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Wednesday, October 23, 2019

Optimized CRISPR-Cas9 genome editing for Leishmania and its use to target a multigene family, induce chromosomal translocation, and study DNA break repair mechanisms.

CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that the Leishmania donovani rRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of…

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Wednesday, October 23, 2019

Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector.

Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers.We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from…

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Wednesday, October 23, 2019

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M.?mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the…

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Wednesday, October 23, 2019

Sites of retroviral DNA integration: From basic research to clinical applications.

One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as…

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Wednesday, October 23, 2019

CRISPR/Cas9-generated p47(phox)-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47(phox)-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (?GT) mutation in p47(phox) encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47(phox)-subunit deficiency represents the second…

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Wednesday, October 23, 2019

Structural determination of the broadly reactive anti-IGHV1-69 anti-idiotypic antibody G6 and its idiotope.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with…

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Wednesday, October 23, 2019

Bioengineered AAV capsids with combined high human liver transduction in vivo and unique humoral seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with…

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Wednesday, October 23, 2019

Chromosomal-level assembly of yellow catfish genome using third-generation DNA sequencing and Hi-C analysis.

The yellow catfish, Pelteobagrus fulvidraco, belonging to the Siluriformes order, is an economically important freshwater aquaculture fish species in Asia, especially in Southern China. The aquaculture industry has recently been facing tremendous challenges in germplasm degeneration and poor disease resistance. As the yellow catfish exhibits notable sex dimorphism in growth, with adult males about two- to three-fold bigger than females, the way in which the aquaculture industry takes advantage of such sex dimorphism is another challenge. To address these issues, a high-quality reference genome of the yellow catfish would be a very useful resource.To construct a high-quality reference genome for…

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Sunday, September 22, 2019

Membrane attack complex-associated molecules from redlip mullet (Liza haematocheila): Molecular characterization and transcriptional evidence of C6, C7, C8ß, and C9 in innate immunity.

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were…

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Sunday, September 22, 2019

Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of…

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Sunday, September 22, 2019

Two phospholipid scramblase 1-related proteins (PLSCR1like-a & -b) from Liza haematocheila: Molecular and transcriptional features and expression analysis after immune stimulation.

Phospholipid scramblases (PLSCRs) are a family of transmembrane proteins known to be responsible for Ca2+-mediated bidirectional phospholipid translocation in the plasma membrane. Apart from the scrambling activity of PLSCRs, recent studies revealed their diverse other roles, including antiviral defense, tumorigenesis, protein-DNA interactions, apoptosis regulation, and cell activation. Nonetheless, the biological and transcriptional functions of PLSCRs in fish have not been discovered to date. Therefore, in this study, two new members related to the PLSCR1 family were identified in the red lip mullet (Liza haematocheila) as MuPLSCR1like-a and MuPLSCR1like-b, and their characteristics were studied at molecular and transcriptional levels. Sequence analysis…

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Sunday, September 22, 2019

Global transcript structure resolution of high gene density genomes through multi-platform data integration.

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus…

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Sunday, September 22, 2019

Draft genome sequence of Sulfurospirillum sp. strain MES, reconstructed from the metagenome of a microbial electrosynthesis system.

A draft genome of Sulfurospirillum sp. strain MES was isolated through taxonomic binning of a metagenome sequenced from a microbial electrosynthesis system (MES) actively producing acetate and hydrogen. The genome contains the nosZDFLY genes, which are involved in nitrous oxide reduction, suggesting the potential role of this strain in denitrification. Copyright © 2015 Ross et al.

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Sunday, September 22, 2019

Moving beyond microbiome-wide associations to causal microbe identification.

Microbiome-wide association studies have established that numerous diseases are associated with changes in the microbiota. These studies typically generate a long list of commensals implicated as biomarkers of disease, with no clear relevance to disease pathogenesis. If the field is to move beyond correlations and begin to address causation, an effective system is needed for refining this catalogue of differentially abundant microbes and to allow subsequent mechanistic studies. Here we demonstrate that triangulation of microbe-phenotype relationships is an effective method for reducing the noise inherent in microbiota studies and enabling identification of causal microbes. We found that gnotobiotic mice harbouring…

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Sunday, September 22, 2019

16S rRNA long-read sequencing of the granulation tissue from nonsmokers and smokers-severe chronic periodontitis patients

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance…

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