A Novel Porcine In Vitro Model of the Blood-Cerebrospinal Fluid Barrier with Strong Barrier Function

Epithelial cells of the plexus choroideus form the structural basis of the blood-cerebrospinal fluid barrier (BCSFB). In vitro models of the BCSFB presenting characteristics of a functional barrier are of significant scientific interest as tools for examination of BCSFB function. Due to a lack of suitable cell lines as in vitro models, primary porcine plexus epithelial cells were subjected to a series of selective cultivation steps until a stable continuous subcultivatable epithelial cell line (PCP-R) was established. PCP-R cells grow in a regular polygonal pattern with a doubling time of 28–36 h. At a cell number of 1.5×105 in a 24-well plate confluence is reached in 56–72 h. Cells are cytokeratin positive and chromosomal analysis revealed 56 chromosomes at peak (84th subculture). Employing reverse transcription PCR mRNA expression of several transporters and components of cell junctions could be detected. The latter includes tight junction components like Claudin-1 and -3, ZO-1, and Occludin, and the adherens junction protein E-cadherin. Cellular localization studies of ZO-1, Occludin and Claudin-1 by immunofluorescence and morphological analysis by electron microscopy demonstrated formation of a dense tight junction structure. Importantly, when grown on cell culture inserts PCP-R developed typical characteristics of a functional BCSFB including high transepithelial electrical resistance above 600 Ω×cm2 as well as low permeability for macromolecules. In summary, our data suggest the PCP-R cell line as a suitable in vitro model of the porcine BCSFB

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis

Polar Invasion and Translocation of Neisseria meningitidis and Streptococcus suis in a Novel Human Model of the Blood-Cerebrospinal Fluid Barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens

Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Cancer Cells

Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of “normalized” oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics

Advances, challenges and future directions for stem cell therapy in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative condition where loss of motor neurons within the brain and spinal cord leads to muscle atrophy, weakness, paralysis and ultimately death within 3–5 years from onset of symptoms. The specific molecular mechanisms underlying the disease pathology are not fully understood and neuroprotective treatment options are minimally effective. In recent years, stem cell transplantation as a new therapy for ALS patients has been extensively investigated, becoming an intense and debated field of study. In several preclinical studies using the SOD1G93A mouse model of ALS, stem cells were demonstrated to be neuroprotective, effectively delayed disease onset and extended survival. Despite substantial improvements in stem cell technology and promising results in preclinical studies, several questions still remain unanswered, such as the identification of the most suitable and beneficial cell source, cell dose, route of delivery and therapeutic mechanisms. This review will cover publications in this field and comprehensively discuss advances, challenges and future direction regarding the therapeutic potential of stem cells in ALS, with a focus on mesenchymal stem cells. In summary, given their high proliferation activity, immunomodulation, multi-differentiation potential, and the capacity to secrete neuroprotective factors, adult mesenchymal stem cells represent a promising candidate for clinical translation. However, technical hurdles such as optimal dose, differentiation state, route of administration, and the underlying potential therapeutic mechanisms still need to be assessed

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p