The proteome and phosphoproteome of circulating extracellular vesicle-enriched preparations are associated with characteristic clinical features in type 1 diabetes.

Introduction: There are no validated clinical or laboratory biomarkers to identify and differentiate endotypes of type 1 diabetes (T1D) or the risk of progression to chronic complications. Extracellular vesicles (EVs) have been studied as biomarkers in several different disease states but have not been well studied in T1D. Methods: As the initial step towards circulating biomarker identification in T1D, this pilot study aimed to provide an initial characterization of the proteomic and phosphoproteomic landscape of circulating EV-enriched preparations in participants with established T1D (N=10) and healthy normal volunteers (Controls) (N=7) (NCT03379792) carefully matched by age, race/ethnicity, sex, and BMI. EV-enriched preparations were obtained using EVtrap® technology. Proteins were identified and quantified by LC-MS analysis. Differential abundance and coexpression network (WGCNA), and pathway enrichment analyses were implemented. Results: The detected proteins and phosphoproteins were enriched (75%) in exosomal proteins cataloged in the ExoCarta database. A total of 181 proteins and 8 phosphoproteins were differentially abundant in participants with T1D compared to controls, including some well-known EVproteins (i.e., CD63, RAB14, BSG, LAMP2, and EZR). Enrichment analyses of differentially abundant proteins and phosphoproteins of EV-enriched preparations identified associations with neutrophil, platelet, and immune response functions, as well as prion protein aggregation. Downregulated proteins were involved in MHC class II signaling and the regulation of monocyte differentiation. Potential key roles in T1D for C1q, plasminogen, IL6ST, CD40, HLA-DQB1, HLA-DRB1, CD74, NUCB1, and SAP, are highlighted. Remarkably, WGCNA uncovered two protein modules significantly associated with pancreas size, which may be implicated in the pathogenesis of T1D. Similarly, these modules showed significant enrichment for membrane compartments, processes associated with inflammation and the immune response, and regulation of viral processes, among others. Discussion: This study demonstrates the potential of proteomic and phosphoproteomic signatures of EV-enriched preparations to provide insight into the pathobiology of T1D. The WGCNA analysis could be a powerful tool to discriminate signatures associated with different pathobiological components of the disease.

Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity.

Insulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous, however, this association remains controversial. The aim of this study was to perform an in-depth multifactorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active, n = 9), individuals with obesity (Obese, n = 9), and individuals with obesity, insulin resistance, and type 2 diabetes (T2D, n = 22). Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic-supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by Blue Native (BN)-PAGE and immunoblot. Tricarboxylic (TCA) cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. We reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity, supercomplex assembly, TCA cycle intermediates, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (body mass index, age, and aerobic capacity). We highlight that lean, active individuals have greater mitochondrial content, mitochondrial capacity, supercomplex assembly, and TCA cycle intermediates. These phenotypical changes are reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance. Clinical trial number: NCT01911104.NEW & NOTEWORTHY Whether impaired mitochondrial capacity contributes to skeletal muscle insulin resistance is debated. Our multifactorial analysis shows no differences in skeletal muscle mitochondrial content, mitochondrial capacity, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity). We highlight that lean, active individuals have enhanced skeletal muscle mitochondrial capacity that is also reflected at the level of DNA methylation and gene transcription.

A single nuclei atlas of aging human abdominal subcutaneous white adipose tissue.

White adipose tissue (WAT) is a robust energy storage and endocrine organ critical for maintaining metabolic health as we age. Our aim was to identify cell-specific transcriptional aberrations that occur in WAT with aging. We leveraged full-length snRNA-Seq to characterize the cellular landscape of human subcutaneous WAT in a prospective cohort of 10 Younger (≤ 30 years) and 10 Older individuals (≥ 65 years) balanced for sex and body mass index (BMI). We highlight that aging WAT is associated with adipocyte hypertrophy, increased proportions of resident macrophages (M2), an upregulated innate immune response and senescence profiles in specific adipocyte populations, highlighting CXCL14 as a biomarker of this process. We also identify novel markers of pre-adipocytes and track their expression levels through pre-adipocyte differentiation. We propose that aging WAT is associated with low-grade inflammation that is managed by a foundation of innate immunity to preserve the metabolic health of the WAT.

Isolation of nuclei from frozen human subcutaneous adipose tissue for full-length single-nuclei transcriptional profiling.

Automated single-cell dispensing is incompatible with white adipose tissue (WAT) due to lipid-laden adipocytes. Single-nuclei RNA-Seq permits transcriptional profiling of all cells from WAT. Human WAT faces unique technical challenges in isolating nuclei compared to rodent tissue due to greater extra-cellular matrix content and larger lipid droplets. In this protocol, we detail how to isolate nuclei from frozen subcutaneous human WAT for single-nuclei RNA-Seq. For complete information on the generation and use of this protocol, please refer to Whytock et al. (2022).1.

Single cell full-length transcriptome of human subcutaneous adipose tissue reveals unique and heterogeneous cell populations.

White adipose tissue (WAT) is a complex mixture of adipocytes and non-adipogenic cells. Characterizing the cellular composition of WAT is critical for identifying where potential alterations occur that impact metabolism. Most single-cell (sc) RNA-Seq studies focused on the stromal vascular fraction (SVF) which does not contain adipocytes and have used technology that has a 3' or 5' bias. Using full-length sc/single-nuclei (sn) RNA-Seq technology, we interrogated the transcriptional composition of WAT using: snRNA-Seq of whole tissue, snRNA-Seq of isolated adipocytes, and scRNA-Seq of SVF. Whole WAT snRNA-Seq provided coverage of major cell types, identified three distinct adipocyte clusters, and was capable of tracking adipocyte differentiation with pseudotime. Compared to WAT, adipocyte snRNA-Seq was unable to match adipocyte heterogeneity. SVF scRNA-Seq provided greater resolution of non-adipogenic cells. These findings provide critical evidence for the utility of sc full-length transcriptomics in WAT and SVF in humans.

Pancreatic Ductal Adenocarcinoma (PDAC) circulating tumor cells influence myeloid cell differentiation to support their survival and immunoresistance in portal vein circulation.

The portal venous circulation provides a conduit for pancreatic ductal adenocarcinoma (PDAC) tumor cells to the liver parenchyma sinusoids, a frequent site of metastasis. Turbulent flow in the portal circulation promotes retention of PDAC shed circulating tumor cells (CTC) and myeloid-derived immunosuppressor cells (MDSC). Excessive colony stimulating factor-1 receptor (CSF1R) signaling can induce myeloid differentiation to MDSC and transformation of MDSC to myeloid-derived fibroblasts (M-FB). Interactions between PDAC CTC and M-FB in the portal blood promotes the formation of immunoresistant clusters that enhance CTC proliferation, migration, and survival. Analysis of portal and peripheral blood samples collected intraoperatively from 30 PDAC patients undergoing pancreatico-duodenectomy showed that PDAC patient plasma contained high levels of macrophage colony stimulating factor (M-CSF/CSF1), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interleukin-8 (IL-8), and interleukin-34 (IL-34) compared to healthy control levels. Moreover, the level of M-CSF in portal blood was significantly higher than that detected in the peripheral blood of PDAC patients. PDAC CTC aseptically isolated by fluorescence activated cell sorting (FACS) out of freshly collected patient portal blood mononuclear cells (PortalBMC) had elevated RNA expression of IL34 (IL-34 gene) and CSF1 (M-CSF/CSF1 gene) which both signal through CSF1R. PDAC CTC also had high levels of RNA expression for CXCL8, the gene encoding chemokine interleukin-8 (IL-8) which can attract myeloid cells through their CXCR2 receptors. FACS-isolated portal PDAC CTC and M-FB co-cultured ex vivo had increased CTC proliferation, motility, and cluster formation compared to CTC cultured alone. CSF1R and CXCR2 cell surface expression were found on PDAC portal blood CTC and M-FB, suggesting that both cell types may respond to M-CSF, IL-34, and IL-8-mediated signaling. Portal PDAC CTC displayed enhanced RNA expression of CSF1 and IL34, while CTC+M-FB+ clusters formed in vivo had increased RNA expression of CSF2 and IL34. Portal M-FB were found to have high CSF1R RNA expression. CTC isolated from ex vivo 7-day cultures of PDAC patient portal blood mononuclear cells (PortalBMC) expressed elevated CSF1, IL34, and IL8 RNA, and CSF1 expression was elevated in M-FB. Treatment with rabbit anti-CSF1R antibodies decreased CTC proliferation. Treatment of PortalBMC cultures with humanized anti-CSF1R, humanized anti-IL-8, or anti-IL-34 antibodies disrupted CTC cluster formation and increased CTC apoptosis. U937 myeloid precursor cell line cultures treated with conditioned media from PortalBMC ex vivo cultures without treatment or treated with anti-IL-8 and/or anti-CSF1R did not prevent myeloid differentiation in the myeloid precursor cell line U937 to macrophage, dendritic cell, MDSC, and M-FB phenotypes; whereas, U937 cultures treated with conditioned media from PortalBMC ex vivo cultures exposed to anti-IL-34 were significantly inhibited in their myeloid differentiation to all but the M-FB phenotype. PDAC patient T cells that were found phenotypically anergic (CD3+CD25+CTLA4+PD1L1+) in PortalBMC could be re-activated (CD3+CD25+CTLA4-PD1L1-), and displayed increased interferon gamma (IFNγ) production when PortalBMC ex vivo cultures were treated with anti-CSF1R, anti-IL-8, and anti-IL-34 antibodies alone or in combination. These findings suggest that PDAC CTC have the potential to influence myeloid differentiation and/or antigen presenting cell activation in the PDAC portal blood microenvironment, and that disruption of CTC/M-FB interactions may be potential targets for reversing the immunosuppression supporting CTC survival in the portal blood.

Skeletal muscle transcriptome response to a bout of endurance exercise in physically active and sedentary older adults.

Age-related declines in cardiorespiratory fitness and physical function are mitigated by regular endurance exercise in older adults. This may be due, in part, to changes in the transcriptional program of skeletal muscle following repeated bouts of exercise. However, the impact of chronic exercise training on the transcriptional response to an acute bout of endurance exercise has not been clearly determined. Here, we characterized baseline differences in muscle transcriptome and exercise-induced response in older adults who were active/endurance trained or sedentary. RNA-sequencing was performed on vastus lateralis biopsy specimens obtained before, immediately after, and 3 h following a bout of endurance exercise (40 min of cycling at 60%-70% of heart rate reserve). Using a recently developed bioinformatics approach, we found that transcript signatures related to type I myofibers, mitochondria, and endothelial cells were higher in active/endurance-trained adults and were associated with key phenotypic features including V̇o2peak, ATPmax, and muscle fiber proportion. Immune cell signatures were elevated in the sedentary group and linked to visceral and intermuscular adipose tissue mass. Following acute exercise, we observed distinct temporal transcriptional signatures that were largely similar among groups. Enrichment analysis revealed catabolic processes were uniquely enriched in the sedentary group at the 3-h postexercise timepoint. In summary, this study revealed key transcriptional signatures that distinguished active and sedentary adults, which were associated with difference in oxidative capacity and depot-specific adiposity. The acute response signatures were consistent with beneficial effects of endurance exercise to improve muscle health in older adults irrespective of exercise history and adiposity.NEW & NOTEWORTHY Muscle transcript signatures associated with oxidative capacity and immune cells underlie important phenotypic and clinical characteristics of older adults who are endurance trained or sedentary. Despite divergent phenotypes, the temporal transcriptional signatures in response to an acute bout of endurance exercise were largely similar among groups. These data provide new insight into the transcriptional programs of aging muscle and the beneficial effects of endurance exercise to promote healthy aging in older adults.

A Metabolomic Signature of Glucagon Action in Healthy Individuals With Overweight/Obesity.

CONTEXT: Glucagon is produced and released from the pancreatic alpha-cell to regulate glucose levels during periods of fasting. The main target for glucagon action is the liver, where it activates gluconeogenesis and glycogen breakdown; however, glucagon is postulated to have other roles within the body. OBJECTIVE: We sought to identify the circulating metabolites that would serve as markers of glucagon action in humans. METHODS: In this study (NCT03139305), we performed a continuous 72-hour glucagon infusion in healthy individuals with overweight/obesity. Participants were randomized to receive glucagon 12.5 ng/kg/min (GCG 12.5), glucagon 25 ng/kg/min (GCG 25), or a placebo control. A comprehensive metabolomics analysis was then performed from plasma isolated at several time points during the infusion to identify markers of glucagon activity. RESULTS: Glucagon (GCG 12.5 and GCG 25) resulted in significant changes in the plasma metabolome as soon as 4 hours following infusion. Pathways involved in amino acid metabolism were among the most affected. Rapid and sustained reduction of a broad panel of amino acids was observed. Additionally, time-dependent changes in free fatty acids and diacylglycerol and triglyceride species were observed. CONCLUSION: These results define a distinct signature of glucagon action that is broader than the known changes in glucose levels. In particular, the robust changes in amino acid levels may prove useful to monitor changes induced by glucagon in the context of additional glucagon-like peptide-1 or gastric inhibitory polypeptide treatment, as these agents also elicit changes in glucose levels.

An improvement in skeletal muscle mitochondrial capacity with short-term aerobic training is associated with changes in Tribbles 1 expression.

Exercise training and physical activity are known to be associated with high mitochondrial content and oxidative capacity in skeletal muscle. Metabolic diseases including obesity and insulin resistance are associated with low mitochondrial capacity in skeletal muscle. Certain transcriptional factors such as PGC-1α are known to mediate the exercise response; however, the precise molecular mechanisms involved in the adaptation to exercise are not completely understood. We performed multiple measurements of mitochondrial capacity both in vivo and ex vivo in lean or overweight individuals before and after an 18-day aerobic exercise training regimen. These results were compared to lean, active individuals. Aerobic training in these individuals resulted in a marked increase in mitochondrial oxidative respiratory capacity without an appreciable increase in mitochondrial content. These adaptations were associated with robust transcriptome changes. This work also identifies the Tribbles pseudokinase 1, TRIB1, as a potential mediator of the exercise response in human skeletal muscle.

Skeletal Muscle Energetics and Mitochondrial Function Are Impaired Following 10 Days of Bed Rest in Older Adults.

BACKGROUND: Older adults exposed to periods of inactivity during hospitalization, illness, or injury lose muscle mass and strength. This, in turn, predisposes poor recovery of physical function upon reambulation and represents a significant health risk for older adults. Bed rest (BR) results in altered skeletal muscle fuel metabolism and loss of oxidative capacity that have recently been linked to the muscle atrophy program. Our primary objective was to explore the effects of BR on mitochondrial energetics in muscle from older adults. A secondary objective was to examine the effect of ß-hydroxy-ß-methylbuturate (HMB) supplementation on mitochondrial energetics. METHODS: We studied 20 older adults before and after a 10-day BR intervention, who consumed a complete oral nutritional supplement (ONS) with HMB (3.0 g/d HMB, n = 11) or without HMB (CON, n = 9). Percutaneous biopsies of the vastus lateralis were obtained to determine mitochondrial respiration and H2O2 emission in permeabilized muscle fibers along with markers of content. RNA sequencing and lipidomics analyses were also conducted. RESULTS: We found a significant up-regulation of collagen synthesis and down-regulation of ribosome, oxidative metabolism and mitochondrial gene transcripts following BR in the CON group. Alterations to these gene transcripts were significantly blunted in the HMB group. Mitochondrial respiration and markers of content were both reduced and H2O2 emission was elevated in both groups following BR. CONCLUSIONS: In summary, 10 days of BR in older adults causes a significant deterioration in mitochondrial energetics, while transcriptomic profiling revealed that some of these negative effects may be attenuated by an ONS containing HMB.

Loss of mitochondrial energetics is associated with poor recovery of muscle function but not mass following disuse atrophy.

Skeletal muscle atrophy is a clinically important outcome of disuse because of injury, immobilization, or bed rest. Disuse atrophy is accompanied by mitochondrial dysfunction, which likely contributes to activation of the muscle atrophy program. However, the linkage of muscle mass and mitochondrial energetics during disuse atrophy and its recovery is incompletely understood. Transcriptomic analysis of muscle biopsies from healthy older adults subject to complete bed rest revealed marked inhibition of mitochondrial energy metabolic pathways. To determine the temporal sequence of muscle atrophy and changes in intramyocellular lipid and mitochondrial energetics, we conducted a time course of hind limb unloading-induced atrophy in adult mice. Mitochondrial respiration and calcium retention capacity were diminished, whereas H2O2 emission was increased within 3 days of unloading before significant muscle atrophy. These changes were associated with a decrease in total cardiolipin and profound changes in remodeled cardiolipin species. Hind limb unloading performed in muscle-specific peroxisome proliferator-activated receptor-γ coactivator-1α/ß knockout mice, a model of mitochondrial dysfunction, did not affect muscle atrophy but impacted muscle function. These data suggest early mitochondrial remodeling affects muscle function but not mass during disuse atrophy. Early alterations in mitochondrial energetics and lipid remodeling may represent novel targets to prevent muscle functional impairment caused by disuse and to enhance recovery from periods of muscle atrophy.

Developmental and transcriptional responses of maize to drought stress under field conditions.

Drought is a common abiotic stress which significantly limits global crop productivity. Maize is an important staple crop and its yield is determined by successful development of the female inflorescence, the ear. We investigated drought stress responses across several developmental stages of the maize B73 inbred line under field conditions. Drought suppressed plant growth, but had little impact on progression through developmental stages. While ear growth was suppressed by drought, the process of spikelet initiation was not significantly affected. Tassel growth was reduced to a lesser extent compared to the observed reduction in ear growth under stress. Parallel RNA-seq profiling of leaves, ears, and tassels at several developmental stages revealed tissue-specific differences in response to drought stress. High temperature fluctuation was an additional environmental factor that also likely influenced gene expression patterns in the field. Drought induced significant transcriptional changes in leaves and ears but only minor changes in the tassel. Additionally, more genes were drought responsive in ears compared to leaves over the course of drought treatment. Genes that control DNA replication, cell cycle, and cell division were significantly down-regulated in stressed ears, which was consistent with inhibition of ear growth under drought. Inflorescence meristem genes were affected by drought to a lesser degree which was consistent with the minimal impact of drought on spikelet initiation. In contrast, genes that are involved in floret and ovule development were sensitive to stress, which is consistent with the detrimental effect of drought on gynoecium development and kernel set.

tassel-less1 encodes a boron channel protein required for inflorescence development in maize.

tassel-less1 (tls1) is a classical maize (Zea mays) inflorescence mutant. Homozygous mutant plants have no tassels or very small tassels, and ear development is also impaired. Using a positional cloning approach, ZmNIP3;1 (a NOD26-like intrinsic protein) was identified as the candidate gene for tls1. The ZmNIP3;1 gene is completely deleted in the tls1 mutant genome. Two Mutator-insertional TUSC alleles of ZmNIP3;1 exhibited tls1-like phenotypes, and allelism tests confirmed that the tls1 gene encodes ZmNIP3;1. Transgenic plants with an RNA interference (RNAi) construct to down-regulate ZmNIP3;1 also showed tls1-like phenotypes, further demonstrating that TLS1 is ZmNIP3;1. Sequence analysis suggests that ZmNIP3;1 is a boron channel protein. Foliar application of boron could rescue the tls1 phenotypes and restore the normal tassel and ear development. Gene expression analysis indicated that in comparison with that of the wild type or tls1 plants treated with boron, the transition from the vegetative to reproductive phase or the development of the floral meristem is impaired in the shoot apical meristem of the tls1 mutant plants. It is concluded that the tls1 mutant phenotypes are caused by impaired boron transport, and boron is essential for inflorescence development in maize.

Gnom(Cmp): a quantitative approach for comparative analysis of closely related genomes of bacterial pathogens.

Comparative genome analysis is a powerful approach to understanding the biology of infectious bacterial pathogens. In this study, a quantitative approach, referred to as Gnom(Cmp), was developed to study the microevolution of bacterial pathogens. Although much more time-consuming than existing tools, this procedure provides a much higher resolution. Gnom(Cmp) accomplishes this by establishing genome-wide heterogeneity genotypes, which are then quantified and comparatively analyzed. The heterogeneity genotypes are defined as chromosomal base positions that have multiple variants within particular genomes, resulted from DNA duplications and subsequent mutations. To prove the concept, the procedure was applied on the genomes of 15 Staphylococcus aureus strains, focusing extensively on two pairs of hVISA/VISA strains. hVISA refers to heteroresistant vancomycin-intermediate S. aureus strains and VISA is their VISA mutants. hVISA/VISA displays some remarkable properties. hVISA is susceptible to vancomycin, but VISA mutants emerge soon after a short period of vancomycin therapy, therefore making the pathogen a great model organism for fast-evolving bacterial pathogens. The analysis indicated that Gnom(Cmp) could reveal variants within the genomes, which can be analyzed within the global genome context. Gnom(Cmp) discovered evolutionary hotspots and their dynamics among many closely related, even isogenic genomes. The analysis thus allows the exploration of the molecular mechanisms behind hVISA/VISA evolution, providing a working hypotheses for experimental testing and validation.

New implications on genomic adaptation derived from the Helicobacter pylori genome comparison.

BACKGROUND: Helicobacter pylori has a reduced genome and lives in a tough environment for long-term persistence. It evolved with its particular characteristics for biological adaptation. Because several H. pylori genome sequences are available, comparative analysis could help to better understand genomic adaptation of this particular bacterium. PRINCIPAL FINDINGS: We analyzed nine H. pylori genomes with emphasis on microevolution from a different perspective. Inversion was an important factor to shape the genome structure. Illegitimate recombination not only led to genomic inversion but also inverted fragment duplication, both of which contributed to the creation of new genes and gene family, and further, homological recombination contributed to events of inversion. Based on the information of genomic rearrangement, the first genome scaffold structure of H. pylori last common ancestor was produced. The core genome consists of 1186 genes, of which 22 genes could particularly adapt to human stomach niche. H. pylori contains high proportion of pseudogenes whose genesis was principally caused by homopolynucleotide (HPN) mutations. Such mutations are reversible and facilitate the control of gene expression through the change of DNA structure. The reversible mutations and a quasi-panmictic feature could allow such genes or gene fragments frequently transferred within or between populations. Hence, pseudogenes could be a reservoir of adaptation materials and the HPN mutations could be favorable to H. pylori adaptation, leading to HPN accumulation on the genomes, which corresponds to a special feature of Helicobacter species: extremely high HPN composition of genome. CONCLUSION: Our research demonstrated that both genome content and structure of H. pylori have been highly adapted to its particular life style.

GenHtr: a tool for comparative assessment of genetic heterogeneity in microbial genomes generated by massive short-read sequencing.

BACKGROUND: Microevolution is the study of short-term changes of alleles within a population and their effects on the phenotype of organisms. The result of the below-species-level evolution is heterogeneity, where populations consist of subpopulations with a large number of structural variations. Heterogeneity analysis is thus essential to our understanding of how selective and neutral forces shape bacterial populations over a short period of time. The Solexa Genome Analyzer, a next-generation sequencing platform, allows millions of short sequencing reads to be obtained with great accuracy, allowing for the ability to study the dynamics of the bacterial population at the whole genome level. The tool referred to as GenHtr was developed for genome-wide heterogeneity analysis. RESULTS: For particular bacterial strains, GenHtr relies on a set of Solexa short reads on given bacteria pathogens and their isogenic reference genome to identify heterogeneity sites, the chromosomal positions with multiple variants of genes in the bacterial population, and variations that occur in large gene families. GenHtr accomplishes this by building and comparatively analyzing genome-wide heterogeneity genotypes for both the newly sequenced genomes (using massive short-read sequencing) and their isogenic reference (using simulated data). As proof of the concept, this approach was applied to SRX007711, the Solexa sequencing data for a newly sequenced Staphylococcus aureus subsp. USA300 cell line, and demonstrated that it could predict such multiple variants. They include multiple variants of genes critical in pathogenesis, e.g. genes encoding a LysR family transcriptional regulator, 23 S ribosomal RNA, and DNA mismatch repair protein MutS. The heterogeneity results in non-synonymous and nonsense mutations, leading to truncated proteins for both LysR and MutS. CONCLUSION: GenHtr was developed for genome-wide heterogeneity analysis. Although it is much more time-consuming when compared to Maq, a popular tool for SNP analysis, GenHtr is able to predict potential multiple variants that pre-exist in the bacterial population as well as SNPs that occur in the highly duplicated gene families. It is expected that, with the proper experimental design, this analysis can improve our understanding of the molecular mechanism underlying the dynamics and the evolution of drug-resistant bacterial pathogens.

An exon-based comparative variant analysis pipeline to study the scale and role of frameshift and nonsense mutation in the human-chimpanzee divergence.

Chimpanzees and humans are closely related but differ in many deadly human diseases and other characteristics in physiology, anatomy, and pathology. In spite of decades of extensive research, crucial questions about the molecular mechanisms behind the differences are yet to be understood. Here I report ExonVar, a novel computational pipeline for Exon-based human-chimpanzee comparative Variant analysis. The objective is to comparatively analyze mutations specifically those that caused the frameshift and nonsense mutations and to assess their scale and potential impacts on human-chimpanzee divergence. Genomewide analysis of human and chimpanzee exons with ExonVar identified a number of species-specific, exon-disrupting mutations in chimpanzees but much fewer in humans. Many were found on genes involved in important biological processes such as T cell lineage development, the pathogenesis of inflammatory diseases, and antigen induced cell death. A "less-is-more" model was previously established to illustrate the role of the gene inactivation and disruptions during human evolution. Here this analysis suggested a different model where the chimpanzee-specific exon-disrupting mutations may act as additional evolutionary force that drove the human-chimpanzee divergence. Finally, the analysis revealed a number of sequencing errors in the chimpanzee and human genome sequences and further illustrated that they could be corrected without resequencing.

Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes.

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.

An evolution-based analysis scheme to identify CO2/O2 specificity-determining factors for ribulose 1,5-bisphosphate carboxylase/oxygenase.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) catalyzes a rate-limiting step in photosynthetic carbon assimilation (reacting with CO2) and its competitive photo-respiratory carbon oxidation (reacting with O2). RuBisCo enzyme with an enhanced CO2/O2 specificity would boost the ability to make great progress in agricultural production and environmental management. RuBisCos in marine non-green algae, resulting from an earlier endo-symbiotic event, diverge greatly from those in green plants and cyanobacteria and, further, have the highest CO2/O2 specificity whereas RuBisCos in cyanobacteria have the lowest. We assumed that there exist different levels of CO2/O2 specificity-determining factors, corresponding to different evolutionary events and specificity levels. Based on this assumption, we devised a scheme to identify these substrate-determining factors. From this analysis, we are able to discover different categories of the CO2/O2 specificity-determining factors that show which residue substitutions account for (relatively) small specificity changes, as happened in green plants, or a tremendous enhancement, as observed in marine non-green algae. Therefore, the analysis can improve our understanding of molecular mechanisms in the substrate specificity development and prioritize candidate specificity-determining surface residues for site-directed mutagenesis.

In silico discovery of enzyme-substrate specificity-determining residue clusters.

The binding between an enzyme and its substrate is highly specific, despite the fact that many different enzymes show significant sequence and structure similarity. There must be, then, substrate specificity-determining residues that enable different enzymes to recognize their unique substrates. We reason that a coordinated, not independent, action of both conserved and non-conserved residues determine enzymatic activity and specificity. Here, we present a surface patch ranking (SPR) method for in silico discovery of substrate specificity-determining residue clusters by exploring both sequence conservation and correlated mutations. As case studies we apply SPR to several highly homologous enzymatic protein pairs, such as guanylyl versus adenylyl cyclases, lactate versus malate dehydrogenases, and trypsin versus chymotrypsin. Without using experimental data, we predict several single and multi-residue clusters that are consistent with previous mutagenesis experimental results. Most single-residue clusters are directly involved in enzyme-substrate interactions, whereas multi-residue clusters are vital for domain-domain and regulator-enzyme interactions, indicating their complementary role in specificity determination. These results demonstrate that SPR may help the selection of target residues for mutagenesis experiments and, thus, focus rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.