Daleengland8709

racterization of oral fungi and bacteria in order to better elucidate the dynamic changes in microbial community structure and interkingdom functional interactions during the injury of chemotherapy and antibiotic exposure as well as the clinical consequences of these interrelated alterations. Copyright © 2020 Robinson et al.Candida albicans adapts to various conditions in different body niches by regulating gene expression, protein synthesis, and metabolic pathways. These adaptive reactions not only allow survival but also influence the interaction with host cells, which is governed by the composition and structure of the fungal cell wall. Numerous studies had shown linkages between mitochondrial functionality, cell wall integrity and structure, and pathogenicity. Thus, we decided to inhibit single complexes of the respiratory chain of C. albicans and to analyze the resultant interaction with macrophages via their phagocytic activity. Remarkably, inhibition of the fungal bc1 complex by antimycin A increased phagocytosis, which correlated with an increased accessibility of β-glucans. To contribute to mechanistic insights, we performed metabolic studies, which highlighted significant changes in the abundance of constituents of the plasma membrane. Collectively, our results reinforce the strong linkage between fungal energy metabolvance of energy metabolism, indirectly also for host-pathogen interactions, without affecting viability. Copyright © 2020 Cui et al.Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11, the latter protein binds to RNA and the RNA polymerase II carboxy terminal domain. Here, we used siRNA mediated knock-down and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3' un-translated regions (3' UTRs) we observed shifts from promoter proximal regions to the 3' UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells co-sedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer. Published by Cold Spring Harbor Laboratory Press for the RNA Society.The nadA motif is the first known NAD+-dependent riboswitch, comprising two similar tandem bulged stem-loop structures. We have determined the structure of the 5' domain 1 of the riboswitch. It has three coaxial helical segments, separated by an ACANCCCC bulge and by an internal loop, with a tertiary contact between them that includes two CG base pairs. We have determined the structure with a number of ligands related to NADH, but in each case only the ADP moiety is observed. The adenosine adopts an anti conformation, and forms multiple hydrogen bonds across the width of the sugar edge of the penultimate CG base pair of the helix preceding the bulge, and the observed contacts have been confirmed by mutagenesis and calorimetry. Two divalent metal ions play a key structural role at the narrow neck of the bulge. One makes direct bonding contacts to the di-phosphate moiety, locking it into position. Thus the nucleobase, ribose and phosphate groups of the ADP moiety are all specifically recognised by the RNA. The NAD+ riboswitch is modular. Domain 1 is an ADP binding domain that may be ancient, and could potentially be used in combination with other ligand binding motifs such as CoA. Published by Cold Spring Harbor Laboratory Press for the RNA Society.Cells adapt to environmental changes, including fluctuations in oxygen levels, through the induction of specific gene expression programs. However, most transcriptomic studies do not distinguish the relative contribution of transcription, RNA processing and RNA degradation processes to cellular homeostasis. click here Here we used metabolic labeling followed by massive parallel sequencing of newly transcribed and preexisting RNA fractions to simultaneously analyze RNA synthesis and decay in primary endothelial cells exposed to low oxygen tension. We found that changes in transcription rates induced by hypoxia are the major determinant of changes in RNA levels. However, degradation rates also had a significant contribution, accounting for 24% of the observed variability in total mRNA. In addition, our results indicated that hypoxia led to a reduction of the overall mRNA stability from a median half-life in normoxia of 8.7 hours, to 5.7 hours in hypoxia. Analysis of RNA content per cell confirmed a decrease of both mRNA and total RNA in hypoxic samples and that this effect is dependent on the EGLN/HIF/TSC2 axis. This effect could potentially contribute to fundamental global responses such as inhibition of translation in hypoxia. In summary, our study provides a quantitative analysis of the contribution of RNA synthesis and stability to the transcriptional response to hypoxia and uncovers an unexpected effect on the latter. Published by Cold Spring Harbor Laboratory Press for the RNA Society.Compendial testing methods are not required to be fully validated but their suitability should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in United States. ANVISA (Agéncia Nacional de Vigiláncia Sanitária) also requires that compendial analytical methods shall have their suitability demonstrated for the intended use by a partial validation study. Suitability verification or partial validation can be divided into two major categories visual and instrumental methods. For visual methods, the color and clarity interferences should be evaluated. If the color or clarity/opalescence of the sample is outside of the range of the Pharmacopeia standards/reference solutions, the validity of test results should be evaluated. Specificity is usually waived since the methods are not specific to products, and accuracy/precision can be addressed by comparing results from analyst to analyst. For instrument methods, specificity can also be waived for certain assays.