The outbreak of the coronavirus disease (COVID-19) pandemic caused by the novel coronavirus (SARS-CoV-2) has been declared an international public health crisis. It is essential to develop diagnostic tests that can quickly identify infected individuals to limit the spread of the virus and assign treatment options. Herein, we report a proof-of-concept label-free electrochemical immunoassay for the rapid detection of SARS-CoV-2 virus via the spike surface protein. The assay consists of a graphene working electrode functionalized with anti-spike antibodies. The concept of the immunosensor is to detect the signal perturbation obtained from ferri/ferrocyanide measurements after binding of the antigen during 45 min of incubation with a sample. The absolute change in the [Fe(CN)6]3−/4− current upon increasing antigen concentrations on the immunosensor surface was used to determine the detection range of the spike protein. The sensor was able to detect a specific signal above 260 nM (20 µg/mL) of subunit 1 of recombinant spike protein. Additionally, it was able to detect SARS-CoV-2 at a concentration of 5.5 × 105 PFU/mL, which is within the physiologically relevant concentration range. The novel immunosensor has a significantly faster analysis time than the standard qPCR and is operated by a portable device which can enable on-site diagnosis of infection.

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

Peptoids are an alternative approach to antimicrobial peptides that offer higher stability towards enzymatic degradation. It is essential when developing new types of peptoids, that mimic the function of antimicrobial peptides, to understand their mechanism of action. Few studies on the specific mechanism of action of antimicrobial peptoids have been described in the literature, despite the plethora of studies on the mode of action of antimicrobial peptides. Here, we investigate the mechanism of action of two short cationic peptoids, rich in lysine and tryptophan side chain functionalities. We demonstrate that both peptoids are able to cause loss of viability in E. coli susceptible cells at their MIC (16–32 μg/ml) concentrations. Dye leakage assays demonstrate slow and low membrane permeabilization for peptoid 1, that is still higher for lipid compositions mimicking bacterial membranes than lipid compositions containing Cholesterol. At concentrations of 4 × MIC (64–128 μg/ml), pore formation, leakage of cytoplasmic content and filamentation were the most commonly observed morphological changes seen by SEM in E. coli treated with both peptoids. Flow cytometry data supports the increase of cell size as observed in the quantification analysis from the SEM images and suggests overall decrease of DNA per cell mass over time.

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.

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Sequencing of primary colorectal tumors has identified a gene fusion in approximately 3% of colorectal cancer patients of the VTI1A and TCF7L2 genes, encoding a VTI1A-TCF4 fusion protein containing a truncated TCF4. As dysregulation of the Wnt signaling pathway is associated with colorectal cancer development and progression, the functional properties and transcriptional regulation of the VTI1A-TCF4 fusion protein may also play a role in these processes. Functional characteristics of the VTI1A-TCF4 fusion protein in Wnt signaling were analyzed in NCI-H508 and LS174T colon cancer cell lines. The NCI-H508 cell line, containing the VTI1A-TCF7L2 fusion gene, showed no active Wnt signaling, and overexpression of the VTI1A-TCF4 fusion protein in LS174T cells along with a Wnt signaling luciferase reporter plasmid showed inhibition of activity. The transcriptional regulation of the VTI1A-TCF4 fusion gene was investigated in LS174T cells where the activity of the VTI1A promoter was compared to that of the TCF7L2 promoter, and the transcription factor CDX2 was analyzed for gene regulatory activity of the VTI1A promoter through luciferase reporter gene assay using colon cancer cell lines as a model. Transfection of LS174T cells showed that the VTI1A promoter is highly active compared to the TCF7L2 promoter, and that CDX2 activates transcription of VTI1A. These results suggest that the VTI1A-TCF4 fusion protein is a dominant negative regulator of the Wnt signaling pathway, and that transcription of VTI1A is activated by CDX2.

The type II membrane-anchored serine protease, matriptase, encoded by suppression of tumorgenicity-14 (ST14) regulates the integrity of the intestinal epithelial barrier in concert with its inhibitor, HAI-1 encoded by serine peptidase inhibitor, Kunitz type -1 (SPINT1). The balance of the protease/inhibitor gene expression ratio is vital in preventing the oncogenic potential of matriptase. The intestinal cell lineage is regulated by a transcriptional regulatory network where the tumor suppressor, Caudal homeobox 2 (CDX2) is considered to be an intestinal master transcription factor. In this study, we show that CDX2 has a dual function in regulating both ST14 and SPINT1, gene expression in intestinal cells. We find that CDX2 is not required for the basal ST14 and SPINT1 gene expression; however changes in CDX2 expression affects the ST14/SPINT1 mRNA ratio. Exploring CDX2 ChIP-seq data from intestinal cell lines, we identified genomic CDX2-enriched enhancer elements for both ST14 and SPINT1, which regulate their corresponding gene promoter activity. We show that CDX2 displays both repressive and enhancing regulatory abilities in a cell specific manner. Together, these data reveal new insight into transcriptional mechanisms controlling the intestinal matriptase/inhibitor balance.

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co‐transcription factor yes‐associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco‐2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase‐expressing YAP1 promoter‐reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco‐2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein‐DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco‐2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.