Time Required for Nanopore Whole-Genome Sequencing of Neisseria gonorrhoeae for Identification of Phylogenetic Relationships.

BACKGROUND: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health challenge. Limitations to AMR surveillance reporting, alongside reduction in culture-based susceptibility testing, has resulted in a need for rapid diagnostics and strain detection. We investigated Nanopore sequencing time, and depth, to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina sequencing. METHODS: N. gonorrhoeae strains collected from a London sexual health clinic were cultured and sequenced with MiSeq and MinION sequencing platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions (37 resistance-associated markers). Accuracy at varying MinION sequencing depths was determined through retrospective time-stamped read analysis. RESULTS: Of 22 MinION-MiSeq pairs reaching sufficient sequencing depth, agreement of variant call positions passing quality control criteria was 185/185 (100%; 95% confidence interval [CI], 98.0%-100.0%), 502/503 (99.8%; 95% CI, 98.9%-99.9%), and 564/565 (99.8%; 95% CI, 99.0%-100.0%) at 10x, 30x, and 40x MinION depth, respectively. Isolates identified as closely related by MiSeq, within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified via MinION. CONCLUSIONS: Nanopore sequencing shows utility as a rapid surveillance tool, identifying closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median time of 29 minutes. This highlights its potential for tracking local transmission and AMR markers.

Resistance gene transfer: induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage.

Objectives: Horizontal gene transfer of antimicrobial resistance (AMR) genes between clinical isolates via transduction is poorly understood. MRSA are opportunistic pathogens resistant to all classes of antimicrobial agents but currently no strains are fully drug resistant. AMR gene transfer between Staphylococcus aureus isolates is predominantly due to generalized transduction via endogenous bacteriophage, and recent studies have suggested transfer is elevated during host colonization. The aim was to investigate whether exposure to sub-MIC concentrations of antimicrobials triggers bacteriophage induction and/or increased efficiency of AMR gene transfer. Methods: Isolates from MRSA carriers were exposed to nine antimicrobials and supernatants were compared for lytic phage particles and ability to transfer an AMR gene. A new technology, droplet digital PCR, was used to measure the concentration of genes in phage particles. Results: All antibiotics tested induced lytic phage and AMR gene transduction, although the ratio of transducing particles to lytic particles differed substantially for each antibiotic. Mupirocin induced the highest ratio of transducing versus lytic particles. Gentamicin and novobiocin reduced UV-induced AMR transduction. The genes carried in phage particles correlated with AMR transfer or lytic particle activity, suggesting antimicrobials influence which DNA sequences are packaged into phage particles. Conclusions: Sub-inhibitory antibiotics induce AMR gene transfer between clinical MRSA, while combination therapy with an inhibiting antibiotic could potentially alter AMR gene packaging into phage particles, reducing AMR transfer. In a continually evolving environment, pathogens have an advantage if they can transfer DNA while lowering the risk of lytic death.

Microarray analysis of bone marrow lesions in osteoarthritis demonstrates upregulation of genes implicated in osteochondral turnover, neurogenesis and inflammation.

OBJECTIVE: Bone marrow lesions (BMLs) are well described in osteoarthritis (OA) using MRI and are associated with pain, but little is known about their pathological characteristics and gene expression. We evaluated BMLs using novel tissue analysis tools to gain a deeper understanding of their cellular and molecular expression. METHODS: We recruited 98 participants, 72 with advanced OA requiring total knee replacement (TKR), 12 with mild OA and 14 non-OA controls. Participants were assessed for pain (using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)) and with a knee MRI (using MOAKS). Tissue was then harvested at TKR for BML analysis using histology and tissue microarray. RESULTS: The mean (SD) WOMAC pain scores were significantly increased in advanced OA 59.4 (21.3) and mild OA 30.9 (20.3) compared with controls 0.5 (1.28) (p<0.0001). MOAKS showed all TKR tissue analysed had BMLs, and within these lesions, bone marrow volume was starkly reduced being replaced by dense fibrous connective tissue, new blood vessels, hyaline cartilage and fibrocartilage. Microarray comparing OA BML and normal bone found a significant difference in expression of 218 genes (p<0.05). The most upregulated genes included stathmin 2, thrombospondin 4, matrix metalloproteinase 13 and Wnt/Notch/catenin/chemokine signalling molecules that are known to constitute neuronal, osteogenic and chondrogenic pathways. CONCLUSION: Our study is the first to employ detailed histological analysis and microarray techniques to investigate knee OA BMLs. BMLs demonstrated areas of high metabolic activity expressing pain sensitisation, neuronal, extracellular matrix and proinflammatory signalling genes that may explain their strong association with pain.

Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy.

INTRODUCTION: Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. METHODS: NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. RESULTS: NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%-98.3%) and 100% (95% CI 94.7%-100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%-100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%-100%), 100% (95% CI 87.2%-100%) and 100% (95% CI 82.4%-100%), respectively. CONCLUSIONS: Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for surveillance.

Increased apoptosis, altered oxygen signaling, and antioxidant defenses in first-trimester pregnancies with high-resistance uterine artery blood flow.

The mechanisms of deficient placentation in the first trimester remain poorly understood, although apoptosis, hypoxia, and oxidative stress have been implicated. High uterine artery Doppler resistance indexes (RIs) are predictive of placental complications of pregnancy, such as preeclampsia, fetal growth restriction, and stillbirth. We provide evidence that even in the first trimester, pregnancies with high uterine artery Doppler RI demonstrate alterations in placental gene and protein expression. Apoptosis was significantly higher in high RI placental tissue, as determined by Western blot analysis of cleaved poly (ADP-ribose) polymerase and caspase 3. Protein expression of the trophoblast survival factor insulin-like growth factor-2 was significantly lower. Both high and normal RI placentas showed evidence of hypoxia and oxidative stress with expression of hypoxia-inducible factors 1α and 2α, heat shock protein 70, presence of nitrotyrosine residues, and lipid peroxidation. We observed no exaggerated placental hypoxia or oxidative stress associated with high RI pregnancies. High RI placental tissue demonstrated an altered balance of antioxidant enzyme activity. Hypoxia and oxidative stress appear to be a physiological state in early pregnancy; our data did not support the hypothesis that they are associated with deficient placentation in the first trimester. Higher levels of apoptosis, reduced insulin-like growth factor-2 expression, and altered antioxidant defenses may contribute to abnormal placentation and the later development of pregnancy complications, such as preeclampsia, fetal growth restriction, and stillbirth.

Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm.

Peroxisome proliferator-activated receptor x03B3; agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor x03B3; agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation.

Performance evaluation of automated urine microscopy as a rapid, non-invasive approach for the diagnosis of non-gonococcal urethritis.

OBJECTIVES: Gram-stained urethral smear (GSUS), the standard point-of-care test for non-gonococcal urethritis (NGU) is operator dependent and poorly specific. The performance of rapid automated urine flow cytometry (AUFC) of first void urine (FVU) white cell counts (UWCC) for predicting Mycoplasma genitalium and Chlamydia trachomatis urethral infections was assessed and its application to asymptomatic infection was evaluated. METHODS: Receiver operating characteristic curve analysis, determining FVU-UWCC threshold for predicting M. genitalium or C. trachomatis infection was performed on 208 'training' samples from symptomatic patients and subsequently validated using 228 additional FVUs obtained from prospective unselected patients. RESULTS: An optimal diagnostic threshold of >29 UWC/µL gave sensitivities and specificities for either infection of 81.5% (95% CI 65.1% to 91.6%) and 85.8% (79.5% to 90.4%), respectively, compared with 86.8% (71.1% to 95%) and 64.7% (56.9% to 71.7%), respectively, for GSUS, using the training set samples. FVU-UWCC demonstrated sensitivities and specificities of 69.2% (95% CI 48.1% to 84.9%) and 92% (87.2% to 95.2%), respectively, when using validation samples. In asymptomatic patients where GSUS was not used, AUFC would have enabled more infections to be detected compared with clinical considerations only (71.4% vs 28.6%; p=0.03). The correlation between UWCC and bacterial load was stronger for M. genitalium compared with C. trachomatis (τ=0.426, p≤0.001 vs τ=0.295, p=0.022, respectively). CONCLUSIONS: AUFC offers improved specificity over microscopy for predicting C. trachomatis or M. genitalium infection. Universal AUFC may enable non-invasive diagnosis of asymptomatic NGU at the PoC. The degree of urethral inflammation exhibits a stronger association with pathogen load for M. genitalium compared with C. trachomatis.

Trophoblast-induced changes in C-x-C motif chemokine 10 expression contribute to vascular smooth muscle cell dedifferentiation during spiral artery remodeling.

OBJECTIVE: During pregnancy, fetal trophoblast disrupt endothelial cell and vascular smooth muscle cell (VSMC) interactions in spiral arteries of the maternal decidua to enable increased nutritional and oxygen delivery to the fetus. Little is known regarding this transformation because of difficulties of studying human pregnancy in vivo. This study investigated how trophoblast-secreted factors affect the interactions of vascular cells and the differentiation status of VSMC during spiral arteries remodeling using 3-dimensional vascular spheroid coculture. METHODS AND RESULTS: Endothelial cell and VSMC were cocultured in hanging droplets to form spheroids representing an inverted vessel lumen. Control or conditioned media from an extravillous trophoblast (EVT) cell line was incubated with vascular spheroids for 24 hours. Spheroid RNA was then analyzed by Illumina Sentrix BeadChip array. Spheroids incubated with EVT conditioned medium showed significant up/downregulation of 101 genes (>1.5-fold; P<0.05), including an upregulation of C-X-C motif chemokine 10 (IP-10). C-X-C motif chemokine 10 expression was confirmed by qualitative real-time PCR and Western blot analysis of spheroids, and immunohistochemistry of first trimester decidua and ex vivo dissected nonplacental bed spiral arteries. EVT conditioned medium reduced VSMC expression of differentiation markers, and both EVT conditioned medium and C-X-C motif chemokine 10 increased motility of VSMC indicating dedifferentiation of VSMC. CONCLUSIONS: EVT-induced C-X-C motif chemokine 10 expression may contribute to spiral arteries remodeling during pregnancy by altering the motility and differentiation status of the VSMC in the vessel.

Elevation of plasma high-density lipoproteins inhibits development of experimental abdominal aortic aneurysms.

OBJECTIVE: Patients with abdominal aortic aneurysms have lower concentrations of high-density lipoproteins (HDLs), leading us to investigate whether increasing plasma HDLs could influence aneurysm formation. METHODS AND RESULTS: Using the angiotensin II-induced hypercholesterolemic and the CaCl(2)-induced normocholesterolemic mouse model of AAA, we investigated the hypothesis that elevation of HDLs inhibits AAA. HDLs elevated before or at the time of AAA induction reduced AAA formation in both models but had no effect on early ruptures. Analysis of protein lysates from specific aortic segments demonstrated site-specific effects of HDLs on early signal transduction and cellular attrition. We found that HDLs reduced extracellular signal related kinases 1/2 activation in the suprarenal segment, while having no effect on p38 mitogen-associated protein kinase activation in any aortic segment and inhibiting c-Jun N-terminal kinase activation in all aortic segments. In addition, HDL elevation inhibited angiotensin II-induced apoptosis while inducing autophagy in the suprarenal segment of the aorta. Using Illumina gene array profiling we investigated the ability of HDL to modulate basal suprarenal aortic gene expression. CONCLUSIONS: Increasing plasma HDLs inhibit experimental AAA formation, independent of hypercholesterolemia via reduced extracellular signal related kinases 1/2 activation and alteration of the balance of cellular attrition. HDLs modulate genes involved in matrix remodelling, cell migration, and proliferation.

Markers of epidemiological success of methicillin-resistant Staphylococcus aureus isolates in European populations.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) infections impose a considerable burden on health systems, yet there is remarkable variation in the global incidence and epidemiology of MRSA. The MACOTRA consortium aimed to identify bacterial markers of epidemic success of MRSA isolates in Europe using a representative MRSA collection originating from France, the Netherlands and the United Kingdom. METHODS: Operational definitions of success were defined in consortium meetings to compose a balanced strain collection of successful and sporadic MRSA isolates. Isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing; genes were identified and phylogenetic trees constructed. Markers of epidemiological success were identified using genome-based time-scaled haplotypic density analysis and linear regression. Antimicrobial usage data from ESAC-Net was compared with national MRSA incidence data. RESULTS: Heterogeneity of MRSA isolate collections across countries hampered the use of a unified operational definition of success; therefore, country-specific approaches were used to establish the MACOTRA strain collection. Phenotypic antimicrobial resistance varied within related MRSA populations and across countries. In time-scaled haplotypic density analysis, fluoroquinolone, macrolide and mupirocin resistance were associated with MRSA success, whereas gentamicin, rifampicin and trimethoprim resistance were associated with sporadicity. Usage of antimicrobials across 29 European countries varied substantially, and ß-lactam, fluoroquinolone, macrolide and aminoglycoside use correlated with MRSA incidence. DISCUSSION: Our results are the strongest yet to associate MRSA antibiotic resistance profiles and antibiotic usage with the incidence of infection and successful clonal spread, which varied by country. Harmonized isolate collection, typing, resistance profiling and alignment with antimicrobial usage over time will aid comparisons and further support country-specific interventions to reduce MRSA burden.

Early Colorectal Responses to HIV-1 and Modulation by Antiretroviral Drugs.

Innate responses during acute HIV infection correlate with disease progression and pathogenesis. However, limited information is available about the events occurring during the first hours of infection in the mucosal sites of transmission. With an ex vivo HIV-1 challenge model of human colorectal tissue we assessed the mucosal responses induced by R5- and X4-tropic HIV-1 isolates in the first 24 h of exposure. Microscopy studies demonstrated virus penetration of up to 39 µm into the lamina propia within 6 h of inoculation. A rapid, 6 h post-challenge, increase in the level of secretion of inflammatory cytokines, chemokines, interferon- γ (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed following exposure to R5- or X4-tropic isolates. This profile persisted at the later time point measured of 24 h. However, exposure to the X4-tropic isolate tested induced greater changes at the proteomic and transcriptomic levels than the R5-tropic. The X4-isolate induced greater levels of CCR5 ligands (RANTES, MIP-1α and MIP-1ß) secretion than R5-HIV-1. Potential drugs candidates for colorectal microbicides, including entry, fusion or reverse transcriptase inhibitors demonstrated differential capacity to modulate these responses. Our findings indicate that in colorectal tissue, inflammatory responses and a Th1 cytokine profile are induced in the first 24 h following viral exposure.

PCR for the detection of pathogens in neonatal early onset sepsis.

BACKGROUND: A large proportion of neonates are treated for presumed bacterial sepsis with broad spectrum antibiotics even though their blood cultures subsequently show no growth. This study aimed to investigate PCR-based methods to identify pathogens not detected by conventional culture. METHODS: Whole blood samples of 208 neonates with suspected early onset sepsis were tested using a panel of multiplexed bacterial PCRs targeting Streptococcus pneumoniae, Streptococcus agalactiae (GBS), Staphylococcus aureus, Streptococcus pyogenes (GAS), Enterobacteriaceae, Enterococcus faecalis, Enterococcus faecium, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium, a 16S rRNA gene broad-range PCR and a multiplexed PCR for Candida spp. RESULTS: Two-hundred and eight samples were processed. In five of those samples, organisms were detected by conventional culture; all of those were also identified by PCR. PCR detected bacteria in 91 (45%) of the 203 samples that did not show bacterial growth in culture. S. aureus, Enterobacteriaceae and S. pneumoniae were the most frequently detected pathogens. A higher bacterial load detected by PCR was correlated positively with the number of clinical signs at presentation. CONCLUSION: Real-time PCR has the potential to be a valuable additional tool for the diagnosis of neonatal sepsis.

Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies.

BACKGROUND: The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers both linked to T7 polymerase in vitro run off transcription. RESULTS: The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5 ng total M. tuberculosis RNA with unamplified RNA from the same source. In addition, as a direct measure of the effectiveness of bacterial amplification for identifying biologically relevant changes in gene expression, a model M. tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5 ng total RNA (~equivalent to 2 x 105 bacilli). Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles using both methods. CONCLUSION: Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling.

Clearance of viable Mycobacterium ulcerans from Buruli ulcer lesions during antibiotic treatment as determined by combined 16S rRNA reverse transcriptase /IS 2404 qPCR assay.

INTRODUCTION: Buruli ulcer (BU) caused by Mycobacterium ulcerans is effectively treated with rifampicin and streptomycin for 8 weeks but some lesions take several months to heal. We have shown previously that some slowly healing lesions contain mycolactone suggesting continuing infection after antibiotic therapy. Now we have determined how rapidly combined M. ulcerans 16S rRNA reverse transcriptase / IS2404 qPCR assay (16S rRNA) became negative during antibiotic treatment and investigated its influence on healing. METHODS: Fine needle aspirates and swab samples were obtained for culture, acid fast bacilli (AFB) and detection of M. ulcerans 16S rRNA and IS2404 by qPCR (16S rRNA) from patients with IS2404 PCR confirmed BU at baseline, during antibiotic and after treatment. Patients were followed up at 2 weekly intervals to determine the rate of healing. The Kaplan-Meier survival analysis was used to analyse the time to clearance of M. ulcerans 16S rRNA and the influence of persistent M ulcerans 16S rRNA on time to healing. The Mann Whitney test was used to compare the bacillary load at baseline in patients with or without viable organisms at week 4, and to analyse rate of healing at week 4 in relation to detection of viable organisms. RESULTS: Out of 129 patients, 16S rRNA was detected in 65% of lesions at baseline. The M. ulcerans 16S rRNA remained positive in 78% of patients with unhealed lesions at 4 weeks, 52% at 8 weeks, 23% at 12 weeks and 10% at week 16. The median time to clearance of M. ulcerans 16S rRNA was 12 weeks. BU lesions with positive 16S rRNA after antibiotic treatment had significantly higher bacterial load at baseline, longer healing time and lower healing rate at week 4 compared with those in which 16S rRNA was not detected at baseline or had become undetectable by week 4. CONCLUSIONS: Current antibiotic therapy for BU is highly successful in most patients but it may be possible to abbreviate treatment to 4 weeks in patients with a low initial bacterial load. On the other hand persistent infection contributes to slow healing in patients with a high bacterial load at baseline, some of whom may need antibiotic treatment extended beyond 8 weeks. Bacterial load was estimated from a single sample taken at baseline. A better estimate could be made by taking multiple samples or biopsies but this was not ethically acceptable.

The use of microarray analysis to determine the gene expression profiles of Mycobacterium tuberculosis in response to anti-bacterial compounds.

The response of Mycobacterium tuberculosis to six anti-microbial agents was determined by microarray analysis in an attempt to define mechanisms of innate resistance in M. tuberculosis. The gene expression profiles of M. tuberculosis after treatment at the minimal inhibitory concentration (MIC) for 4 h with isoniazid, isoxyl, tetrahydrolipstatin, SRI#221, SR1#967 and SR1#9190 were compared to untreated M. tuberculosis. A common response to drug exposure was defined, and this expression profile overlapped with a number of other mycobacterial stress responses recently identified by microarray analysis. Compound-specific responses were also distinguished including a number of putative transcriptional regulators and translocation-related genes. These genes may contribute to the intrinsic resistance of M. tuberculosis to anti-microbial compounds. Further investigation into these mechanisms may elucidate novel pathways contributing to mycobacterial drug resistance and influence anti-mycobacterial drug development strategies.

Stationary phase gene expression of Mycobacterium tuberculosis following a progressive nutrient depletion: a model for persistent organisms?

The majority of individuals infected with TB develop a latent infection, in which organisms survive within the body while evading the host immune system. Such persistent bacilli are capable of surviving several months of combinatorial antibiotic treatment. Evidence suggests that stationary phase bacteria adapt to increase their tolerance to environmental stresses. We have developed a unique in vitro model of dormancy based on the characterization of a single, large volume fermenter culture of M. tuberculosis, as it adapts to stationary phase. Cells are maintained in controlled and defined aerobic conditions (50% dissolved oxygen tension), using probes that measure dissolved oxygen tension, temperature, and pH. Microarray analysis has been used in conjunction with viability and nutrient depletion assays to dissect differential gene expression. Following exponential phase growth the gradual depletion of glucose/glycerol resulted in a small population of survivors that were characterized for periods in excess of 100 days. Bacilli adapting to nutrient depletion displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the up-regulation of genes involved in beta-oxidation of fatty acids and virulence. A reduced population of non-replicating bacilli went on to adapt sufficiently to re-initiate cellular division.

Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways.

Anosmin-1, encoded by the KAL1 gene, is an extracellular matrix (ECM)-associated protein which plays essential roles in the establishment of olfactory and GNRH neurons during early brain development. Loss-of-function mutations of KAL1 results in Kallmann syndrome with delayed puberty and anosmia. There is, however, little comprehension of its role in the developed brain. As reactivation of developmental signal pathways often takes part in tumorigenesis, we investigated if anosmin-1-mediated cellular mechanisms associated with brain tumors. Our meta-analysis of gene expression profiles of patients' samples and public microarray datasets indicated that KAL1 mRNA was significantly upregulated in high-grade primary brain tumors compared with the normal brain and low-grade tumors. The tumor-promoting capacity of anosmin-1 was demonstrated in the glioblastoma cell lines, where anosmin-1 enhanced cell motility and proliferation. Notably, anosmin-1 formed a part of active ß1 integrin complex, inducing downstream signaling pathways. ShRNA-mediated knockdown of anosmin-1 attenuated motility and growth of tumor cells and induced apoptosis. Anosmin-1 may also enhance the invasion of tumor cells within the ECM by modulating cell adhesion and activating extracellular proteases. In a mouse xenograft model, anosmin-1-expressing tumors grew faster, indicating the role of anosmin-1 in tumor microenvironment in vivo. Combined, these data suggest that anosmin-1 can facilitate tumor cell proliferation, migration, invasion, and survival. Therefore, although the normal function of anosmin-1 is required in the proper development of GNRH neurons, overexpression of anosmin-1 in the developed brain may be an underlying mechanism for some brain tumors.

Characterisation of bovine leukocyte Ig-like receptors.

Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.

Examining the basis of isoniazid tolerance in nonreplicating Mycobacterium tuberculosis using transcriptional profiling.

BACKGROUND: Understanding how growth state influences Mycobacterium tuberculosis responses to antibiotic exposure provides a window into drug action during patient chemotherapy. In this article, we describe the transcriptional programs mediated by isoniazid (INH) during the transition from log-phase to nonreplicating bacilli, from INH-sensitive to INH-tolerant bacilli respectively, using the Wayne model. RESULTS: INH treatment did not elicit a transcriptional response from nonreplicating bacteria under microarophilic conditions (NRP2), unlike the induction of a robust and well-characterized INH signature in log-phase bacilli. CONCLUSION: The differential regulation (between drug-free NRP2 and log-phase bacilli) of genes directly implicated in INH resistance could not account for the abrogation of INH killing in nongrowing bacilli. Thus, factors affecting the requirement for mycolic acids and the redox status of bacilli are likely responsible for the reduction in INH efficacy. We speculate on additional mechanisms revealed by transcriptome analysis that might account for INH tolerance.

Immunomodulation by herpesvirus U51A chemokine receptor via CCL5 and FOG-2 down-regulation plus XCR1 and CCR7 mimicry in human leukocytes.

Human herpesvirus-6A (HHV-6A) betachemokine-receptor U51A binds inflammatory modulators CCL2, CCL5, CCL11, CCL7, and CCL13. This unique specificity overlaps that of human chemokine receptors CCR1, CCR2, CCR3, and CCR5. In model cell lines, expression leads to CCL5 down-regulation with both constitutive and inducible signaling. Here, immunomodulation pathways are investigated in human leukocytes permissive for infection. Constitutive signaling was shown using inositol phosphate assays and inducible calcium signaling by response to CCL2, CCL5 and CCL11. Constitutive signaling targets were examined using an immune response-related microarray and RT-PCR, showing down-regulation of CCL5 and FOG-2, a hematopoietic transcriptional repressor. By RT-PCR and siRNA reversion, CCL5 and FOG-2 were shown down-regulated, during peak U51A expression post infection. Two further active ligands, XCL1 and CCL19, were identified, making U51A competitor to their human receptors, XCR1 and CCR7, on T lymphocytes, NK and dendritic cells. Finally, U51A-expressing cell lines and infected ex vivo leukocytes, showed migration towards chemokine-gradients, and chemokine internalization. Consequently, U51A may affect virus dissemination or host transmission by chemotaxis of infected cells to sites of chemokine secretion specific for U51A (for example the lymph node or lung, by CCL19 or CCL11, respectively) and evade immune-effector cells by chemokine diversion and down-regulation, affecting virus spread and inflammatory pathology.