Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink ( Neovison vison ) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.

The World Health Organization has declared COVID-19 caused by the newly discovered SARS-CoV-2 a pandemic. Due to growing demand for reagents and/ or kits to extract SARS-CoV-2 RNA for subsequent RT-qPCR diagnostics, there is a worldwide risk of shortages. With a detection sensitivity of 97.4% (95% CI: 86.2-99.9%), we describe a simple, fast, alternative workflow for molecular detection of SARS-CoV-2, where samples are simply heat-processed for 5 min at 98 °C before a commonly-used RT-qPCR procedure.Coronavirus disease (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), was first detected in Wuhan, China in December 2019 and then spread worldwide in a few months [1]. There is currently a global shortage of viral nucleic acid (NA) extraction kits, which is affecting the diagnosis of an increasing number of suspected COVID-19 cases. The aim of this study was to investigate a new simplified workflow for molecular detection of SARS-CoV-2 that does not require NA extraction and could serve as an alternative in diagnostic laboratories to overcome chemical-based kit-shortages. Direct approach for molecular detection of SARS-CoV-2NA purification before PCR/reverse transcription (RT)-PCR is the gold standard for molecular diagnostics. The MagNa Pure 96 system (Roche Molecular Biochemicals, Indianapolis, Indiana, United States (US)) is a widely used system for high-throughput NA purification in many public health laboratories worldwide [2]. However, with Roche's announcement of emerging kitshortages and bottlenecks in kit production processes [3], we investigated if real-time RT-PCR (RT-qPCR) analysis could be performed with minimal pre-treatment of samples. We used the most common sample type (oropharyngeal swabs) collected from patients suspected of COVID-19 in Denmark.Three simplified approaches, which involved minimal handling of the samples before the RT-qPCR for SARS-CoV-2 were employed to avoid the NA purification step. The first approach was direct: 5 µL of the saline/transport solution from the throat-swab were added to the RT-qPCR reaction without any treatment. The second was a phosphate-buffered saline (PBS) diluted approach: the saline/transport solution was further diluted 1:1 with PBS before adding 5 µL directly to the RT-qPCR reaction. The third was a heat-processed method: we compared four different heat-processes on 10 µL of the saline/transport solution from the throat swab, (i) 5 min at 95 °C, (ii) 10 min at 95 °C, (iii) 5 min at 98 °C and (iv) 10 min at 98 °C, respectively. All heat-processed clinical samples were cooled for 2 min at 4 °C before 5 µL were used in the RT-qPCR reaction. Two SARS-CoV-2 RT-qPCR assays were used: (i) the published and widely used RT-qPCR assay for the envelope (E)-gene [4,5] combined with the SensiFAST Probe No-ROX One-Step Real-time PCR kit (Bioline Meridian BioScience, Cincinnati, Ohio, US), and (ii) the commercial RealStar SARS-CoV-2 RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany). We employed 87 patient samples, comprising 6...

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3 UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

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.

The World Health Organisation has declared a pandemic caused by the newly discovered SARS-CoV-2.Due to growing demand for reagents used for SARS-CoV-2 RNA extraction for subsequent molecular diagnostics, there is a worldwide risk of kit-and/or reagent-shortages for extraction. With a detection sensitivity of 97.4% (95% CI=86.2-99.9%), we describe a simple, fast, alternative workflow for molecular detection of SARS-CoV-2, where samples are simply heat-processed for 5 minutes at 98˚C prior to the RT-qPCR reaction.

Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 potassium channels. The inhibition is found on channels expressed heterologously in both Xenopus oocytes and mammalian HEK293 cells. mKCNE4 does not inhibit Kv1.2, Kv1.4, Kv1.5, or Kv4.3 homomeric complexes, but it does significantly reduce current through Kv1.1/Kv1.2 and Kv1.2/Kv1.3 heteromeric complexes. Confocal microscopy and Western blotting reveal that Kv1.1 is present at the cell surface together with KCNE4. Real-time RT-PCR shows a relatively high presence of mKCNE4 mRNA in several organs, including uterus, kidney, lung, intestine, and in embryo, whereas a much lower mRNA level is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials.

Persistent infection with high-risk human papillomavirus (HPV) and expression of the proteins E6 and E7 is a prerequisite for development of cervical cancer. The distal non-coding part of E6/E7 messengers from several HPV types is able to downregulate synthesis of a reporter gene through mechanisms with involvement of cytoplasmic polyadenylation elements (CPEs) in the messengers. We here show that the mRNA levels of one of the four known CPE-binding proteins (CPEBs), the CPEB3, were downregulated in HPV-positive cervical cancers, whereas in ovarian cancer the CPEB1 mRNA level was downregulated. In addition, we showed that the RNA levels of the widely used reference marker GAPDH were upregulated in both cancer forms, and the level of the reference marker U6snRNA was upregulated in cervical cancers. Moreover, a possible correlation between the degree of U6snRNA upregulation and cervical cancer propagation was shown. These changes observed in CPEB1 and CPEB3 might indicate regulatory functions of CPEBs in cancer development of HPV-positive and HPV-negative tumors, respectively, and the U6snRNA, GAPDH mRNA and CPEB1 mRNA levels may be useful as tumor markers for genital cancers although further investigations are needed.

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.