Publications by authors named "Pawel Michalak"

64 Publications

Coinfection of cattle in Virginia with Ikeda genotype and .

J Vet Diagn Invest 2021 Nov 11:10406387211057627. Epub 2021 Nov 11.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA.

Ikeda is a newly identified agent of bovine infectious anemia in the United States. Although Ikeda is transmitted by ticks other than the tick that transmits -a bacterial etiology of bovine infectious anemia-the geographic distributions of these 2 infectious organisms overlap, with coinfection reported in some cattle. Only anaplasmosis has an approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex real-time PCR (rtPCR) for and . With a cutoff of 38 cycles, the duplex assay has a sensitivity of 97.0% and a specificity of 100% for ; with a cutoff of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for , compared to existing tests. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, our rtPCR facilitates the study of coinfection of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for , 125 were positive for , and 12 samples were positive for both and Hence, coinfection by these 2 agents of bovine infectious anemia does occur within Virginia. It is likely that this pattern of infection will be seen in other regions where and infections are endemic, despite the difference in tick vectors.
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http://dx.doi.org/10.1177/10406387211057627DOI Listing
November 2021

A Draft Genome Assembly for the Eastern Fox Squirrel, Sciurus niger.

G3 (Bethesda) 2021 Sep 22. Epub 2021 Sep 22.

Virginia Museum of Natural History, 21 Starling Avenue, Martinsville, Virginia 24112, USA. 276.634.4177.

The eastern fox squirrel, Sciurus niger, exhibits marked geographic variation in size and coat color, is a model organism for studies of behavior and ecology, and a potential model for investigating physiological solutions to human porphyrias. We assembled a genome using Illumina HiSeq, PacBio SMRT, and Oxford Nanopore MinION sequencing platforms. Together, the sequencing data resulted in a draft genome of 2.99 Gb, containing 32,830 scaffolds with an average size of 90.9Kb and N50 of 183.8 Kb. Genome completeness was estimated to be 93.78%. A total of 24,443 protein-encoding genes were predicted from the assembly, and 23,079 (94.42%) were annotated. Repeat elements comprised an estimated 38.49% of the genome, with the majority being LINEs (13.92%), SINEs (6.04%), and LTR elements. The topology of the species tree reconstructed using maximum likelihood phylogenetic analysis was congruent with those of previous studies. This genome assembly can prove useful for comparative studies of genome structure and function in this rapidly diversifying lineage of mammals, for studies of population genomics and adaptation, and for biomedical research. Predicted amino acid sequence alignments for genes affecting heme biosynthesis, color vision and hibernation showed point mutations and indels that may affect protein function and ecological adaptation.
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http://dx.doi.org/10.1093/g3journal/jkab315DOI Listing
September 2021

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation.

Cell 2021 08 7;184(17):4392-4400.e4. Epub 2021 Jul 7.

Department of Biomedical Sciences and Pathobiology, VA-MD Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24060, USA. Electronic address:

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.
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http://dx.doi.org/10.1016/j.cell.2021.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260498PMC
August 2021

Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster.

Heredity (Edinb) 2021 09 23;127(3):278-287. Epub 2021 Jun 23.

Institute of Evolution, University of Haifa, Haifa, Israel.

Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. Here, we test for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. We show that winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. We ascribe the observed differences to indirect selection on recombination caused by directional selection on desiccation tolerance. Our findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8-10 generations).
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http://dx.doi.org/10.1038/s41437-021-00449-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405755PMC
September 2021

Ethnically biased microsatellites contribute to differential gene expression and glutathione metabolism in Africans and Europeans.

PLoS One 2021 25;16(3):e0249148. Epub 2021 Mar 25.

Edward Via College of Osteopathic Medicine, Blacksburg, Virginia, United States of America.

Approximately three percent of the human genome is occupied by microsatellites: a type of short tandem repeat (STR). Microsatellites have well established effects on (a) the genetic structure of diverse human populations and (b) expression of nearby genes. These lines of inquiry have uncovered 3,984 ethnically biased microsatellite loci (EBML) and 28,375 expression STRs (eSTRs), respectively. We hypothesize that a combination of EBML, eSTRs, and gene expression data (RNA-seq) can be used to show that microsatellites contribute to differential gene expression and phenotype in human populations. In fact, our previous study demonstrated a degree of mutual overlap between EBML and eSTRs but fell short of quantifying effects on gene expression. The present work aims to narrow the gap. First, we identify 313 overlapping EBML/eSTRs and recapitulate their mutual overlap. The 313 EBML/eSTRs are then characterized across ethnicity and tissue type. We use RNA-seq data to pursue validation of 49 regions that affect whole blood gene expression; 32 out of 54 affected genes are differentially expressed in Africans and Europeans. We quantify the relative contribution of these 32 genes to differential expression; fold change tends to be less than other differentially expressed genes. Repeat length correlates with expression for 15 of the 32 genes; two are conspicuously involved in glutathione metabolism. Finally, we repurpose a mathematical model of glutathione metabolism to investigate how a single polymorphic microsatellite affects phenotype. We conclude with a testable prediction that microsatellite polymorphisms affect GPX7 expression and oxidative stress in Africans and Europeans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249148PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993785PMC
October 2021

DNA sequencing of anatomy lab cadavers to provide hands-on precision medicine introduction to medical students.

BMC Med Educ 2020 Nov 16;20(1):437. Epub 2020 Nov 16.

Edward Via College of Osteopathic Medicine, (VCOM), VA, Biomedical Sciences, 2265 Kraft Drive, Blacksburg, VA, 24060, USA.

Background: Medical treatment informed by Precision Medicine is becoming a standard practice for many diseases, and patients are curious about the consequences of genomic variants in their genome. However, most medical students' understanding of Precision Medicine derives from classroom lectures. This format does little to foster an understanding for the potential and limitations of Precision Medicine. To close this gap, we implemented a hands-on Precision Medicine training program utilizing exome sequencing to prepare a clinical genetic report of cadavers studied in the anatomy lab. The program reinforces Precision Medicine related learning objectives for the Genetics curriculum.

Methods: Pre-embalmed blood samples and embalmed tissue were obtained from cadavers (donors) used in the anatomy lab. DNA was isolated and sequenced and illustrative genetic reports provided to the students. The reports were used to facilitate discussion with students on the implications of pathogenic genomic variants and the potential correlation of these variants in each "donor" with any anatomical anomalies identified during cadaver dissection.

Results: In 75% of cases, analysis of whole exome sequencing data identified a variant associated with increased risk for a disease/abnormal condition noted in the donor's cause of death or in the students' anatomical findings. This provided students with real-world examples of the potential relationship between genomic variants and disease risk. Our students also noted that diseases associated with 92% of the pathogenic variants identified were not related to the anatomical findings, demonstrating the limitations of Precision Medicine.

Conclusion: With this study, we have established protocols and classroom procedures incorporating hands-on Precision Medicine training in the medical student curriculum and a template for other medical educators interested in enhancing their Precision Medicine training program. The program engaged students in discovering variants that were associated with the pathophysiology of the cadaver they were studying, which led to more exposure and understanding of the potential risks and benefits of genomic medicine.
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http://dx.doi.org/10.1186/s12909-020-02366-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670733PMC
November 2020

A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in Agrobacterium tumefaciens.

Sci Rep 2020 09 11;10(1):14968. Epub 2020 Sep 11.

Center for One Health Research, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24060, USA.

LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is defective in motility, biofilm formation, and tumorigenesis of potato discs. RNA-sequencing analyses revealed that more than 250 genes are dysregulated in the ∆vtlR strain, and importantly, VtlR directly controls the expression of three sRNAs in A. tumefaciens. Taken together, these data support a model in which VtlR indirectly regulates hundreds of genes via manipulation of sRNA pathways in A. tumefaciens, and moreover, while the VtlR/LsrB protein is present and structurally conserved in many members of the Alphaproteobacteria, the VtlR/LsrB regulatory circuitry has diverged in order to accommodate the unique environmental niche of each organism.
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http://dx.doi.org/10.1038/s41598-020-72117-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486931PMC
September 2020

Analysis of Genomic Sequence Data Reveals the Origin and Evolutionary Separation of Hawaiian Hoary Bat Populations.

Genome Biol Evol 2020 09;12(9):1504-1514

U.S. Geological Survey, Pacific Island Ecosystems Research Center, Hawai'i National Park, HI.

We examine the genetic history and population status of Hawaiian hoary bats (Lasiurus semotus), the most isolated bats on Earth, and their relationship to northern hoary bats (Lasiurus cinereus), through whole-genome analysis of single-nucleotide polymorphisms mapped to a de novo-assembled reference genome. Profiles of genomic diversity and divergence indicate that Hawaiian hoary bats are distinct from northern hoary bats, and form a monophyletic group, indicating a single ancestral colonization event 1.34 Ma, followed by substantial divergence between islands beginning 0.51 Ma. Phylogenetic analysis indicates Maui is central to the radiation across the archipelago, with the southward expansion to Hawai'i and westward to O'ahu and Kaua'i. Because this endangered species is of conservation concern, a clearer understanding of the population genetic structure of this bat in the Hawaiian Islands is of timely importance.
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http://dx.doi.org/10.1093/gbe/evaa137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543519PMC
September 2020

Characterizing the transport and utilization of the neurotransmitter GABA in the bacterial pathogen Brucella abortus.

PLoS One 2020 26;15(8):e0237371. Epub 2020 Aug 26.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia, United States of America.

The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this non-proteinogenic amino acid plays multiple physiological roles in biology. In the present study, the transport and function of GABA is studied in the highly infectious intracellular bacterium Brucella abortus. The data show that 3H-GABA is imported by B. abortus under nutrient limiting conditions and that the small RNAs AbcR1 and AbcR2 negatively regulate this transport. A specific transport system, gts, is responsible for the transport of GABA as determined by measuring 3H-GABA transport in isogenic deletion strains of known AbcR1/2 regulatory targets; however, this locus is unnecessary for Brucella infection in BALB/c mice. Similar assays revealed that 3H-GABA transport is uninhibited by the 20 standard proteinogenic amino acids, representing preference for the transport of 3H-GABA. Metabolic studies did not show any potential metabolic utilization of GABA by B. abortus as a carbon or nitrogen source, and RNA sequencing analysis revealed limited transcriptional differences between B. abortus 2308 with or without exposure to GABA. While this study provides evidence for GABA transport by B. abortus, questions remain as to why and when this transport is utilized during Brucella pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237371PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449393PMC
October 2020

The tuatara genome reveals ancient features of amniote evolution.

Nature 2020 08 5;584(7821):403-409. Epub 2020 Aug 5.

Department of Anatomy, University of Otago, Dunedin, New Zealand.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana-is an iconic species that is endemic to New Zealand. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.
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http://dx.doi.org/10.1038/s41586-020-2561-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116210PMC
August 2020

The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.

BMC Genomics 2019 Aug 30;20(1):684. Epub 2019 Aug 30.

Department of Chemistry, George Mason University, Manassas, VA, 20110, USA.

Background: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals. .

Results: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple β-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon β-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 β-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome.

Conclusions: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon.
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http://dx.doi.org/10.1186/s12864-019-6029-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716921PMC
August 2019

Desiccation-induced changes in recombination rate and crossover interference in Drosophila melanogaster: evidence for fitness-dependent plasticity.

Genetica 2019 Aug 25;147(3-4):291-302. Epub 2019 Jun 25.

Institute of Evolution, University of Haifa, 3498838, Haifa, Israel.

Meiotic recombination is evolutionarily ambiguous, as being associated with both benefits and costs to its bearers, with the resultant dependent on a variety of conditions. While existing theoretical models explain the emergence and maintenance of recombination, some of its essential features remain underexplored. Here we focus on one such feature, recombination plasticity, and test whether recombination response to stress is fitness-dependent. We compare desiccation stress effects on recombination rate and crossover interference in chromosome 3 between desiccation-sensitive and desiccation-tolerant Drosophila lines. We show that relative to desiccation-tolerant genotypes, desiccation-sensitive genotypes exhibit a significant segment-specific increase in single- and double-crossover frequencies across the pericentromeric region of chromosome 3. Significant changes (relaxation) in crossover interference were found for the interval pairs flanking the centromere and extending to the left arm of the chromosome. These results indicate that desiccation is a recombinogenic factor and that desiccation-induced changes in both recombination rate and crossover interference are fitness-dependent, with a tendency of less fitted individuals to produce more variable progeny. Such dependence may play an important role in the regulation of genetic variation in populations experiencing environmental challenges.
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http://dx.doi.org/10.1007/s10709-019-00070-6DOI Listing
August 2019

Genomic divergence and adaptive convergence in from Evolution Canyon, Israel.

Proc Natl Acad Sci U S A 2019 06 24;116(24):11839-11844. Epub 2019 May 24.

Edward Via College of Osteopathic Medicine, Blacksburg, VA 24060;

Biodiversity refugia formed by unique features of the Mediterranean arid landscape, such as the dramatic ecological contrast of "Evolution Canyon," provide a natural laboratory in which local adaptations to divergent microclimate conditions can be investigated. Significant insights have been provided by studies of diversifying along the thermal gradient in Evolution Canyon, but a comparative framework to survey adaptive convergence across sister species at the site has been lacking. To fill this void, we present an analysis of genomic polymorphism and evolutionary divergence of , a close relative of with which it co-occurs on both slopes of the canyon. Our results show even deeper interslope divergence in than in , with extensive signatures of selective sweeps present in flies from both slopes but enhanced in the population from the hotter and drier south-facing slope. Interslope divergence was enriched for genes related to electrochemical balance and transmembrane transport, likely in response to increased selection for dehydration resistance on the hotter slope. Both species shared genomic regions that underwent major selective sweeps, but the overall level of adaptive convergence was low, demonstrating no shortage of alternative genomic solutions to cope with the challenges of the microclimate contrast. Mobile elements were a major source of genetic polymorphism and divergence, affecting all parts of the genome, including coding sequences of mating behavior-related genes.
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http://dx.doi.org/10.1073/pnas.1720938116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576144PMC
June 2019

Dysfunctional DNA repair pathway via defective FANCD2 gene engenders multifarious exomic and transcriptomic effects in Fanconi anemia.

Mol Genet Genomic Med 2018 11 18;6(6):1199-1208. Epub 2018 Nov 18.

Primary Care Research Network and the Center for Bioinformatics and Genetics, Edward Via College of Osteopathic Medicine, Blacksburg, Virginia.

Background: Fanconi anemia (FA) affects only one in 130,000 births, but has severe and diverse clinical consequences. It has been theorized that defects in the FA DNA cross-link repair complex lead to a spectrum of variants that are responsible for those diverse clinical phenotypes.

Methods: Using NextGen sequencing, we show that a clinically derived FA cell line had accumulated numerous genetic variants, including high-impact mutations, such as deletion of start codons, introduction of premature stop codons, missense mutations, and INDELs.

Results: About 65% of SNPs and 55% of INDELs were found to be commonly present in both the FA dysfunctional and retrovirally corrected cell lines, showing their common origin. The number of INDELs, but not SNPs, is decreased in FANCD2-corrected samples, suggesting that FANCD2 deficiency preferentially promotes the origin of INDELs. These genetic modifications had a considerable effect on the transcriptome, with statistically significant changes in the expression of 270 genes. These genetic and transcriptomic variants significantly impacted pathways and molecular functions, spanning a diverse spectrum of disease phenotypes/symptoms, consistent with the disease diversity seen in FA patients.

Conclusion: These results underscore the consequences of defects in the DNA cross-link repair mechanism and indicate that accumulating diverse mutations from individual parent cells may make it difficult to anticipate the longitudinal clinical behavior of emerging disease states in an individual with FA.
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http://dx.doi.org/10.1002/mgg3.502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305641PMC
November 2018

Unique divergence of the breast cancer 2 () gene in Neanderthals.

Hereditas 2018 3;155:34. Epub 2018 Nov 3.

2Edward Via College of Osteopathic Medicine, Blacksburg, VA 24060 USA.

Unique divergence of the BRCA2, a tumor suppressor gene, in Neanderthals relative to other primates, including modern humans, is highlighted. This divergence with potentially pathogenic consequences raises a question about cancer susceptibility in the archaic species that was replaced by modern humans about 40,000 years ago.
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http://dx.doi.org/10.1186/s41065-018-0073-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215347PMC
November 2018

Genomic signatures of experimental adaptive radiation in Drosophila.

Mol Ecol 2019 02 4;28(3):600-614. Epub 2018 Dec 4.

Department of Bioscience, Aarhus University, Aarhus, Denmark.

Abiotic environmental factors play a fundamental role in determining the distribution, abundance and adaptive diversification of species. Empowered by new technologies enabling rapid and increasingly accurate examination of genomic variation in populations, researchers may gain new insights into the genomic background of adaptive radiation and stress resistance. We investigated genomic variation across generations of large-scale experimental selection regimes originating from a single founder population of Drosophila melanogaster, diverging in response to ecologically relevant environmental stressors: heat shock, heat knock down, cold shock, desiccation and starvation. When compared to the founder population, and to parallel unselected controls, there were more than 100,000 single nucleotide polymorphisms (SNPs) displaying consistent allelic changes in response to selective pressures across generations. These SNPs were found in both coding and noncoding sequences, with the highest density in promoter regions, and involved a broad range of functionalities, including molecular chaperoning by heat-shock proteins. The SNP patterns were highly stressor-specific despite considerable variation among line replicates within each selection regime, as reflected by a principal component analysis, and co-occurred with selective sweep regions. Only ~15% of SNPs with putatively adaptive changes were shared by at least two selective regimes, while less than 1% of SNPs diverged in opposite directions. Divergent stressors driving evolution in the experimental system of adaptive radiation left distinct genomic signatures, most pronounced in starvation and heat-shock selection regimes.
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http://dx.doi.org/10.1111/mec.14917DOI Listing
February 2019

CAGm: a repository of germline microsatellite variations in the 1000 genomes project.

Nucleic Acids Res 2019 01;47(D1):D39-D45

Edward Via College of Osteopathic Medicine, 2265 Kraft Drive, Blacksburg, VA 24060, USA.

The human genome harbors an abundance of repetitive DNA; however, its function continues to be debated. Microsatellites-a class of short tandem repeat-are established as an important source of genetic variation. Array length variants are common among microsatellites and affect gene expression; but, efforts to understand the role and diversity of microsatellite variation has been hampered by several challenges. Without adequate depth, both long-read and short-read sequencing may not detect the variants present in a sample; additionally, large sample sizes are needed to reveal the degree of population-level polymorphism. To address these challenges we present the Comparative Analysis of Germline Microsatellites (CAGm): a database of germline microsatellites from 2529 individuals in the 1000 genomes project. A key novelty of CAGm is the ability to aggregate microsatellite variation by population, ethnicity (super population) and gender. The database provides advanced searching for microsatellites embedded in genes and functional elements. All data can be downloaded as Microsoft Excel spreadsheets. Two use-case scenarios are presented to demonstrate its utility: a mononucleotide (A) microsatellite at the BAT-26 locus and a dinucleotide (CA) microsatellite in the coding region of FGFRL1. CAGm is freely available at http://www.cagmdb.org/.
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http://dx.doi.org/10.1093/nar/gky969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323891PMC
January 2019

Characterization of Three Small Proteins in Brucella abortus Linked to Fucose Utilization.

J Bacteriol 2018 09 24;200(18). Epub 2018 Aug 24.

Department of Biomedical Sciences and Pathobiology, Center for One Health Research, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

Elucidating the function of proteins <50 amino acids in length is no small task. Nevertheless, small proteins can play vital roles in the lifestyle of bacteria and influence the virulence of pathogens; thus, the investigation of the small proteome is warranted. Recently, our group identified the protein VtlR as a transcriptional activator of four genes, one of which is the well-studied small regulatory RNA AbcR2, while the other three genes encode hypothetical small proteins, two of which are highly conserved among the order This study provides evidence that all three genes encode authentic small proteins and that all three are highly expressed under oxidative stress, low-pH, and stationary-phase growth conditions. Fractionation of the cells revealed that the proteins are localized to the membranes of We demonstrate that the small proteins under the transcriptional control of VtlR are not accountable for attenuation observed with the deletion strain. However, there is an association between VtlR-regulated genes and growth inhibition in the presence of the sugar l-fucose. Subsequent transcriptomic analyses revealed that initiates the transcription of a locus encoding a putative sugar transport and utilization system when the bacteria are cultured in the presence of l-fucose. Altogether, our observations characterize the role of the VtlR-controlled small proteins BAB1_0914, BAB2_0512, and BAB2_0574 in the biology of , particularly in the capacity of the bacteria to utilize l-fucose. Despite being one of the most common zoonoses worldwide, there is currently no human vaccine to combat brucellosis. Therefore, a better understanding of the pathogenesis and biology of spp., the causative agent of brucellosis, is essential for the discovery of novel therapeutics against these highly infectious bacteria. In this study, we further characterize the virulence-associated transcriptional regulator VtlR in Our findings not only shed light on our current understanding of a virulence related genetic system in spp. but also increase our knowledge of small proteins in the field of bacteriology.
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http://dx.doi.org/10.1128/JB.00127-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112010PMC
September 2018

Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates.

Nat Commun 2017 11 17;8(1):1570. Epub 2017 Nov 17.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. Here we study Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. We identify several hundred differentially expressed genes and dozens of differentially edited sites between flies from each slope, correlate these changes with genetic differences, and use CRISPR mutagenesis to validate that an intronic SNP in prominin regulates its editing levels. We also demonstrate that while temperature affects editing levels at more sites than genetic differences, genetically regulated sites tend to be less affected by temperature. This work shows the extent to which gene expression and RNA editing differ between flies from different microclimates, and provides insights into the regulation responsible for these differences.
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http://dx.doi.org/10.1038/s41467-017-01658-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691062PMC
November 2017

Mapping Genomic Scaffolds to Chromosomes Using Laser Capture Microdissection in Application to Hawaiian Picture-Winged Drosophila.

Cytogenet Genome Res 2017 8;152(4):204-212. Epub 2017 Nov 8.

Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA.

Next-generation sequencing technologies have led to a decreased cost and an increased throughput in genome sequencing. Yet, many genome assemblies based on short sequencing reads have been assembled only to the scaffold level due to the lack of sufficient chromosome mapping information. Traditional ways of mapping scaffolds to chromosomes require a large amount of laboratory work and time to generate genetic and/or physical maps. To address this problem, we conducted a rapid technique which uses laser capture microdissection and enables mapping scaffolds of de novo genome assemblies directly to chromosomes in Hawaiian picture-winged Drosophila. We isolated and sequenced intact chromosome arms from larvae of D. differens. By mapping the reads of each chromosome to the recently assembled scaffolds from 3 Hawaiian picture-winged Drosophila species, at least 67% of the scaffolds were successfully assigned to chromosome arms. Even though the scaffolds are not ordered within a chromosome, the fast-generated chromosome information allows for chromosome-related analyses after genome assembling. We utilize this new information to test the faster-X evolution effect for the first time in these Hawaiian picture-winged Drosophila species.
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http://dx.doi.org/10.1159/000481790DOI Listing
December 2017

A Test for Gene Flow among Sympatric and Allopatric Hawaiian Picture-Winged Drosophila.

J Mol Evol 2017 06 10;84(5-6):259-266. Epub 2017 May 10.

Biocomplexity Institute, Virginia Tech, Washington Street, MC 0477, Blacksburg, VA, 24061-0477, USA.

The Hawaiian Drosophila are one of the most species-rich endemic groups in Hawaii and a spectacular example of adaptive radiation. Drosophila silvestris and D. heteroneura are two closely related picture-winged Drosophila species that occur sympatrically on Hawaii Island and are known to hybridize in nature, yet exhibit highly divergent behavioral and morphological traits driven largely through sexual selection. Their closest-related allopatric species, D. planitibia from Maui, exhibits hybrid male sterility and reduced behavioral reproductive isolation when crossed experimentally with D. silvestris or D. heteroneura. A modified four-taxon test for gene flow was applied to recently obtained genomes of the three Hawaiian Drosophila species. The analysis indicates recent gene flow in sympatry, but also, although less extensive, between allopatric species. This study underscores the prevalence of gene flow, even in taxonomic groups considered classic examples of allopatric speciation on islands. The potential confounding effects of gene flow in phylogenetic and population genetics inference are discussed, as well as the implications for conservation.
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http://dx.doi.org/10.1007/s00239-017-9795-7DOI Listing
June 2017

Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.

J Proteome Res 2017 04 24;16(4):1470-1482. Epub 2017 Feb 24.

School of Systems Biology, George Mason University , 10920 George Mason Circle, 1H8, Manassas, Virginia 20110, United States.

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.
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http://dx.doi.org/10.1021/acs.jproteome.6b00857DOI Listing
April 2017

Nucleotide diversity inflation as a genome-wide response to experimental lifespan extension in Drosophila melanogaster.

BMC Genomics 2017 01 14;18(1):84. Epub 2017 Jan 14.

Department of Bioscience, Aarhus University, Ny Munkegade 114-116, Aarhus, DK-8000, Denmark.

Background: Evolutionary theory predicts that antagonistically selected alleles, such as those with divergent pleiotropic effects in early and late life, may often reach intermediate population frequencies due to balancing selection, an elusive process when sought out empirically. Alternatively, genetic diversity may increase as a result of positive frequency-dependent selection and genetic purging in bottlenecked populations.

Results: While experimental evolution systems with directional phenotypic selection typically result in at least local heterozygosity loss, we report that selection for increased lifespan in Drosophila melanogaster leads to an extensive genome-wide increase of nucleotide diversity in the selected lines compared to replicate control lines, pronounced in regions with no or low recombination, such as chromosome 4 and centromere neighborhoods. These changes, particularly in coding sequences, are most consistent with the operation of balancing selection and the antagonistic pleiotropy theory of aging and life history traits that tend to be intercorrelated. Genes involved in antioxidant defenses, along with multiple lncRNAs, were among those most affected by balancing selection. Despite the overwhelming genetic diversification and the paucity of selective sweep regions, two genes with functions important for central nervous system and memory, Ptp10D and Ank2, evolved under positive selection in the longevity lines.

Conclusions: Overall, the 'evolve-and-resequence' experimental approach proves successful in providing unique insights into the complex evolutionary dynamics of genomic regions responsible for longevity.
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http://dx.doi.org/10.1186/s12864-017-3485-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237518PMC
January 2017

Transcriptomic imprints of adaptation to fresh water: parallel evolution of osmoregulatory gene expression in the Alewife.

Mol Ecol 2017 Feb 27;26(3):831-848. Epub 2017 Jan 27.

Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, 06269-3043, USA.

Comparative approaches in physiological genomics offer an opportunity to understand the functional importance of genes involved in niche exploitation. We used populations of Alewife (Alosa pseudoharengus) to explore the transcriptional mechanisms that underlie adaptation to fresh water. Ancestrally anadromous Alewives have recently formed multiple, independently derived, landlocked populations, which exhibit reduced tolerance of saltwater and enhanced tolerance of fresh water. Using RNA-seq, we compared transcriptional responses of an anadromous Alewife population to two landlocked populations after acclimation to fresh (0 ppt) and saltwater (35 ppt). Our results suggest that the gill transcriptome has evolved in primarily discordant ways between independent landlocked populations and their anadromous ancestor. By contrast, evolved shifts in the transcription of a small suite of well-characterized osmoregulatory genes exhibited a strong degree of parallelism. In particular, transcription of genes that regulate gill ion exchange has diverged in accordance with functional predictions: freshwater ion-uptake genes (most notably, the 'freshwater paralog' of Na /K -ATPase α-subunit) were more highly expressed in landlocked forms, whereas genes that regulate saltwater ion secretion (e.g. the 'saltwater paralog' of NKAα) exhibited a blunted response to saltwater. Parallel divergence of ion transport gene expression is associated with shifts in salinity tolerance limits among landlocked forms, suggesting that changes to the gill's transcriptional response to salinity facilitate freshwater adaptation.
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http://dx.doi.org/10.1111/mec.13983DOI Listing
February 2017

Nucleolar Dominance and Repression of 45S Ribosomal RNA Genes in Hybrids between Xenopus borealis and X. muelleri (2n = 36).

Cytogenet Genome Res 2016 12;149(4):290-296. Epub 2016 Oct 12.

Biocomplexity Institute, Virginia Tech, Blacksburg, Va., USA.

Nucleolar dominance is a dramatic disruption in the formation of nucleoli and the expression of ribosomal RNA (rRNA) genes, characteristic of some plant and animal hybrids. Here, we report that F1 hybrids produced from reciprocal crosses between 2 sister species of Xenopus clawed frogs, X. muelleri and X. borealis, undergo nucleolar dominance somewhat distinct from a pattern previously reported in hybrids between phylogenetically more distant Xenopus species. Patterns of nucleolar development, 45S rRNA expression, and gene copy inheritance were investigated using a combination of immunostaining, pyrosequencing, droplet digital PCR, flow cytometry, and epigenetic inhibition. In X. muelleri × X. borealis hybrids, typically only 1 nucleolus is formed, and 45S rRNA genes are predominantly expressed from 1 progenitor's alleles, X. muelleri, regardless of the cross-direction. These changes are accompanied by an extensive (∼80%) loss of rRNA gene copies in the hybrids relative to their parents, with the transcriptionally underdominant variant (X. borealis) being preferentially lost. Chemical treatment of hybrid larvae with a histone deacetylase inhibitor resulted in a partial derepression of the underdominant variant. Together, these observations shed light on the genetic and epigenetic basis of nucleolar dominance as an underappreciated manifestation of genetic conflicts within a hybrid genome.
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http://dx.doi.org/10.1159/000450665DOI Listing
January 2017

Genomic Signatures of Speciation in Sympatric and Allopatric Hawaiian Picture-Winged Drosophila.

Genome Biol Evol 2016 05 30;8(5):1482-8. Epub 2016 May 30.

Biocomplexity Institute, Virginia Tech, Blacksburg, Virginia

The Hawaiian archipelago provides a natural arena for understanding adaptive radiation and speciation. The Hawaiian Drosophila are one of the most diverse endemic groups in Hawaiì with up to 1,000 species. We sequenced and analyzed entire genomes of recently diverged species of Hawaiian picture-winged Drosophila, Drosophila silvestris and Drosophila heteroneura from Hawaiì Island, in comparison with Drosophila planitibia, their sister species from Maui, a neighboring island where a common ancestor of all three had likely occurred. Genome-wide single nucleotide polymorphism patterns suggest the more recent origin of D. silvestris and D. heteroneura, as well as a pervasive influence of positive selection on divergence of the three species, with the signatures of positive selection more prominent in sympatry than allopatry. Positively selected genes were significantly enriched for functional terms related to sensory detection and mating, suggesting that sexual selection played an important role in speciation of these species. In particular, sequence variation in Olfactory receptor and Gustatory receptor genes seems to play a major role in adaptive radiation in Hawaiian pictured-winged Drosophila.
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http://dx.doi.org/10.1093/gbe/evw095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4898809PMC
May 2016

Rapid genomic changes in Drosophila melanogaster adapting to desiccation stress in an experimental evolution system.

BMC Genomics 2016 Mar 15;17:233. Epub 2016 Mar 15.

Biocomplexity Institute, Virginia Tech, Blacksburg, VA, 24061, USA.

Background: Experimental evolution studies, coupled with whole genome resequencing and advances in bioinformatics, have become a powerful tool for exploring how populations respond to selection at the genome-wide level, complementary to genome-wide association studies (GWASs) and linkage mapping experiments as strategies to connect genotype and phenotype. In this experiment, we analyzed genomes of Drosophila melanogaster from lines evolving under long-term directional selection for increased desiccation resistance in comparison with control (no-selection) lines.

Results: We demonstrate that adaptive responses to desiccation stress have exerted extensive footprints on the genomes, manifested through a high degree of fixation of alleles in surrounding neighborhoods of eroded heterozygosity. These patterns were highly convergent across replicates, consistent with signatures of 'soft' selective sweeps, where multiple alleles present as standing genetic variation become beneficial and sweep through the replicate populations at the same time. Albeit much less frequent, we also observed line-unique sweep regions with zero or near-zero heterozygosity, consistent with classic, or 'hard', sweeps, where novel rather than pre-existing adaptive mutations may have been driven to fixation. Genes responsible for cuticle and protein deubiquitination seemed to be central to these selective sweeps. High divergence within coding sequences between selected and control lines was also reflected by significant results of the McDonald-Kreitman and Ka/Ks tests, showing that as many as 347 genes may have been under positive selection.

Conclusions: Desiccation stress, a common challenge to many organisms inhabiting dry environments, proves to be a very potent selecting factor having a big impact on genome diversity.
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http://dx.doi.org/10.1186/s12864-016-2556-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791783PMC
March 2016

Ancient forest: keep the logging ban.

Authors:
Pawel Michalak

Nature 2016 Feb;530(7591):419

Virginia Polytechnic Institute and State University, Blacksburg, USA.

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http://dx.doi.org/10.1038/530419cDOI Listing
February 2016

Nucleolar dominance and maternal control of 45S rDNA expression.

Proc Biol Sci 2015 Dec;282(1820):20152201

Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061, USA

Using a system of interspecies hybrids, trihybrids, and recombinants with varying proportions of genomes from three distinct Xenopus species, we provide evidence for de novo epigenetic silencing of paternal 45 S ribosomal ribonucleic acid (rRNA) genes and their species-dependent expression dominance that escapes transcriptional inactivation after homologous recombination. The same pattern of imprinting is maintained in the offspring from mothers being genetic males (ZZ) sex-reversed to females, indicating that maternal control of ribosomal deoxyribonucleic acid (rDNA) expression is not sex-chromosome linked. Nucleolar dominance (nucleolus underdevelopment) in Xenopus hybrids appears to be associated with a major non-Mendelian reduction in the number of 45 S rDNA gene copies rather than a specific pattern of their expression. The loss of rRNA gene copies in F1 hybrids was non-random with respect to the parental species, with the transcriptionally dominant variant preferentially removed from hybrid zygotes. This dramatic disruption in the structure and function of 45 S rDNA impacts transcriptome patterns of small nucleolar RNAs and messenger RNAs, with genes from the ribosome and oxidative stress pathways being among the most affected. Unorthodoxies of rDNA inheritance and expression may be interpreted as hallmarks of genetic conflicts between parental genomes, as well as defensive epigenetic mechanisms employed to restore genome integrity.
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http://dx.doi.org/10.1098/rspb.2015.2201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685780PMC
December 2015
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