LEDGF/p75 is critical but not essential for multiple-round HIV 1 replication

status: publishe

Alteration of Cardiac Progenitor Cell Potency in GRMD Dogs

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Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis

Wheat Domestication Accelerated Evolution and Triggered Positive Selection in the β-Xylosidase Enzyme of Mycosphaerella graminicola

Plant cell wall degrading enzymes (PCWDEs) of plant pathogens are receiving increasing interest for their potential to trigger plant defense reactions. In an antagonistic co-evolutionary arms race between host and pathogen, PCWDEs could be under strong selection. Here, we tested the hypothesis that PCWDEs in the fungal wheat pathogen Mycosphaerella graminicola have been positively selected by analyzing ratios of non-synonymous and synonymous nucleotide changes in the genes encoding these enzymes. Analyses of five PCWDEs demonstrated that one (β-xylosidase) has been under strong positive selection and experienced an accelerated rate of evolution. In contrast, PCWDEs in the closest relatives of M. graminicola collected from wild grasses did not show evidence for selection or deviation from a molecular clock. Since the genealogical divergence of M. graminicola from these latter species coincided with the onset of agriculture, we hypothesize that the recent domestication of the host plant and/or agricultural practices triggered positive selection in β-xylosidase and that this enzyme played a key role in the emergence of a host-specialized pathogen

LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones

BACKGROUND The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue

Track A Basic Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138319/1/jia218438.pd