CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8⁺ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19–MIC25–MIC60 MICOS subcomplex that regulates cristae morphology. Full article

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography–mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD. Full article

Activin A (ActA) is considered to play a major role in cancer-induced cachexia (CC). Indeed, circulating ActA levels are elevated and predict survival in patients with CC. However, the mechanisms by which ActA mediates CC development and in particular skeletal muscle (SM) atrophy in humans are not yet fully understood. In this work, we aimed to investigate the effects of ActA on human SM and in mouse models of CC. We used a model of human muscle cells in culture to explore how ActA acts towards human SM. In this model, recombinant ActA induced myotube atrophy associated with the decline of MyHC-β/slow, the main myosin isoform in human muscle cells studied. Moreover, ActA inhibited the expression and activity of MEF2C, the transcription factor regulating MYH7, the gene which codes for MyHC-β/slow. This decrease in MEF2C was involved in the decline of MyHC-β/slow expression, since inhibition of MEF2C by a siRNA leads to the decrease in MyHC-β/slow expression. The relevance of this ActA/MEF2C pathway in vivo was supported by the parallel decline of MEF2C expression and SM mass, which are both blunted by ActA inhibition, in animal models of CC. In this work, we showed that ActA is a potent negative regulator of SM mass by inhibiting MyHC-β/slow synthesis through downregulation of MEF2C. This observation highlights a novel interaction between ActA signaling and MEF2C transcriptional activity which contributes to SM atrophy in CC models. Full article

Although neuropsychiatric symptoms (NPS) are common and severely affect older people with cognitive decline, little is known about their underlying molecular mechanisms and relationships with Alzheimer’s disease (AD). The aim of this study was to identify and characterize cerebrospinal fluid (CSF) proteome alterations related to NPS. In a longitudinally followed-up cohort of subjects with normal cognition and patients with cognitive impairment (MCI and mild dementia) from a memory clinic setting, we quantified a panel of 790 proteins in CSF using an untargeted shotgun proteomic workflow. Regression models and pathway enrichment analysis were used to investigate protein alterations related to NPS, and to explore relationships with AD pathology and cognitive decline at follow-up visits. Regression analysis selected 27 CSF proteins associated with NPS. These associations were independent of the presence of cerebral AD pathology (defined as CSF p-tau181/Aβ1–42 > 0.0779, center cutoff). Gene ontology enrichment showed abundance alterations of proteins related to cell adhesion, immune response, and lipid metabolism, among others, in relation to NPS. Out of the selected proteins, three were associated with accelerated cognitive decline at follow-up visits after controlling for possible confounders. Specific CSF proteome alterations underlying NPS may both represent pathophysiological processes independent from AD and accelerate clinical disease progression. Full article

The aim of this study was to validate the use of human brain organoids (hBOs) to investigate the therapeutic potential and mechanism of human-neural-crest-derived nasal turbinate stem cells (hNTSCs) in models of Alzheimer’s disease (AD). We generated hBOs from human induced pluripotent stem cells, investigated their characteristics according to neuronal markers and electrophysiological features, and then evaluated the protective effect of hNTSCs against amyloid-β peptide (Aβ_1–42) neurotoxic activity in vitro in hBOs and in vivo in a mouse model of AD. Treatment of hBOs with Aβ_1–42 induced neuronal cell death concomitant with decreased expression of neuronal markers, which was suppressed by hNTSCs cocultured under Aβ_1–42 exposure. Cytokine array showed a significantly decreased level of osteopontin (OPN) in hBOs with hNTSC coculture compared with hBOs only in the presence of Aβ_1–42. Silencing OPN via siRNA suppressed Aβ-induced neuronal cell death in cell culture. Notably, compared with PBS, hNTSC transplantation significantly enhanced performance on the Morris water maze, with reduced levels of OPN after transplantation in a mouse model of AD. These findings reveal that hBO models are useful to evaluate the therapeutic effect and mechanism of stem cells for application in treating AD. Full article

Background: Hepatitis C virus (HCV) constitutes a global health problem, while hepatitis E virus (HEV) is the major cause of acute viral hepatitis globally. HCV/HEV co-infections have been poorly characterized, as they are hampered by the lack of robust HEV cell culture systems. This study developed experimental models to study HCV/HEV co-infections and investigate viral interference in cells and humanized mice. Methods: We used state-of-the art human hepatocytes tissue culture models to assess HEV and HCV replication in co- or super-transfection settings. Findings were confirmed by co- and super-infection experiments in human hepatocytes and in vivo in human liver chimeric mice. Results: HEV was inhibited by concurrent HCV replication in human hepatocytes. This exclusion phenotype was linked to the protease activity of HCV. These findings were corroborated by the fact that in HEV on HCV super-infected mice, HEV viral loads were reduced in individual mice. Similarly, HCV on HEV super-infected mice showed reduced HCV viral loads. Conclusion: Direct interference of both viruses with HCV NS3/4A as the determinant was observed. In vivo, we detected reduced replication of both viruses after super-infection in individual mice. These findings provide new insights into the pathogenesis of HCV-HEV co-infections and should contribute to its clinical management in the future. Full article

Aggregated alpha-synuclein (α-synuclein) is the main component of Lewy bodies (LBs), Lewy neurites (LNs), and glial cytoplasmic inclusions (GCIs), which are pathological hallmarks of idiopathic Parkinson’s disease (IPD) and multiple system atrophy (MSA). Initiating factors that culminate in forming LBs/LNs/GCIs remain elusive. Several species of α-synuclein exist, including phosphorylated and nitrated forms. It is unclear which α-synuclein post-translational modifications (PTMs) appear within aggregates throughout disease pathology. Herein we aimed to establish the predominant α-synuclein PTMs in postmortem IPD and MSA pathology using immunohistochemistry. We examined the patterns of three α-synuclein PTMs (pS87, pS129, nY39) simultaneously in pathology-affected regions of 15 IPD cases, 5 MSA cases, and 6 neurologically normal controls. All antibodies recognized LBs, LNs, and GCIs, albeit to a variable extent. pS129 α-synuclein antibody was particularly immunopositive for LNs and synaptic dot-like structures, followed by nY39 α-synuclein antibody. GCIs, neuronal inclusions, and small threads were positive for nY39 α-synuclein in MSA. Quantification of the LB scores revealed that pS129 α-synuclein was the dominant and earliest α-synuclein PTM, followed by nY39 α-synuclein, while lower amounts of pSer87 α-synuclein appeared later in disease progression in PD. These results may have implications for novel biomarker and therapeutic developments. Full article

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis. Full article

Background and aims: Non-alcoholic steatohepatitis (NASH) is a life-threatening stage of non-alcoholic fatty liver disease (NAFLD) for which no drugs have been approved. We have previously shown that human-derived hepatic in vitro models can be used to mimic key cellular mechanisms involved in the progression of NASH. In the present study, we first characterize the transcriptome of multiple in vitro NASH models. Subsequently, we investigate how elafibranor, which is a peroxisome proliferator-activated receptor (PPAR)-α/δ agonist that has recently failed a phase 3 clinical trial as a potential anti-NASH compound, modulates the transcriptome of these models. Finally, we compare the elafibranor-induced gene expression modulation to transcriptome data of patients with improved/resolved NAFLD/NASH upon bariatric surgery, which is the only proven clinical NASH therapy. Methods: Human whole genome microarrays were used for the transcriptomics evaluation of hepatic in vitro models. Comparison to publicly available clinical datasets was conducted using multiple bioinformatic application tools. Results: Primary human hepatocytes (PHH), HepaRG, and human skin stem cell-derived hepatic progenitors (hSKP-HPC) exposed to NASH-inducing triggers exhibit up to 35% overlap with datasets of liver samples from NASH patients. Exposure of the in vitro NASH models to elafibranor partially reversed the transcriptional modulations, predicting an inhibition of toll-like receptor (TLR)-2/4/9-mediated inflammatory responses, NFκB-signaling, hepatic fibrosis, and leukocyte migration. These transcriptomic changes were also observed in the datasets of liver samples of patients with resolved NASH. Peroxisome Proliferator Activated Receptor Alpha (PPARA), PPARG Coactivator 1 Alpha (PPARGC1A), and Sirtuin 1 (SIRT1) were identified as the major common upstream regulators upon exposure to elafibranor. Analysis of the downstream mechanistic networks further revealed that angiopoietin Like 4 (ANGPTL4), pyruvate dehydrogenase kinase 4 (PDK4), and perilipin 2 (PLIN2), which are involved in the promotion of hepatic lipid accumulation, were also commonly upregulated by elafibranor in all in vitro NASH models. Contrarily, these genes were not upregulated in liver samples of patients with resolved NASH. Conclusion: Transcriptomics comparison between in vitro NASH models exposed to elafibranor and clinical datasets of NAFLD patients after bariatric surgery reveals commonly modulated anti-inflammatory responses, but discordant modulations of key factors in lipid metabolism. This discordant adverse effect of elafibranor deserves further investigation when assessing PPAR-α/δ agonism as a potential anti-NASH therapy. Full article

Along with insulin resistance and increased risk of type 2 diabetes (T2D), lean first-degree relatives of T2D subjects (FDR) feature impaired adipogenesis in subcutaneous adipose tissue (SAT) and subcutaneous adipocyte hypertrophy well before diabetes onset. The molecular mechanisms linking these events have only partially been clarified. In the present report, we show that silencing of the transcription factor Homeobox A5 (HOXA5) in human preadipocytes impaired differentiation in mature adipose cells in vitro. The reduced adipogenesis was accompanied by inappropriate WNT-signaling activation. Importantly, in preadipocytes from FDR individuals, HOXA5 expression was attenuated, with hypermethylation of the HOXA5 promoter region found responsible for its downregulation, as revealed by luciferase assay. Both HOXA5 gene expression and DNA methylation were significantly correlated with SAT adipose cell hypertrophy in FDR, whose increased adipocyte size marks impaired adipogenesis. In preadipocytes from FDR, the low HOXA5 expression negatively correlated with enhanced transcription of the WNT signaling downstream genes NFATC1 and WNT2B. In silico evidence indicated that NFATC1 and WNT2B were directly controlled by HOXA5. The HOXA5 promoter region also was hypermethylated in peripheral blood leukocytes from these same FDR individuals, which was further revealed in peripheral blood leukocytes from an independent group of obese subjects. Thus, HOXA5 controlled adipogenesis in humans by suppressing WNT signaling. Altered DNA methylation of the HOXA5 promoter contributed to restricted adipogenesis in the SAT of lean subjects who were FDR of type 2 diabetics and in obese individuals. Full article

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species. Full article

Apart from its role in the metabolism of carcinogens, the aryl hydrocarbon receptor (AhR) has been suggested to be involved in the control of inflammatory responses within the respiratory tract. However, the mechanisms responsible for this are only partially known. In this study, we used A549 cell line, as a human model of lung alveolar type II (ATII)-like cells, to study the functional role of the AhR in control of inflammatory responses. Using IL-1β as an inflammation inducer, we found that the induction of cyclooxygenase-2 and secretion of prostaglandins, as well as expression and release of pro-inflammatory cytokines, were significantly higher in the AhR-deficient A549 cells. This was linked with an increased nuclear factor-κB (NF-κB) activity, and significantly enhanced phosphorylation of its regulators, IKKα/β, and their target IκBα, in the AhR-deficient A549 cells. In line with this, when we mimicked the exposure to a complex mixture of airborne pollutants, using an organic extract of reference diesel exhaust particle mixture, an exacerbated inflammatory response was observed in the AhR-deficient cells, as compared with wild-type A549 cells. Together, the present results indicate that the AhR may act as a negative regulator of the inflammatory response in the A549 model, via a direct modulation of NF-κB signaling. Its role(s) in the control of inflammation within the lung alveoli exposed to airborne pollutants, especially those which simultaneously activate the AhR, thus deserve further attention. Full article

Advances in sequencing and assembly technology have led to the creation of genome assemblies for a wide variety of non-model organisms. The rapid production and proliferation of updated, novel assembly versions can create vexing problems for researchers when multiple-genome assembly versions are available at once, requiring researchers to work with more than one reference genome. Multiple-genome assemblies are especially problematic for researchers studying the genetic makeup of individual cells, as single-cell RNA sequencing (scRNAseq) requires sequenced reads to be mapped and aligned to a single reference genome. Using the Astyanax mexicanus, this study highlights how the interpretation of a single-cell dataset from the same sample changes when aligned to its two different available genome assemblies. We found that the number of cells and expressed genes detected were drastically different when aligning to the different assemblies. When the genome assemblies were used in isolation with their respective annotations, cell-type identification was confounded, as some classic cell-type markers were assembly-specific, whilst other genes showed differential patterns of expression between the two assemblies. To overcome the problems posed by multiple-genome assemblies, we propose that researchers align to each available assembly and then integrate the resultant datasets to produce a final dataset in which all genome alignments can be used simultaneously. We found that this approach increased the accuracy of cell-type identification and maximised the amount of data that could be extracted from our single-cell sample by capturing all possible cells and transcripts. As scRNAseq becomes more widely available, it is imperative that the single-cell community is aware of how genome assembly alignment can alter single-cell data and their interpretation, especially when reviewing studies on non-model organisms. Full article

Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy. Full article

Although the optimal therapy for myocardial infarction includes reperfusion to restore blood flow to the ischemic area, myocardial injury after ischemia/reperfusion usually leads to an inflammatory response, oxidative stress, and cardiomyocyte apoptosis. In this study, rat adipose-derived stem cells were differentiated into low-thermogenic beige adipocytes (LBACs) and high-thermogenic beige adipocytes (HBACs) to study the different cardioprotective effects of heterogeneous expression of brown adipocytes. We found that antioxidant and antiapoptotic factors in H9c2 cardiomyocytes were upregulated by high levels of secreted FGF21 in HBAC conditioned medium (HBAC-CM), whereas FGF21 in HBAC-CM did not affect antioxidative or antiapoptotic cell death in H9c2 cardiomyocytes with Nrf2 knockdown. These results show that NRF2 mediates antioxidative and antiapoptotic effects through the HBAC-secreted factor FGF21. Consistent with this finding, the expression of antioxidant and antiapoptotic genes was upregulated by highly secreted FGF21 after HBAC-CM treatment compared to LBAC-CM treatment in H9c2 cardiomyocytes via NRF2 activation. Furthermore, HBAC-CM significantly attenuated ischemic rat heart tissue injury via NRF2 activation. Based on these findings, we propose that HBAC-CM exerts beneficial effects in rat cardiac ischemia/reperfusion injury by modulating NRF2 and has potential as a promising therapeutic agent for myocardial infarction. Full article

Mitochondria–endoplasmic reticulum (ER) contact sites (MERCS) have been emerging as a multifaceted subcellular region of the cell which affects several physiological and pathological mechanisms. A thus far underexplored aspect of MERCS is their contribution to exocytosis. Here, we set out to understand the role of these contacts in exocytosis and find potential mechanisms linking these structures to vesicle release in human neuroblastoma SH-SY5Y cells. We show that increased mitochondria to ER juxtaposition through Mitofusin 2 (Mfn2) knock-down resulted in a substantial upregulation of the number of MERCS, confirming the role of Mfn2 as a negative regulator of these structures. Furthermore, we report that both vesicle numbers and vesicle protein levels were decreased, while a considerable upregulation in exocytotic events upon cellular depolarization was detected. Interestingly, in Mfn2 knock-down cells, the inhibition of the inositol 1,4,5-trisphosphate receptor (IP3R) and the mitochondrial calcium (Ca²⁺) uniporter (MCU) restored vesicle protein content and attenuated exocytosis. We thus suggest that MERCS could be targeted to prevent increased exocytosis in conditions in which ER to mitochondria proximity is upregulated. Full article

Mesenchymal stromal cells (MSC) loaded on biphasic calcium phosphate biomaterial (MSC + BCP) have been used as an advanced therapy medicinal product to treat complex maxillofacial bone defects in patients. Further, MSC-derived extracellular vesicles (EVs) are established vehicles of paracrine factors, supporting inter-cellular communication between MSC and other interacting cell types, such as monocytes/macrophages. However, the information about the immunomodulatory potential of EVs derived from MSC and biomaterial constructs (MSC + BCP:EV) and inflammatory primed constructs (MSCp + BCP:EV) are scarce. Hence, we isolated and characterized EVs from these different systems, and compared their cytokine contents with plastic-adherent MSC-derived EVs (MSC:EV). When EVs from all three MSC systems were added to the primary blood-derived macrophages in vitro, significantly higher numbers of M0 (naive) macrophages shifted to M2-like (anti-inflammatory) by MSCp + BCP:EV treatment. Further, this treatment led to enhanced switching of M1 polarized macrophages to M2 polarized, and conversely, M2 to M1, as evaluated by determining the M1/M2 ratios after treatment. The enhanced macrophage modulation by MSCp + BCP:EV was attributed to their higher immunomodulatory (TNFα, IL1β, IL5), angiogenic (VEGF), and chemokine-rich (RANTES, MCP1, MIP1β) cytokine cargo. In conclusion, we successfully isolated and characterized EVs from MSC + BCP constructs and demonstrated that, depending upon the tissue microenvironment, these EVs contribute towards modulating the macrophage-mediated inflammation and healing responses. The study offers new insights into the use of biomaterial-induced EVs for MSC secretome delivery, as a step towards future ‘cell-free’ bone regenerative therapies. Full article

Amniotic epithelial stem cells (AECs) are largely studied for their pro-regenerative properties. However, it remains undetermined if low oxygen (O₂) levels that AECs experience in vivo can be of value in maintaining their biological properties after isolation. To this aim, the present study has been designed to evaluate the effects of a hypoxia-mimetic agent, cobalt chloride (CoCl₂), on AECs’ stemness and angiogenic activities. First, a CoCl₂ dose-effect was performed to select the concentration able to induce hypoxia, through HIF-1α stabilization, without promoting any cytotoxicity effect assessed through the analysis of cell vitality, proliferation, and apoptotic-related events. Then, the identified CoCl₂ dose was evaluated on the expression and angiogenic properties of AECs’ stemness markers (OCT-4, NANOG, SOX-2) by analysing VEGF expression, angiogenic chemokines’ profiles, and AEC-derived conditioned media activity through an in vitro angiogenic xeno-assay. Results demonstrated that AECs are sensitive to the cytotoxicity effects of CoCl₂. The unique concentration leading to HIF-1α stabilization and nuclear translocation was 10 µM, preserving cell viability and proliferation up to 48 h. CoCl₂ exposure did not modulate stemness markers in AECs while progressively decreasing VEGF expression. On the contrary, CoCl₂ treatment promoted a significant short-term release of angiogenic chemokines in culture media (CM). The enrichment in bio-active factors was confirmed by the ability of CoCl₂-derived CM to induce HUVEC growth and the cells’ organization in tubule-like structures. These findings demonstrate that an appropriate dose of CoCl₂ can be adopted as a hypoxia-mimetic agent in AECs. The short-term, chemical-induced hypoxic condition can be targeted to enhance AECs’ pro-angiogenic properties by providing a novel approach for stem cell-free therapy protocols. Full article

The leptin receptor (LepR) acts as a signaling nexus for the regulation of glucose uptake and obesity, among other metabolic responses. The functional role of LepR under leptin-deficient conditions remains unclear. This study reports that epiregulin (EREG) governed glucose uptake in vitro and in vivo in Lep^ob mice by activating LepR under leptin-deficient conditions. Single and long-term treatment with EREG effectively rescued glucose intolerance in comparative insulin and EREG tolerance tests in Lep^ob mice. The immunoprecipitation study revealed binding between EREG and LepR in adipose tissue of Lep^ob mice. EREG/LepR regulated glucose uptake without changes in obesity in Lep^ob mice via mechanisms, including ERK activation and translocation of GLUT4 to the cell surface. EREG-dependent glucose uptake was abolished in Lepr^db mice which supports a key role of LepR in this process. In contrast, inhibition of the canonical epidermal growth factor receptor (EGFR) pathway implicated in other EREG responses, increased glucose uptake. Our data provide a basis for understanding glycemic responses of EREG that are dependent on LepR unlike functions mediated by EGFR, including leptin secretion, thermogenesis, pain, growth, and other responses. The computational analysis identified a conserved amino acid sequence, supporting an evolutionary role of EREG as an alternative LepR ligand. Full article

Background: Atherosclerosis is a chronic inflammatory disease where macrophages participate in the progression of the disease. However, the role of resident-like macrophages (res-like) in the atherosclerotic aorta is not completely understood. Methods: A single-cell RNA sequencing analysis of CD45⁺ leukocytes in the atherosclerotic aorta of apolipoprotein E–deficient (Apoe^−/−) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the side-to-specific predisposition to atherosclerosis, was performed. A population of res-like macrophages expressing hyaluronan receptor LYVE-1 was investigated via flow cytometry, co-culture experiments, and immunofluorescence in human atherosclerotic plaques from carotid artery disease patients (CAD). Results: We identified 12 principal leukocyte clusters with distinct atherosclerosis disease-relevant gene expression signatures. LYVE-1⁺ res-like macrophages, expressing a high level of CC motif chemokine ligand 24 (CCL24, eotaxin-2), expanded under hypercholesteremia in Apoe^−/− mice and promoted VSMC phenotypic modulation to osteoblast/chondrocyte-like cells, ex vivo, in a CCL24-dependent manner. Moreover, the abundance of LYVE-1⁺CCL24⁺ macrophages and elevated systemic levels of CCL24 were associated with vascular calcification and CAD events. Conclusions: LYVE-1 res-like macrophages, via the secretion of CCL24, promote the transdifferentiation of VSMC to osteogenic-like cells with a possible role in vascular calcification and likely a detrimental role in atherosclerotic plaque destabilization. Full article

Alzheimer’s disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ_1-40 and Aβ_1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ_1-40 and Aβ_1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ_1-42 had more adverse effects than Aβ_1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ_1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ_1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ_1-40 and Aβ_1-42 significantly increased Ca²⁺ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells. Full article

Exposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP₃R2-Ca²⁺-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation. Full article

Mast cells (MCs) function as a component of the tumor microenvironment (TME) and have both pro- and anti-tumorigenic roles depending on the tumor type and its developmental stage. Several reports indicate the involvement of MCs in angiogenesis in the TME by releasing angiogenic mediators. Tumor cells and other cells in the TME may interact by releasing extracellular vesicles (EVs) that affect the cells in the region. We have previously shown that tumor-derived microvesicles (TMVs) from non-small-cell lung cancer (NSCLC) cells interact with human MCs and activate them to release several cytokines and chemokines. In the present study, we characterized the MC expression of other mediators after exposure to TMVs derived from NSCLC. Whole-genome expression profiling disclosed the production of several chemokines, including CC chemokine ligand 18 (CCL18). This chemokine is expressed in various types of cancer, and was found to be associated with extensive angiogenesis, both in vitro and in vivo. We now show that CCL18 secreted from MCs activated by NSCLC-TMVs increased the migration of human umbilical cord endothelial cells (HUVECs), tube formation and endothelial- to-mesenchymal transition (EndMT), thus promoting angiogenesis. Our findings support the conclusion that TMVs have the potential to influence MC activity and may affect angiogenesis in the TME. Full article

Niemann-Pick type C1 (NP-C1) is a fatal, progressive neurodegenerative disease caused by mutations in the NPC1 gene. Mutations of NPC1 can result in a misfolded protein that is subsequently marked for proteasomal degradation. Such loss-of-function mutations lead to cholesterol accumulation in late endosomes and lysosomes. Pharmacological chaperones (PCs) are described to protect misfolded proteins from proteasomal degradation and are being discussed as a treatment strategy for NP-C1. Here, we used a combinatorial approach of high-throughput in silico screening of FDA-approved drugs and in vitro biochemical assays to identify potential PCs. The effects of the hit compounds identified by molecular docking were compared in vitro with 25-hydroxycholesterol (25-HC), which is known to act as a PC for NP-C1. We analyzed cholesterol accumulation, NPC1 protein content, and lysosomal localization in patient-specific fibroblasts, as well as in neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells (iPSCs). One compound, namely abiraterone acetate, showed comparable results to 25-HC and restored NPC1 protein level, corrected the intracellular localization of NPC1, and consequently decreased cholesterol accumulation in NPC1-mutated fibroblasts and iPSC-derived neural differentiated and hepatocyte-like cells. The discovered PC altered not only the pathophysiological phenotype of cells carrying the I1061T mutation— known to be responsive to treatment with PCs—but an effect was also observed in cells carrying other NPC1 missense mutations. Therefore, we hypothesize that the PCs studied here may serve as an effective treatment strategy for a large group of NP-C1 patients. Full article

Advances in the regenerative stem cell field have propelled the generation of tissue-specific cells in the culture dish for subsequent transplantation, drug screening purposes, or the elucidation of disease mechanisms. One major obstacle is the heterogeneity of these cultures, in which the tissue-specific cells of interest usually represent only a fraction of all generated cells. Direct identification of the cells of interest and the ability to specifically isolate these cells in vitro is, thus, highly desirable for these applications. The type VI intermediate filament protein NESTIN is widely used as a marker for neural stem/progenitor cells (NSCs/NPCs) in the developing and adult central and peripheral nervous systems. Applying CRISPR-Cas9 technology, we have introduced a red fluorescent reporter (mScarlet) into the NESTIN (NES) locus of a human induced pluripotent stem cell (hiPSC) line. We describe the generation and characterization of NES-mScarlet reporter hiPSCs and demonstrate that this line is an accurate reporter of NSCs/NPCs during their directed differentiation into human midbrain dopaminergic (mDA) neurons. Furthermore, NES-mScarlet hiPSCs can be used for direct identification during live cell imaging and for flow cytometric analysis and sorting of red fluorescent NSCs/NPCs in this paradigm. Full article

Doxorubicin (Dox) is an effective antineoplastic drug with serious cardiotoxic side effects that persist after drug withdrawal and can lead to heart failure. Dysregulation of vascular endothelium has been linked to the development of Dox-induced cardiotoxicity, but it is unclear whether and how transient exposure to Dox leads to long-term downregulation of Endothelial Vascular Endothelial Growth Factor Receptor type2 (VEGFR2), essential for endothelial cells function. Using an in vitro model devised to study the long-lasting effects of brief endothelial cells exposure to Dox, we show that Dox leads to sustained protein synthesis inhibition and VEGFR2 downregulation. Transient Dox treatment led to the development of long-term senescence associated with a reduction in VEGFR2 levels that persisted days after drug withdrawal. By analyzing VEGFR2 turnover, we ruled out that its downregulation was depended on Dox-induced autophagy. Conversely, Dox induced p53 expression, reduced mTOR-dependent translation, and inhibited global protein synthesis. Our data contribute to a mechanistic basis to the permanent damage caused to endothelial cells by short-term Dox treatment. Full article

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors—classical LDLR and alternative LOX-1—in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis. Full article

Alzheimer’s disease (AD) is the most common form of dementia globally; however, the aetiology of AD remains elusive hindering the development of effective therapeutics. MicroRNAs (miRNAs) are regulators of gene expression and have been of growing interest in recent studies in many pathologies including AD not only for their use as biomarkers but also for their implications in the therapeutic field. In this study, miRNA and protein profiles were obtained from brain tissues of different stage (Braak III-IV and Braak V-VI) of AD patients and compared to matched controls. The aim of the study was to identify in the late stage of AD, the key dysregulated pathways that may contribute to pathogenesis and then to evaluate whether any of these pathways could be detected in the early phase of AD, opening new opportunity for early treatment that could stop or delay the pathology. Six common pathways were found regulated by miRNAs and proteins in the late stage of AD, with one of them (Rap1 signalling) activated since the early phase. MiRNAs and proteins were also compared to explore an inverse trend of expression which could lead to the identification of new therapeutic targets. These results suggest that specific miRNA changes could represent molecular fingerprint of neurodegenerative processes and potential therapeutic targets for early intervention. Full article

Hypermobile Ehlers-Danlos syndrome (hEDS) is the most frequent type of EDS and is characterized by generalized joint hypermobility and musculoskeletal manifestations which are associated with chronic pain, and mild skin involvement along with the presence of more than a few comorbid conditions. Despite numerous research efforts, no causative gene(s) or validated biomarkers have been identified and insights into the disease-causing mechanisms remain scarce. Variability in the spectrum and severity of symptoms and progression of hEDS patients’ phenotype likely depend on a combination of age, gender, lifestyle, and the probable multitude of genes involved in hEDS. However, considering the clinical overlap with other EDS forms, which lead to abnormalities in extracellular matrix (ECM), it is plausible that the mechanisms underlying hEDS pathogenesis also affect the ECM to a certain extent. Herein, we performed a series of in vitro studies on the secretome of hEDS dermal fibroblasts that revealed a matrix metalloproteinases (MMPs) dysfunction as one of the major disease drivers by causing a detrimental feedback loop of excessive ECM degradation coupled with myofibroblast differentiation. We demonstrated that doxycycline-mediated inhibition of MMPs rescues in hEDS cells a control-like ECM organization and induces a partial reversal of their myofibroblast-like features, thus offering encouraging clues for translational studies confirming MMPs as a potential therapeutic target in hEDS with the expectation to improve patients’ quality of life and alleviate their disabilities. Full article

Background: Ovarian cancer progression and invasiveness are promoted by a range of soluble factors released by cancer cells and stromal cells within the tumor microenvironment. Our previous studies demonstrated that resveratrol (RV), a nutraceutical and caloric restriction mimetic with tumor-suppressive properties, counteracts cancer cell motility induced by stromal IL-6 by upregulating autophagy. Lysophosphatidic acid (LPA), a bioactive phospholipid that shows elevated levels in the tumor microenvironment and the ascites of ovarian cancers, stimulates the growth and tissue invasion of cancer cells. Whether LPA elicits these effects by inhibiting autophagy and through which pathway and whether RV can counteract the same remain obscure. Aims: To investigate the molecular pathways involved in LPA-induced ovarian cancer malignancy, particularly focusing on the role of autophagy, and the ability of RV to counteract LPA activity. Results: LPA stimulated while RV inhibited ovarian cancer cell migration. Transcriptomic and bioinformatic analyses showed an opposite regulation by LPA and RV of genes linked to epithelial-to-mesenchymal transition (EMT) and autophagy with involvement of the PI3K-AKT, JAK-STAT and Hedgehog (Hh) pathways. LPA upregulated the Hh and EMT members GLI1, BMI-1, SNAIL-1 and TWIST1 and inhibited autophagy, while RV did the opposite. Similar to the inhibitors of the Hh pathway, RV inhibited LPA-induced cancer cell migration and 3D growth of ovarian cancer cells. BMI-1 silencing prevented LPA-induced EMT, restored autophagy and hampered cell migration, resembling the effects of RV. TCGA data analyses indicated that patients with low expression of Hh/EMT-related genes together with active autophagy flux tended to have a better prognosis and this correlates with a more effective response to platinum therapy. In in vitro 3D spheroids, LPA upregulated BMI-1, downregulated autophagy and inhibited platinum toxicity while RV and Hh inhibitors restored autophagy and favored BAX-mediated cell death in response to platinum. Conclusions: By inhibiting the Hh pathway and restoration of autophagy, RV counteracts LPA-induced malignancy, supporting its inclusion in the therapy of ovarian cancer for limiting metastasis and chemoresistance. Full article

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA. Full article

Endothelial dysfunction is a key hallmark of hypertension, which is a leading risk factor for cognitive decline in older adults with or without frailty. Similarly, hyperglycemia is known to impair endothelial function and is a predictor of severe cardiovascular outcomes, independent of the presence of diabetes. On these grounds, we designed a study to assess the effects of high-glucose and metformin on brain microvascular endothelial cells (ECs) and on cognitive impairment in frail hypertensive patients. We tested the effects of metformin on high-glucose-induced cell death, cell permeability, and generation of reactive oxygen species in vitro, in human brain microvascular ECs. To investigate the consequences of hyperglycemia and metformin in the clinical scenario, we recruited frail hypertensive patients and we evaluated their Montreal Cognitive Assessment (MoCA) scores, comparing them according to the glycemic status (normoglycemic vs. hyperglycemic) and the use of metformin. We enrolled 376 patients, of which 209 successfully completed the study. We observed a significant correlation between MoCA score and glycemia. We found that hyperglycemic patients treated with metformin had a significantly better MoCA score than hyperglycemic patients treated with insulin (18.32 ± 3.9 vs. 14.94 ± 3.8; p < 0.001). Our in vitro assays confirmed the beneficial effects of metformin on human brain microvascular ECs. To our knowledge, this is the first study correlating MoCA score and glycemia in frail and hypertensive older adults, showing that hyperglycemia aggravates cognitive impairment. Full article

The aim of this study was to investigate the kinetics of neutralizing antibodies (NAbs) and anti-SARS-CoV-2 anti-S-RBD IgGs up to three months after the second vaccination dose with the BNT162b2 mRNA vaccine. NAbs and anti-S-RBD levels were measured on days 1 (before the first vaccine shot), 8, 22 (before the second shot), 36, 50, and three months after the second vaccination (D111) (NCT04743388). 283 health workers were included in this study. NAbs showed a rapid increase from D8 to D36 at a constant rate of about 3% per day and reached a median (SD) of 97.2% (4.7) at D36. From D36 to D50, a slight decrease in NAbs values was detected and it became more prominent between D50 and D111 when the rate of decline was determined at −0.11 per day. The median (SD) NAbs value at D111 was 92.7% (11.8). A similar pattern was also observed for anti-S-RBD antibodies. Anti-S-RBDs showed a steeper increase during D22–D36 and a lower decline rate during D36–D111. Prior COVID-19 infection and younger age were associated with superior antibody responses over time. In conclusion, we found a persistent but declining anti-SARS-CoV-2 humoral immunity at 3 months following full vaccination with BNT162b2 in healthy individuals. Full article

The extracellular matrix (ECM) plays a key role in synaptogenesis and the regulation of synaptic functions in the central nervous system. Recent studies revealed that in addition to dopaminergic and serotoninergic neuromodulatory systems, microglia also contribute to the regulation of ECM remodeling. In the present work, we investigated the physiological role of microglia in the remodeling of perineuronal nets (PNNs), predominantly associated with parvalbumin-immunopositive (PV+) interneurons, and the perisynaptic ECM around pyramidal neurons in the hippocampus. Adult mice were treated with PLX3397 (pexidartinib), as the inhibitor of colony-stimulating factor 1 receptor (CSF1-R), to deplete microglia. Then, confocal analysis of the ECM and synapses was performed. Although the elimination of microglia did not alter the overall number or intensity of PNNs in the CA1 region of the hippocampus, it decreased the size of PNN holes and elevated the expression of the surrounding ECM. In the neuropil area in the CA1 str. radiatum, the depletion of microglia increased the expression of perisynaptic ECM proteoglycan brevican, which was accompanied by the elevated expression of presynaptic marker vGluT1 and the increased density of dendritic spines. Thus, microglia regulate the homeostasis of pre- and postsynaptic excitatory terminals and the surrounding perisynaptic ECM as well as the fine structure of PNNs enveloping perisomatic—predominantly GABAergic—synapses. Full article

Glomerulonephritis are renal inflammatory processes characterized by increased permeability of the Glomerular Filtration Barrier (GFB) with consequent hematuria and proteinuria. Glomerular endothelial cells (GEC) and podocytes are part of the GFB and contribute to the maintenance of its structural and functional integrity through the release of paracrine mediators. Activation of the complement cascade and pro-inflammatory cytokines (CK) such as Tumor Necrosis Factor α (TNF-α) and Interleukin-6 (IL-6) can alter GFB function, causing acute glomerular injury and progression toward chronic kidney disease. Endothelial Progenitor Cells (EPC) are bone-marrow-derived hematopoietic stem cells circulating in peripheral blood and able to induce angiogenesis and to repair injured endothelium by releasing paracrine mediators including Extracellular Vesicles (EVs), microparticles involved in intercellular communication by transferring proteins, lipids, and genetic material (mRNA, microRNA, lncRNA) to target cells. We have previously demonstrated that EPC-derived EVs activate an angiogenic program in quiescent endothelial cells and renoprotection in different experimental models. The aim of the present study was to evaluate in vitro the protective effect of EPC-derived EVs on GECs and podocytes cultured in detrimental conditions with CKs (TNF-α/IL-6) and the complement protein C5a. EVs were internalized in both GECs and podocytes mainly through a L-selectin-based mechanism. In GECs, EVs enhanced the formation of capillary-like structures and cell migration by modulating gene expression and inducing the release of growth factors such as VEGF-A and HGF. In the presence of CKs, and C5a, EPC-derived EVs protected GECs from apoptosis by decreasing oxidative stress and prevented leukocyte adhesion by inhibiting the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin). On podocytes, EVs inhibited apoptosis and prevented nephrin shedding induced by CKs and C5a. In a co-culture model of GECs/podocytes that mimicked GFB, EPC-derived EVs protected cell function and permeselectivity from inflammatory-mediated damage. Moreover, RNase pre-treatment of EVs abrogated their protective effects, suggesting the crucial role of RNA transfer from EVs to damaged glomerular cells. In conclusion, EPC-derived EVs preserved GFB integrity from complement- and cytokine-induced damage, suggesting their potential role as therapeutic agents for drug-resistant glomerulonephritis. Full article

The genetic code that dictates how nucleic acids are translated into proteins is well known, however, the code through which proteins recognize membranes remains mysterious. In eukaryotes, this code is mediated by hundreds of membrane readers that recognize unique phosphatidylinositol phosphates (PIPs), which demark organelles to initiate localized trafficking and signaling events. The only superfamily which specifically detects all seven PIPs are the Phox homology (PX) domains. Here, we reveal that throughout evolution, these readers are universally regulated by the phosphorylation of their PIP binding surfaces based on our analysis of existing and modelled protein structures and phosphoproteomic databases. These PIP-stops control the selective targeting of proteins to organelles and are shown to be key determinants of high-fidelity PIP recognition. The protein kinases responsible include prominent cancer targets, underscoring the critical role of regulated membrane readership. Full article

Alzheimer’s disease (AD), a progressive neurodegenerative disorder characterized by memory loss and cognitive decline, is a major cause of death and disability among the older population. Despite decades of scientific research, the underlying etiological triggers are unknown. Recent studies suggested that gut microbiota can influence AD progression; however, potential mechanisms linking the gut microbiota with AD pathogenesis remain obscure. In the present study, we provided a potential mechanistic link between dysbiotic gut microbiota and neuroinflammation associated with AD progression. Using a mouse model of AD, we discovered that unfavorable gut microbiota are correlated with abnormally elevated expression of gut NLRP3 and lead to peripheral inflammasome activation, which in turn exacerbates AD-associated neuroinflammation. To this end, we observe significantly altered gut microbiota compositions in young and old 5xFAD mice compared to age-matched non-transgenic mice. Moreover, 5xFAD mice demonstrated compromised gut barrier function as evident from the loss of tight junction and adherens junction proteins compared to non-transgenic mice. Concurrently, we observed increased expression of NLRP3 inflammasome and IL-1β production in the 5xFAD gut. Consistent with our hypothesis, increased gut–microbial–inflammasome activation is positively correlated with enhanced astrogliosis and microglial activation, along with higher expression of NLRP3 inflammasome and IL-1β production in the brains of 5xFAD mice. These data indicate that the elevated expression of gut–microbial–inflammasome components may be an important trigger for subsequent downstream activation of inflammatory and potentially cytotoxic mediators, and gastrointestinal NLRP3 may promote NLRP3 inflammasome-mediated neuroinflammation. Thus, modulation of the gut microbiota may be a potential strategy for the treatment of AD-related neurological disorders in genetically susceptible hosts. Full article

The study examines the effect of acclimation on the antioxidant system and proline metabolism in cucumber leaves subjected to 100 and 150 NaCl stress. The levels of protein carbonyl group, thiobarbituric acid reactive substances, α-tocopherol, and activity of ascorbate and glutathione peroxidases, catalase, glutathione S-transferase, pyrroline-5-carboxylate: synthetase and reductase as well as proline dehydrogenase were determined after 24 and 72 h periods of salt stress in the acclimated and non-acclimated plants. Although both groups of plants showed high α-tocopherol levels, in acclimated plants was observed higher constitutive concentration of these compounds as well as after salt treatment. Furthermore, the activity of enzymatic antioxidants grew in response to salt stress, mainly in the acclimated plants. In the acclimated plants, protein carbonyl group levels collapsed on a constitutive level and in response to salt stress. Although both groups of plants showed a decrease in proline dehydrogenase activity, they differed with regard to the range and time. Differences in response to salt stress between the acclimated and non-acclimated plants may suggest a relationship between increased tolerance in acclimated plants and raised activity of antioxidant enzymes, high-level of α-tocopherol as well, as decrease enzyme activity incorporates in proline catabolism. Full article

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape. Full article

Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival at diagnosis of 16–20 months. Metabolism represents a new attractive therapeutic target; however, due to high intratumoral heterogeneity, the application of metabolic drugs in GBM is challenging. We characterized the basal bioenergetic metabolism and antiproliferative potential of metformin (MF), dichloroacetate (DCA), sodium oxamate (SOD) and diazo-5-oxo-L-norleucine (DON) in three distinct glioma stem cells (GSCs) (GBM18, GBM27, GBM38), as well as U87MG. GBM27, a highly oxidative cell line, was the most resistant to all treatments, except DON. GBM18 and GBM38, Warburg-like GSCs, were sensitive to MF and DCA, respectively. Resistance to DON was not correlated with basal metabolic phenotypes. In combinatory experiments, radiomimetic bleomycin exhibited therapeutically relevant synergistic effects with MF, DCA and DON in GBM27 and DON in all other cell lines. MF and DCA shifted the metabolism of treated cells towards glycolysis or oxidation, respectively. DON consistently decreased total ATP production. Our study highlights the need for a better characterization of GBM from a metabolic perspective. Metabolic therapy should focus on both glycolytic and oxidative subpopulations of GSCs. Full article

Akkermansia muciniphila is considered as one of the next-generation beneficial bacteria in the context of obesity and associated metabolic disorders. Although a first proof-of-concept of its beneficial effects has been established in the context of metabolic syndrome in humans, mechanisms are not yet fully understood. This study aimed at deciphering whether the bacterium exerts its beneficial properties through the modulation of the endocannabinoidome (eCBome). Circulating levels of 25 endogenous endocannabinoid-related lipids were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in the plasma of overweight or obese individuals before and after a 3 months intervention consisting of the daily ingestion of either alive or pasteurized A. muciniphila. Results from multivariate analyses suggested that the beneficial effects of A. muciniphila were not linked to an overall modification of the eCBome. However, subsequent univariate analysis showed that the decrease in 1-Palmitoyl-glycerol (1-PG) and 2-Palmitoyl-glycerol (2-PG), two eCBome lipids, observed in the placebo group was significantly counteracted by the alive bacterium, and to a lower extent by the pasteurized form. We also discovered that 1- and 2-PG are endogenous activators of peroxisome proliferator-activated receptor alpha (PPARα). We hypothesize that PPARα activation by mono-palmitoyl-glycerols may underlie part of the beneficial metabolic effects induced by A. muciniphila in human metabolic syndrome. Full article

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a mutation in LMNA. A G608G mutation in exon 11 of LMNA is responsible for most HGPS cases, generating a truncated protein called “progerin”. Progerin is permanently farnesylated and accumulates in HGPS cells, causing multiple cellular defects such as nuclear dysmorphism, a thickened lamina, loss of heterochromatin, premature senescence, and clustering of Nuclear Pore Complexes (NPC). To identify the mechanism of NPC clustering in HGPS cells, we evaluated post-mitotic NPC assembly in control and HGPS cells and found no defects. Next, we examined the occurrence of NPC clustering in control and HGPS cells during replicative senescence. We reported that NPC clustering occurs solely in the dysmorphic nuclei of control and HGPS cells. Hence, NPC clustering occurred at a higher frequency in HGPS cells compared to control cells at early passages; however, in late cultures with similar senescence index, NPCs clustering occurred at a similar rate in both control and HGPS. Our results show that progerin does not disrupt post-mitotic reassembly of NPCs. However, NPCs frequently cluster in dysmorphic nuclei with a high progerin content. Additionally, nuclear envelope defects that arise during replicative senescence cause NPC clustering in senescent cells with dysmorphic nuclei. Full article

Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1–100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa. Full article

Alpha synuclein has been linked to both sporadic and familial forms of Parkinson’s disease (PD) and is the most abundant protein in Lewy bodies a hallmark of Parkinson’s disease. The function of this protein and the molecular mechanisms underlying its toxicity are still unclear, but many studies have suggested that the mechanism of α-synuclein toxicity involves alterations to mitochondrial function. Here we expressed human α-synuclein and two PD-causing α-synuclein mutant proteins (with a point mutation, A53T, and a C-terminal 20 amino acid truncation) in the eukaryotic model Dictyostelium discoideum. Mitochondrial disease has been well studied in D. discoideum and, unlike in mammals, mitochondrial dysfunction results in a clear set of defective phenotypes. These defective phenotypes are caused by the chronic hyperactivation of the cellular energy sensor, AMP-activated protein kinase (AMPK). Expression of α-synuclein wild type and mutant forms was toxic to the cells and mitochondrial function was dysregulated. Some but not all of the defective phenotypes could be rescued by down regulation of AMPK revealing both AMPK-dependent and -independent mechanisms. Importantly, we also show that the C-terminus of α-synuclein is required and sufficient for the localisation of the protein to the cell cortex in D. discoideum. Full article

COVID-19, caused by SARS-CoV-2 virus, emerged as a pandemic disease posing a severe threat to global health. To date, sporadic studies have demonstrated that innate immune mechanisms, specifically neutrophilia, NETosis, and neutrophil-associated cytokine responses, are involved in COVID-19 pathogenesis; however, our understanding of the exact nature of this aspect of host–pathogen interaction is limited. Here, we present a detailed dissection of the features and functional profiles of neutrophils, dendritic cells, and monocytes in COVID-19. We portray the crucial role of neutrophils as drivers of hyperinflammation associated with COVID-19 disease via the shift towards their immature forms, enhanced degranulation, cytokine production, and augmented interferon responses. We demonstrate the impaired functionality of COVID-19 dendritic cells and monocytes, particularly their low expression of maturation markers, increased PD-L1 levels, and their inability to upregulate phenotype upon stimulation. In summary, our work highlights important data that prompt further research, as therapeutic targeting of neutrophils and their associated products may hold the potential to reduce the severity of COVID-19. Full article

Alzheimer’s disease (AD) is an age-related detrimental dementia. Amyloid beta peptides (Aβ) play a crucial role in the pathology of AD. In familial AD, Aβ are generated from the full-length amyloid beta precursor protein (APP) via dysregulated proteolytic processing; however, in the case of sporadic AD, the mechanism of Aβ biogenesis remains elusive. circRNAs are a class of transcripts preferentially expressed in brain. We identified a circRNA harboring the Aβ-coding region of the APP gene termed circAβ-a. This circular RNA was detected in the brains of AD patients and non-dementia controls. With the aid of our recently established approach for analysis of circRNA functions, we demonstrated that circAβ-a is efficiently translated into a novel Aβ-containing Aβ175 polypeptide (19.2 KDa) in both cultured cells and human brain. Furthermore, Aβ175 was shown to be processed into Aβ peptides—a hallmark of AD. In summary, our analysis revealed an alternative pathway of Aβ biogenesis. Consequently, circAβ-a and its corresponding translation product could potentially represent novel therapeutic targets for AD treatment. Importantly, our data point to yet another evolutionary route for potentially increasing proteome complexity by generating additional polypeptide variants using back-splicing of primary transcripts that yield circular RNA templates. Full article

In this work, polythiophene nanoparticles (PTh–NPs) were synthesized by a surfactant-free oxidative chemical polymerization method at 60 °C, using ammonium persulphate as an oxidant. Various physicochemical properties were studied in terms of field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), Fourier transform infra-red (FT-IR) spectroscopy, and differential scanning calorimetry (DSC)/thermogravimetric analysis (TGA). Photothermal performance of the as-synthesized PTh–NPs was studied by irradiating near infra-red of 808 nm under different concentration of the substrate and power supply. The photothermal stability of PTh–NPs was also studied. Photothermal effects of the as-synthesized PTh–NPs on colorectal cancer cells (CT-26) were studied at 100 µg/mL concentration and 808 nm NIR irradiation of 2.0 W/cm² power. Our in vitro results showed remarkable NIR laser-triggered photothermal apoptotic cell death by PTh–NPs. Based on the experimental findings, it is revealed that PTh–NPs can act as a heat mediator and can be an alternative material for photothermal therapy in cancer treatment. Full article

N4-methylcytosine as one kind of modification of DNA has a critical role which alters genetic performance such as protein interactions, conformation, stability in DNA as well as the regulation of gene expression same cell developmental and genomic imprinting. Some different 4mC site identifiers have been proposed for various species. Herein, we proposed a computational model, DNC4mC-Deep, including six encoding techniques plus a deep learning model to predict 4mC sites in the genome of F. vesca, R. chinensis, and Cross-species dataset. It was demonstrated by the 10-fold cross-validation test to get superior performance. The DNC4mC-Deep obtained 0.829 and 0.929 of MCC on F. vesca and R. chinensis training dataset, respectively, and 0.814 on cross-species. This means the proposed method outperforms the state-of-the-art predictors at least 0.284 and 0.265 on F. vesca and R. chinensis training dataset in turn. Furthermore, the DNC4mC-Deep achieved 0.635 and 0.565 of MCC on F. vesca and R. chinensis independent dataset, respectively, and 0.562 on cross-species which shows it can achieve the best performance to predict 4mC sites as compared to the state-of-the-art predictor. Full article

Stroke triggers neurogenesis in the striatum in mice, with new neurons deriving in part from the nearby subventricular zone and in part from parenchymal astrocytes. The initiation of neurogenesis by astrocytes within the striatum is triggered by reduced Notch-signaling, and blocking this signaling pathway by deletion of the gene encoding the obligate Notch coactivator Rbpj is sufficient to activate neurogenesis by striatal astrocytes in the absence of an injury. Here we report that blocking Notch-signaling in stroke increases the neurogenic response to stroke 3.5-fold in mice. Deletion of Rbpj results in the recruitment of a larger number of parenchymal astrocytes to neurogenesis and over larger areas of the striatum. These data suggest inhibition of Notch-signaling as a potential translational strategy to promote neuronal regeneration after stroke. Full article

Extracellular vesicles (EVs) are considered as promising nanoparticle theranostic tools in many pathological contexts. The increasing clinical employment of therapeutic nanoparticles is contributing to the development of a new research area related to the design of artificial EVs. To this aim, different approaches have been described to develop mimetic biologically functional nanovescicles. In this paper, we suggest a simplified procedure to generate plasma membrane-derived nanovesicles with the possibility to efficiently encapsulate different drugs during their spontaneously assembly. After physical and molecular characterization by Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy, and flow cytometry, as a proof of principle, we have loaded into mimetic EVs the isoquinoline alkaloid Berberine chloride and the chemotherapy compounds Temozolomide or Givinostat. We demonstrated the fully functionality of these nanoparticles in drug encapsulation and cell delivery, showing, in particular, a similar cytotoxic effect of direct cell culture administration of the anticancer drugs. In conclusion, we have documented the possibility to easily generate scalable nanovesicles with specific therapeutic cargo modifications useful in different drug delivery contexts. Full article