Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.     

Are all bony fishes oxygen regulators? Evidence for oxygen regulation in a putative oxygen conformer,the swamp eel Synbranchus marmoratus

This study investigated the oxygen consumption of the putative oxygen conformer marbled swamp eel Synbranchus marmoratus during progressive hypoxia. Earlier studies have not reached an agreement on whether S. marmoratus is a conformer or a regulator. Our results support the view that S. marmoratus is an oxygen regulator, like most bony fishes.     

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia

The histology‐based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label‐free LC‐MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate‐derived proteins including IGKV3D‐20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D‐20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.     

MS‐Rescue: A Computational Pipeline to Increase the Quality and Yield of Immunopeptidomics Experiments

LC–MS/MS has become the standard platform for the characterization of immunopeptidomes, the collection of peptides naturally presented by major histocompatibility complex molecules to the cell surface. The protocols and algorithms used for immunopeptidomics data analysis are based on tools developed for traditional bottom‐up proteomics that address the identification of peptides generated by tryptic digestion. Such algorithms are generally not tailored to the specific requirements of MHC ligand identification and, as a consequence, immunopeptidomics datasets suffer from dismissal of informative spectral information and high false discovery rates. Here, a new pipeline for the refinement of peptide‐spectrum matches (PSM) is proposed, based on the assumption that immunopeptidomes contain a limited number of recurring peptide motifs, corresponding to MHC specificities. Sequence motifs are learned directly from the individual peptidome by training a prediction model on high‐confidence PSMs. The model is then applied to PSM candidates with lower confidence, and sequences that score significantly higher than random peptides are rescued as likely true ligands. The pipeline is applied to MHC class I immunopeptidomes from three different species, and it is shown that it can increase the number of identified ligands by up to 20–30%, while effectively removing false positives and products of co‐precipitation. Spectral validation using synthetic peptides confirms the identity of a large proportion of rescued ligands in the experimental peptidome.     

Response to starvation and microbial community composition in microbial fuel cells enriched on different electron donors

In microbial fuel cells (MFCs), microorganisms generate electrical current by oxidizing organic compounds. MFCs operated with different electron donors harbour different microbial communities, and it is unknown how that affects their response to starvation. We analysed the microbial communities in acetate- and glucose-fed MFCs and compared their responses to 10 days starvation periods. Each starvation period resulted in a 4.2 ± 1.4% reduction in electrical current in the acetate-fed MFCs and a 10.8 ± 3.9% reduction in the glucose-fed MFCs. When feed was resumed, the acetate-fed MFCs recovered immediately, whereas the glucose-fed MFCs required 1 day to recover. The acetate-fed bioanodes were dominated by Desulfuromonas spp. converting acetate into electrical current. The glucose-fed bioanodes were dominated by Trichococcus sp., functioning as a fermenter, and a member of Desulfuromonadales, using the fermentation products to generate electrical current. Suspended biomass and biofilm growing on non-conductive regions within the MFCs had different community composition than the bioanodes. However, null models showed that homogenizing dispersal of microorganisms within the MFCs affected the community composition, and in the glucose-fed MFCs, the Trichococcus sp. was abundant in all locations. The different responses to starvation can be explained by the more complex pathway requiring microbial interactions to convert glucose into electrical current.     

Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla

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A single-vector EYFP reporter gene assay for G protein-coupled receptors

We here present an improved and simplified assay to study signal transduction of the G_s class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any G_s protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2).