VTechWorks Repository :: Browsing by Author "Michalak, Pawel"

Browsing by Author "Michalak, Pawel"

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CAGm: A repository of germline microsatellite variations in the 1000 genomes project
Kinney, N.; Titus-Glover, K.; Wren, J.D.; Varghese, Ronnie; Michalak, Pawel; Liao, H.; Anandakrishnan, Ramu; Pulenthiran, A.; Kang, L.; Garner, Harold R. (Oxford University Press, 2019-01-08)
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/. © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in Agrobacterium tumefaciens
Budnick, James A.; Sheehan, Lauren M.; Ginder, Miranda. J.; Failor, Kevin C.; Perkowski, Julia. M.; Pinto, John. F.; Kohl, Kirsten A.; Kang, Lin; Michalak, Pawel; Luo, Li; Heindl, Jason E.; Caswell, Clayton C. (2020-09-11)
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.
Characterizing the transport and utilization of the neurotransmitter GABA in the bacterial pathogen Brucella abortus
Budnick, James A.; Sheehan, Lauren M.; Benton, Angela H.; Pitzer, Joshua E.; Kang, Lin; Michalak, Pawel; Roop, R. Martin II; Caswell, Clayton C. (PLoS, 2020-08-26)
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.
Chromosome evolution and mechanisms of speciation in the Anopheles gambiae complex
Liang, Jiang-tao (Virginia Tech, 2020-06-01)
Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Understanding the chromosome evolution and mechanisms of speciation can shed light on developing novel ecological-friendly vector control techniques. Sibling species of the An. gambiae complex provide an excellent model system for these topics. To understand the mechanisms of speciation, we investigated the cellular basis and phenotypes of hybrid male sterility in species crosses of the An. gambiae complex. By performing inter-species crosses of An. coluzzii/An. gambiae and An. merus lab strains, we found an asymmetric pattern of hybrid male sterility existed in sons from reciprocal interspecies crosses. Compared with pure species, hybrid males from crosses of ♀An. merus  ♂An. gambiae/An. coluzzii were normal in the morphology of male reproductive tracts; however, the testes of which that process the reductional meiotic division failed to produce primary spermatocytes and were accompanied with unpaired and insufficiently condensed chromosomes. As a result, primary spermatocytes undergo a mitosis-like anaphase division, producing nonmotile and malfunctional diploid sperm with two tails. However, individuals can mate with females normally and form the mating plug to induce the female monogamy. In contrast, hybrid males from the opposite crosses manifest severely underdeveloped reproductive tracts and a premeiotic arrest of germline stem cells in the testis, accompanied by a strong suppression of premeiotic and meiotic genes. In addition, hybrid males from this cross suffered from a shorter copulation time and failed to form mating plugs to induce female monogamous behaviors, albeit the expression of male accessory gland specific genes were similar between hybrids and pure species. To figure out chromosome evolution in the An. gambiae complex, we studied the molecular organization of heterochromatin and investigated the spatial organizations of autosomal regions of polytene chromosomes in soma and germline cells. We found that molecular composition of pericentrometric autosome and sex chromosome repetitive DNA differs among sibling species of An. gambiae complex with highly similarity between An. coluzzii and An. arabiensis. In addition, heterochromatin blocks of chromosomes have distinct compositions of satellite DNA sequences. Next, in order to address the relationship between inter-chromosomal (Chr-Chr) contacts and chromosome-nuclear envelope (Chr-NE) attachments during the development of the organism, we conducted microscopic analyses of the 3D organization of polytene chromosome in An. gambiae, An. coluzzii, and An. merus. Our quantitative study on chromosome territories in larval salivary gland cells and adult ovarian nurse cells showed that, compared with autosomal arms, the X chromosome has a significantly smaller volume and occupies more compact territories. The number of Chr-Chr contacts and the percentage of Chr-NE attachment were conserved among the species within the same cell type. Our data also demonstrated that there is a significantly and consistently inverse relationship between the frequencies of Chr–NE and Chr–Chr attachments on autosomes of two cell types in all tested species.
Comparative Analysis of Heterochromatin in the Anopheles gambiae Complex
Sharma, Atashi (Virginia Tech, 2016-05-10)
Mosquito borne diseases continue to be a big threat to human health worldwide. Despite using various vector control methods, we lose a great number of lives to this malicious disease in tropical and subtropical countries each year. Not surprisingly, mosquito is considered as the deadliest animal on the earth, because mortality rates from mosquito-vectored infections only lag behind other major diseases such as HIV and tuberculosis. Current approaches of vector control are mostly limited to using insecticidal bed nets, thus novel techniques are required to prevent a staggering loss to human health and quality of life. Advances in the genome sequencing in the past decade have helped to uncover numerous secrets of diverse genomes. The genome of malaria mosquito Anopheles gambiae was first sequenced in 2002 and since then has been updated to include additional scaffolds, their orientations and correction of mis-assemblies. Yet, the greatest challenge remains in assembling the heterochromatin regions, that are repeat rich part and contain relatively low-gene density. Although previously neglected by scientific studies due to its characteristic paucity of genes, heterochromatin is now recognized to be crucial for several processes such as cell viability, chromosome pairing, meiosis, longevity etc. It is therefore not surprising that heterochromatin comprises of a significant portion of the genome in many species. The efforts to analyze the genome of malaria mosquito in order to identify potential new leads for vector control warrant a better understanding of the heterochromatin. Mosquitoes diploid chromosome number equal 6. While autosomes 2 and 3 are submetacentric and present in both sexes, females are homogametic with XX and males are heterogametic with XY sex chromosomes. To achieve a better understanding of the Anopheles heterochromatin, we investigated heterochromatic region of the X chromosome. Despite one arm of the X chromosome being completely heterochromatic, few studies have investigated the molecular content of this region. Protocols were developed for performing fluorescent in situ hybridization (FISH) on mitotic X chromosomes in An. gambiae. Using cytogenetics and molecular biology techniques, we characterized the X chromosome heterochromatin in members of the An. gambiae complex. Specific satellite DNA and 18S ribosomal DNA probes (major components of heterochromatin) were mapped to X chromosomes enabling their differentiation and characterization in the An. gambiae complex. Microarray studies have highlighted the importance of X chromosome during investigation of nascent species An. gambiae and An. coluzzii. Here for the first time qualitative differences in heterochromatin in between nascent species are described. Cytogenetic idiograms are developed as to include the molecular and qualitative differences between the species of the An. gambiae complex. These idiograms are expected to provide a better resolution of the X chromosome heterochromatin for comparison in major malaria vectors, closing some of the gaps present due to poor sequencing of unassembled repeat rich regions in An. gambiae complex. The current understanding of Y chromosome for transgenic manipulation is poor and limited to very few genes. Due to its near total heterochromatic composition, it is the hardest part of the genome to assemble. In collaboration with other researchers, the Y chromosome content was characterized among sibling species of the An. gambiae complex. Our data revealed the swift changes the Y chromosome has undergone in a relatively short evolutionary time period. These include a rapid rate of turnover not only in heterochromatin but also in euchromatin. In addition to previously described repeats, a novel highly repetitive element called Zanzibar was discovered and mapped to the males of various Anopheles sibling species. Our data can form the basis for evolutionary studies in heterochromatin for male mosquitoes within the An. gambiae complex while also help identify novel targets to create successful transgenic male populations. Along with the X chromosome heterochromatin, to our knowledge this is the most extensive contribution to improve the understanding of mitotic chromosome heterochromatin in malaria mosquitoes. This study also investigated if epigenetics play role in mosquito development, fecundity and heterochromatin formation. DNA methylation, histone modifications and small noncoding RNAs are among the epigenetic mechanisms scrutinized in mammals. However, knowledge about epigenetic mechanisms and their effects is sparse in mosquitoes. A protocol for testing the various effects of epigenetics on different stages of malaria mosquito was developed. An epigenetic drug was utilized to probe the effects on immature and adult malaria mosquitoes. Different concentrations of DZNep, a histone methyltransferase inhibitor, were administered to An. coluzzii larvae. Total survivorship and pupation were compared for treated and untreated groups. The drug was also administered to adult blood feeding females to determine any effects on fecundity and egg morphology, revealing a negative association with an increase in drug concentration. A dose dependent decrease in SAH hydrolase concentration in An. coluzzii was also noticed. These results suggest epigenetics plays a critical role in mosquito pupation and ovarian development. Our work lays the groundwork for future investigations into the field of epigenetics in mosquitoes by revealing its effect on several important developmental stages in malaria mosquitoes. Although genomics and next-gen sequencing technology have come a long way in the last decade since the first Anopheles genome was sequenced, considerable gaps still exist in case characterization of heterochromatin function in an organism. Through our work, we have endeavored to elucidate a few of the major roles that heterochromatin may play in organization, evolution and adaptation of the malaria mosquitoes.
Comparative Genomics Insights into Speciation and Evolution of Hawaiian Drosophila
Kang, Lin (Virginia Tech, 2017-05-01)
Speciation and adaptation have always been of great interest to biologists. The Hawaiian archipelago provides a natural arena for understanding adaptive radiation and speciation, and genomics and bioinformatics offer new approaches for studying these fundamental processes. The mode of speciation should have profound impacts on the genomic architecture and patterns of reproductive isolation of new species. The Hawaiian Drosophila are a spectacular example of sequential colonization, adaptive radiation, and speciation in the islands with nearly 1,000 estimated species, of which more than 500 have been described to date. This dissertation gives an overview of the Hawaiian Drosophila system (Chapter 1), new insights into genomes of three recently diverged species of Hawaiian picture-winged Drosophila (Chapter 2), as well as estimated gene flow patterns (Chapter 3). Additionally, I present a new approach of mapping genomic scaffolds onto chromosomes, based on NextGen sequencing from chromosomal microdissections (Chapter 4), and gene expression profiles of backcross hybrids and their parental forms (Chapter 5). Overall, obtained results were used to address such fundamental questions as the role of adaptive changes, founder effects (small effective population size in isolation), and genetic admixture during speciation.
Development of Molecular and Morphological Resources for Identification and Monitoring of Freshwater Mussel Species in the Genera Fusconaia and Pleurobema in the Green River, Kentucky
Hyde, Miluska Olivera (Virginia Tech, 2021-01-11)
Freshwater mussel species in the genera Fusconaia and Pleurobema are particularly challenging to identify in the field. In this study, mussels from these genera were collected from the Green River, Kentucky for genetic and morphological analyses. I used molecular markers to detect any cryptic species within these genera and to test for genetic differentiation between two closely related nominal taxa P. rubrum and P. sintoxia using both mitochondrial (ND1, COI, 16S rRNA) and nuclear (ITS1) DNA sequences. After species identification, I used microsatellite DNA markers to estimate genetic diversity and effective population sizes (Ne) of species of Pleurobema. I used microsatellite primers that were developed for P. clava and P. pyriforme in previous studies, as well as microsatellites that I developed for P. plenum. Finally, I assessed morphological variation in my study species and developed dichotomous keys for the identification of both live mussels and shells. My results suggest that P. rubrum and P. sintoxia are the same species based on the mitochondrial DNA analyses, as there were few genetic differences between them. My results showed phylogenetically distinct lineages for F. flava, F. subrotunda, P. cordatum and P. plenum but no cryptic species were detected in the Green River. Current and contemporary Ne showed that these species have large population sizes that should allow for avoiding inbreeding and maintaining their evolutionary potential. Large genetic diversity as well as long-term effective population size could be the result of these species historically occurring as much larger assemblages that extended into the Ohio River and its numerous tributaries. The last objective was to assess morphometrical differences among these species. Using Canonical Variate Analysis, I found discernable morphological differences between the investigated species of Fusconaia and Pleurobema. The two Fusconaia species were morphologically different from the Pleurobema species. However, the Canonical Variate Analysis did not show differences among the Pleurobema species. I used decision tree analysis to develop a dichotomous tree, and random forest analysis was used to aid in the development of a dichotomous key by finding the most important diagnostic characters to distinguish these mussels. I then used the less subjective and easier to identify characters for the development of my dichotomous keys for live mussels and shells. However, both keys need to be tested in the field to determine their effectiveness. I could not separate P. rubrum and P. sintoxia mussels for morphometric analysis due to the lack of genetic differentiation and the inconsistent identification by the experts. However, I did describe a few individuals that look like P. rubrum and P. sintoxia to the eye of the experts. The description of these individuals matched previous descriptions of these mussels. Future studies need to assess taxonomic relationships among these species using genomics approaches, which might result in better node resolution. High genetic diversity and large effective population numbers for Pleurobema species suggest that these species' populations are genetically healthy. However, these results need to be interpreted carefully, and I therefore recommend additional studies to assess life history, habitat, host-fish availability, and current reproduction of these mussels in the Green River.
DNA Metabarcoding-based Evaluation of the Diet of Big Brown Bats (Eptesicus fuscus) in the Mid-Atlantic Region
Deeley, Sabrina; Kang, Lin; Michalak, Pawel; Hallerman, Eric M.; Ford, W. Mark (Eagle Hill Institute, 2022-12)
High-throughput DNA sequencing can generate large genetic datasets in a cost-effective manner. Although the diet of Eptesicus fuscus (Big Brown Bat) has been studied widely in natural and rural systems using visual identification of prey items in feces, our aim was to more completely assess diet using a metabarcoding approach across a wide urban-natural landscape gradient in the mid-Atlantic region. Concordant with our expectations and previous Big Brown Bat diet studies from visual identification, we observed a high abundance of Coleoptera (beetles) relative to other insect orders. Although a possible improvement over visual techniques for studying food habits, we suggest caution in interpreting metabarcoding results in diet studies. We noted observations of environmental or contaminant taxa within these data, and designed a stringent filtering method that we used to eliminate these taxa, but that also removed previously documented prey taxa from our dataset.
A draft genome assembly for the eastern fox squirrel, Sciurus niger
Kang, Lin; Michalak, Pawel; Hallerman, Eric M.; Moncrief, Nancy D. (Oxford University Press, 2021-12-01)
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 90.9 Kb 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.
The Epigenetic Role of EGR1 during Postnatal Mammalian Brain Development
Sun, Zhixiong (Virginia Tech, 2018-08-03)
DNA methylation is an epigenetic mechanism critical for tissue development, cell specification and cellular function. Mammalian brains consist of millions to billions of neurons and glial cells that can be subdivided into many distinct types of cells. We hypothesize that brain methylomes are heterogeneously methylated across different types of cells and the transcription factors play key roles in brain methylome programming. To dissect brain methylome heterogeneity, in Chapter 2, we first focused on the identification of cell-subset specific methylated (CSM) loci which demonstrate bipolar DNA methylation pattern, i.e., hypermethylated in one cell subset but hypomethylated in others. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Importantly, we found that putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. It suggests the important role of putative CSM loci during brain development, implying that dramatic changes in functions and complexities of the brain may be companied by a rapid change in epigenetic heterogeneity. To explore epigenetic regulatory mechanisms during brain development, as described in Chapter 3, we adopted unbiased data-driven approaches to re-analyze methylomes for human and mouse frontal cortices at different developmental stages. We predicted Egr1, a transcriptional factor with important roles in neuron maturation, synaptic plasticity, long-term memory formation and learning, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated that thousands of EGR1 binding sites are with cell-type specific methylation patterns established during postnatal frontal cortex development. More specifically, the CpG dinucleotides within these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks at EGR1 binding sites and activate downstream genes. Also, we found that the frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. Collectively, the study in this dissertation reveals EGR1 programs the brain methylome together with TET1 during postnatal development. This study also provides new insights into how life experience and neuronal activity may shape the brain methylome.
EvoCor: a platform for predicting functionally related genes using phylogenetic and expression profiles
Dittmar, W. James; McIver, Lauren; Michalak, Pawel; Garner, Harold R.; Valdez, Gregorio (2014-07-01)
The wealth of publicly available gene expression and genomic data provides unique opportunities for computational inference to discover groups of genes that function to control specific cellular processes. Such genes are likely to have co-evolved and be expressed in the same tissues and cells. Unfortunately, the expertise and computational resources required to compare tens of genomes and gene expression data sets make this type of analysis difficult for the average end-user. Here, we describe the implementation of a web server that predicts genes involved in affecting specific cellular processes together with a gene of interest. We termed the server 'EvoCor', to denote that it detects functional relationships among genes through evolutionary analysis and gene expression correlation. This web server integrates profiles of sequence divergence derived by a Hidden Markov Model (HMM) and tissue-wide gene expression patterns to determine putative functional linkages between pairs of genes.
Genetic Divergence between Freshwater and Marine Morphs of Alewife (Alosa pseudoharengus): A ‘Next-Generation’ Sequencing Analysis
Czesny, Sergiusz; Epifanio, John; Michalak, Pawel (PLoS ONE, 2012-03-15)
Alewife Alosa pseudoharengus, a small clupeid fish native to Atlantic Ocean, has recently (,150 years ago) invaded the North American Great Lakes and despite challenges of freshwater environment its populations exploded and disrupted local food web structures. This range expansion has been accompanied by dramatic changes at all levels of organization. Growth rates, size at maturation, or fecundity are only a few of the most distinct morphological and life history traits that contrast the two alewife morphs. A question arises to what extent these rapidly evolving differences between marine and freshwater varieties result from regulatory (including phenotypic plasticity) or structural mutations. To gain insights into expression changes and sequence divergence between marine and freshwater alewives, we sequenced transcriptomes of individuals from Lake Michigan and Atlantic Ocean. Population specific single nucleotide polymorphisms were rare but interestingly occurred in sequences of genes that also tended to show large differences in expression. Our results show that the striking phenotypic divergence between anadromous and lake alewives can be attributed to massive regulatory modifications rather than coding changes.
Genomic divergence and adaptive convergence in Drosophila simulans from Evolution Canyon, Israel
Kang, Lin; Rashkovetsky, Eugenia; Michalak, Katarzyna; Garner, Harold R.; Mahaney, James E.; Rzigalinski, Beverly A.; Korol, Abraham B.; Nevo, Eviatar; Michalak, Pawel (2019-06-11)
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 Drosophila melanogaster 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 Drosophila simulans, a close relative of Drosophila melanogaster with which it co-occurs on both slopes of the canyon. Our results show even deeper interslope divergence in D. simulans than in D. melanogaster, 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.
Hybridization between Aedes aegypti and Aedes mascarensis mosquitoes leads to disruption of male sex determination
Liang, Jiangtao; Kang, Lin; Michalak, Pawel; Sharakhov, Igor V. (Springer Nature, 2024-07-22)
Understanding the sex determination pathway and its disruptions in mosquitoes is critical for the effective control of disease vectors through genetic manipulations based on sex separation. When male hybrids of Aedes aegypti females and Ae. mascarensis males are backcrossed to Ae. aegypti females, a portion of the backcross progeny manifests as males with abnormal sexual differentiation. We discovered a significant correlation between pupal abnormalities and the feminization of subsequent adults exemplified by the relative abundance of ovarian and testicular tissues. All intersex individuals were genetic males as they expressed a male determining factor, Nix. Further, our analysis of the sex-specific splicing of doublesex and fruitless transcripts demonstrated the presence of both male and female splice variants indicating that sex determination is disrupted. A comparative transcriptomic analysis revealed similar expression levels of most female-associated genes in reproductive organs and carcasses between intersexual males and normal females. Moreover, intersexes had largely normal gene expression in testes but significant gene downregulation in male accessory glands when compared with normal males. We conclude that evolving hybrid incompatibilities between Ae. aegypti and Ae. mascarensis involve disruption of sex determination and are accompanied by changes in gene expression associated with sexual differentiation.
Insights into the Evolution of Longevity from the Bowhead Whale Genome
Keane, Michael; Semeiks, Jeremy; Webb, Andrew E.; Li, Yang I.; Quesada, Victor; Craig, Thomas; Madsen, Lone Bruhn; van Dam, Sipko; Brawand, David; Marques, Patricia I.; Michalak, Pawel; Kang, Lin; Bhak, Jong; Yim, Hyung-Soon; Grishin, Nick V.; Nielsen, Nynne Hjort; Heide-Jorgensen, Mads Peter; Oziolor, Elias M.; Matson, Cole W.; Church, George M.; Stuart, Gary W.; Patton, John C.; George, J. Craig; Suydam, Robert; Larsen, Knud; Lopez-Otin, Carlos; O'Connell, Mary J.; Bickham, John W.; Thomsen, Bo; de Magalhaes, Joao Pedro (Cell Press, 2015-01-06)
The bowhead whale (Balaena mysticetus) is estimated to live over 200 years and is possibly the longest-living mammal. These animals should possess protective molecular adaptations relevant to age-related diseases, particularly cancer. Here, we report the sequencing and comparative analysis of the bowhead whale genome and two transcriptomes from different populations. Our analysis identifies genes under positive selection and bow-head-specific mutations in genes linked to cancer and aging. In addition, we identify gene gain and loss involving genes associated with DNA repair, cell-cycle regulation, cancer, and aging. Our results expand our understanding of the evolution of mammalian longevity and suggest possible players involved in adaptive genetic changes conferring cancer resistance. We also found potentially relevant changes in genes related to additional processes, including thermoregulation, sensory perception, dietary adaptations, and immune response. Our data are made available online (http://www.bowhead-whale.org) to facilitate research in this long-lived species.
The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters
van Hoek, Monique L.; Prickett, M. D.; Settlage, Robert E.; Kang, Lin; Michalak, Pawel; Vliet, Kent A.; Bishop, Barney M. (2019-08-30)
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.
Modeling the Regulatory Mechanisms by Which NLRX1 Modulates Innate Immune Responses to Helicobacter pylori Infection
Philipson, Casandra W.; Bassaganya-Riera, Josep; Viladomiu, Monica; Kronsteiner, Barbara; Abedi, Vida; Hoops, Stefan; Michalak, Pawel; Kang, Lin; Girardin, Stephen E.; Hontecillas, Raquel (PLOS, 2015-09-14)
Helicobacter pylori colonizes half of the world’s population as the dominant member of the gastric microbiota resulting in a lifelong chronic infection. Host responses toward the bacterium can result in asymptomatic, pathogenic or even favorable health outcomes; however, mechanisms underlying the dual role of H. pylori as a commensal versus pathogenic organism are not well characterized. Recent evidence suggests mononuclear phagocytes are largely involved in shaping dominant immunity during infection mediating the balance between host tolerance and succumbing to overt disease. We combined computational modeling, bioinformatics and experimental validation in order to investigate interactions between macrophages and intracellular H. pylori. Global transcriptomic analysis on bone marrow-derived macrophages (BMDM) in a gentamycin protection assay at six time points unveiled the presence of three sequential host response waves: an early transient regulatory gene module followed by sustained and late effector responses. Kinetic behaviors of pattern recognition receptors (PRRs) are linked to differential expression of spatiotemporal response waves and function to induce effector immunity through extracellular and intracellular detection of H. pylori. We report that bacterial interaction with the host intracellular environment caused significant suppression of regulatory NLRC3 and NLRX1 in a pattern inverse to early regulatory responses. To further delineate complex immune responses and pathway crosstalk between effector and regulatory PRRs, we built a computational model calibrated using time-series RNAseq data. Our validated computational hypotheses are that: 1) NLRX1 expression regulates bacterial burden in macrophages; and 2) early host response cytokines down-regulate NLRX1 expression through a negative feedback circuit. This paper applies modeling approaches to characterize the regulatory role of NLRX1 in mechanisms of host tolerance employed by macrophages to respond to and/or to co-exist with intracellular H. pylori.
Modeling the Role of Peroxisome Proliferator-Activated Receptor c and MicroRNA-146 in Mucosal Immune Responses to Clostridium difficile
Viladomiu, Monica; Hontecillas, Raquel; Pedragosa, Mireia; Carbo, Adria; Hoops, Stefan; Michalak, Pawel; Michalak, Katarzyna; Guerrant, Richard L.; Roche, James K.; Warren, Cirle A.; Bassaganya-Riera, Josep (Public Library of Science, 2012-10-11)
Clostridium difficile is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) c has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to C. difficile and its toxins remains largely unknown. To characterize the role of PPARc in C. difficileassociated disease (CDAD), immunity and gut pathology, we used a mouse model of C. difficile infection in wild-type and T cell-specific PPARc null mice. The loss of PPARc in T cells increased disease activity and colonic inflammatory lesions following C. difficile infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cellspecific PPARc null mice. Also, both the loss of PPARc in T cells and C. difficile infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of C. difficile infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARc co-activator NCOA4, and PPARc, leading to upregulation of IL-17. Oral treatment of C. difficile-infected mice with the PPARc agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARc activation may be implicated in the regulation of Th17 responses and colitis in C. difficile-infected mice.
Novel Microsatellite Detection, Microsatellite Based Biomarker Discovery In Lung Cancer And The Exome-Wide Effects Of A Dysfunctional DNA Repair Mechanism
Velmurugan, Karthik Raja (Virginia Tech, 2017-05-02)
Since the dawn of the genomics era, the genetics of numerous human disorders has been understood which has led to improvements in targeted therapeutics. However, the focus of most research has been primarily on protein coding genes, which account for only 2% of the entire genome, leaving much of the remaining genome relatively unstudied. In particular, repetitive sequences, called microsatellites (MST), which are tandem repeats of 1 to 6 bases, are known to be mutational hotspots and have been linked to diseases, such as Huntington disease and Fragile X syndrome. This work represents a significant effort towards closing this knowledge gap. Specifically, we developed a next generation sequencing based enrichment method along with the supporting computational pipeline for detecting novel MST sequences in the human genome. Using this global MST enrichment protocol, we have identified 790 novel sequences. Analysis of these novel sequences has identified previously unknown functional elements, demonstrating its potential for aiding in the completion of the euchromatic DNA. We also developed a disease risk diagnostic using a novel target specific enrichment method that produces high resolution MST sequencing data that has the potential to validate, for the first time, the link between MST genotype variation and cancer. Combined with publicly available exome datasets of non-small cell lung cancer and 1000 genomes project, the target specific MST enrichment method uncovered a signature set of 21 MST loci that can differentiate between lung cancer and non-cancer control samples with a sensitivity ratio of 0.93. Finally, to understand the molecular causes of MST instability, we analyzed genomic variants and gene expression data for an autosomal recessive disorder, Fanconi anemia (FA). This first of its kind study quantified the heterogeneity of FA cells and demonstrated the possibility of utilizing the DNA crosslink repair dysfunctional FA cells as a suitable system to further study the causes of MST instability.
Novel Prognostic Markers and Therapeutic Targets for Glioblastoma
Varghese, Robin (Virginia Tech, 2016-06-23)
Glioblastoma is the most common and lethal malignant brain tumor with a survival rate of 14.6 months and a tumor recurrence rate of ninety percent. Two key causes for glioblastomas grim outcome derive from the lack of applicable prognostic markers and effective therapeutic targets. By employing a loss of function RNAi screen in glioblastoma cells we found a list of 20 kinases that can be considered glioblastoma survival kinases. These survival kinases which we term as survival kinase genes, (SKGs) were investigated to find prognostic markers as well as therapeutic targets for glioblastoma. Analyzing these survival kinases in The Cancer Genome Atlas patient database, we found that CDCP1, CDKL5, CSNK1𝜀, IRAK3, LATS2, PRKAA1, STK3, TBRG4, and ULK4 genes could be used as prognostic markers for glioblastoma with or without temozolomide chemotherapeutic treatment as a covariate. For the first time, we found that patients with increased levels of NEK9 and PIK3CB mRNA expression had a higher probability of recurrent tumors. We also discovered that expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary glioblastoma tumors was associated with tumor recurrence prognosis. To note, of these recurrent prognostic candidates, PIK3CB expression in recurrent tumor tissue had much higher expression compared to primary tissue. Further investigation in the PI3K pathway showed a strong correlation with recurrence rate, days to recurrence and survival emphasizing the role of PIK3CB in tumor recurrence in glioblastoma. In efforts to find effective therapeutic targets for glioblastoma we used SKGs as potential candidates. We chose the serine/threonine kinase, Casein Kinase 1 Epsilon (CSNK1𝜀) as a target for glioblastoma because multiple shRNAs targeted this gene in our loss of function screen and multiple commercially available inhibitors of this gene are available. Casein kinase 1 epsilon protein and mRNA expression were investigated using computational tools. It was revealed that CSNK1𝜀 expression has higher expression in glioblastoma than normal tissue. To further examine this gene we knocked down (KD) or inhibited CSNK1𝜀 in glioblastoma cells lines and noticed a significant increase in cell death without any significant effect on normal cell lines. KD and inhibition of CSNK1𝜀 in cancer stem cells, a culprit of tumor recurrence, also revealed limited self-renewal and proliferation in cancer stem cells and a significant decrease in cell survival without affecting normal stem cells. Further analysis of downstream effects of CSNK1𝜀 knockdown and inhibition indicate a significant increase in the protein expression of β-catenin (CTNNB1). We found that CSNK1𝜀 KD activated β-catenin, which increased GBM cell death, but can be rescued using CTNNB1 shRNA. Our survival kinase screen, computational analyses, patient database analyses and experimental methods contributed to the discovery of novel prognostic markers and therapeutic targets for glioblastoma.

Browsing by Author "Michalak, Pawel"

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