HIV-1-induced translocation of CPSF6 to biomolecular condensates.

Cleavage and polyadenylation specificity factor subunit 6 (CPSF6, also known as CFIm68) is a 68 kDa component of the mammalian cleavage factor I (CFIm) complex that modulates mRNA alternative polyadenylation (APA) and determines 3' untranslated region (UTR) length, an important gene expression control mechanism. CPSF6 directly interacts with the HIV-1 core during infection, suggesting involvement in HIV-1 replication. Here, we review the contributions of CPSF6 to every stage of the HIV-1 replication cycle. Recently, several groups described the ability of HIV-1 infection to induce CPSF6 translocation to nuclear speckles, which are biomolecular condensates. We discuss the implications for CPSF6 localization in condensates and the potential role of condensate-localized CPSF6 in the ability of HIV-1 to control the protein expression pattern of the cell.

Electrochemical sensors based on metal nanoparticles with biocatalytic activity.

Biosensors have attracted a great deal of attention, as they allow for the translation of the standard laboratory-based methods into small, portable devices. The field of biosensors has been growing, introducing innovations into their design to improve their sensing characteristics and reduce sample volume and user intervention. Enzymes are commonly used for determination purposes providing a high selectivity and sensitivity; however, their poor shelf-life is a limiting factor. Researchers have been studying the possibility of substituting enzymes with other materials with an enzyme-like activity and improved long-term stability and suitability for point-of-care biosensors. Extra attention is paid to metal and metal oxide nanoparticles, which are essential components of numerous enzyme-less catalytic sensors. The bottleneck of utilising metal-containing nanoparticles in sensing devices is achieving high selectivity and sensitivity. This review demonstrates similarities and differences between numerous metal nanoparticle-based sensors described in the literature to pinpoint the crucial factors determining their catalytic performance. Unlike other reviews, sensors are categorised by the type of metal to study their catalytic activity dependency on the environmental conditions. The results are based on studies on nanoparticle properties to narrow the gap between fundamental and applied research. The analysis shows that the catalytic activity of nanozymes is strongly dependent on their intrinsic properties (e.g. composition, size, shape) and external conditions (e.g. pH, type of electrolyte, and its chemical composition). Understanding the mechanisms behind the metal catalytic activity and how it can be improved helps designing a nanozyme-based sensor with the performance matching those of an enzyme-based device.

Lithium preserves peritoneal membrane integrity by suppressing mesothelial cell αB-crystallin.

Life-saving renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. However, the pathophysiological mechanisms involved are incompletely understood, limiting identification of therapeutic targets. We report that addition of lithium chloride (LiCl) to PDF is a translatable intervention to counteract PDF-induced mesothelial cell death, peritoneal membrane fibrosis, and angiogenesis. LiCl improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most consistently counter-regulated by LiCl. In vitro and in vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like up-regulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGF-ß-independent activation of TGF-ß-regulated targets. In contrast, αB-crystallin knockdown decreased VEGF expression and early mesothelial-to-mesenchymal transition. LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically up-regulated in response to PDF and increased in peritoneal mesothelial cells from biopsies from pediatric patients undergoing PD, correlating with markers of angiogenesis and fibrosis. LiCl-supplemented PDF promoted morphological preservation of mesothelial cells and the submesothelial zone in a mouse model of chronic PD. Thus, repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy.

APOE genetic variants and apoE, miR-107 and miR-650 levels in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative dementia in adults. Pathogenesis of AD depends on various factors, including APOE genetic variants, apolipoprotein E (apoE) phenotype and oxidative stress, which may promote both DNA and RNA damage, including non-coding RNA (ncRNA). Among ncRNAs, microRNA (miRNA) is known to contribute to pathologic processes in AD. The aim of the study was to analyse the plasma concentration of apoE by ELISA as well as the plasma levels of miR-107 and miR-650 by qPCR in relation to APOE genetic variants and clinical features including the age of onset and dementia severity in 64 AD patients and 132 controls. Our data showed that a low apoE plasma concentration was a risk factor for developing AD (OR = 5.18, p = 6.58E-06) and was particularly pronounced in severe dementia (p < 0.001) and correlated with cognitive functions (R = 0.295, p = 0.020), similarly as the level of miR-650 (R = 0.385, p = 0.033). The presence of APOE E4 allele in both AD patients and controls led to a reduction in apoE, while APOE E3/E3 genotype was associated with an increased apoE concentration and level of miR-107 in AD (p < 0.05) which was inversely correlated with the number of APOE E4 alleles (R = -0.448, p = 0.009). Additionally, patients with the onset at 60-69 years of age showed a reduced level of miR-107 (p < 0.05, as compared to AD above 80 years of age). Changed levels of plasma apoE, miR-107 and miR-650 may be a marker of the neurodegenerative process in the course of AD, associated with amyloid ß metabolism and inordinate cell cycle.

Biothiols and oxidative stress markers and polymorphisms of TOMM40 and APOC1 genes in Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive disease, with frequently observed improper biothiols turnover, homocysteine (Hcy) and glutathione (GSH). GSH protects cells from oxidative stress and may be determined by 8-oxo-2'-deoxyguanosine (8-oxo2dG) level and its repair enzyme 8-oxoguanine DNA glycosylase (OGG1). The presence of unfavorable alleles, e.g., in APOE cluster, TOMM40 or APOC1 is known to facilitate the dementia onset under oxidative stress. The aim of the study was to analyze rs1052452, rs2075650 TOMM40 polymorphisms, rs4420638 APOC1, and their correlation with Hcy, GSH, 8-oxo2dG, OGG1 levels in plasma of AD patients and controls. We recruited 230 individuals: 88 AD, 80 controls without (UC), 62 controls with (RC) positive family history of AD. The TOMM40 genotype was determined by HRM and capillary electrophoresis, while APOC1 by HRM. The concentrations of OGG1, 8-oxo2dG were determined by ELISA, whereas Hcy, GSH by HPLC/EC. We showed that over 60% of AD patients had increased Hcy levels (p<0.01 vs. UC, p<0.001 vs. RC), while GSH (p<0.01 vs. UC), 8-oxo2dG (p<0.01 vs. UC, p<0.001 vs. RC) were reduced. Minor variants: rs10524523-L, rs4420638-G, rs2075650-G were significantly overrepresented in AD. For rs4420638-G, rs2075650-G variants, the association remained significant in APOE E4 non-carriers. The misbalance of analyzed biothiols, and 8-oxo2dG, OGG1 were more pronounced in carriers of major variants: rs10524523-S/VL, rs4420638-A, rs2075650-A. We showed, for the first time, that APOC1 and TOMM40 rs2075650 polymorphisms may be independent risk factors of developing AD, whose major variants are accompanied by disruption of biothiols metabolism and inefficient removal of DNA oxidation.

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Cross-omics comparison of stress responses in mesothelial cells exposed to heat- versus filter-sterilized peritoneal dialysis fluids.

Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses.

Dynamic O-linked N-acetylglucosamine modification of proteins affects stress responses and survival of mesothelial cells exposed to peritoneal dialysis fluids.

The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD.