Auto Tag: University of Rochester

September 22, 2019  |  

Daily HIV pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate-emtricitabine reduced Streptococcus and increased Erysipelotrichaceae in rectal microbiota.

Daily PrEP is highly effective at preventing HIV-1 acquisition, but risks of long-term tenofovir disoproxil fumarate plus emtricitabine (TDF-FTC) include renal decline and bone mineral density decrease in addition to initial gastrointestinal side effects. We investigated the impact of TDF-FTC on the enteric microbiome using rectal swabs collected from healthy MSM before PrEP initiation and after 48 to 72 weeks of adherent PrEP use. The V4 region of the 16S ribosomal RNA gene sequencing showed that Streptococcus was significantly reduced from 12.0% to 1.2% (p?=?0.036) and Erysipelotrichaceae family was significantly increased from 0.79% to 3.3% (p?=?0.028) after 48-72 weeks of daily PrEP. Catenibacterium mitsuokai, Holdemanella biformis and Turicibacter sanguinis were increased within the Erysipelotrichaceae family and Streptococcus agalactiae, Streptococcus oralis, Streptococcus mitis were reduced. These changes were not associated with host factors including PrEP duration, age, race, tenofovir diphosphate blood level, any drug use and drug abuse, suggesting that the observed microbiome shifts were likely induced by daily PrEP use. Long-term PrEP resulted in increases of Catenibacterium mitsuokai and Holdemanella biformis, which have been associated with gut microbiome dysbiosis. Our observations can aid in characterizing PrEP’s side effects, which is likely to improve PrEP adherence, and thus HIV-1 prevention.

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July 19, 2019  |  

Genomic changes following the reversal of a Y chromosome to an autosome in Drosophila pseudoobscura

Robertsonian translocations resulting in fusions between sex chromosomes and autosomes shape karyotype evolution by creating new sex chromosomes from autosomes. These translocations can also reverse sex chromosomes back into autosomes, which is especially intriguing given the dramatic differences between autosomes and sex chromosomes. To study the genomic events following a Y chromosome reversal, we investigated an autosome-Y translocation in Drosophila pseudoobscura. The ancestral Y chromosome fused to a small autosome (the dot chromosome) approximately 10–15 Mya. We used single molecule real-time sequencing reads to assemble the D. pseudoobscura dot chromosome, including this Y-to-dot translocation. We find that the intervening sequence between the ancestral Y and the rest of the dot chromosome is only ~78 Kb and is not repeat-dense, suggesting that the centromere now falls outside, rather than between, the fused chromosomes. The Y-to-dot region is 100 times smaller than the D. melanogaster Y chromosome, owing to changes in repeat landscape. However, we do not find a consistent reduction in intron sizes across the Y-to-dot region. Instead, deletions in intergenic regions and possibly a small ancestral Y chromosome size may explain the compact size of the Y-to-dot translocation.

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July 19, 2019  |  

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster.

Highly repetitive satellite DNA (satDNA) repeats are found in most eukaryotic genomes. SatDNAs are rapidly evolving and have roles in genome stability and chromosome segregation. Their repetitive nature poses a challenge for genome assembly and makes progress on the detailed study of satDNA structure difficult. Here, we use single-molecule sequencing long reads from Pacific Biosciences (PacBio) to determine the detailed structure of all major autosomal complex satDNA loci in Drosophila melanogaster, with a particular focus on the 260-bp and Responder satellites. We determine the optimal de novo assembly methods and parameter combinations required to produce a high-quality assembly of these previously unassembled satDNA loci and validate this assembly using molecular and computational approaches. We determined that the computationally intensive PBcR-BLASR assembly pipeline yielded better assemblies than the faster and more efficient pipelines based on the MHAP hashing algorithm, and it is essential to validate assemblies of repetitive loci. The assemblies reveal that satDNA repeats are organized into large arrays interrupted by transposable elements. The repeats in the center of the array tend to be homogenized in sequence, suggesting that gene conversion and unequal crossovers lead to repeat homogenization through concerted evolution, although the degree of unequal crossing over may differ among complex satellite loci. We find evidence for higher-order structure within satDNA arrays that suggest recent structural rearrangements. These assemblies provide a platform for the evolutionary and functional genomics of satDNAs in pericentric heterochromatin. © 2017 Khost et al.; Published by Cold Spring Harbor Laboratory Press.

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July 19, 2019  |  

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.© 2018, Fallon et al.

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July 7, 2019  |  

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis.

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019  |  

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2016;9999: 1-13. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

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July 7, 2019  |  

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

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July 7, 2019  |  

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans.

We report the whole genome sequence of the serotype e Cbm+ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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July 7, 2019  |  

Moving forward: recent developments for the ferret biomedical research model.

Since the initial report in 1911, the domestic ferret has become an invaluable biomedical research model. While widely recognized for its utility in influenza virus research, ferrets are used for a variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens. However, there are limitations to the model that must be overcome for maximal utility for the scientific community. Here, we describe important recent advances that will accelerate biomedical research with this animal model. Copyright © 2018 Albrecht et al.

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July 7, 2019  |  

Meeting report: mobile genetic elements and genome plasticity 2018

The Mobile Genetic Elements and Genome Plasticity conference was hosted by Keystone Symposia in Santa Fe, NM USA, February 11–15, 2018. The organizers were Marlene Belfort, Evan Eichler, Henry Levin and Lynn Maquat. The goal of this conference was to bring together scientists from around the world to discuss the function of transposable elements and their impact on host species. Central themes of the meeting included recent innovations in genome analysis and the role of mobile DNA in disease and evolution. The conference included 200 scientists who participated in poster presentations, short talks selected from abstracts, and invited talks. A total of 58 talks were organized into eight sessions and two workshops. The topics varied from mechanisms of mobilization, to the structure of genomes and their defense strategies to protect against transposable elements.

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July 7, 2019  |  

Bridging gaps in transposable element research with single-molecule and single-cell technologies

More than half of the genomic landscape in humans and many other organisms is composed of repetitive DNA, which mostly derives from transposable elements (TEs) and viruses. Recent technological advances permit improved assessment of the repetitive content across genomes and newly developed molecular assays have revealed important roles of TEs and viruses in host genome evolution and organization. To update on our current understanding of TE biology and to promote new interdisciplinary strategies for the TE research community, leading experts gathered for the 2nd Uppsala Transposon Symposium on October 4–5, 2018 in Uppsala, Sweden. Using cutting-edge single-molecule and single-cell approaches, research on TEs and other repeats has entered a new era in biological and biomedical research.

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