There has been an immense effort by global pharmaceutical companies to develop anti-COVID-19 drugs, including small molecule-based RNA replication inhibitors via drug repositioning and antibody-based spike protein blockers related to cell entry by SARS-CoV-2. However, several limitations to their clinical use have emerged in addition to a lack of progress in the development of small molecule-based cell entry inhibitors from natural products. In this study, we tested the effectiveness of kuwanon C (KC), which has mainly been researched using in silico docking simulation and can serve as an effective building block for developing anti-COVID-19 drugs, in blocking the spike S1 RBD:ACE2 receptor interaction. KC is a natural product derived from Morus alba L., commonly known as mulberry, which has known antiviral efficacy. Molecular interaction studies using competitive ELISA and the BLItz system revealed that KC targets both the spike S1 RBD and the ACE2 receptor, successfully disrupting their interaction, as supported by the in silico docking simulation. Furthermore, we established a mechanism of action by observing how KC prevents the infection of SARS-CoV-2 spike pseudotyped virus in ACE2/TPRSS2-overexpressing HEK293T cells. Finally, we demonstrated that KC inhibits clinical isolates of SARS-CoV-2 in Vero cells. Future combinations of small molecule-based cell entry inhibitors, such as KC, with the currently prescribed RNA replication inhibitors are anticipated to significantly enhance the efficacy of COVID-19 therapies. Full article

Hippocampus is the brain area where aluminum (Al) accumulates in abundance and is widely associated with learning and memory. In the present study, we evaluate behavioral, tissue, and proteomic changes in the hippocampus of Wistar rats caused by exposure to doses that mimic human consumption of aluminum chloride (AlCl₃) in urban areas. For this, male Wistar rats were divided into two groups: Control (distilled water) and AlCl₃ (8.3 mg/kg/day), both groups were exposed orally for 60 days. After the Al exposure protocol, cognitive functions were assessed by the Water maze test, followed by a collection for analysis of the global proteomic profile of the hippocampus by mass spectrometry. Aside from proteomic analysis, we performed a histological analysis of the hippocampus, to the determination of cell body density by cresyl violet staining in Cornu Ammonis fields (CA) 1 and 3, and hilus regions. Our results indicated that exposure to low doses of aluminum chloride triggered a decreased cognitive performance in learning and memory, being associated with the deregulation of proteins expression, mainly those related to the regulation of the cytoskeleton, cellular metabolism, mitochondrial activity, redox regulation, nervous system regulation, and synaptic signaling, reduced cell body density in CA1, CA3, and hilus. Full article

Human mitochondrial transcription termination factor 1 (MTERF1) has been demonstrated to play an important role in mitochondrial gene expression regulation. However, the molecular mechanism of MTERF1 in colorectal cancer (CRC) remains largely unknown. Here, we found that MTERF1 expression was significantly increased in colon cancer tissues compared with normal colorectal tissue by Western blotting, immunohistochemistry, and tissue microarrays (TMA). Overexpression of MTERF1 in the HT29 cell promoted cell proliferation, migration, invasion, and xenograft tumor formation, whereas knockdown of MTERF1 in HCT116 cells appeared to be the opposite phenotype to HT29 cells. Furthermore, MTERF1 can increase mitochondrial DNA (mtDNA) replication, transcription, and protein synthesis in colorectal cancer cells; increase ATP levels, the mitochondrial crista density, mitochondrial membrane potential, and oxygen consumption rate (OCR); and reduce the ROS production in colorectal cancer cells, thereby enhancing mitochondrial oxidative phosphorylation (OXPHOS) activity. Mechanistically, we revealed that MTERF1 regulates the AMPK/mTOR signaling pathway in cancerous cell lines, and we also confirmed the involvement of the AMPK/mTOR signaling pathway in both xenograft tumor tissues and colorectal cancer tissues. In summary, our data reveal an oncogenic role of MTERF1 in CRC progression, indicating that MTERF1 may represent a new therapeutic target in the future. Full article

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endometrial microbiome is of particular interest given the potential impact on the embryo implantation. To date, all endometrial microbiome studies have applied a metagenomics approach. A sequencing-based technique, however, has its limitations, more specifically in adequately exploring low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were included after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab^®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial microbiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab^®-assisted culturomics to investigate the low biomass endometrial microbiome at a species level. Full article

Neuroglial apoptosis and neuroinflammation play an important role in epileptogenesis. The aim of this study is to evaluate neuronal and glial apoptosis in association with neuroinflammation in brain epileptic focus and inflammatory changes in blood in patients with focal drug-resistant epilepsy (DRE). Pathological changes in the temporal lobe in epilepsy (histology, transmission electron microscopy), levels of apoptotic and neuroinflammatory proteins: active caspase-3 (immunohistochemistry), full-length form caspase-3, caspase-9, FAS, FAS-L, NF-kB, TNF-α, p53 (Western blot), and cytokine levels in blood: IL-1β, IL-2, IL-4, IL-7, TNF-α, etc. (multiplex analysis) were studied. In the present work, ultrastructural and immunohistochemical apoptotic signs were found in neurons and oligodendrocytes in the temporal lobe of DRE patients. Levels of proinflammatory cytokines that play a role in apoptosis (TNF-α, FAS, NF-kB) were increased. The blood concentration of IL-4, IL-7, TNF-α is increased and IL-2 is reduced. Oligodendroglial apoptosis has been shown to play an important role in DRE pathogenesis and to explain demyelination. Thus, a comprehensive analysis of revealed changes in the blood and brain in DRE patients showed the neuroinflammation in the epileptic focus, which was combined with the development of apoptosis of glial cells and neurons. This creates conditions for the development of drug resistance and the epilepsy progression. Full article

Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP. Full article

Biological therapy, with its multifaceted applications, has revolutionized the treatment of tumors, mainly due to its ability to exclusively target cancer cells and reduce the adverse effects on normal tissues. This review focuses on the therapies targeting the CXCR4 and CD47 receptors. We surveyed the results of early clinical trials testing compounds classified as nonpeptides, small peptides, CXCR4 antagonists or specific antibodies whose activity reduces or completely blocks the intracellular signaling pathways and cell proliferation. We then examined antibodies and fusion proteins against CD47, the receptor that acts as a “do not eat me” signal to phagocytes escaping immune surveillance. Despite these molecules being tested in early clinical trials, some drawbacks are emerging that impair their use in practice. Finally, we examined the ImmunoGenic Surrender mechanism that involves crosstalk and co-internalization of CXCR4 and CD47 upon engagement of CXCR4 by ligands or other molecules. The favorable effect of such compounds is dual as CD47 surface reduction impact on the immune response adds to the block of CXCR4 proliferative potential. These results suggest that a combination of different therapeutic approaches has more beneficial effects on patients’ survival and may pave the way for new accomplishments in personalized anticancer therapy. Full article

Diabetic retinopathy (DR) is a chronic disease associated with diabetes mellitus and is a leading cause of visual impairment among the working population in the US. Clinically, DR has been diagnosed and treated as a vascular complication, but it adversely impacts both neural retina and retinal vasculature. Degeneration of retinal neurons and microvasculature manifests in the diabetic retina and early stages of DR. Retinal photoreceptors undergo apoptosis shortly after the onset of diabetes, which contributes to the retinal dysfunction and microvascular complications leading to vision impairment. Chronic inflammation is a hallmark of diabetes and a contributor to cell apoptosis, and retinal photoreceptors are a major source of intraocular inflammation that contributes to vascular abnormalities in diabetes. As the levels of microRNAs (miRs) are changed in the plasma and vitreous of diabetic patients, miRs have been suggested as biomarkers to determine the progression of diabetic ocular diseases, including DR. However, few miRs have been thoroughly investigated as contributors to the pathogenesis of DR. Among these miRs, miR-150 is downregulated in diabetic patients and is an endogenous suppressor of inflammation, apoptosis, and pathological angiogenesis. In this review, how miR-150 and its downstream targets contribute to diabetes-associated retinal degeneration and pathological angiogenesis in DR are discussed. Currently, there is no effective treatment to stop or reverse diabetes-caused neural and vascular degeneration in the retina. Understanding the molecular mechanism of the pathogenesis of DR may shed light for the future development of more effective treatments for DR and other diabetes-associated ocular diseases. Full article

Metabolic disorders, mainly characterized as the marked alteration of the lipid and carbohydrate profile, in addition to the clinical presence of the direct consequences of these alterations, are pathological conditions that have considerably increased in prevalence in recent years. They are directly linked to the onset of various pathologies, including cancer, particularly breast cancer, and are hormone-responsive. Alongside the known conditions responsible for this scenario, such as nutrition and lifestyle in general, the importance of both the colonic microbiota and the various organs and systems is becoming increasingly evident. In fact, it is now evident that microbial dysbiosis plays a fundamental role in the onset of these metabolic disorders, and therefore how these conditions are indirectly responsible for the onset and progression of neoplasms. Indirect mechanisms such as an altered Firmicutes/Bacteroidetes ratio; the formation of metabolites such as short-chain fatty acids (SCFAs), in particular, butyrate, which is capable of acting as a tumor suppressor; and the glucuronidase activity of estroboloma (bacteria responsible for estrogen metabolism) are just some of the most important mechanisms that contribute to the history of breast cancer. It is therefore understandable that in clinical terms, it is essential to associate the modulation of metabolic disorders and the microbial conditions that contribute to generating them with common therapies, preferably using compounds and solutions that are effective and acceptable for the patient without side effects. Nutraceuticals such as berberine (active both in metabolic scenarios and in the microbiota) and interventions modulating the microbial structure such as the use of probiotics and prebiotics seem to be ideal solutions for these preventive and no-longer-ignorable strategies in the light of numerous data now present in the literature. Full article

The most prevalent malignant bone tumor, osteosarcoma, affects the growth plates of long bones in adolescents and young adults. Standard chemotherapeutic methods showed poor response rates in patients with recurrent and metastatic phases. Therefore, it is critical to develop novel and efficient targeted therapies to address relapse cases. In this regard, RNA interference technologies are encouraging options in cancer treatment, in which small interfering RNAs regulate the gene expression following RNA interference pathways. The determination of target tissue is as important as the selection of tissue-specific promoters. Moreover, small interfering RNAs should be delivered effectively into the cytoplasm. Lentiviral vectors could encapsulate and deliver the desired gene into the cell and integrate it into the genome, providing long-term regulation of targeted genes. Silencing overexpressed genes promote the tumor cells to lose invasiveness, prevents their proliferation, and triggers their apoptosis. The uniqueness of cancer cells among patients requires novel therapeutic methods that treat patients based on their unique mutations. Several studies showed the effectiveness of different approaches such as microRNA, drug- or chemotherapy-related methods in treating the disease; however, identifying various targets was challenging to understanding disease progression. In this regard, the patient-specific abnormal gene might be targeted using genomics and molecular advancements such as RNA interference approaches. Here, we review potential therapeutic targets for the RNA interference approach, which is applicable as a therapeutic option for osteosarcoma patients, and we point out how the small interfering RNA method becomes a promising approach for the unmet challenge. Full article

Protein arginine methyltransferase 7 (PRMT7) regulates various cellular responses, including gene expression, cell migration, stress responses, and stemness. In this study, we investigated the biological role of PRMT7 in cell cycle progression and DNA damage response (DDR) by inhibiting PRMT7 activity with either SGC8158 treatment or its specific siRNA transfection. Suppression of PRMT7 caused cell cycle arrest at the G₁ phase, resulting from the stabilization and subsequent accumulation of p21 protein. In addition, PRMT7 activity is closely associated with DNA repair pathways, including both homologous recombination and non-homologous end-joining. Interestingly, SGC8158, in combination with doxorubicin, led to a synergistic increase in both DNA damage and cytotoxicity in MCF7 cells. Taken together, our data demonstrate that PRMT7 is a critical modulator of cell growth and DDR, indicating that it is a promising target for cancer treatment. Full article

E2F4 was initially described as a transcription factor with a key function in the regulation of cell quiescence. Nevertheless, a number of recent studies have established that E2F4 can also play a relevant role in cell and tissue homeostasis, as well as tissue regeneration. For these non-canonical functions, E2F4 can also act in the cytoplasm, where it is able to interact with many homeostatic and synaptic regulators. Since E2F4 is expressed in the nervous system, it may fulfill a crucial role in brain function and homeostasis, being a promising multifactorial target for neurodegenerative diseases and brain aging. The regulation of E2F4 is complex, as it can be chemically modified through acetylation, from which we present evidence in the brain, as well as methylation, and phosphorylation. The phosphorylation of E2F4 within a conserved threonine motif induces cell cycle re-entry in neurons, while a dominant negative form of E2F4 (E2F4DN), in which the conserved threonines have been substituted by alanines, has been shown to act as a multifactorial therapeutic agent for Alzheimer’s disease (AD). We generated transgenic mice neuronally expressing E2F4DN. We have recently shown using this mouse strain that expression of E2F4DN in 5xFAD mice, a known murine model of AD, improved cognitive function, reduced neuronal tetraploidization, and induced a transcriptional program consistent with modulation of amyloid-β (Aβ) peptide proteostasis and brain homeostasis recovery. 5xFAD/E2F4DN mice also showed reduced microgliosis and astrogliosis in both the cerebral cortex and hippocampus at 3-6 months of age. Here, we analyzed the immune response in 1 year-old 5xFAD/E2F4DN mice, concluding that reduced microgliosis and astrogliosis is maintained at this late stage. In addition, the expression of E2F4DN also reduced age-associated microgliosis in wild-type mice, thus stressing its role as a brain homeostatic agent. We conclude that E2F4DN transgenic mice represent a promising tool for the evaluation of E2F4 as a therapeutic target in neuropathology and brain aging. Full article

Proteomics has been widely used to study muscle biology and meat quality traits from different species including beef. Beef proteomics studies allow a better understanding of the biological processes related to meat quality trait determination. This study aimed to decipher by means of two-dimensional electrophoresis (2D-PAGE), mass spectrometry and bioinformatics the changes in post-mortem muscle with a focus on proteins differentially expressed in the Longissimus thoracis (LT) muscle of immunocastrated young heifers and steers. Carcass traits, chemical composition, pH, instrumental color (L*, a*, b*), cooking loss and Warner-Bratzler shear force (WBSF) of meat from F1 Montana-Nellore cattle were also evaluated. Backfat thickness (BFT) and intramuscular fat content (IMF) were 46.8% and 63.6% higher in heifers (p < 0.05), respectively, while evaporation losses (EL) were 10.22% lower compared to steers. No differences (p > 0.05) were observed for tenderness evaluated by WBSF (3, 10, and 17 days post-mortem), pH, and color traits (L*, a* and b*) between the experimental groups. The study revealed several proteins to be differentially expressed proteins in heifers compared steers (p < 0.05). In heifers, proteins involved in nutrient transport (TF, ALB, and MB), energy metabolism (ALDOA, GAPDH, and PKM), and oxidative stress and response to stress (HSPA8 and CA3) were associated with a greater BFT and IMF deposition. The higher expression of these proteins indicated greater oxidative capacity and lower glycolytic activity in the LT muscle of heifers. In steers, there was greater abundance of protein expression related to muscle contraction and proteins of structure (ACTA1, TPM2 and TNNT3), energy metabolism (ENO1, ENO3, PYGM, PGM1 and TPI1) and ATP metabolism (ATP5F1B, PEBP1 and AK1), indicating greater glycogenolysis in LT muscle, suggesting a shift in the glycolytic/oxidative fibers of steers. Full article

Cumulative studies have indicated that high-dose vitamin C has antitumor effects against a variety of cancers. However, the molecular mechanisms underlying these inhibitory effects against tumorigenesis and metastasis, particularly in relation to pancreatic cancer, are unclear. Here, we report that vitamin C at high concentrations impairs the growth and survival of pancreatic ductal adenocarcinoma (PDAC) cells by inhibiting glucose metabolism. Vitamin C was also found to trigger apoptosis in a caspase-independent manner. We further demonstrate that it suppresses the invasion and metastasis of PDAC cells by inhibiting the Wnt/β-catenin-mediated epithelial-mesenchymal transition (EMT). Taken together, our results suggest that vitamin C has therapeutic effects against pancreatic cancer. Full article

Ovarian cancer is the fifth leading cause of cancer, followed by front line is mostly platinum agents and PARP inhibitors, and very limited option in later lines. Therefore, there is a need for alternative therapeutic options. Nectin-2, which is overexpressed in ovarian cancer, is a known immune checkpoint that deregulates immune cell function. In this study, we generated a novel anti-nectin-2 antibody (chimeric 12G1, c12G1), and further characterized it using epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization assays. The c12G1 antibody specifically bound to the C2 domain of human nectin-2 with high affinity (K_D = 2.90 × 10⁻¹⁰ M), but not to mouse nectin-2. We then generated an antibody-drug conjugate comprising the c12G1 antibody conjugated to DM1 and investigated its cytotoxic effects against cancer cells in vitro and in vivo. c12G1-DM1 induced cell cycle arrest at the mitotic phase in nectin-2-positive ovarian cancer cells, but not in nectin-2-negative cancer cells. c12G1-DM1 induced ~100-fold cytotoxicity in ovarian cancer cells, with an IC₅₀ in the range of 0.1 nM~7.4 nM, compared to normal IgG-DM1. In addition, c12G1-DM1 showed ~91% tumor growth inhibition in mouse xenograft models transplanted with OV-90 cells. These results suggest that c12G1-DM1 could be used as a potential therapeutic agent against nectin-2-positive ovarian cancers. Full article