Identification of innate immunity genes and pathways using a comparative genomics approach

To reveal regulators of innate immunity, we used RNAi assays to monitor the immune response when genes are inhibited in Caenorhabditis elegans and mouse macrophages. Genes that altered innate immune responsiveness in C. elegans were validated in murine macrophages, resulting in the discovery of 11 genes that regulate the innate immune response in both systems and the subsequent identification of a protein interaction network with a conserved role in innate immunity regulation. We confirmed the role of four of these 11 genes in antimicrobial gene regulation using available mutants in C. elegans. Several of these genes (acy-1, tub-2, and tbc-1) also regulate susceptibility to the pathogen Pseudomonas aeruginosa. These genes may prove critical to understanding host defense and represent potential therapeutic targets for infectious and immunological diseases.     

Population genomics of Mycobacterium tuberculosis in the Inuit

Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain.The tubercule bacillus, Mycobacterium tuberculosis, is a highly successful, medically important human-adapted pathogen. Studies of diverse strain collections reveal a geographic aggregation of the principal M. tuberculosis lineages (1) consistent with a dissemination of this organism around the world with the paleo migration (2). Ancient DNA studies also support the notion that M. tuberculosis has caused disease in humans for thousands of years. Thus, it can be inferred that M. tuberculosis has evolved in step with its human host, successfully responding to changes in the host and its environment that could affect the capacity to cause transmissible disease.In contrast to the global diversity of M. tuberculosis strains (1–3), we have previously observed limited genetic diversity in the Nunavik region of Québec (4). One possible explanation is a founder strain, wherein genetic similarity is due to a single recent introduction of a bacterium and may not necessarily represent ongoing spread between communities. In this scenario, isolates might have indistinguishable genotypes by conventional genotyping modalities (restriction fragment length polymorphism, mycobacterial interspersed repetitive units, spoligotyping) but distinct genotypes when assessed using a higher-resolution method, namely whole-genome sequencing (WGS) (5). An additional explanation is that a single clone of M. tuberculosis is currently spreading both within and between villages; however, the great distances between these communities that are not linked by roads make intervillage spread less likely. These possible explanations need not be mutually exclusive.To evaluate these possibilities, we conducted WGS on M. tuberculosis isolates from Nunavik isolated over 23 y. Estimation of the divergence date of the most recent common ancestor (MRCA) provided evidence that tuberculosis (TB) was introduced into this region in the early 20th century, following which time there has been substantial ongoing transmission, predominantly within villages. This setting provides a unique opportunity to study the genomic characteristics of an epidemiologically successful strain of M. tuberculosis over time.     

Comparative study of mutations in SNP loci of K-RAS,hMLH1 and hMSH2 genes in neoplastic intestinal polyps and colorectal cancer

AIM: To clarify the molecular mechanism involved in pathogenesis of colorectal cancer as well as clinical significance of genetic analysis of histological samples.METHODS: A total of 480 blood and tissue specimens were collected in our hospital from January 2011 to October 2012. In the observation group, there were 120 blood specimens and 120 intestinal tract tissue specimens collected from patients with neoplastic intestinal polyps. In the control group I there were 80 blood specimens and 80 intestinal tract tissue specimens collected from patients with colorectal cancer. In the control group II there were 40 blood specimens and 40 intestinal tract tissue specimens collected from healthy individuals. The gene segments were amplified using PCR and DNA gel electrophoresis along with DNA sequence analysis were employed for the detection of the following single nucleotide polymorphisms (SNPs): K-RAS codons 12 and 13; hMLH1 (human mutS homolog 1) gene missense mutation at Va1384Asp; hMSH2 (human mutS homolog 2) gene missense mutation at 2783C/A.RESULTS: The mutation rate of the SNP at Va1384Asp locus of the hMLH1 gene from blood and tissue specimens in the observation group showed no statistical difference from those in the control group I. The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene were significantly lower in the observation group than in the control group I (χ² = 15.476, 29.670, 10.811, 16.618, 33.538, 7.898, P < 0.05). The mutation rate of SNP at Va1384Asp locus of the hMLH1 gene was significantly higher in the observation group when compared to the control group II (χ² = 10.486, 4.876, P < 0.05). The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene did not show any statistical difference from those in the control group II.CONCLUSION: There may be important clinical significance and relevance between neoplastic intestinal polyps and colorectal cancer in terms of the mechanisms involved in the pathogenesis.     

Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.     

Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion

Cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. To facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. However, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self-stimulation. We hypothesized that such cells must alternate their physiological state between signal sending and receiving to allow recognition and behavioral changes. To test this hypothesis, we studied cell communication in the filamentous fungus Neurospora crassa, a simple and experimentally amenable model system. In N. crassa, germinating asexual spores (germlings) of identical genotype chemotropically sense others in close proximity, show attraction-mediated directed growth, and ultimately undergo cell fusion. Here, we report that two proteins required for cell fusion, a MAP kinase (MAK-2) and a protein of unknown molecular function (SO), exhibit rapid oscillatory recruitment to the plasma membranes of interacting germlings undergoing chemotropic interactions via directed growth. Using an inhibitable MAK-2 variant, we show that MAK-2 kinase activity is required both for chemotropic interactions and for oscillation of MAK-2 and SO to opposing cell tips. Thus, N. crassa germlings undergoing chemotropic interactions rapidly alternate between two different physiological states, associated with signal delivery and response. Such spatiotemporal coordination of signaling allows genetically identical and developmentally equivalent cells to avoid self-stimulation and to coordinate their behavior to achieve the beneficial physiological outcome of cell fusion.     

Multiple-stress analysis for isolation of Drosophila longevity genes

Long-lived organisms tend to be more resistant to various forms of environmental stress. An example is the Drosophila longevity mutant, methuselah, which has enhanced resistance to heat, oxidants, and starvation. To identify genes regulated by these three stresses, we made a cDNA library for each by subtraction of "unstressed" from "stressed" cDNA and used DNA hybridization to identify genes that are regulated by all three. This screen indeed identified 13 genes, some already known to be involved in longevity, plus candidate genes. Two of these, hsp26 and hsp27, were chosen to test for their effects on lifespan by generating transgenic lines and by using the upstream activating sequence/GAL4 system. Overexpression of either hsp26 or hsp27 extended the mean lifespan by 30%, and the flies also displayed increased stress resistance. The results demonstrate that multiple-stress screening can be used to identify new longevity genes.     

Germline promoter hypermethylation of tumor suppressor genes in gastric cancer

AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1, CDH1 and P16^INK4a in suspected cases of hereditary gastric cancer (GC).METHODS: A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1, CDH1 and P16^INK4a tumor suppressor genes. Genomic DNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfite DNA sequencing for a specific region of the MLH1 promoter. The methylation status of CDH1 or P16^INK4a was assayed using methylation-specific PCR. Clonal bisulfite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions.RESULTS: Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16^INK4a genes was detected.CONCLUSION: Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16^INK4a gene is not a frequent event.     

SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host-symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant-fungal and plant-bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.     

Comparative evolutionary genetics of spontaneous mutations affecting fitness in rhabditid nematodes

Deleterious mutations are of fundamental importance to all aspects of organismal biology. Evolutionary geneticists have expended tremendous effort to estimate the genome-wide rate of mutation and the effects of new mutations on fitness, but the degree to which genomic mutational properties vary within and between taxa is largely unknown, particularly in multicellular organisms. Beginning with two highly inbred strains from each of three species in the nematode family Rhabditidae (Caenorhabditis briggsae, Caenorhabditis elegans, and Oscheius myriophila), we allowed mutations to accumulate in the relative absence of natural selection for 200 generations. We document significant variation in the rate of decay of fitness because of new mutations between strains and between species. Estimates of the per-generation mutational decay of fitness were very consistent within strains between assays 100 generations apart. Rate of mutational decay in fitness was positively associated with genomic mutation rate and negatively associated with average mutational effect. These results provide unambiguous experimental evidence for substantial variation in genome-wide properties of mutation both within and between species and reinforce conclusions from previous experiments that the cumulative effects on fitness of new mutations can differ markedly among related taxa.     

Conserved role for autophagy in Rho1-mediated cortical remodeling and blood cell recruitment

Dynamic regulation of cell shape underlies many developmental and immune functions. Cortical remodeling is achieved under the central control of Rho GTPase pathways that modulate an exquisite balance in the dynamic assembly and disassembly of the cytoskeleton and focal adhesions. Macroautophagy (autophagy), associated with bulk cytoplasmic remodeling through lysosomal degradation, has clearly defined roles in cell survival and death. Moreover, it is becoming apparent that proteins, organelles, and pathogens can be targeted for autophagic clearance by selective mechanisms, although the extent and roles of such degradation are unclear. Here we report a conserved role for autophagy specifically in the cortical remodeling of Drosophila blood cells (hemocytes) and mouse macrophages. Continuous autophagy was required for integrin-mediated hemocyte spreading and Rho1-induced cell protrusions. Consequently, hemocytes disrupted for autophagy were impaired in their recruitment to epidermal wounds. Cell spreading required ref(2)P, the Drosophila p62 multiadaptor, implicating selective autophagy as a novel mechanism for modulating cortical dynamics. These results illuminate a specific and conserved role for autophagy as a regulatory mechanism for cortical remodeling, with implications for immune cell function.     

Chimpanzees modify recruitment screams as a function of audience composition

Wild chimpanzees produce acoustically distinct scream vocalizations depending on their social role during agonistic interactions with other group members. Here, we show that victims during such agonistic interactions alter the acoustic structure of their screams depending on the severity of aggression experienced, providing nearby listeners with important cues about the nature of the attack. However, we also found that victims of severe attacks produced screams that significantly exaggerated the true level of aggression experienced, but they did so only if there was at least one listener in the audience who matched or surpassed the aggressor in rank. Our results are consistent with the more general hypothesis that chimpanzees possess sophisticated understanding of third-party relationships, so-called triadic awareness, and that this knowledge influences their vocal production.     

High frequency of hotspot mutations in core genes of Escherichia coli due to short-term positive selection

Core genes comprising the ubiquitous backbone of bacterial genomes are not subject to frequent horizontal transfer and generally are not thought to contribute to the adaptive evolution of bacterial pathogens. We determined, however, that at least one-third and possibly more than one-half of the core genes in Escherichia coli genomes are targeted by repeated replacement substitutions in the same amino acid positions—hotspot mutations. Occurrence of hotspot mutations is driven by positive selection, as their rate is significantly higher than expected by random chance alone, and neither intragenic recombination nor increased mutability can explain the observed patterns. Also, commensal E. coli strains have a significantly lower frequency of mutated genes and mutations per genome than pathogenic strains. E. coli strains causing extra-intestinal infections accumulate hotspot mutations at the highest rate, whereas the highest total number of mutated genes has been found among Shigella isolates, suggesting the pathoadaptive nature of such mutations. The vast majority of hotspot mutations are of recent evolutionary origin, implying short-term positive selection, where adaptive mutations emerge repeatedly but are not sustained in natural circulation for long. Such pattern of dynamics is consistent with source-sink model of virulence evolution.     

Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson's disease model

Genomic multiplication of the locus-encoding human alpha-synuclein (alpha-syn), a polypeptide with a propensity toward intracellular misfolding, results in Parkinson's disease (PD). Here we report the results from systematic screening of nearly 900 candidate genetic targets, prioritized by bioinformatic associations to existing PD genes and pathways, via RNAi knockdown. Depletion of 20 gene products reproducibly enhanced misfolding of alpha-syn over the course of aging in the nematode Caenorhabditis elegans. Subsequent functional analysis of seven positive targets revealed five previously unreported gene products that significantly protect against age- and dose-dependent alpha-syn-induced degeneration in the dopamine neurons of transgenic worms. These include two trafficking proteins, a conserved cellular scaffold-type protein that modulates G protein signaling, a protein of unknown function, and one gene reported to cause neurodegeneration in knockout mice. These data represent putative genetic susceptibility loci and potential therapeutic targets for PD, a movement disorder affecting approximately 2% of the population over 65 years of age.