Comparison of some DNA extraction methods from monovarietal must and wines

The methods applied for DNA extraction from must and wine samples with monovarietal origin are presented and discussed aiming to prove the quality of extracted DNA and its good properties for further use in molecular tests. In the present research were compared four different DNA extraction methods from must and wine samples obtained from eleven V. vinifera varieties (five grapevine varieties for white wines and six grapevine varieties for red wines, respectively). Taking into consideration the amounts of obtained DNA, the concentrations and purities of the final DNA extracts, were stood out two modified methods. For all must samples, very efficient was the second method, which allowed obtaining a mean value of 87.9 ng µl-1 for the DNA concentration with 1.55 purity. Among the tested procedures, for monovarietal wine samples, the fourth method proved to be the most efficient which brought a mean value of 64.7 ng μl-1 for DNA concentration with 1.66 purity. This method adequate for wine samples involves two CTAB solution treatments and the RNase treatment applied before DNA resuspension. The DNA from must and wine extracts and the DNA from leaves of the corresponding grapevine varieties were amplified with five specific microsatellite primers (VVS2, VVMD27, VVMD32, VrZAG79 and VrZAG62). The aspects of pattern profiles were analysed in parallel and proved that the extracted DNA was suitable for amplification with these specific V. vinifera primers. The two selected extraction procedures are considered good for research purposes and ensure obtaining of good-quality extracted DNA from musts and one-year old wines.

Identification of the RNase-binding site of SARS-CoV-2 RNA for anchor primer-PCR detection of viral loading in 306 COVID-19 patients

The pandemic of coronavirus disease 2019 (COVID-19) urgently calls for more sensitive molecular diagnosis to improve sensitivity of current viral nuclear acid detection. We have developed an anchor primer (AP)-based assay to improve viral RNA stability by bioinformatics identification of RNase-binding site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and implementing AP dually targeting the N gene of SARS-CoV-2 RNA and RNase 1, 3, 6. The arbitrarily primed polymerase chain reaction (AP-PCR) improvement of viral RNA integrity was supported by (a) the AP increased resistance of the targeted gene (N gene) of SARS-CoV-2 RNA to RNase treatment; (b) the detection of SARS-CoV-2 RNA by AP-PCR with lower cycle threshold values (−2.7 cycles) compared to two commercially available assays; (c) improvement of the viral RNA stability of the ORF gene upon targeting of the N gene and RNase. Furthermore, the improved sensitivity by AP-PCR was demonstrated by detection of SARS-CoV-2 RNA in 70–80% of sputum, nasal, pharyngeal swabs and feces and 36% (4/11) of urine of the confirmed cases (n = 252), 7% convalescent cases (n = 54) and none of 300 negative cases. Lastly, AP-PCR analysis of 306 confirmed and convalescent cases revealed prolonged presence of viral loading for >20 days after the first positive diagnosis. Thus, the AP dually targeting SARS-CoV-2 RNA and RNase improves molecular detection by preserving SARS-CoV-2 RNA integrity and reveals the prolonged viral loading associated with older age and male gender in COVID-19 patients.

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

ABSTRACTRNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalogue of microbial RBPs.

P2587Cardiosomal microRNAs are essential in post-infarction myofibroblast phenoconversion

Background Circulating microRNAs (miRNAs) are actively transported, via inclusion in extracellular microvesicles/exosomes (herein named cardiosomes since they are deriving from cardiomyocytes), allowing protection from degradation and underlying cell-cell communications. After myocardial infarction (MI), quiescent cardiac fibroblasts can acquire an activated phenotype, and a recognized marker of such activation is the expression of α-smooth muscle actin (α-SMA); such cells are commonly known as myofibroblasts. Hypothesis We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Methods Using an established murine model of MI, obtained via permanent ligation of the left anterior descending coronary artery, we tested our hypothesis by measuring in primary isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, which we found to be upregulated in cardiomyocytes post-MI and to be involved in myofibroblast phenoconversion. Results Bioinformatic studies validated by biological assays identified three cardiomyocyte-specific miRNAs (namely miR-92a, miR-195, and miR-494) that are upregulated following cardiac ischemia and are also known to regulate α-SMA expression. We demonstrated that these miRNAs are upregulated in fibroblasts post-ischemia and are also contained in cardiosomes; to ensure that these miRNAs were confined inside cardiosomes, the samples were treated with RNase, showing that the level of miRNAs was not affected by RNase treatment, unless in presence of the detergent Triton X-100. The activation of myofibroblasts was confirmed by the increased expression of α-SMA. Using a luciferase assay, we also validated that miR-92a, miR-195, and mir-494 target the 3'UTR of α-SMA and regulate its transcription. Moreover, we observed that primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effects were detected following incubation with cardiosomes or medium from sham cardiomyocytes. Strikingly, myofibroblast phenoconversion was markedly attenuated when GW4869, an inhibitor of neutral sphingomyelinase 2 responsible for cardiosomes secretion, was added to cultured post-MI cardiomyocytes. Conclusion Taken together, our findings indicate that three cardiomyocyte-specific miRNAs (miR-92a, miR-195, and miR-494) are crucial in the activation of myofibroblasts.

Isolation and characterization of BpL1, a broad acting lytic bacteriophage against Brucella

We have isolated a new broad acting lytic brucellaphage (BpL1) from the sewage of a dairy farm. The phage lysed all the 12Brucella abortusfield isolates,B. abortusstrain 99 andBrucella melitensisbut did not lyse any of the heterologous species tested viz.Staphylococcus aureus, Pasteurella multocida, Escherichia coli,andSalmonella species. Streaking the plaques onBrucellalawn gave clear zones along the streak lines. The plaques were circular with a diameter of 0.5- 3.0 mm. At a concentration of 10−4the phage count was 4.5 × 106plaques per ml. It was a tailed phage with icosahedral head (62.2 nm in diameter and 73.71 nm in length), and the head to tail length was 229.21 nm. The phage belonged to the order Caudovirales and familySiphoviridae. It was inactivated within one hour at 55°C and within 4 hours at −20°C. Treatment at pH 2 for 4 hours and at pH 4 for 12 hours inactivated it. It was inactivated after 4 hours exposure to sunlight, and within 4 minutes by UV light. Chloroform and Sodium Dodecyl Sulfate inactivated it within 15 minutes. Lysozyme inactivated it within 1 hour whereas RNase treatment did not affect its activity.

The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL

The mammalian cell nucleus contains membraneless suborganelles referred to as nuclear bodies (NBs). Some NBs are formed with an architectural RNA (arcRNA) as the structural core. Here, we searched for new NBs that are built on unidentified arcRNAs by screening for ribonuclease (RNase)-sensitive NBs using 32,651 fluorescently tagged human cDNA clones. We identified 32 tagged proteins that required RNA for their localization in distinct nuclear foci. Among them, seven RNA-binding proteins commonly localized in the Sam68 nuclear body (SNB), which was disrupted by RNase treatment. Knockdown of each SNB protein revealed that SNBs are composed of two distinct RNase-sensitive substructures. One substructure is present as a distinct NB, termed the DBC1 body, in certain conditions, and the more dynamic substructure including Sam68 joins to form the intact SNB. HNRNPL acts as the adaptor to combine the two substructures and form the intact SNB through the interaction of two sets of RNA recognition motifs with the putative arcRNAs in the respective substructures.

Destruction of the Capsid and Genome of GII.4 Human Norovirus Occurs during Exposure to Metal Alloys Containing Copper

ABSTRACTHuman norovirus (HuNoV) represents a significant public health burden worldwide and can be environmentally transmitted. Copper surfaces have been shown to inactivate the cultivable surrogate murine norovirus, but no such data exist for HuNoV. The purpose of this study was to characterize the destruction of GII.4 HuNoV and virus-like particles (VLPs) during exposure to copper alloy surfaces. Fecal suspensions positive for a GII.4 HuNoV outbreak strain or GII.4 VLPs were exposed to copper alloys or stainless steel for 0 to 240 min and recovered by elution. HuNoV genome integrity was assessed by reverse transcription-quantitative PCR (RT-qPCR) (without RNase treatment), and capsid integrity was assessed by RT-qPCR (with RNase treatment), transmission electron microscopy (TEM), SDS-PAGE/Western blot analysis, and a histo-blood group antigen (HBGA) binding assay. Exposure of fecal suspensions to pure copper for 60 min reduced the GII.4 HuNoV RNA copy number by ∼3 log10units when analyzed by RT-qPCR without RNase treatment and by 4 log10units when a prior RNase treatment was used. The rate of reduction of the HuNoV RNA copy number was approximately proportional to the percentage of copper in each alloy. Exposure of GII.4 HuNoV VLPs to pure-copper surfaces resulted in noticeable aggregation and destruction within 240 min, an 80% reduction in the VP1 major capsid protein band intensity in 15 min, and a near-complete loss of HBGA receptor binding within 8 min. In all experiments, HuNoV remained stable on stainless steel. These results suggest that copper surfaces destroy HuNoV and may be useful in preventing environmental transmission of the virus in at-risk settings.

A Novel Mechanism of Cellular Activation Mediated By Antiphospholipid Antibody-Induced Extracellular Vesicles

Introduction Antiphospholipid Syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). Elevated levels of endothelial cell-derived extracellular vesicles (EV) are present in the plasma of patients with APLA, and APLA, particularly those directed against β2-glycoprotein I (β2GPI), stimulate EV release from endothelial cells. However, the constituents or activity of these EV are not well studied. Objective To determine whether EV derived from anti-β2GPI antibody treated endothelial cells activate quiescent endothelial cells, and define the mechanism. Methods EV released by endothelial cells in response to exposure to β2GPI and either anti-β2GPI antibodies or control IgG were isolated by ultra-centrifugation. Endothelial cells treated in this manner, as well as released EV and EV-free conditioned medium were analyzed for interleukin-1β (IL-1β) content. Both EC and EV were also probed for expression of components of the NLRP3 inflammasome. The ability of EV isolated from treated cells to activate endothelial cells in an autocrine/paracrine manner was assessed through measurement of E-selectin expression on the endothelial cell surface as well as phosphorylation of IRAK4. To define the mechanism of endothelial cell activation by EV, endothelial cells were pretreated with several inhibitors of the IL-1β signaling pathway, or with siRNA against IRAK4, TLR2, TLR4, TLR7 and TLR9, or EV. We also examined the effect of pretreating EV with RNase A before addition to endothelial cells. Results EV released from endothelial cells in response to anti-β2GPI antibodies, but not control IgG, were enriched in mature interleukin-1β (IL-1β) and induced EC activation. However, the ability of these EV to activate EC was not inhibited by a neutralizing IL-1 antibody, IL-1 receptor antagonist, or IL-1 receptor siRNA. To define the signaling cascade activated by EC-derived EV, we examined downstream components of the IL-1 receptor (IL-1R)/Toll-like receptor (TLR) pathway, finding that activation was associated with and dependent upon phosphorylation of IRAK4. To determine which members of the IL-1R/TLR family mediated IRAK4 phosphorylation and cellular activation in response to APLA-induced EV, we inhibited the expression of TLR2, TLR4, TLR7 and TLR9 using specific siRNAs. Inhibition of TLR7, but not other TLRs, blocked EC activation in response to EV (Figure: Panel A). Since a ligand of TLR7 is single-stranded RNA (ssRNA), we also pretreated EV with RNase A, which inhibited activation to a similar extent as TLR7 knockdown (Figure: Panel B). Finally, we also observed that anti-β2GPI antibodies increased the expression of endothelial cell TLR7 (Figure: Panel C). Conclusions APLA/anti-β2GPI antibodies cause EC activation and inflammasome formation, leading to release of IL-1β enriched EV. These EV induce EC activation; however, activation is mediated primarily through interactions of EV-associated ssRNA with TLR7 rather than through an IL-1β receptor-dependent pathway. These EV may contribute to vascular activation in an autocrine/paracrine manner and contribute to the prothrombotic phenotype in APS. Acknowledgment: This work was supported by an ASH Bridge Grant. Figure 1 Figure: (A) Effect of siRNA to TLR siRNA on endothelial cell activation, (B) Effect of RNase treatment of EV on endothelial cell activation, (C) anti-β2GPI antibodies induce endothelial cell TLR7 expression. Figure 1. Figure: (A) Effect of siRNA to TLR siRNA on endothelial cell activation, (B) Effect of RNase treatment of EV on endothelial cell activation, (C) anti-β2GPI antibodies induce endothelial cell TLR7 expression. Disclosures No relevant conflicts of interest to declare.

The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation

How cells shape and remodel organelles in response to cellular signals is a poorly understood process. Using Xenopus laevis egg extract, we found that increases in cytosolic calcium lead to the activation of an endogenous ribonuclease, XendoU. A fraction of XendoU localizes to the endoplasmic reticulum (ER) and is required for nuclear envelope assembly and ER network formation in a catalysis-dependent manner. Using a purified vesicle fusion assay, we show that XendoU functions on the surface of ER membranes to promote RNA cleavage and ribonucleoprotein (RNP) removal. Additionally, RNA removal from the surface of vesicles by RNase treatment leads to increased ER network formation. Using human tissue culture cells, we found that hEndoU localizes to the ER, where it promotes the formation of ER tubules in a catalysis-dependent manner. Together, these results demonstrate that calcium-activated removal of RNA from membranes by XendoU promotes and refines ER remodeling and the formation of tubular ER.