulatory response in T2D occurs even in patients with short duration of disease. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03102801?term=NCT03102801&draw=2&rank=1, identifier NCT03102801.

Signal Transducer and Activator of Transcription 3 (STAT3) Suppresses STAT1/Interferon Signaling Pathway and Inflammation in Senescent Preadipocytes.

Obesity promotes premature aging and dysfunction of white adipose tissue (WAT) through the accumulation of cellular senescence. The senescent cells burden in WAT has been linked to inflammation, insulin-resistance (IR), and type 2 diabetes (T2D). There is limited knowledge about molecular mechanisms that sustain inflammation in obese states. Here, we describe a robust and physiologically relevant in vitro system to trigger senescence in mouse 3T3-L1 preadipocytes. By employing transcriptomics analyses, we discovered up-regulation of key pro-inflammatory molecules and activation of interferon/signal transducer and activator of transcription (STAT)1/3 signaling in senescent preadipocytes, and expression of downstream targets was induced in epididymal WAT of obese mice, and obese human adipose tissue. To test the relevance of STAT1/3 signaling to preadipocyte senescence, we used Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to delete STAT1/3 and discovered that STAT1 promoted growth arrest and cooperated with cyclic Guanosine Monophosphate-Adenosine Monophosphate (GMP-AMP) synthase-stimulator of interferon genes (cGAS-STING) to drive the expression of interferon ß (IFNß), C-X-C motif chemokine ligand 10 (CXCL10), and interferon signaling-related genes. In contrast, we discovered that STAT3 was a negative regulator of STAT1/cGAS-STING signaling-it suppressed senescence and inflammation. These data provide insights into how STAT1/STAT3 signaling coordinates senescence and inflammation through functional interactions with the cGAS/STING pathway.

SIRT1 promotes lipid metabolism and mitochondrial biogenesis in adipocytes and coordinates adipogenesis by targeting key enzymatic pathways.

The NAD+-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD+ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD+ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD+ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.

Dromedary camels as a natural source of neutralizing nanobodies against SARS-CoV-2.

The development of prophylactic and therapeutic agents for coronavirus disease 2019 (COVID-19) is a current global health priority. Here, we investigated the presence of cross-neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in dromedary camels that were Middle East respiratory syndrome coronavirus (MERS-CoV) seropositive but MERS-CoV free. The tested 229 dromedaries had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins, including the S trimer and M, N, and E proteins. Using SARS-CoV-2 competitive immunofluorescence immunoassays and pseudovirus neutralization assays, we found medium-to-high titers of cross-neutralizing antibodies against SARS-CoV-2 in these animals. Through linear B cell epitope mapping using phage immunoprecipitation sequencing and a SARS-CoV-2 peptide/proteome microarray, we identified a large repertoire of Betacoronavirus cross-reactive antibody specificities in these dromedaries and demonstrated that the SARS-CoV-2-specific VHH antibody repertoire is qualitatively diverse. This analysis revealed not only several SARS-CoV-2 epitopes that are highly immunogenic in humans, including a neutralizing epitope, but also epitopes exclusively targeted by camel antibodies. The identified SARS-CoV-2 cross-neutralizing camel antibodies are not proposed as a potential treatment for COVID-19. Rather, their presence in nonimmunized camels supports the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.