Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis. Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes.

Long non-coding RNAs (lncRNAs) are emerging as important players in regulation of gene expression in higher eukaryotes. DDX5/p68 RNA helicase protein which is involved in splicing of precursor mRNAs also interacts with lncRNAs like, SRA and mrhl, to modulate gene expression. We performed RIP-seq analysis in HEK293T cells to identify the complete repertoire of DDX5/p68 interacting transcripts including 73 single exonic (SE) lncRNAs. The LOC284454 lncRNA is the second top hit of the list of SE lncRNAs which we have characterized in detail for its molecular features and cellular functions. The RNA is located in the same primary transcript harboring miR-23a∼27a∼24-2 cluster. LOC284454 is a stable, nuclear restricted and chromatin associated lncRNA. The sequence is conserved only in primates among 26 different species and is expressed in multiple human tissues. Expression of LOC284454 is significantly reduced in breast, prostate, uterus and kidney cancer and also in breast cancer cell lines (MCF7 and T47D). Global gene expression studies upon loss and gain of function of LOC284454 revealed perturbation of genes related to cancer-related pathways. Focal adhesion and cell migration pathway genes are downregulated under overexpression condition, and these genes are significantly upregulated in breast cancer cell lines as well as breast cancer tissue samples suggesting a functional role of LOC284454 lncRNA in breast cancer pathobiology.

ASCL1 is a basic Helix-Loop-Helix transcription factor (TF), which is involved in various cellular processes like neuronal development and signaling pathways. Transcriptome profiling has shown that ASCL1 overexpression plays an important role in the development of glioma and Small Cell Lung Carcinoma (SCLC), but distinct and common molecular mechanisms regulated by ASCL1 in these cancers are unknown. In order to understand how it drives the cellular functional network in these two tumors, we generated a gene expression profile in a glioma cell line (U87MG) to identify ASCL1 gene targets by an si RNA silencing approach and then compared this with a publicly available dataset of similarly silenced SCLC (NCI-H1618 cells). We constructed TF-TF and gene-gene interactions, as well as protein interaction networks of ASCL1 regulated genes in glioma and SCLC cells. Detailed network analysis uncovered various biological processes governed by ASCL1 target genes in these two tumor cell lines. We find that novel ASCL1 functions related to mitosis and signaling pathways influencing development and tumor growth are affected in both glioma and SCLC cells. In addition, we also observed ASCL1 governed functional networks that are distinct to glioma and SCLC.

BACKGROUND The COVID-19 pandemic necessitated rapid real-time surveillance of epidemiological data to advise governments and the public, but the accuracy of these data depend on myriad auxiliary assumptions, not least accurate reporting of cases by the public. Wastewater monitoring has emerged internationally as an accurate and objective means for assessing disease prevalence with reduced latency and less dependence on public vigilance, reliability, and engagement. How public interest aligns with COVID-19 personal testing data and wastewater monitoring is, however, very poorly characterised. OBJECTIVE This study assesses the associations between internet search volume data relevant to COVID-19, public healthcare statistics and national-scale wastewater monitoring of SARS-CoV-2 across South Wales, UK over time to investigate how interest in the pandemic may reflect the prevalence of SARS-CoV-2, as detected by national testing and wastewater monitoring. METHODS Relative search volume data from Google Trends for search terms linked to the COVID-19 pandemic were extracted and compared against government-reported COVID-19 statistics and RT-qPCR SARS-CoV-2 data generated from wastewater in South Wales, UK, using multivariate linear models and correlation analysis. RESULTS Wastewater monitoring data suggests that prevalence of the virus exceeded that reported in self-testing national reports. Google search volumes surrounding the COVID-19 pandemic also decreased across the same period, potentially suggesting a reduction in public interest is reflected in lower volumes of self-testing and reporting with consequential loss of accuracy of those data. CONCLUSIONS Wastewater monitoring presents a valuable means for assessing population-level prevalence of SARS-CoV-2. The importance of such monitoring is increasingly clear as a means of objectively assessing the prevalence of COVID-19 despite the dynamic interest and participation of the public. Increased accessibility of wastewater monitoring data to the public, as is the case for other national data, may enhance public engagement with these forms of monitoring.

Nanopore sequencing facilitates the rapid and cost-effective sequencing of long fragment DNA for a massive range of applications. When looking to holistically analyse low-yield DNA samples using nanopore sequencing, the optimal method is likely to involve the PCR Barcoding Kit. This effectively involves blunt end ligation of priming sites onto all extant DNA for holistic amplification to achieve yields suitable for nanopore sequencing. The currently available kits from nanopore facilitate the multiplexing of 96 samples in one sequencing run using this method, but the reagent costs are inherently multiplicative. This protocol is designed to streamline (in terms of cost, reagents and time) the process of sequencing up to 96 samples of genomic DNA through nanopore sequencing. This protocol is best applied to large numbers of samples (up to 96). For smaller numbers of samples, consider the smaller "PCR Barcoding" kits provided by nanopore which similarly achieve holistic DNA amplification and sequencing, but without the need for additional adapter ligation. The protocol is best suited to samples with low DNA yields (100 ng input is recommended). If you can input 1000 ng of DNA from each of your samples, consider using the 96-well Ligation Kit from Oxford Nanopore which can similarly be streamlined in terms of cost and time, but avoids the amplification step.