Multiple Novel Nesprin-1 and Nesprin-2 Variants Act as Versatile Tissue-Specific Intracellular Scaffolds

<div><h3>Background</h3><p>Nesprins (<u>N</u>uclear <u>e</u>nvelope <u>s</u>pectrin-<u>r</u>epeat <u>p</u>roteins) are a novel family of giant spectrin-repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms.</p> <h3>Results</h3><p>In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types.</p> <h3>Conclusions</h3><p>These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.</p> </div

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

The RNA binding protein La promotes RIG-I-mediated type I and type III IFN responses following Sendai viral infection

Abstract La/SS-B (or La) is a 48 kDa RNA-binding protein and an autoantigen in autoimmune disorders such as systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SS). La involvement in regulating the type I interferon (IFN) response is controversial - acting through both positive and negative regulatory mechanisms; inhibiting the IFN response and enhancing viral growth, or directly inhibiting viral replication. We therefore sought to clarify how La regulates IFN production in response to viral infection. ShRNA knockdown of La in HEK 293 T cells increased Sendai virus infection efficiency, decreased IFN-β, IFN-λ1, and interferon-stimulated chemokine gene expression. In addition, knockdown attenuated CCL-5 and IFN-λ1 secretion. Thus, La has a positive role in enhancing type I and type III IFN production. Mechanistically, we show that La directly binds RIG-I and have mapped this interaction to the CARD domains of RIG-I and the N terminal domain of La. In addition, we showed that this interaction is induced following RIG-I activation and that overexpression of La enhances RIG-I-ligand binding. Together, our results demonstrate a novel role for La in mediating RIG-I-driven responses downstream of viral RNA detection, ultimately leading to enhanced type I and III IFN production and positive regulation of the anti-viral response

Drug discovery in advanced prostate cancer: translating biology into therapy.

Castration-resistant prostate cancer (CRPC) is associated with a poor prognosis and poses considerable therapeutic challenges. Recent genetic and technological advances have provided insights into prostate cancer biology and have enabled the identification of novel drug targets and potent molecularly targeted therapeutics for this disease. In this article, we review recent advances in prostate cancer target identification for drug discovery and discuss their promise and associated challenges. We review the evolving therapeutic landscape of CRPC and discuss issues associated with precision medicine as well as challenges encountered with immunotherapy for this disease. Finally, we envision the future management of CRPC, highlighting the use of circulating biomarkers and modern clinical trial designs