A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

Sp110 transcription is induced and required by Anaplasma phagocytophilum for infection of human promyelocytic cells

<p>Abstract</p> <p>Background</p> <p>The tick-borne intracellular pathogen, <it>Anaplasma phagocytophilum </it>(Rickettsiales: Anaplasmataceae) causes human granulocytic anaplasmosis after infection of polymorphonuclear leucocytes. The human Sp110 gene is a member of the nuclear body (NB) components that functions as a nuclear hormone receptor transcriptional coactivator and plays an important role in immunoprotective mechanisms against pathogens in humans. In this research, we hypothesized that Sp110 may be involved in the infection of human promyelocytic HL-60 cells with <it>A. phagocytophilum</it>.</p> <p>Methods</p> <p>The human Sp110 and <it>A. phagocytophilum msp4 </it>mRNA levels were evaluated by real-time RT-PCR in infected human HL-60 cells sampled at 0, 12, 24, 48, 72 and 96 hours post-infection. The effect of Sp110 expression on <it>A. phagocytophilum </it>infection was determined by RNA interference (RNAi). The expression of Sp110 was silenced in HL-60 cells by RNAi using pre-designed siRNAs using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Gaithersburg, MD, USA). The <it>A. phagocytophilum </it>infection levels were evaluated in HL-60 cells after RNAi by real-time PCR of <it>msp4 </it>and normalizing against human <it>Alu </it>sequences.</p> <p>Results</p> <p>While Sp110 mRNA levels increased concurrently with <it>A. phagocytophilum </it>infections in HL-60 cells, the silencing of Sp110 expression by RNA interference resulted in decreased infection levels.</p> <p>Conclusion</p> <p>These results demonstrated that Sp110 expression is required for <it>A. phagocytophilum </it>infection and multiplication in HL-60 cells, and suggest a previously undescribed mechanism by which <it>A. phagocytophilum </it>modulates Sp110 mRNA levels to facilitate establishment of infection of human HL-60 cells.</p

Genome Analysis of Planctomycetes Inhabiting Blades of the Red Alga

Porphyra is a macrophytic red alga of the Bangiales that is important ecologically and economically. We describe the genomes of three bacteria in the phylum Planctomycetes (designated P1, P2 and P3) that were isolated from blades of Porphyra umbilicalis (P.um.1). These three Operational Taxonomic Units (OTUs) belong to distinct genera; P2 belongs to the genus Rhodopirellula, while P1 and P3 represent undescribed genera within the Planctomycetes. Comparative analyses of the P1, P2 and P3 genomes show large expansions of distinct gene families, which can be widespread throughout the Planctomycetes (e.g., protein kinases, sensors/response regulators) and may relate to specific habitat (e.g., sulfatase gene expansions in marine Planctomycetes) or phylogenetic position. Notably, there are major differences among the Planctomycetes in the numbers and sub-functional diversity of enzymes (e.g., sulfatases, glycoside hydrolases, polysaccharide lyases) that allow these bacteria to access a range of sulfated polysaccharides in macroalgal cell walls. These differences suggest that the microbes have varied capacities for feeding on fixed carbon in the cell walls of P.um.1 and other macrophytic algae, although the activities among the various bacteria might be functionally complementary in situ. Additionally, phylogenetic analyses indicate augmentation of gene functions through expansions arising from gene duplications and horizontal gene transfers; examples include genes involved in cell wall degradation (e.g., κ-carrageenase, alginate lyase, fucosidase) and stress responses (e.g., efflux pump, amino acid transporter). Finally P1 and P2 contain various genes encoding selenoproteins, many of which are enzymes that ameliorate the impact of environmental stresses that occur in the intertidal habitat

Genome of the red alga Porphyridium purpureum

The limited knowledge we have about red algal genomes comes from the highly specialized extremophiles, Cyanidiophyceae. Here, we describe the first genome sequence from a mesophilic, unicellular red alga, Porphyridium purpureum. The 8,355 predicted genes in P. purpureum, hundreds of which are likely to be implicated in a history of horizontal gene transfer, reside in a genome of 19.7 Mbp with 235 spliceosomal introns. Analysis of light-harvesting complex proteins reveals a nuclear-encoded phycobiliprotein in the alga. We uncover a complex set of carbohydrate-active enzymes, identify the genes required for the methylerythritol phosphate pathway of isoprenoid biosynthesis, and find evidence of sexual reproduction. Analysis of the compact, function-rich genome of P. purpureum suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life

Correction of Population Stratification in Large Multi-Ethnic Association Studies

The vast majority of genetic risk factors for complex diseases have, taken individually, a small effect on the end phenotype. Population-based association studies therefore need very large sample sizes to detect significant differences between affected and non-affected individuals. Including thousands of affected individuals in a study requires recruitment in numerous centers, possibly from different geographic regions. Unfortunately such a recruitment strategy is likely to complicate the study design and to generate concerns regarding population stratification.We analyzed 9,751 individuals representing three main ethnic groups - Europeans, Arabs and South Asians - that had been enrolled from 154 centers involving 52 countries for a global case/control study of acute myocardial infarction. All individuals were genotyped at 103 candidate genes using 1,536 SNPs selected with a tagging strategy that captures most of the genetic diversity in different populations. We show that relying solely on self-reported ethnicity is not sufficient to exclude population stratification and we present additional methods to identify and correct for stratification.Our results highlight the importance of carefully addressing population stratification and of carefully “cleaning” the sample prior to analyses to obtain stronger signals of association and to avoid spurious results

A Tale of Four Stories: Soil Ecology, Theory, Evolution and the Publication System

International audienceBACKGROUND: Soil ecology has produced a huge corpus of results on relations between soil organisms, ecosystem processes controlled by these organisms and links between belowground and aboveground processes. However, some soil scientists think that soil ecology is short of modelling and evolutionary approaches and has developed too independently from general ecology. We have tested quantitatively these hypotheses through a bibliographic study (about 23000 articles) comparing soil ecology journals, generalist ecology journals, evolutionary ecology journals and theoretical ecology journals. FINDINGS: We have shown that soil ecology is not well represented in generalist ecology journals and that soil ecologists poorly use modelling and evolutionary approaches. Moreover, the articles published by a typical soil ecology journal (Soil Biology and Biochemistry) are cited by and cite low percentages of articles published in generalist ecology journals, evolutionary ecology journals and theoretical ecology journals. CONCLUSION: This confirms our hypotheses and suggests that soil ecology would benefit from an effort towards modelling and evolutionary approaches. This effort should promote the building of a general conceptual framework for soil ecology and bridges between soil ecology and general ecology. We give some historical reasons for the parsimonious use of modelling and evolutionary approaches by soil ecologists. We finally suggest that a publication system that classifies journals according to their Impact Factors and their level of generality is probably inadequate to integrate "particularity" (empirical observations) and "generality" (general theories), which is the goal of all natural sciences. Such a system might also be particularly detrimental to the development of a science such as ecology that is intrinsically multidisciplinary

Association of narcolepsy-cataplexy with HLA-DRB1 and DQB1 in Mexican patients: A relationship between HLA and gender is suggested

<p>Abstract</p> <p>Background</p> <p>Narcolepsy-cataplexy is characterized by excessive daytime sleepiness with recurrent episodes of irresistible sleep, cataplexy, hallucinations and sleep paralysis. Its aetiology is unknown, but it is positively associated with the human leukocyte antigens (HLA) in all studied populations. The purpose of the present study was to investigate the association of HLA class II <it>DRB1</it>/<it>DQB1 </it>alleles with narcolepsy-cataplexy in Mexican Mestizo patients.</p> <p>Methods</p> <p>This is a case-control study of consecutive patients and ethnically matched controls. We included 32 patients diagnosed with typical narcolepsy-cataplexy, of the National Institute of Neurology, of the Institute of Psychiatry and at the Center of Narcolepsy at Stanford University. As healthy controls, 203 Mexican Mestizos were included. <it>DRB1 </it>alleles were identified using sequence based typing. A PCR-SSOP reverse dot blot was used for <it>DQB1 </it>typing. Allele frequency was calculated by direct counting and the significance of the differences was assessed using the Yates Chi square. Odds ratio and confidence intervals were evaluated.</p> <p>Results</p> <p>HLA-<it>DRB1</it>*1501 (OR = 8.2; pc < 0.0001) and <it>DQB1</it>*0602 (OR = 8.4; pc < 0.0001) were found positively associated with narcolepsy. When deleting <it>DQB1</it>*0602+ patients from the analysis, <it>DQB1</it>*0301 was also found increased (OR = 2.7; p = 0.035; pc = NS). <it>DQB1</it>*0602/<it>DQB1</it>*0301 genotype was present in 15.6% of the cases (OR = 11.5; p = 0.00035), conferring a high risk. <it>DRB1</it>*0407 (OR = 0.2; p = 0.016 pc = NS) and <it>DQB1</it>*0302(OR = 0.4; p = 0.017, pc = NS) were found decreased in the patients. The gender stratification analysis showed a higher risk in females carrying <it>DRB1</it>*1501 (OR = 15.8, pc < 0.0001) and <it>DQB1</it>*0602 (OR = 19.8, pc < 0.0001) than in males (OR = 5.0 for both alleles; p = 0.012, pc = NS for <it>DRB1 </it>& p = 0.0012, pc = 0.017 for <it>DQB1</it>). The susceptibility alleles found in Mexicans with narcolepsy are also present in Japanese and Caucasians; <it>DRB1</it>*04 linked protection has also been shown in Koreans. A stronger HLA association is suggested in females, in accordance with the sexual dimorphism claimed previously.</p> <p>Conclusion</p> <p>This knowledge may contribute to a better understanding of the disease pathogenesis in different populations. The evaluation of the risk to develop narcolepsy-cataplexy in carriers of the described alleles/genotypes may also be possible. A larger sample should be analysed in Mexican and in other Hispanic patients to confirm these results.</p

Altered sensory-weighting mechanisms is observed in adolescents with idiopathic scoliosis

BACKGROUND: Scoliosis is the most common type of spinal deformity. In North American children, adolescent idiopathic scoliosis (AIS) makes up about 90% of all cases of scoliosis. While its prevalence is about 2% to 3% in children aged between 10 to 16 years, girls are more at risk than boys for severe progression with a ratio of 3.6 to 1. The aim of the present study was to test the hypothesis that idiopathic scoliosis interferes with the mechanisms responsible for sensory-reweighting during balance control. METHODS: Eight scoliosis patients (seven female and one male; mean age: 16.4 years) and nine healthy adolescents (average age 16.5 years) participated in the experiment. Visual and ankle proprioceptive information was perturbed (eyes closed and/or tendon vibration) suddenly and then returned to normal (eyes open and/or no tendon vibration). An AMTI force platform was used to compute centre of pressure root mean squared velocity and sway density curve. RESULTS: For the control condition (eyes open and no tendon vibration), adolescent idiopathic scoliosis patients had a greater centre of pressure root mean squared velocity (variability) than control participants. Reintegration of ankle proprioception, when vision was either available or removed, led to an increased centre of pressure velocity variability for the adolescent idiopathic scoliosis patients whereas the control participants reduced their centre of pressure velocity variability. Moreover, in the absence of vision, adolescent idiopathic scoliosis exhibited an increased centre of pressure velocity variability when ankle proprioception was returned to normal (i.e. tendon vibration stopped). The analysis of the sway density plot suggests that adolescent idiopathic scoliosis patients, during sensory reintegration, do not scale appropriately their balance control commands. CONCLUSION: Altogether, the present results demonstrate that idiopathic scoliosis adolescents have difficulty in reweighting sensory inputs following a brief period of sensory deprivation

In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As a result, proper diagnosis of PCD is difficult and may require repeated sampling of patients’ tissue, which is not always possible. The culturing of differentiated cells and tissues derived from the human RE seems to be the best way to diagnose PCD, to study genotype–phenotype relations of genes involved in ciliary dysfunction, as well as other aspects related to the functioning of the RE. In this review, different methods of culturing differentiated cells and tissues derived from the human RE, along with their potential and limitations, are summarized. Several considerations with respect to the factors influencing the process of in vitro differentiation (cell-to-cell interactions, medium composition, cell-support substrate) are also discussed