Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.     

Inhibition of GSK3 promotes replication and survival of pancreatic beta cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.     

Eccentric exercise increases EMG amplitude and force fluctuations during submaximal contractions of elbow flexor muscles.

The purpose of this study was to determine the effect of eccentric exercise on the ability to exert steady submaximal forces with muscles that cross the elbow joint. Eight subjects performed two tasks requiring isometric contraction of the right elbow flexors: a maximum voluntary contraction (MVC) and a constant-force task at four submaximal target forces (5, 20, 35, 50% MVC) while electromyography (EMG) was recorded from elbow flexor and extensor muscles. These tasks were performed before, after, and 24 h after a period of eccentric (fatigue and muscle damage) or concentric exercise (fatigue only). MVC force declined after eccentric exercise (45% decline) and remained depressed 24 h later (24%), whereas the reduced force after concentric exercise (22%) fully recovered the following day. EMG amplitude during the submaximal contractions increased in all elbow flexor muscles after eccentric exercise, with the greatest change in the biceps brachii at low forces (3-4 times larger at 5 and 20% MVC) and in the brachialis muscle at moderate forces (2 times larger at 35 and 50% MVC). Eccentric exercise resulted in a twofold increase in coactivation of the triceps brachii muscle during all submaximal contractions. Force fluctuations were larger after eccentric exercise, particularly at low forces (3-4 times larger at 5% MVC, 2 times larger at 50% MVC), with a twofold increase in physiological tremor at 8-12 Hz. These data indicate that eccentric exercise results in impaired motor control and altered neural drive to elbow flexor muscles, particularly at low forces, suggesting altered motor unit activation after eccentric exercise.     

Global secretome analysis of resident cardiac progenitor cells from wild-type and transgenic heart failure mice: Why ambience matters

Resident cardiac progenitor cells (CPCs) have gained attention in cardiac regenerative medicine primarily due to their paracrine activity. In our current study we determined the role of pathological conditions such as heart failure on the autocrine-paracrine action of stem cell antigen-1 (Sca-1) expressing CPC. This comparative secretome profiling of Sca-1⁺ cells derived from transgenic heart failure (αMHC–cyclin-T1/Gαq overexpression [Cyc] cells) versus healthy (wild-type [Wt] cells) mice, achieved via mass-spectrometric quantification, enabled the identification of over 700 proteins. Our results demonstrate that the heart failure milieu caused a 2-fold enrichment of extracellular matrix proteins (ECM) like biglycan, versican, collagen XII, and angiogenic factors like heparan sulfate proteoglycan 2, plasminogen activator inhibitor 1 in the secretome. We further elucidated the direct influence of the secretome on the functional behavior of Sca-1 ⁺ cells via in vitro tube forming assay. Secreted factors present in the diseased milieu induced tube formation in Cyc cells (1.7-fold; p < 0.01) when compared with Wt cells after 24 hr of exposure. The presence of conditioned media moderately increased the proliferation of Cyc cells but had a more pronounced effect on Wt cells. Overall, these findings revealed global modifications in the secretory activity of adult Sca-1 ⁺ cells in the heart failure milieu. The secretion of ECM proteins and angiogenic factors, which are crucial for cardiac remodeling and recovery, was notably enriched in the supernatant of Cyc cells. Thus, during heart failure the microenvironment of Sca-1 ⁺ cells might favor angiogenesis and proliferation suggesting their potential to recover the damaged heart.