Resveratrol mediates mitochondrial function through the sirtuin 3 pathway to improve abnormal metabolic remodeling in atrial fibrillation.

This study investigated the impact of resveratrol on abnormal metabolic remodeling in atrial fibrillation (AF) and explored potential molecular mechanisms. An AF cell model was established by high-frequency electrical stimulation of HL-1 atrial muscle cells. Resveratrol concentrations were optimized using CCK-8 and flow cytometry. AF-induced increases in ROS and mitochondrial calcium, along with decreased adenosine triphosphate (ATP) and mitochondrial membrane potential, were observed. Resveratrol mitigated these changes and maintained normal mitochondrial morphology. Moreover, resveratrol acted through the SIRT3-dependent pathway, as evidenced by its ability to suppress AF-induced acetylation of key metabolic enzymes. SIRT3 overexpression controls acetylation modifications, suggesting its regulatory role. In conclusion, resveratrol's SIRT3-dependent pathway intervenes in AF-induced mitochondrial dysfunction, presenting a potential therapeutic avenue for AF-related metabolic disorders. This study sheds light on the role of resveratrol in mitigating AF-induced mitochondrial remodeling and highlights its potential as a novel treatment for AF.

Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor.

About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete cytokines, i.e., the pro-inflammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I) dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting compounds have been demonstrated to reduce the release of pro-inflammatory cytokines through functional involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin would be cleaved into anti-inflammatory bitter peptides during gastric digestion. Using immortalized human parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-evoked IL-17A release by up to 89.7 ± 21.9% (p ≤ 0.01). Functional involvement of TAS2R16 was demonstrated by the study of specific antagonists and siRNA knock-down experiments.

Mouse models to explore the biological and organismic role of DNA polymerase beta.

Gene knock-out (KO) mouse models for DNA polymerase beta (Polß) revealed that loss of Polß leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models. This technology was exploited to investigate roles for Polß in V(D)J recombination (variable-diversity-joining rearrangement), DNA demethylation, gene complementation, SPO11-induced DNA double-strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin-induced DNA damage, and cancer onset. The revolution in knock-in (KI) mouse models was made possible by CRISPR/cas9-mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polß. Such KI mouse models have helped uncover the importance of key Polß active site residues or specific Polß enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polß protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polß protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polß.

Vascular mimicry as a facilitator of melanoma brain metastasis.

Melanoma has the highest propensity among solid tumors to metastasize to the brain. Melanoma brain metastases (MBM) are a leading cause of death in melanoma and affect 40-60% of patients with late-stage disease. Therefore, uncovering the molecular mechanisms behind MBM is necessary to enhance therapeutic interventions. Vascular mimicry (VM) is a form of neovascularization linked to invasion, increased risk of metastasis, and poor prognosis in many tumor types, but its significance in MBM remains poorly understood. We found that VM density is elevated in MBM compared to paired extracranial specimens and is associated with tumor volume and CNS edema. In addition, our studies indicate a relevant role of YAP and TAZ, two transcriptional co-factors scarcely studied in melanoma, in tumor cell-vasculogenesis and in brain metastasis. We recently demonstrated activation of the Hippo tumor suppressor pathway and increased degradation of its downstream targets YAP and TAZ in a metastasis impaired cell line model. In the current study we establish the utility of anti-YAP/TAZ therapy in mouse models of metastatic melanoma whereby treatment effectively inhibits VM and prolongs survival of mice with MBM. The data presented herein suggest that VM may be an important and targetable mechanism in melanoma and that VM inhibition might be useful for treating MBM, an area of high unmet clinical need, thus having important implications for future treatment regimens for these patients.

The effect of continuous long-term illumination with visible light in different spectral ranges on mammalian cells.

One of the biggest challenges in tissue engineering and regenerative medicine is to ensure oxygen supply of cells in the (temporary) absence of vasculature. With the vision to exploit photosynthetic oxygen production by microalgae, co-cultivated in close vicinity to oxygen-consuming mammalian cells, we are searching for culture conditions that are compatible for both sides. Herein, we investigated the impact of long-term illumination on mammalian cells which is essential to enable photosynthesis by microalgae: four different cell types-primary human fibroblasts, dental pulp stem cells, and osteoblasts as well as the murine beta-cell line INS-1-were continuously exposed to warm white light, red or blue light over seven days. We observed that illumination with red light has no adverse effects on viability, metabolic activity and growth of the cells whereas exposure to white light has deleterious effects that can be attributed to its blue light portion. Quantification of intracellular glutathione did not reveal a clear correlation of this effect with an enhanced production of reactive oxygen species. Finally, our data indicate that the cytotoxic effect of short-wavelength light is predominantly a direct effect of cell illumination; photo-induced changes in the cell culture media play only a minor role.

From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept.

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

TP53INP2 knockdown inhibits inflammatory response and apoptosis after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a traumatic neurological disorder with limited therapeutic options. Tumor protein p53-inducible nuclear protein 2 (TP53INP2) is involved in the occurrence and development of various diseases, and it may play a role during SCI via affecting inflammation and neuronal apoptosis. This study investigated the associated roles and mechanisms of TP53INP2 in SCI. METHODS: Mouse and lipopolysaccharide (LPS)-induced SCI BV-2 cell models were constructed to explore the role of TP53INP2 in SCI and the associated mechanisms. Histopathological evaluation of spinal cord tissue was detected by hematoxylin and eosin staining. The Basso, Beattie, and Bresnahan score was used to measure the motor function of the mice, while the spinal cord water content was used to assess spinal cord edema. The expression of TP53INP2 was measured using RT-qPCR. In addition, inflammatory factors in the spinal cord tissue of SCI mice and LPS-treated BV-2 cells were measured using enzyme-linked immunosorbent assay. Apoptosis and related protein expression levels were detected by flow cytometry and western blot analysis, respectively. RESULTS: TP53INP2 levels increased in SCI mice and LPS-treated BV-2 cells. The results of in vivo and in vitro experiments showed that TP53INP2 knockdown inhibited the inflammatory response and neuronal apoptosis in mouse spinal cord tissue or LPS-induced BV-2 cells. CONCLUSIONS: After spinal cord injury, TP53INP2 was upregulated, and TP53INP2 knockdown inhibited the inflammatory response and apoptosis.

Downregulation of Glis3 in INS1 cells exposed to chronically elevated glucose contributes to glucotoxicity-associated ß cell dysfunction.

Chronically elevated levels of glucose are deleterious to pancreatic ß cells and contribute to ß cell dysfunction, which is characterized by decreased insulin production and a loss of ß cell identity. The Krüppel-like transcription factor, Glis3 has previously been shown to positively regulate insulin transcription and mutations within the Glis3 locus have been associated with the development of several pathologies including type 2 diabetes mellitus. In this report, we show that Glis3 is significantly downregulated at the transcriptional level in INS1 832/13 cells within hours of being subjected to high glucose concentrations and that diminished expression of Glis3 is at least partly attributable to increased oxidative stress. CRISPR/Cas9-mediated knockdown of Glis3 indicated that the transcription factor was required to maintain normal levels of both insulin and MafA expression and reduced Glis3 expression was concomitant with an upregulation of ß cell disallowed genes. We provide evidence that Glis3 acts similarly to a pioneer factor at the insulin promoter where it permissively remodels the chromatin to allow access to a transcriptional regulatory complex including Pdx1 and MafA. Finally, evidence is presented that Glis3 can positively regulate MafA transcription through its pancreas-specific promoter and that MafA reciprocally regulates Glis3 expression. Collectively, these results suggest that decreased Glis3 expression in ß cells exposed to chronic hyperglycemia may contribute significantly to reduced insulin transcription and a loss of ß cell identity.

Investigating the Impact of IL6 on Insulin Secretion: Evidence from INS-1 Cells, Human Pancreatic Islets, and Serum Analysis.

Interleukin-6 (IL6) is a pleiotropic cytokine implicated in metabolic disorders and inflammation, yet its precise influence on insulin secretion and glucose metabolism remains uncertain. This study examined IL6 expression in pancreatic islets from individuals with/without diabetes, alongside a series of functional experiments, including siRNA silencing; IL6 treatment; and assessments of glucose uptake, cell viability, apoptosis, and expression of key ß-cell genes, which were conducted in both INS-1 cells and human islets to elucidate the effect of IL6 on insulin secretion. Serum levels of IL6 from Emirati patients with type 2 diabetes (T2D) were measured, and the effect of antidiabetic drugs on IL6 levels was studied. The results revealed that IL6 mRNA expression was higher in islets from diabetic and older donors compared to healthy or young donors. IL6 expression correlated negatively with PDX1, MAFB, and NEUROD1 and positively with SOX4, HES1, and FOXA1. Silencing IL6 in INS-1 cells reduced insulin secretion and glucose uptake independently of apoptosis or oxidative stress. Reduced expression of IL6 was associated with the downregulation of Ins, Pdx1, Neurod1, and Glut2 in INS-1 cells. In contrast, IL6 treatment enhanced insulin secretion in INS-1 cells and human islets and upregulated insulin expression. Serum IL6 levels were elevated in patients with T2D and associated with higher glucose, HbA1c, and triglycerides, regardless of glucose-lowering medications. This study provides a new understanding of the role of IL6 in ß-cell function and the pathophysiology of T2D. Our data highlight differences in the response to IL6 between INS-1 cells and human islets, suggesting the presence of species-specific variations across different experimental models. Further research is warranted to unravel the precise mechanisms underlying the observed effects of IL-6 on insulin secretion.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer.

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Study of the effect of sFRP1 protein on molecules involved in the regulation of DNA methylation in CML cell line.

Wnt-signaling pathway plays a crucial role in the pathogenesis and progression of Chronic Myeloid Leukemia (CML). sFRP1 is involved in the suppression of the Wnt-signaling pathway and has been shown to be epigenetically silenced by promoter hypermethylation during CML progression. DNMT3A plays a crucial role in promoter hypermethylation and is responsible for establishing methylation patterns. We aimed to analyze the relationship between sFRP1 expression and DNMT3A, TET1, TET2 and TET3 proteins that are responsible for maintaining cellular methylation patterns; along with miRNAs miR144-3p and miR-767-5p that are known to be associated with these proteins. CML cell lines K562 and K562S which stably expresses sFRP1, were used to compare the changes in miR144-3p and miR-767-5p expression. DNMT3A, TET1, TET2 and TET3 protein levels were analyzed by Western blot. In K562S cells the expression of miR-144-3p and miR-767-5p were decreased along with DNMT3A and TET1 protein levels. On the contrary, TET2 protein was increased. Our results support other reports involving sFRP1 and methylation dynamics; as well as opening new avenues of exploration. Our data supports the conclusion that re-expression of sFRP1 protein alters the expression of factors that play important roles in the overall methylation patterns in the leukemic cell line K562.

MFSynDCP: multi-source feature collaborative interactive learning for drug combination synergy prediction.

Drug combination therapy is generally more effective than monotherapy in the field of cancer treatment. However, screening for effective synergistic combinations from a wide range of drug combinations is particularly important given the increase in the number of available drug classes and potential drug-drug interactions. Existing methods for predicting the synergistic effects of drug combinations primarily focus on extracting structural features of drug molecules and cell lines, but neglect the interaction mechanisms between cell lines and drug combinations. Consequently, there is a deficiency in comprehensive understanding of the synergistic effects of drug combinations. To address this issue, we propose a drug combination synergy prediction model based on multi-source feature interaction learning, named MFSynDCP, aiming to predict the synergistic effects of anti-tumor drug combinations. This model includes a graph aggregation module with an adaptive attention mechanism for learning drug interactions and a multi-source feature interaction learning controller for managing information transfer between different data sources, accommodating both drug and cell line features. Comparative studies with benchmark datasets demonstrate MFSynDCP's superiority over existing methods. Additionally, its adaptive attention mechanism graph aggregation module identifies drug chemical substructures crucial to the synergy mechanism. Overall, MFSynDCP is a robust tool for predicting synergistic drug combinations. The source code is available from GitHub at https://github.com/kkioplkg/MFSynDCP .

Expression and functional characterization of chimeric recombinant bovine follicle-stimulating hormone produced in Leishmania tarentolae.

Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the ß- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO2 or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction.

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Clinical translation of pluripotent stem cell-based therapies: successes and challenges.

The translational stem cell research field has progressed immensely in the past decade. Development and refinement of differentiation protocols now allows the generation of a range of cell types, such as pancreatic ß-cells and dopaminergic neurons, from human pluripotent stem cells (hPSCs) in an efficient and good manufacturing practice-compliant fashion. This has led to the initiation of several clinical trials using hPSC-derived cells to replace lost or dysfunctional cells, demonstrating evidence of both safety and efficacy. Here, we highlight successes from some of the hPSC-based trials reporting early signs of efficacy and discuss common challenges in clinical translation of cell therapies.

Development, synthesis and validation of improved c-Myc/Max inhibitors.

The pathophysiological foundations of various diseases are often subject to alteration through the utilization of small compounds, rendering them invaluable tools for the exploration and advancement of novel therapeutic strategies. Within the scope of this study, we meticulously curated a diverse library of novel small compounds meticulously designed to specifically target the c-Myc/Max complex. We conducted in vitro examinations of novel c-Myc inhibitors across a spectrum of cancer cell lines, including PANC1 (pancreatic adenocarcinoma), MCF7 (breast carcinoma), DU-145 (prostate carcinoma), and A549 (lung cancer). The initial analysis involved a 25 µM dose, which enabled the identification of potent anticancer compounds effective against a variety of tumour types. We identified c-Myc inhibitors with remarkable potency, featuring IC50 values as low as 1.6 µM and up to 40 times more effective than the reference molecule in diminishing cancer cell viability. Notably, c-Myc-i7 exhibited exceptional selectivity, displaying 37-fold and 59-fold preference for targeting prostate and breast cancers, respectively, over healthy cells. Additionally, we constructed drug-likeness models. This study underscores the potential for in vitro investigations of various tumour types using novel c-Myc inhibitors to yield ground-breaking and efficacious anticancer compounds.

High-Throughput Cell-Based Screening of Small Molecule KRAS Signaling Inhibitors Using a Homogeneous Time-Resolved Fluorescence (HTRF) Assay.

With recent advances proving that effective inhibition of KRAS is possible, there have been significant efforts made to develop inhibitors of specific mutant alleles. Here we describe a detailed protocol that employs homogeneous time-resolved fluorescence (HTRF) to identify compounds acting on KRAS signaling in malignant cell lines. This method allows for high-throughput, cell-based screens of large compound libraries for the development of RAS-targeted therapeutics.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Quality Control of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel.

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype.

AP39, a novel mitochondria-targeted hydrogen sulfide donor ameliorates doxorubicin-induced cardiotoxicity by regulating the AMPK/UCP2 pathway.

Doxorubicin (DOX) is a broad-spectrum, highly effective antitumor agent; however, its cardiotoxicity has greatly limited its use. Hydrogen sulfide (H2S) is an endogenous gaseous transmitter that exerts cardioprotective effects via the regulation of oxidative stress and apoptosis and maintenance of mitochondrial function, among other mechanisms. AP39 is a novel mitochondria-targeted H2S donor that, at appropriate concentrations, attenuates intracellular oxidative stress damage, maintains mitochondrial function, and ameliorates cardiomyocyte injury. In this study, DOX-induced cardiotoxicity models were established using H9c2 cells and Sprague-Dawley rats to evaluate the protective effect of AP39 and its mechanisms of action. Both in vivo and in vitro experiments showed that DOX induces oxidative stress injury, apoptosis, and mitochondrial damage in cardiomyocytes and decreases the expression of p-AMPK/AMPK and UCP2. All DOX-induced changes were attenuated by AP39 treatment. Furthermore, the protective effect of AP39 was significantly attenuated by the inhibition of AMPK and UCP2. The results suggest that AP39 ameliorates DOX-induced cardiotoxicity by regulating the expression of AMPK/UCP2.