Biology subjects

dos Santos, T.

Publications and source records attributed to dos Santos, T..

11 recordsLinked to original sources

bioRxiv2025

Supervised machine learning identifies impaired mitochondrial quality control in β cells with development of type 2 diabetes

In type 2 diabetes (T2D), molecular pathways driving {beta} cell failure are difficult to resolve with standard single cell analysis. Here we developed an interpretable, supervised machine learning framework that couples sparse rule-based classification (SnakeClassifier), pathway constrained modelling (BlackSwanClassifier), and {beta} cell mitochondrial fitness stratification (Kolmogorov-Arnold Neural Networks KANN), linking and integrating them into disease mechanisms in single cell RNA sequencing (scRNA-seq) from 52 human donors. SnakeClassifier trained on 50 genes accurately predicted T2D at single cell resolution, outperforming classical ensemble machine learning classifier models, and yielded donor level diabetes scores that correlated with chronic hyperglycemia. The clustering of {beta} cell populations ({beta}1-4) revealed a resilient non-diabetic (ND) {beta}1 subtype characterized by preserved {beta} cell identity genes and lower disease risk, whereas T2D {beta}2-4 subtypes exhibited upregulation of genes involved in cellular and mitochondrial stress and suppression of genes promoting oxidative phosphorylation and insulin secretion. Mitophagy emerged as the dominant program linked to T2D and a mitophagy focused BlackSwanClassifier nominated PINK1, BNIP3, and FUNDC1 as key regulators. PINK1 was enriched in ND {beta}1, decreased with T2D disease score and connected sex stratified mitophagy. We generated a KANN derived mitochondrial fitness index (MFI) integrating mitophagy, mitochondrial proteostasis, biogenesis and oxidative phosphorylation into a single interpretable score (R2 = 0.934 vs module-based mitochondria quality index), which identified mitophagy PINK1, SQSTM1, PRKN and BNIP3 as top contributors to T2D progression. These transparent models unify prediction with T2D disease mechanism and identify the mitophagy receptor PINK1 as a central determinant of {beta} cell metabolic fitness

systems biology↗

bioRxiv2025

Linking molecular pathways and islet cell dysfunction in human type 1 diabetes

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing {beta}-cells, along with dysregulated glucagon secretion from pancreatic -cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors to investigate the mechanisms underlying their dysfunction. Surviving {beta}-cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In -cells, we observed hyper-responsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to -cell dysfunction, including MHC class I which were closely linked with -cell dysfunction. Our data provide novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual {beta}-cell function and modulate -cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D. HighlightsO_LISurviving {beta}-cells in T1D show disrupted electrical function linked to metabolic reprogramming and immune stress. C_LIO_LITranscripts associated with -cell dysfunction are enriched in genetic risk alleles for T1D. C_LIO_LIUpregulated MHC class I and impaired nuclear localization of key transcription factors associate with -cell dysfunction in T1D. C_LIO_LIT1D -cells exhibit increased hyper-activity, lysosomal imbalance and impaired mTORC1 signaling, which promotes dysregulated glucagon secretion. C_LI

cell biology↗

bioRxiv2024

TMEM55A-mediated PI5P signaling regulates α-cell actin depolymerization and glucagon secretion

Diabetes is associated with the dysfunction of glucagon-producing pancreatic islet -cells, although the underlying mechanisms regulating glucagon secretion and -cell dysfunction remain unclear. While insulin secretion from pancreatic {beta}-cells has long been known to be partly controlled by intracellular phospholipid signaling, very little is known about the role of phospholipids in glucagon secretion. Here we show that TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-5-phosphate (PI5P), regulates -cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse -cells reduces exocytosis at low glucose, and this is rescued by the direct reintroduction of PI5P. This does not occur through an effect on Ca2+ channel activity, but through a re-modelling of cortical F-actin dependent upon TMEM55A lipid phosphatase activity which occurs in response to oxidative stress. In summary, we reveal a novel pathway by which TMEM55A regulates -cell exocytosis by manipulating intracellular PI5P level and the F-actin network.

cell biology↗

bioRxiv2024

Molecular Correlates of Glycine Receptor Activity in Human β Cells

ObjectivesGlycine acts in an autocrine positive feedback loop in human {beta} cells through its ionotropic receptors (GlyRs). In type 2 diabetes (T2D), islet GlyR activity is impaired by unknown mechanisms. We sought to investigate if the GlyR dysfunction in T2D is replicated by hyperglycemia per se, and to further characterize its action in {beta} cells and islets. MethodsGlyR-mediated currents were measured using whole-cell patch-clamp in human {beta} cells from donors with or without T2D, or after high glucose (15 mM) culture. We also correlated glycine-induced current amplitude with transcript expression levels through patch-seq. The expression of the GlyR 1, 3, and {beta} subunit mRNA splice variants was compared between islets from donors with and without T2D, and after high glucose culture. Insulin secretion from human islets was measured in the presence or absence of the GlyR antagonist strychnine. ResultsAlthough gene expression of GlyRs was decreased in T2D islets, and {beta} cell GlyR-mediated currents were smaller, we found no evidence for a shift in GlyR subunit splicing. Glycine-induced currents are also reduced after 48 hours culture of islets from donors without diabetes in high glucose, where we also find the reduction of the 1 subunit expression, but an increase in the 3 subunit. We discovered that glycine-evoked currents are highly heterogeneous amongst {beta} cells, inversely correlate with donor HbA1c, and are significantly correlated to the expression of 92 different transcripts and gene regulatory networks (GRNs) that include CREB3(+), RREB1(+) and ZNF697(+). Finally, glucose-stimulated insulin secretion is decreased in the presence of the GlyR antagonist strychnine. ConclusionsWe demonstrate that glucose can modulate GlyR expression, and that the current decrease in T2D is likely due to the receptor gene expression downregulation, and not a change in transcript splicing. Moreover, we define a previously unknown set of genes and regulons that are correlated to GlyR-mediated currents and could be involved in GlyR downregulation in T2D. HighlightsO_LIGlycine-evoked currents in {beta} cells are lower in T2D and after high glucose culture C_LIO_LIGlyR gene expression is reduced in T2D, with no shift in splice variant expression C_LIO_LIGlyR inhibition with strychnine decreases insulin secretion C_LIO_LIWe define transcripts and gene regulatory networks correlated to glycine-induced currents C_LI

cell biology↗

bioRxiv2024

HumanIslets: An integrated platform for human islet data access and analysis

Comprehensive molecular and cellular phenotyping of human islets can enable deep mechanistic insights for diabetes research. We established the Human Islet Data Analysis and Sharing (HI-DAS) consortium to advance goals in accessibility, usability, and integration of data from human islets isolated from donors with and without diabetes at the Alberta Diabetes Institute (ADI) IsletCore. Here we introduce HumanIslets.com, an open resource for the research community. This platform, which presently includes data on 547 human islet donors, allows users to access linked datasets describing molecular profiles, islet function and donor phenotypes, and to perform various statistical and functional analyses at the donor, islet and single-cell levels. As an example of the analytic capacity of this resource we show a dissociation between cell culture effects on transcript and protein expression, and an approach to correct for exocrine contamination found in hand-picked islets. Finally, we provide an example workflow and visualization that highlights links between type 2 diabetes status, SERCA3b Ca2+-ATPase levels at the transcript and protein level, insulin secretion and islet cell phenotypes. HumanIslets.com provides a growing and adaptable set of resources and tools to support the metabolism and diabetes research community.

systems biology↗

bioRxiv2023

Integration of single-cell multiomic measurements across disease states with genetics identifies mechanisms of beta cell dysfunction in type 2 diabetes

Altered function and gene regulation of pancreatic islet beta cells is a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of mechanisms driving T2D is still missing. Here we integrate information from measurements of chromatin activity, gene expression and function in single beta cells with genetic association data to identify disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 non-diabetic, pre-T2D and T2D donors, we robustly identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift in T2D. Subtype-defining active chromatin is enriched for T2D risk variants, suggesting a causal contribution of subtype identity to T2D. Both subtypes exhibit activation of a stress-response transcriptional program and functional impairment in T2D, which is likely induced by the T2D-associated metabolic environment. Our findings demonstrate the power of multimodal single-cell measurements combined with machine learning for identifying mechanisms of complex diseases.

systems biology↗

bioRxiv2022

FK506-binding protein 2 participates in proinsulin folding

Apart from chaperoning, disulphide bond formation and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94, and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high molecular weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic {beta}-cells increased apoptosis without detectable upregulation of ER-stress response genes. Interestingly, FKBP2 mRNA was overexpressed in {beta}-cells from pancreatic islets of T2D patients. Based on molecular modelling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B chain (P28) is the substrate of FKBP2s isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

molecular biology↗

bioRxiv2022

Reovirus uses temporospatial compartmentalization to orchestrate core versus outercapsid assembly.

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins {micro}1, {sigma}3 and {sigma}1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a {micro}1-dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC {micro}1 expression with siRNAs led to large factories that remained further from the nucleus, implicating {micro}1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions. NON-TECHNICAL AUTHOR SUMMARYIt is important to understand how viruses replicate and assemble to discover antiviral therapies and to modify viruses for applications like gene therapy or cancer therapy. Reovirus is a harmless virus being tested as a cancer therapy. Reovirus has two coats of proteins, an inner coat and an outer coat. To replicate, reovirus particles need only the inner coat, but to become infectious they require the outer coat. Strangely, inner and outer coat proteins are all made by the virus at once, so it was unknown what determines whether newly made viruses will contain just the inner coat to continue to replicate, or both coats to transmit to new hosts. Our experiments reveal that the inner coat proteins are located in a different area of an infected cell versus the outer coat proteins. The location therefore determines if the newly made viruses contain just the inner coat versus both coats. Reoviruses have evolved extravagant mechanisms to be able to efficiently take on the best composition required for replication and transmission.

microbiology↗

bioRxiv2021

Impacts of the COVID-19 Pandemic on a Human Research Islet Program

Designated a pandemic in March 2020, the spread of severe acute respiratory syndrome virus 2 (SARS-CoV2), the virus responsible for coronavirus disease 2019 (COVID-19), led to new guidelines and restrictions being implemented for individuals, businesses, and societies in efforts to limit the impacts of COVID-19 on personal health and healthcare systems. Here we report the impacts of the COVID-19 pandemic on pancreas processing and islet isolation/distribution outcomes at the Alberta Diabetes Institute IsletCore, a facility specialising in the processing and distribution of human pancreatic islets for research. While the number of organs processed was significantly reduced, organ quality and the function of cellular outputs were minimally impacted during the pandemic when compared to an equivalent period immediately prior. Despite the maintained quality of isolated islets, recipient groups reported poorer feedback regarding the samples. Our findings suggest this is likely due to disrupted distribution which led to increased transit times to recipient labs, particularly those overseas. Thus, to improve overall outcomes in a climate of limited research islet supply, prioritization of tissue recipients based on likely tissue transit times may be needed.

physiology↗

bioRxiv2021

Heterogenous impairment of α-cell function in type 2 diabetes is linked to cell maturation state

In diabetes, glucagon secretion from pancreatic -cells is dysregulated. We examined -cells from human donors and mice using combined electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses -cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat-feeding of mice, -cells shift towards a {beta}-cell-like electrophysiologic profile in concert with an up-regulation of the {beta}-cell Na+ channel isoform Scn9a and indications of impaired -cell identity. In human -cells we identify links between cell membrane properties and cell surface signalling receptors, mitochondrial respiratory complex assembly, and cell maturation. Cell type classification using machine learning of electrophysiology data demonstrates a heterogenous loss of electrophysiologic identity in -cells from donors with T2D. Indeed, a sub-set of -cells with impaired exocytosis is defined by an enrichment in progenitor markers suggesting important links between -cell maturation state and dysfunction in T2D. Key findingsO_LI-cell exocytosis is suppressed by glucose-dependent inhibition of P/Q-type Ca2+ currents C_LIO_LIDysfunction of -cells in type 2 diabetes is associated with a {beta}-cell-like electrophysiologic signature C_LIO_LIPatch-seq links maturation state, the mitochondrial respiratory chain, and cell surface receptor expression to -cell function C_LIO_LI-cell dysfunction occurs preferentially in cells enriched in endocrine lineage markers C_LI

cell biology↗

bioRxiv2021

Follow-up of a hospital cohort during the first 3,530 suspected cases of COVID-19 in Sao Jose do Rio Preto, Sao Paulo, Brazil

IntroductionIn a global context, COVID-19 is the most significant health threat in the present days, evidenced by the fact that, in just over four months, SARS-CoV-2 has spread to 171 countries, reaching a Pandemic status. Most patients with COVID-19 have a mild course of the disease. However, approximately 20% develop severe illness with a high mortality rate which is associated with age, comorbidities, and immunosuppression. Epidemiological studies are used to reveal the extent of viral spread in homes, communities, and hospitals. Thus, preventive and control measures can be established by the authorities. ObjectiveIn this study, patients with suspect COVID-19 symptoms who search for hospital care at the city of Sao Jose do Rio Preto (Sao Paulo, Brazil) were monitored, in order to identify the first case of this new disease in the region. In the first two months (March and April), more than 3000 individuals looked for the public and private health system with suspected respiratory symptoms, but only 164 (8.4%) were COVID-19 confirmed. ResultsFrom those, males (56.1%) and patients of the age distribution of 16-59 (91.2%), with diarrhea (22.2%), runny nose (25%), altered taste (15.9%), and anosmia (11.6%) presented statistical significance, although none comorbidities were related with COVID-19 occurrence. The odds ratio analysis supports this finding. Days of onset of symptoms are positively associated with whit viral load, and the same happens with the occurrence of symptoms (dyspnea and low saturation).

molecular biology↗