Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Inositol polyphosphate multikinase is a metformin target that regulates cell migration.

Metformin has been shown to alter cell adhesion protein expression, which is thought to play a role in its observed antitumor properties. We found that metformin treatment down-regulated integrin ß1 concomitant with the loss of inositol polyphosphate multikinase (IPMK) in murine myocytes, adipocytes, and hepatocytes. To determine if IPMK was upstream of integrin ß1 expression, we examined IPMK-/- mouse embryonic fibroblast cells and found that integrins ß1 and ß3 gene expression was reduced by half, relative to wild-type cells, whereas focal adhesion kinase (FAK) activity and Rho/Rac/Cdc42 protein levels were increased, resulting in migration defects. Using nanonet force microscopy, we determined that cell:extracellular matrix adhesion and cell contractility forces were decreased, confirming the functional relevance of integrin and Rho protein dysregulation. Pharmacological studies showed that inhibition of both FAK1 and proline-rich tyrosine kinase 2 partially restored integrin ß1 expression, suggesting negative regulation of integrin ß1 by FAK. Together our data indicate that IPMK participates in the regulation of cell migration and provides a potential link between metformin and wound healing impairment.-Tu-Sekine, B., Padhi, A., Jin, S., Kalyan, S., Singh, K., Apperson, M., Kapania, R., Hur, S. C., Nain, A., Kim, S. F. Inositol polyphosphate multikinase is a metformin target that regulates cell migration.

Cattle Sex-Specific Recombination and Genetic Control from a Large Pedigree Analysis.

Meiotic recombination is an essential biological process that generates genetic diversity and ensures proper segregation of chromosomes during meiosis. From a large USDA dairy cattle pedigree with over half a million genotyped animals, we extracted 186,927 three-generation families, identified over 8.5 million maternal and paternal recombination events, and constructed sex-specific recombination maps for 59,309 autosomal SNPs. The recombination map spans for 25.5 Morgans in males and 23.2 Morgans in females, for a total studied region of 2,516 Mb (986 kb/cM in males and 1,085 kb/cM in females). The male map is 10% longer than the female map and the sex difference is most pronounced in the subtelomeric regions. We identified 1,792 male and 1,885 female putative recombination hotspots, with 720 hotspots shared between sexes. These hotspots encompass 3% of the genome but account for 25% of the genome-wide recombination events in both sexes. During the past forty years, males showed a decreasing trend in recombination rate that coincided with the artificial selection for milk production. Sex-specific GWAS analyses identified PRDM9 and CPLX1 to have significant effects on genome-wide recombination rate in both sexes. Two novel loci, NEK9 and REC114, were associated with recombination rate in both sexes, whereas three loci, MSH4, SMC3 and CEP55, affected recombination rate in females only. Among the multiple PRDM9 paralogues on the bovine genome, our GWAS of recombination hotspot usage together with linkage analysis identified the PRDM9 paralogue on chromosome 1 to be associated in the U.S. Holstein data. Given the largest sample size ever reported for such studies, our results reveal new insights into the understanding of cattle and mammalian recombination.

Ruminant-specific multiple duplication events of PRDM9 before speciation.

BACKGROUND: Understanding the genetic and evolutionary mechanisms of speciation genes in sexually reproducing organisms would provide important insights into mammalian reproduction and fitness. PRDM9, a widely known speciation gene, has recently gained attention for its important role in meiotic recombination and hybrid incompatibility. Despite the fact that PRDM9 is a key regulator of recombination and plays a dominant role in hybrid incompatibility, little is known about the underlying genetic and evolutionary mechanisms that generated multiple copies of PRDM9 in many metazoan lineages. RESULTS: The present study reports (1) evidence of ruminant-specific multiple gene duplication events, which likely have had occurred after the ancestral ruminant population diverged from its most recent common ancestor and before the ruminant speciation events, (2) presence of three copies of PRDM9, one copy (lineages I) in chromosome 1 (chr1) and two copies (lineages II & III) in chromosome X (chrX), thus indicating the possibility of ancient inter- and intra-chromosomal unequal crossing over and gene conversion events, (3) while lineages I and II are characterized by the presence of variable tandemly repeated C2H2 zinc finger (ZF) arrays, lineage III lost these arrays, and (4) C2H2 ZFs of lineages I and II, particularly the amino acid residues located at positions -1, 3, and 6 have evolved under strong positive selection. CONCLUSIONS: Our results demonstrated two gene duplication events of PRDM9 in ruminants: an inter-chromosomal duplication that occurred between chr1 and chrX, and an intra-chromosomal X-linked duplication, which resulted in two additional copies of PRDM9 in ruminants. The observation of such duplication between chrX and chr1 is rare and may possibly have happened due to unequal crossing-over millions of years ago when sex chromosomes were independently derived from a pair of ancestral autosomes. Two copies (lineages I & II) are characterized by the presence of variable sized tandem-repeated C2H2 ZFs and evolved under strong positive selection and concerted evolution, supporting the notion of well-established Red Queen hypothesis. Collectively, gene duplication, concerted evolution, and positive selection are the likely driving forces for the expansion of ruminant PRDM9 sub-family.

Characterization of genome-wide segmental duplications reveals a common genomic feature of association with immunity among domestic animals.

BACKGROUND: Segmental duplications (SDs) commonly exist in plant and animal genomes, playing crucial roles in genomic rearrangement, gene innovation and the formation of copy number variants. However, they have received little attention in most livestock species. RESULTS: Aiming at characterizing SDs across the genomes of diverse livestock species, we mapped genome-wide SDs of horse, rabbit, goat, sheep and chicken, and also enhanced the existing SD maps of cattle and pig genomes based on the most updated genome assemblies. We adopted two different detection strategies, whole genome analysis comparison and whole genome shotgun sequence detection, to pursue more convincing findings. Accordingly we identified SDs for each species with the length of from 21.7 Mb to 164.1 Mb, and 807 to 4,560 genes were harboured within the SD regions across different species. More interestingly, many of these SD-related genes were involved in the process of immunity and response to external stimuli. We also found the existence of 59 common genes within SD regions in all studied species except goat. These common genes mainly consisted of both UDP glucuronosyltransferase and Interferon alpha families, implying the connection between SDs and the evolution of these gene families. CONCLUSIONS: Our findings provide insights into livestock genome evolution and offer rich genomic sources for livestock genomic research.

Life History of the Oldest Lentivirus: Characterization of ELVgv Integrations in the Dermopteran Genome.

Endogenous retroviruses are genomic elements formed by germline infiltration by originally exogenous viruses. These molecular fossils provide valuable information about the evolution of the retroviral family. Lentiviruses are an extensively studied genus of retroviruses infecting a broad range of mammals. Despite a wealth of information on their modern evolution, little is known about their origins. This is partially due to the scarcity of their endogenous forms. Recently, an endogenous lentivirus, ELVgv, was discovered in the genome of the Malayan colugo (order Dermoptera). This represents the oldest lentiviral evidence available and promises to lead to further insights into the history of this genus. In this study, we analyzed ELVgv integrations at several genomic locations in four distinct colugo specimens covering all the extant dermopteran species. We confirmed ELVgv integrations in all the specimens examined, which implies that the virus originated before the dermopteran diversification. Using a locus-specific dermopteran substitution rate, we estimated that the proviral integrations occurred 21-40 Ma. Using phylogenetic analysis, we estimated that ELVgv invaded an ancestor of today's Dermoptera in an even more distant past. We also provide evidence of selective pressure on the TRIM5 antiviral restriction factor, something usually taken as indirect evidence of past retroviral infections. Interestingly, we show that TRIM5 was under strong positive selection pressure only in the common dermopteran ancestor, where the ELVgv endogenization occurred. Further experiments are required to determine whether ELVgv participated in the TRIM5 selection.

A testis-specific gene within a widely expressed gene: Contrasting evolutionary patterns of two differentially expressed mammalian proteins encoded by a single gene, CAMK4.

Understanding the patterns of genetic variations within fertility-related genes and the evolutionary forces that shape such variations is crucial in predicting the fitness landscapes of subsequent generations. This study reports distinct evolutionary features of two differentially expressed mammalian proteins [CaMKIV (Ca(2+) /calmodulin-dependent protein kinase IV) and CaS (calspermin)] that are encoded by a single gene, CAMK4. The multifunctional CaMKIV, which is expressed in multiple tissues including testis and ovary, is evolving at a relatively low rate (0.46-0.64 × 10(-9) nucleotide substitutions/site/year), whereas the testis-specific CaS gene, which is predominantly expressed in post-meiotic cells, evolves at least three to four times faster (1.48-1.98 × 10(-9) substitutions/site/year). Concomitantly, maximum-likelihood-based selection analyses revealed that the ubiquitously expressed CaMKIV is constrained by intense purifying selection and, therefore, remained functionally highly conserved throughout the mammalian evolution, whereas the testis-specific CaS gene is under strong positive selection. The substitution rates of different mammalian lineages within both genes are positively correlated with GC content, indicating the possible influence of GC-biased gene conversion on the estimated substitution rates. The observation of such unusually high GC content of the CaS gene (≈74%), particularly in the lineage that comprises the bovine species, suggests the possible role of GC-biased gene conversion in the evolution of CaS that mimics positive selection.

Positive selection drives rapid evolution of certain amino acid residues in an evolutionarily highly conserved interferon-inducible antiviral protein of fishes.

Viperin, an evolutionarily highly conserved interferon-inducible multifunctional protein, has previously been reported to exhibit antiviral activity against a wide range of DNA and RNA viruses. Utilizing the complete nucleotide coding sequence data of fish viperin antiviral genes, and employing the maximum likelihood-based codon substitution models, the present study reports the pervasive role of positive selection in the evolution of viperin antiviral protein in fishes. The overall rate of nonsynonymous (dN) to synonymous (dS) substitutions (dN/dS) for the three functional domains of viperin (N-terminal, central domain and C-terminal) were 1.1, 0.12, and 0.24, respectively. Codon-by-codon substitution analyses have revealed that while most of the positively selected sites were located at the N-terminal amphipathic α-helix domain, few amino acid residues at the C-terminal domain were under positive selection. However, none of the sites in the central domain were under positive selection. These results indicate that, although viperin is evolutionarily highly conserved, the three functional domains experienced differential selection pressures. Taken together with the results of previous studies, the present study suggests that the persistent antagonistic nature of surrounding infectious viral pathogens might be the likely cause for such adaptive evolutionary changes of certain amino acids in fish viperin antiviral protein.

Polymorphic integrations of an endogenous gammaretrovirus in the mule deer genome.

Endogenous retroviruses constitute a significant genomic fraction in all mammalian species. Typically they are evolutionarily old and fixed in the host species population. Here we report on a novel endogenous gammaretrovirus (CrERVγ; for cervid endogenous gammaretrovirus) in the mule deer (Odocoileus hemionus) that is insertionally polymorphic among individuals from the same geographical location, suggesting that it has a more recent evolutionary origin. Using PCR-based methods, we identified seven CrERVγ proviruses and demonstrated that they show various levels of insertional polymorphism in mule deer individuals. One CrERVγ provirus was detected in all mule deer sampled but was absent from white-tailed deer, indicating that this virus originally integrated after the split of the two species, which occurred approximately one million years ago. There are, on average, 100 CrERVγ copies in the mule deer genome based on quantitative PCR analysis. A CrERVγ provirus was sequenced and contained intact open reading frames (ORFs) for three virus genes. Transcripts were identified covering the entire provirus. CrERVγ forms a distinct branch of the gammaretrovirus phylogeny, with the closest relatives of CrERVγ being endogenous gammaretroviruses from sheep and pig. We demonstrated that white-tailed deer (Odocoileus virginianus) and elk (Cervus canadensis) DNA contain proviruses that are closely related to mule deer CrERVγ in a conserved region of pol; more distantly related sequences can be identified in the genome of another member of the Cervidae, the muntjac (Muntiacus muntjak). The discovery of a novel transcriptionally active and insertionally polymorphic retrovirus in mammals could provide a useful model system to study the dynamic interaction between the host genome and an invading retrovirus.

Pathogen-driven adaptive evolution of myxovirus resistance (Mx) genes in fishes.

Myxovirus resistance (Mx) proteins, which belong to the dynamin super-family, are known to inhibit RNA viral replication in a wide range of taxonomic groups, including fishes. Given their crucial role in host immune defense, the key amino acid residues in the GTP effector domain (GED) near the C-terminus are expected to evolve adaptively in order to protect the host against invading viral pathogens. The present study reveals the role of recombination and positive selection in the evolution of Mx proteins in fishes. While the GTP-binding domain in the N-terminal domain has experienced purifying selection, several amino acid residues in GED have evolved under positive selection, thus indicating adaptive evolution. Given the antiviral activity of GED, the adaptive evolutionary changes that were observed in this region are therefore predicted to be pathogen-driven.

The effect of vaccination on the evolution and population dynamics of avian paramyxovirus-1.

Newcastle Disease Virus (NDV) is a pathogenic strain of avian paramyxovirus (aPMV-1) that is among the most serious of disease threats to the poultry industry worldwide. Viral diversity is high in aPMV-1; eight genotypes are recognized based on phylogenetic reconstruction of gene sequences. Modified live vaccines have been developed to decrease the economic losses caused by this virus. Vaccines derived from avirulent genotype II strains were developed in the 1950s and are in use globally, whereas Australian strains belonging to genotype I were developed as vaccines in the 1970s and are used mainly in Asia. In this study, we evaluated the consequences of attenuated live virus vaccination on the evolution of aPMV-1 genotypes. There was phylogenetic incongruence among trees based on individual genes and complete coding region of 54 full length aPMV-1 genomes, suggesting that recombinant sequences were present in the data set. Subsequently, five recombinant genomes were identified, four of which contained sequences from either genotype I or II. The population history of vaccine-related genotype II strains was distinct from other aPMV-1 genotypes; genotype II emerged in the late 19(th) century and is evolving more slowly than other genotypes, which emerged in the 1960s. Despite vaccination efforts, genotype II viruses have experienced constant population growth to the present. In contrast, other contemporary genotypes showed population declines in the late 1990s. Additionally, genotype I and II viruses, which are circulating in the presence of homotypic vaccine pressure, have unique selection profiles compared to nonvaccine-related strains. Collectively, these data show that vaccination with live attenuated viruses has changed the evolution of aPMV-1 by maintaining a large effective population size of a vaccine-related genotype, allowing for coinfection and recombination of vaccine and wild type strains, and by applying unique selective pressures on viral glycoproteins.

Adaptive evolution of crustin antimicrobial peptides in decapods.

Antimicrobial peptides (AMPs) are present in a wide range of taxonomic groups and played a crucial role in host adaptation to a diverse array of ever-changing pathogens. Crustin, a cysteine-rich cationic AMP, is known to exhibit antimicrobial activity against Gram-positive and Gram-negative bacteria in decapods. Given their important role in host-immune defense, a large proportion of amino acid substitutions in crustin AMPs are expected to be fixed by natural selection. Utilizing the complete coding nucleotide sequence data of crustin, the present study revealed the pervasive role of positive Darwinian selection in the evolution and divergence of crustin AMPs in decapods. Approximately, 20-35 % of codons in two phylogenetically distinct groups of closely related crustins in Penaeid shrimps are shown to have evolved under positive selection. Interestingly, several of these positively selected sites are located at the carboxyl-terminal region, the region that directly interacts with the invading pathogens. Pathogen-mediated selection pressure could be the likely cause for such an accelerated rate of amino acid substitutions and could have contributed to the structural and functional diversification of crustin AMPs in several taxa.

Dynamic Heterochromatin States in Anisotropic Nuclei of Cells on Aligned Nanofibers.

The cancer cell nucleus deforms as it invades the interstitial spaces in tissues and the tumor microenvironment. While alteration of the chromatin structure in a deformed nucleus is expected and documented, the chromatin structure in the nuclei of cells on aligned matrices has not been elucidated. In this work we elucidate the spatiotemporal organization of heterochromatin in the elongated nuclei of cells on aligned nanofibers with stimulated emission depletion nanoscopy and fluorescence correlation spectroscopy. We show that the anisotropy of nuclei is sufficient to drive H3K9me3-heterochromatin alterations, with enhanced H3K9me3 nanocluster compaction and aggregation states that otherwise are indistinguishable from diffraction-limited microscopy. We interrogated the higher-order heterochromatin structures within major chromatin compartments in anisotropic nuclei and discovered a wider spatial dispersion of nanodomain clusters in the nucleoplasm and condensed larger nanoclusters near the periphery and pericentromeric heterochromatin. Upon examining the spatiotemporal dynamics of heterochromatin in anisotropic nuclei, we observed reduced mobility of the constitutive heterochromatin mark H3K9me3 and the associated heterochromatin protein 1 (HP1α) at the nucleoplasm and periphery regions, correlating with increased viscosity and changes in gene expression. Since heterochromatin remodeling is crucial to genome integrity, our results reveal an unconventional H3K9me3 heterochromatin distribution, providing cues to an altered chromatin state due to perturbations of the nuclei in aligned fiber configurations.

Evidence for positive selection in the extracellular domain of human cytomegalovirus encoded G protein-coupled receptor US28.

The human cytomegalovirus (HCMV)-encoded chemokine receptor US28 is also a seven-transmembrane G-protein coupled receptor, whose signaling pathway is known for its involvement in host immune system evasion. HCMV infection can result in serious disease in immunocompromised individuals and is also linked to atherosclerosis and cardiovascular disease. Identifying amino acid residues that play a crucial role in successful viral adaptation in response to the host's immune defense is critical for effective drug design. In this study maximum likelihood-based codon substitution analyses were carried out to determine whether any codon of US28 has evolved adaptively. If the rate of nonsynonymous (dn) to the rate of synonymous (ds) nucleotide substitutions (ω = dn/ds) is greater than one, the codon is said to be under positive selection, indicating adaptive evolution. Although the overall ω for US28 gene was 0.154, indicating that most codon sites were subject to strong purifying selection, five codon sites are under strong positive selection. Three (E18D/L, D19A/E/G, and R267K/Q) of these positively selected sites are located in extracellular domains, the domains that play a crucial role for successful viral adaptation in response to the host's immune defense. The C-terminal (R329Q/W) and the fifth transmembrane domain (V190I), each have one positively selected site. These results suggest that relative to the extracellular domains, amino acid residues present in intracellular domains are more selectively constrained. A few amino acid residues in extracellular domains of US28 evolved more rapidly, presumably due to positive selection pressure resulting from ligand-binding and pathogen interactions of extracellular domains.

Genomic evidence of intraspecific recombination in sugarcane mosaic virus.

The sugarcane mosaic virus (SCMV) of the genus potyvirus, which primarily affects maize, sugarcane, sorghum, abaca, and grasses, occurs worldwide and causes significant economic loss. Using the full genome sequences of SCMV and several recombination detection methods, in this study we report that recombination is the major driving force in the evolution and emergence of several new variants of SCMV. We reported eight highly significant (P < 0.001) recombination break points, majority of which are located within 6K1-VPg-NIaPro-NIb region, thus indicating a region for recombination hotspot. The observation of commonalities of same recombination events among the SCMV isolates between the countries (Spain and Mexico), and within the country (within China, and within Mexico), suggests common origin of the isolates in respective regions.

Cell Fragment Formation, Migration, and Force Exertion on Extracellular Mimicking Fiber Nanonets.

Cell fragments devoid of the nucleus play an essential role in intercellular communication. Mostly studied on flat 2D substrates, their origins and behavior in native fibrous environments remain unknown. Here, cytoplasmic fragments' spontaneous formation and behavior in suspended extracellular matrices mimicking fiber architectures (parallel, crosshatch, and hexagonal) are described. After cleaving from the parent cell body, the fragments of diverse shapes on fibers migrate faster compared to 2D. Furthermore, while fragments in 2D are mostly circular, a higher number of rectangular and blob-like shapes are formed on fibers, and, interestingly, each shape is capable of forming protrusive structures. Absent in 2D, fibers' fragments display oscillatory migratory behavior with dramatic shape changes, sometimes remarkably sustained over long durations (>20 h). Immunostaining reveals paxillin distribution along fragment body-fiber length, while Forster Resonance Energy Transfer imaging of vinculin reveals mechanical loading of fragment adhesions comparable to whole cell adhesions. Using nanonet force microscopy, the forces exerted by fragments are estimated, and peculiarly small area fragments can exert forces similar to larger fragments in a Rho-associated kinase dependent manner. Overall, fragment dynamics on 2D substrates are insufficient to describe the mechanosensitivity of fragments to fibers, and the architecture of fiber networks can generate entirely new behaviors.

Population dynamics and rates of molecular evolution of a recently emerged paramyxovirus, avian metapneumovirus subtype C.

We report the existence of two distinct sublineages of avian metapneumovirus (MPV) subtype C, a virus which has caused serious economic loss in commercial turkey farms in the United States. This subtype is closely related to human MPV, infects multiple avian species, and is globally distributed. The evolutionary rates of this virus are estimated to be 1.3 x 10(-3) to 7 x 10(-3) substitutions per site per year, and coalescent estimates place its emergence between 1991 and 1996. The four genes examined show a concordant demographic pattern which is characterized by a rapid increase in population size followed by stable population grown until the present.

Natural infection of vertebrate hosts by different lineages of Buggy Creek virus (family Togaviridae, genus Alphavirus).

Buggy Creek virus (BCRV; family Togaviridae, genus Alphavirus) is an arbovirus transmitted by the ectoparasitic swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius) to cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus). BCRV occurs in two lineages (A and B) that are sympatric in bird nesting colonies in the central Great Plains, USA. Previous work on lineages isolated exclusively from swallow bugs suggested that lineage A relies on amplification by avian hosts, in contrast to lineage B, which is maintained mostly among bugs. We report the first data on the BCRV lineages isolated from vertebrate hosts under natural conditions. Lineage A was overrepresented among isolates from nestling house sparrows, relative to the proportions of the two lineages found in unfed bug vectors at the same site at the start of the summer transmission season. Haplotype diversity of each lineage was higher in bugs than in sparrows, indicating reduced genetic diversity of virus amplified in the vertebrate host. BCRV appears to have diverged into two lineages based on different modes of transmission.

ECM in Differentiation: A Review of Matrix Structure, Composition and Mechanical Properties.

Stem cell regenerative potential owing to the capacity to self-renew as well as differentiate into other cell types is a promising avenue in regenerative medicine. Stem cell niche not only provides physical scaffolding but also possess instructional capacity as it provides a milieu of biophysical and biochemical cues. Extracellular matrix (ECM) has been identified as a major dictator of stem cell lineage, thus understanding the structure of in vivo ECM pertaining to specific tissue differentiation will aid in devising in vitro strategies to improve the differentiation efficiency. In this review, we summarize details about the native architecture, composition and mechanical properties of in vivo ECM of the early embryonic stages and the later adult stages. Native ECM from adult tissues categorized on their origin from respective germ layers are discussed while engineering techniques employed to facilitate differentiation of stem cells into particular lineages are noted. Overall, we emphasize that in vitro strategies need to integrate tissue specific ECM biophysical cues for developing accurate artificial environments for optimizing stem cell differentiation.

Author Correction: Force-exerting perpendicular lateral protrusions in fibroblastic cell contraction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.