Browsing by Author "Kang, Lin"

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    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.
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    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.
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    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.
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    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.
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    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.
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    Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation
    Roy, Tithi; Banang-Mbeumi, Sergette; Boateng, Samuel T.; Ruiz, Emmanuelle M.; Chamcheu, Roxane-Cherille N.; Kang, Lin; King, Judy A.; Walker, Anthony L.; Nagalo, Bolni Marius; Kousoulas, Konstantin G.; Esnault, Stephane; Huang, Shile; Chamcheu, Jean Christopher (Frontiers, 2023-01-18)
    Psoriasis is a chronic autoimmune inflammatory skin disorder characterized by epidermal hyperplasia and aberrant immune response. In addition to aberrant cytokine production, psoriasis is associated with activation of the Akt/mTOR pathway. mTOR/S6K1 regulates T-lymphocyte activation and migration, keratinocytes proliferation and is upregulated in psoriatic lesions. Several drugs that target Th1/Th17 cytokines or their receptors have been approved for treating psoriasis in humans with variable results necessitating improved therapies. Fisetin, a natural dietary polyphenol with anti-oxidant and anti-proliferative properties, covalently binds mTOR/S6K1. The effects of fisetin on psoriasis and its underlying mechanisms have not been clearly defined. Here, we evaluated the immunomodulatory effects of fisetin on Th1/Th17-cytokine-activated adult human epidermal keratinocytes (HEKa) and anti-CD3/CD28-stimulated inflammatory CD4(+) T cells and compared these activities with those of rapamycin (an mTOR inhibitor). Transcriptomic analysis of HEKa revealed 12,713 differentially expressed genes (DEGs) in the fisetin-treated group compared to 7,374 DEGs in the rapamycin-treated group, both individually compared to a cytokine treated group. Gene ontology analysis revealed enriched functional groups related to PI3K/Akt/mTOR signaling pathways, psoriasis, and epidermal development. Using in silico molecular modeling, we observed a high binding affinity of fisetin to IL-17A. In vitro, fisetin significantly inhibited mTOR activity, increased the expression of autophagy markers LC3A/B and Atg5 in HEKa cells and suppressed the secretion of IL-17A by activated CD4(+) T lymphocytes or T lymphocytes co-cultured with HEKa. Topical administration of fisetin in an imiquimod (IMQ)-induced mouse psoriasis model exhibited a better effect than rapamycin in reducing psoriasis-like inflammation and Akt/mTOR phosphorylation and promoting keratinocyte differentiation and autophagy in mice skin lesions. Fisetin also significantly inhibited T-lymphocytes and F4/80(+) macrophage infiltration into skin. We conclude that fisetin potently inhibits IL-17A and the Akt/mTOR pathway and promotes keratinocyte differentiation and autophagy to alleviate IMQ-induced psoriasis-like disease in mice. Altogether, our findings suggest fisetin as a potential treatment for psoriasis and possibly other inflammatory skin diseases.
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    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.
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    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.
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    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.
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    Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems
    Auguste, A. Jonathan; Langsjoen, Rose M.; Porier, Danielle L.; Erasmus, Jesse H.; Bergren, Nicholas A.; Bolling, Bethany G.; Luo, Huanle; Singh, Ankita; Guzman, Hilda; Popov, Vsevolod L.; da Rosa, Amelia P. A. Travassos; Wang, Tian; Kang, Lin; Allen, Irving C.; Carrington, Christine V. F.; Tesh, Robert B.; Weaver, Scott C. (2021-10)
    We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.
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    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.
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    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.
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    Nucleotide diversity inflation as a genome-wide response to experimental lifespan extension in Drosophila melanogaster
    Michalak, Pawel; Kang, Lin; Sarup, Pernille M.; Schou, Mads F.; Loeschcke, Volker (2017-01-14)
    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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    Rapid genomic changes in Drosophila melanogaster adapting to desiccation stress in an experimental evolution system
    Kang, Lin; Aggarwal, Dau Dayal; Rashkovetsky, Eugenia; Korol, Abraham B.; Michalak, Pawel (BMC, 2016-03-15)
    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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    Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates
    Yablonovitch, Arielle L.; Fu, Jeremy; Li, Kexin; Mahato, Simpla; Kang, Lin; Rashkovetsky, Eugenia; Korol, Abraham B.; Tang, Hua; Michalak, Pawel; Zelhof, Andrew C.; Nevo, Eviatar; Li, Jin Billy (Springer Nature, 2017-11-17)
    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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    A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation
    Kang, Lin; He, Guijuan; Sharp, Amanda K.; Wang, Xiaofeng; Brown, Anne M.; Michalak, Pawel; Weger-Lucarelli, James (Cell Press, 2021-08-19)
    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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    A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation
    Kang, Lin; He, Guijuan; Sharp, Amanda K.; Wang, Xiaofeng; Brown, Anne M.; Michalak, Pawel; Weger-Lucarelli, James (Virginia Tech, 2021-03-05)
    While SARS-CoV-2 likely has animal origins, the viral genetic changes necessary to adapt this animal-derived ancestral virus to humans are largely unknown, mostly due to low levels of sequence polymorphism and the notorious difficulties in experimental manipulations of coronavirus genomes. We scanned more than 182,000 SARS-CoV-2 genomes for selective sweep signatures and found that a distinct footprint of positive selection is located around a non-synonymous change (A1114G; T372A) within the Receptor-Binding Domain of the Spike protein, which likely played a critical role in overcoming species barriers and accomplishing interspecies transmission from animals to humans. Structural analysis indicated that the substitution of threonine with an alanine in SARS-CoV-2 concomitantly removes a predicted glycosylation site at N370, resulting in more favorable binding predictions to human ACE2, the cellular receptor. Using a novel bacteria-free cloning system for manipulating RNA virus genomes, we experimentally validated that this SARS-CoV-2-unique substitution significantly increases replication in human cells relative to its putative ancestral variant. Notably, this mutation’s impact on virus replication in human cells was much greater than that of the Spike D614G mutant, which has been widely reported to have been selected for during human-to-human transmission.
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    Unique divergence of the breast cancer 2 (BRCA2) gene in Neanderthals
    Michalak, Pawel; Kang, Lin (2018-11-03)
    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.