Integrative Bioinformatics-Gene Network Approach Reveals Linkage between Estrogenic Endocrine Disruptors and Vascular Remodeling in Peripheral Arterial Disease.

Vascular diseases, including peripheral arterial disease (PAD), pulmonary arterial hypertension, and atherosclerosis, significantly impact global health due to their intricate relationship with vascular remodeling. This process, characterized by structural alterations in resistance vessels, is a hallmark of heightened vascular resistance seen in these disorders. The influence of environmental estrogenic endocrine disruptors (EEDs) on the vasculature suggests a potential exacerbation of these alterations.

Supervised Parametric Learning in the Identification of Composite Biomarker Signatures of Type 1 Diabetes in Integrated Parallel Multi-Omics Datasets.

Background: Type 1 diabetes (T1D) is a devastating autoimmune disease, and its rising prevalence in the United States and around the world presents a critical problem in public health. While some treatment options exist for patients already diagnosed, individuals considered at risk for developing T1D and who are still in the early stages of their disease pathogenesis without symptoms have no options for any preventive intervention. This is because of the uncertainty in determining their risk level and in predicting with high confidence who will progress, or not, to clinical diagnosis.

A Composite Biomarker Signature of Type 1 Diabetes Risk Identified via Augmentation of Parallel Multi-Omics Data from a Small Cohort.

Background: Biomarkers of early pathogenesis of type 1 diabetes (T1D) are crucial to enable effective prevention measures in at-risk populations before significant damage occurs to their insulin producing beta-cell mass. We recently introduced the concept of integrated parallel multi-omics and employed a novel data augmentation approach which identified promising candidate biomarkers from a small cohort of high-risk T1D subjects. We now validate selected biomarkers to generate a potential composite signature of T1D risk.

Exploring Computational Data Amplification and Imputation for the Discovery of Type 1 Diabetes (T1D) Biomarkers from Limited Human Datasets.

Background: Type 1 diabetes (T1D) is a devastating disease with serious health complications. Early T1D biomarkers that could enable timely detection and prevention before the onset of clinical symptoms are paramount but currently unavailable. Despite their promise, omics approaches have so far failed to deliver such biomarkers, likely due to the fragmented nature of information obtained through the single omics approach.

RNA aptamers specific for transmembrane p24 trafficking protein 6 and Clusterin for the targeted delivery of imaging reagents and RNA therapeutics to human β cells.

The ability to detect and target β cells in vivo can substantially refine how diabetes is studied and treated. However, the lack of specific probes still hampers a precise characterization of human β cell mass and the delivery of therapeutics in clinical settings. Here, we report the identification of two RNA aptamers that specifically and selectively recognize mouse and human β cells.

Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein-Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2.

Inhibitors of the protein-protein interaction (PPI) between the SARS-CoV-2 spike protein and human ACE2 (hACE2), which acts as a ligand-receptor pair that initiates the viral attachment and cellular entry of this coronavirus causing the ongoing COVID-19 pandemic, are of considerable interest as potential antiviral agents. While blockade of such PPIs with small molecules is more challenging than that with antibodies, small-molecule inhibitors (SMIs) might offer alternatives that are less strain- and mutation-sensitive, suitable for oral or inhaled administration, and more controllable/less immunogenic. Here, we report the identification of SMIs of this PPI by screening our compound library focused around the chemical space of organic dyes.

Parallel Multi-Omics in High-Risk Subjects for the Identification of Integrated Biomarker Signatures of Type 1 Diabetes.

Background: Biomarkers are crucial for detecting early type-1 diabetes (T1D) and preventing significant β-cell loss before the onset of clinical symptoms. Here, we present proof-of-concept studies to demonstrate the potential for identifying integrated biomarker signature(s) of T1D using parallel multi-omics.

Methods: Blood from human subjects at high risk for T1D (and healthy controls; = 4 + 4) was subjected to parallel unlabeled proteomics, metabolomics, lipidomics, and transcriptomics.

Methylene Blue Inhibits the SARS-CoV-2 Spike-ACE2 Protein-Protein Interaction-a Mechanism that can Contribute to its Antiviral Activity Against COVID-19.

Due to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay.

Longitudinal proteomics analysis in the immediate microenvironment of islet allografts during progression of rejection.

The applicability and benefits of pancreatic islet transplantation are limited due to various issues including the need to avoid immune-mediated rejection. Here, we used our experimental platform of allogeneic islet transplant in the anterior chamber of the eye (ACE-platform) to longitudinally monitor the progress of rejection in mice and obtain aqueous humor samples representative of the microenvironment of the graft for accurately-timed proteomic analyses. LC-MS/MS-based proteomics performed on such mass-limited samples (~5 μL) identified a total of 1296 proteins.

The Effect of Recovery Warm-up Time Following Cold Storage on the Dynamic Glucose-stimulated Insulin Secretion of Isolated Human Islets.

Standardized islet characterization assays that can provide results in a timely manner are essential for successful islet cell transplantation. A critical component of islet cell quality is β-cell function, and perifusion-based assessments of dynamic glucose-stimulated insulin secretion (GSIS) are the most informative method to assess this, as they provide the most complex in vitro evaluation of GSIS. However, protocols used vary considerably among centers and investigators as they often use different low- and high-glucose concentrations, exposure-times, flow-rates, oxygen concentrations, islet numbers, analytical methods, measurement units, and instruments, which result in different readouts and make comparisons across platforms difficult.

Concentration-Dependency and Time Profile of Insulin Secretion: Dynamic Perifusion Studies With Human and Murine Islets.

The detailed characterization and quantification of the kinetics of glucose-stimulated insulin secretion (GSIS) by normal pancreatic islets is of considerable interest for characterizing β-cell dysfunction, assessing the quality of isolated islets, and improving the design of artificial pancreas devices. Here, we performed dynamic evaluation of GSIS by human and mouse islets at high temporal resolution (every minute) in response to different glucose steps using an automated multichannel perifusion instrument. In both species, insulin responses were biphasic (a transient first-phase peak followed by a sustained second-phase), and the amount of insulin released showed a sigmoid-type dependence on glucose concentration.

Feasibility of Localized Metabolomics in the Study of Pancreatic Islets and Diabetes.

(1) Background: Disruption of insulin production by native or transplanted pancreatic islets caused by auto/allo-immunity leads to hyperglycemia, a serious health condition and important therapeutic challenge due to the lifelong need for exogeneous insulin administration. Early metabolic biomarkers can prompt timely interventions to preserve islet function, but reliable biomarkers are currently lacking. We explored the feasibility of "localized metabolomics" where initial biomarker discovery is made in aqueous humor samples for further validation in the circulation.

A Double Fail-Safe Approach to Prevent Tumorigenesis and Select Pancreatic β Cells from Human Embryonic Stem Cells.

The transplantation of human embryonic stem cell (hESC)-derived insulin-producing β cells for the treatment of diabetes is finally approaching the clinical stage. However, even with state-of-the-art differentiation protocols, a significant percentage of undefined non-endocrine cell types are still generated. Most importantly, there is the potential for carry-over of non-differentiated cell types that may produce teratomas.

Design, Synthesis, and Evaluation of Novel Immunomodulatory Small Molecules Targeting the CD40⁻CD154 Costimulatory Protein-Protein Interaction.

We report the design, synthesis, and testing of novel small-molecule compounds targeting the CD40⁻CD154 (CD40L) costimulatory interaction for immunomodulatory purposes. This protein-protein interaction (PPI) is a TNF-superfamily (TNFSF) costimulatory interaction that is an important therapeutic target since it plays crucial roles in the activation of T cell responses, and there is resurgent interest in its modulation with several biologics in development. However, this interaction, just as all other PPIs, is difficult to target by small molecules.

A negative allosteric modulator of PDE4D enhances learning after traumatic brain injury.

Traumatic brain injury (TBI) significantly decreases cyclic AMP (cAMP) signaling which produces long-term synaptic plasticity deficits and chronic learning and memory impairments. Phosphodiesterase 4 (PDE4) is a major family of cAMP hydrolyzing enzymes in the brain and of the four PDE4 subtypes, PDE4D in particular has been found to be involved in memory formation. Although most PDE4 inhibitors target all PDE4 subtypes, PDE4D can be targeted with a selective, negative allosteric modulator, D159687.

Osteopontin has a protective role in prostate tumor development in mice.

Osteopontin (OPN) is a protein, generally considered to play a pro-tumorigenic role, whereas several reports have demonstrated the anti-tumorigenic function of OPN during tumor development. These opposing anti- and pro-tumorigenic functions are not fully understood. Here, we report that host-derived OPN plays an anti-tumorigenic role in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model and a TRAMP tumor transplant model.

Differential effects of low-dose decitabine on immune effector and suppressor responses in melanoma-bearing mice.

Background: Low doses of the demethylating agent decitabine have been shown to enhance the sensitivity of tumors to immune effector cells and molecules through upregulation of tumor antigen presentation and apoptotic pathways. Effects on host immune effector and suppressor responses have not been well characterized.

Methods: Mice bearing B16 melanoma were treated with low-dose decitabine, cytokine, interleukin-2 (IL-2), toll-like receptor 9 agonist ODN1826, and/or a viral vectored vaccine targeting the melanoma antigen Trp2.

Epigenetic regulation by decitabine of melanoma differentiation in vitro and in vivo.

Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells.

Angiotensin II-induced MMP-2 activity and MMP-14 and basigin protein expression are mediated via the angiotensin II receptor type 1-mitogen-activated protein kinase 1 pathway in retinal pigment epithelium: implications for age-related macular degeneration.

Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD.

Noncytotoxic differentiation treatment of renal cell cancer.

Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis and/or through differentiation pathways. At low doses, the cytosine analogue decitabine (DAC) can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin, and alter differentiation without causing apoptosis (cytotoxicity).

(Pro)renin receptor is expressed in human retinal pigment epithelium and participates in extracellular matrix remodeling.

The (pro)renin receptor (PRR) is believed to potentiate the renin-angiotensin system (RAS), conferring to prorenin, a likely pathological role at tissue level. The PRR has been identified in the microvascular endothelial cells of the retina, in which it seems to be involved in pathological neovascularization processes. In the present study, we sought to explore PRR expression and prorenin action in human retinal pigment epithelium (RPE) cells, as well as its potential implication in extracellular matrix (ECM) turnover.

Proteomics characterization of cell membrane blebs in human retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE).

Regulation of angiotensin II receptors and extracellular matrix turnover in human retinal pigment epithelium: role of angiotensin II.

The early stage of age-related macular degeneration (AMD) is characterized by the formation of subretinal pigment epithelium (RPE) deposits as a result of the dysregulation in the turnover of extracellular matrix (ECM) molecules. However, the mechanism involved remains unclear. Hypertension (HTN) is an important risk factor for AMD, and angiotensin II (ANG II) is the most important hormone associated with HTN.