Rapid identification of SARS-CoV-2 variants: Validation of the simplexa SARS-CoV-2 variant direct assay.

The circulation of certain SARS-CoV-2 variants may have a great impact on the epidemiological status of a geographical area; therefore, it is important that their presence is monitored. Currently, the gold standard method used to identify newly emerged variants is sequencing of either genes or whole genomes. However, since this method is relatively expensive and has a long turnaround time, other rapid strategies should also be employed. The current study aimed to evaluate the performance of the Simplexa® SARS-CoV-2 Variants Direct assay, which is a RT-PCR that determines the variant present in a nasopharyngeal swab sample in approximately two hours. Totally, 527 positive samples for SARS-CoV-2 were analyzed from January until December 2022 and next-generation sequencing (NGS) was used as the reference method. The assay showed high sensitivity, ranging from 94.12 % to 100 %, depending on the variant. The assay also showed high specificity, reaching 100 % for Delta and BA.1 variants, and 99.74 % and 98.67 % for BA.2 and BA.4/BA.5 variants, respectively. Moreover, the assay was able to identify the correct variant category in the presence of any subvariant in the sample. We conclude that the assay can be used to facilitate faster monitoring of circulating SARS-CoV-2 variants, however sequencing cannot be completely replaced, since new variants always emerge, and constant updates are needed, so that the user is able to interpret the melting curve patterns.

Genome-Wide Analysis of lncRNA-mRNA Co-Expression Networks in CD133+/CD44+ Stem-like PDAC Cells.

Pancreatic ductal adenocarcinoma (PDAC), the second most prevalent gastrointestinal malignancy and the most common type of pancreatic cancer is linked with poor prognosis and, eventually, with high mortality rates. Early detection is seldom, while tumor heterogeneity and microarchitectural alterations benefit PDAC resistance to conventional therapeutics. Although emerging evidence suggest the core role of cancer stem cells (CSCs) in PDAC aggressiveness, unique stem signatures are poorly available, thus limiting the efforts of anti-CSC-targeted therapy. Herein, we report the findings of the first genome-wide analyses of mRNA/lncRNA transcriptome profiling and co-expression networks in PDAC cell line-derived CD133+/CD44+ cells, which were shown to bear a CSC-like phenotype in vitro and in vivo. Compared to CD133-/CD44- cells, the CD133+/CD44+ population demonstrated significant expression differences in both transcript pools. Using emerging bioinformatic tools, we performed lncRNA target coding gene prediction analysis, which revealed significant Gene Ontology (GO), pathway, and network enrichments in many dyregulated lncRNA nearby (cis or trans) mRNAs, with reported involvement in the regulation of CSC phenotype and functions. In this context, the construction of lncRNA/mRNA networks by ingenuity platforms identified the lncRNAs ATF2, CHEK1, DCAF8, and PAX8 to interact with "hub" SC-associated mRNAs. In addition, the expressions of the above lncRNAs retrieved by TCGA-normalized RNAseq gene expression data of PAAD were significantly correlated with clinicopathological features of PDAC, including tumor grade and stage, nodal metastasis, and overall survival. Overall, our findings shed light on the identification of CSC-specific lncRNA signatures with potential prognostic and therapeutic significance in PDAC.

Long non-coding RNAs (lncRNAs) signaling in cancer chemoresistance: From prediction to druggability.

The acquisition of cancer cell resistance to conventional chemotherapeutics is considered the major driver of treatment failure and disease recurrence in most solid and hematological malignancies. The molecular basis of tumor chemoresistance has been extensively investigated and newly identified gene signatures have eventually paved the way towards the development of novel therapeutic interventions in the era of precision medicine in oncology. Long non-coding RNAs (lncRNAs) are defined as a class of transcripts longer than 200 nucleotides that lack translational activities and are highly abundant across the human genome. LncRNAs show higher tissue and cell subtype specificities than most mRNAs, while their biological relevance has been associated with the regulation of coding gene expression at the epigenetic, transcriptional, and post-transcriptional levels, regulation of DNA replication timing and chromosome stability, as well as aging and disease. Given their specific expression and functional diversities in a variety of human cancers, lncRNAs have currently received extensive attention regarding their implications in the disease pathophysiology and their potential applications as diagnostic/prognostic biomarkers and/or therapeutic targets in cancer. Over the last decade, different lncRNAs were found to play pivotal regulatory roles in drug resistance of certain cancer cell types via mechanisms that include among others, alterations in drug efflux, metabolism and targeting, cell death machinery, DNA damage repair, epithelial to mesenchymal transition (EMT), autophagy and oxidative stress management, as well as modifications in epigenetic regulators, oncogenes, and miRNAs. The present review discusses the current state of knowledge on the emerging research into lncRNAs as drug resistance regulators and predictors in various tumors, emphasizing lncRNA patterns associated with cancer stemness, certain drug classes and common underlying mechanisms of action. The review further reveals cutting edge strategies for lncRNA modulation and the current progress on lncRNA-targeting molecules designed to overcome cancer resistance. Our input is a reference for future research investigations on cancer chemosensitivity and provides new insights into the clinical development of lncRNA-targeted pharmacological interventions.

A novel small-molecule selective activator of homomeric GIRK4 channels.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel-phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.

Long Non-Coding RNAs (lncRNAs) in Response and Resistance to Cancer Immunosurveillance and Immunotherapy.

Long non-coding RNAs (lncRNAs) are critical regulatory elements in cellular functions in states of both normalcy and disease, including cancer. LncRNAs can influence not only tumorigenesis but also cancer features such as metastasis, angiogenesis and resistance to chemo-and immune-mediated apoptotic signals. Several lncRNAs have been demonstrated to control directly or indirectly the number, type and activities of distinct immune cell populations of adaptive and innate immunities within and without the tumor microenvironment. The disruption of lncRNA expression in both cancer and immune cells may reflect alterations in tumor responses to cancer immunosurveillance and immunotherapy, thus providing new insights into lncRNA biomarker-based prognostic and therapeutic cancer assessment. Here we present an overview on lncRNAs' functions and underlying molecular mechanisms related to cancer immunity and conventional immunotherapy, with the expectation that any elucidations may lead to a better understanding and management of cancer immune escape and response to current and future immunotherapeutics.

A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels.

Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects.