Enhanced self-renewal of human pluripotent stem cells by simulated microgravity.

A systematic study on the biological effects of simulated microgravity (sµg) on human pluripotent stem cells (hPSC) is still lacking. Here, we used a fast-rotating 2-D clinostat to investigate the sµg effect on proliferation, self-renewal, and cell cycle regulation of hPSCs. We observed significant upregulation of protein translation of pluripotent transcription factors in hPSC cultured in sµg compared to cells cultured in 1g conditions. In addition to a significant increase in expression of telomere elongation genes. Differentiation experiments showed that hPSC cultured in sµg condition were less susceptible to differentiation compared to cells in 1g conditions. These results suggest that sµg enhances hPSC self-renewal. Our study revealed that sµg enhanced the cell proliferation of hPSCs by regulating the expression of cell cycle-associated kinases. RNA-seq analysis indicated that in sµg condition the expression of differentiation and development pathways are downregulated, while multiple components of the ubiquitin proteasome system are upregulated, contributing to an enhanced self-renewal of hPSCs. These effects of sµg were not replicated in human fibroblasts. Taken together, our results highlight pathways and mechanisms in hPSCs vulnerable to microgravity that imposes significant impacts on human health and performance, physiology, and cellular and molecular processes.

Simulated Leaching of Foliar Applied Copper Bactericides on the Soil Microbiome Utilizing Various Beta Diversity Resemblance Measurements.

Copper bactericides are routinely used to control Xanthomonas perforans (XP), causal agent of bacterial spot of tomato. Given the widespread tolerance to copper in XP strains in FL, USA, nanotechnology-based elemental composites have gained interest for their potential applications in agriculture in part due to their enhanced antimicrobial properties and toxicity to copper-tolerant strains. However, little is known about the potential impact of conventional copper bactericides as well as nano-based elemental composites on soil microbial communities, as determined by high-throughput sequencing of the 16S rDNA. We compared the effects of 2 and 200 µg/mL of core-shell (CS), a metallic copper composite, and a conventional copper bactericide + mancozeb (Cu+Man) on the soil microbiome. These treatments were compared to three controls, the microbial profile of the soil prior to application of copper products, a water application, and spiking the soil with a soilborne phytobacterium, Ralstonia solanacearum (RS). The RS treatment was included to determine if downstream analysis could detect the artificial inoculation. Utilizing multiple ß diversity measurements, each emphasizing various tenets of ecology, provided a greater perspective of the effects the treatments had on the microbiome. Analysis of HTS data revealed that the two treatments containing field applied rates of metallic copper, CS 200 and Cu+Man, had the largest impact on the soil microbiome at seven-days posttreatment compared to water. However, we simulated field applied rates of CS 200 entering the soil by treating soil with CS 2 and determined this concentration had a negligible effect on the soil microbiome. IMPORTANCE Nanotechnology-based elemental composites have gained popularity for their potential applications in plant disease management due to their enhanced antimicrobial properties. However, little is known about their potential impact on the environment. Foliar applications of nano metallic composites upon leaching into the soil have the potential to impact soil microbial populations that in turn influence soil health. Utilizing multiple ß diversity measurements, high-throughput sequencing analysis revealed that field applied rates of metallic copper (200 µg/mL) from an advanced copper composite (core-shell [CS]) and a conventional copper bactericide in combination with mancozeb had the largest impact on the soil microbiome compared to water and nontreated control. To simulate leaching from the leaf surface, a lower concentration (2 µg/mL) of CS was also applied to the soil and had a negligible effect on the soil microbiome. Thus, field applied rates of CS may have a minimal effect on soil microbial communities.

Independent Evolution with the Gene Flux Originating from Multiple Xanthomonas Species Explains Genomic Heterogeneity in Xanthomonas perforans.

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria for that of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of 2 decades, including a shift in race and diversification into three phylogenetic groups driven by genome-wide homologous-recombination events derived from X. euvesicatoria In our sampling of Xanthomonas strains associated with bacterial spot disease in Alabama, we were readily able to isolate X. perforans from symptomatic pepper plants grown in several Alabama counties, indicating a recent shift in the host range of the pathogen. To investigate the diversity of these pepper-pathogenic strains and their relation to populations associated with tomatoes grown in the southeast United States, we sequenced the genomes of eight X. perforans strains isolated from tomatoes and peppers grown in Alabama and compared them with previously published genome data available from GenBank. Surprisingly, reconstruction of the X. perforans core genome revealed the presence of two novel genetic groups in Alabama that each harbored a different transcription activation-like effector (TALE). While one TALE, AvrHah1, was associated with an emergent lineage pathogenic to both tomato and pepper, the other was identified as a new class within the AvrBs3 family, here designated PthXp1, and was associated with enhanced symptom development on tomato. Examination of patterns of homologous recombination across the larger X. euvesicatoria species complex revealed a dynamic pattern of gene flow, with multiple donors of Xanthomonas spp. associated with diverse hosts of isolation.IMPORTANCE Bacterial leaf spot of tomato and pepper is an endemic plant disease with a global distribution. In this study, we investigated the evolutionary processes leading to the emergence of novel X. perforans lineages identified in Alabama. While one lineage was isolated from symptomatic tomato and pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, here designated PthXp1. Functional analysis of PthXp1 indicated that it does not induce Bs4-mediated resistance in tomato and contributes to virulence, providing an adaptive advantage to strains on tomato. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts that results in the emergence of novel pathogen lineages and associated phenotypes, including host range.

Reclassification of Xanthomonas gardneri (ex Sutic 1957) Jones et al. 2006 as a later heterotypic synonym of Xanthomonas cynarae Trébaol et al. 2000 and description of X. cynarae pv. cynarae and X. cynarae pv. gardneri based on whole genome analyses.

Multilocus sequence analysis of Xanthomonas species revealed a very close relationship between Xanthomonas cynarae, an artichoke pathogen and Xanthomonas gardneri, a tomato and pepper pathogen. Results of whole genome sequence comparisons using average nucleotide identity between representative strains of X. gardneri and X. cynarae were well above the threshold of 95-96 %. Inoculations of X. gardneri strains in artichoke leaves caused mild disease symptoms, but only weak symptoms were observed in the bracts. Both X. cynarae and X. gardneri grew equally and caused typical bacterial spot symptoms in pepper after artificial inoculation. However, X. cynarae induced a hypersensitive reaction in tomato, while X. gardneri strains were virulent. Pathogenicity-associated gene clusters, including the protein secretion systems, type III effector profiles, and lipopolysaccharide cluster were nearly identical between the two species. Based on our results from whole genome sequence comparison, X. gardneri and X. cynarae belong to the same species. The name X. cynarae has priority and X. gardneri should be considered as a later heterotypic synonym. An emended description of X. cynarae (type strain=CFBP 4188T, =DSM 16794T) is given. However, due to the host specificity in artichoke and tomato, two pathovars, X. cynarae pv. cynarae and X. cynarae pv. gardneri, are proposed.

Design and Rationale for the Veterans Affairs "Cooperative Study Program 594 Comparative Effectiveness in Gout: Allopurinol vs. Febuxostat" Trial.

BACKGROUND: Gout patients do not routinely achieve optimal outcomes related in part to suboptimal administration of urate lowering therapy (ULT) including first-line xanthine oxidase inhibitors allopurinol or febuxostat. Studies leading to the approval of febuxostat compared this agent to allopurinol in inappropriately low, fixed doses. We will compare allopurinol with febuxostat in gout using appropriately titrated doses of both agents and a "treat-to-target" strategy congruent with specialty guidelines. METHODS: We have planned and initiated the Veterans Affairs (VA) Cooperative Study Program (CSP) 594, Comparative Effectiveness in Gout: Allopurinol vs Febuxostat study. This large double-blind, non-inferiority trial will enroll 950 gout patients randomized to receive allopurinol or febuxostat. Patients will be followed for a total of 72 weeks encompassing 3 distinct 24-week study phases. During Phase I (0-24 weeks), participants will undergo gradual dose titration of ULT until achievement of serum uric acid (sUA) <6.0 mg/dL or <5.0 mg/dL if tophi are present. Dose escalation will not be allowed during final three study visits of Phase 2 (24-48 weeks) and during Phase 3 (48-72 weeks). The primary study outcome is the proportion of participants experiencing at least one gout flare during Phase 3. Subsequent to the 72-week study, participants will be followed passively for up to 10 years after the study to assess long-term health outcomes. CONCLUSION: With its completion, the VA Comparative Effectiveness in Gout: Allopurinol vs Febuxostat study will demonstrate the central role of gradual ULT dose escalation and a treat-to-target strategy in gout management.

A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato.

Bacterial spot of tomato, a major problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hrpB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB7 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB7 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB7 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.

Multilocus Sequence Analysis Reveals Genetic Diversity in Xanthomonads Associated With Poinsettia Production.

Xanthomonas axonopodis pv. poinsettiicola is traditionally identified as the primary causal agent of bacterial leaf spot on poinsettia (family Euphorbiaceae). Sixty-seven strains of xanthomonads isolated from lesions associated with several species within the family Euphorbiaceae were collected over a 64-year period. The pathogenicity of these strains was compared on several potential hosts and they were analyzed by multilocus sequence analysis (MLSA) using six housekeeping genes. The 67 Xanthomonas strains associated with poinsettia production were separated into three distinct clades based on MLSA. The first clade identified contained the X. axonopodis pv. poinsettiicola reference strain (LMG849PT). A second clade was more closely related to X. hortorum pv. pelargonii (LMG7314PT) and the third clade contained the X. codiaei type strain (LMG8678T). This analysis indicated that there may also be other closely related pathovars or species of Xanthomonas that can infect poinsettia. Strains from the three clades could not be distinguished by symptoms or virulence on poinsettia plants. Strains capable of infecting geranium were found in all three clades, although the extent of leaf spot formation and number of systemic infections were significantly less than those produced by X. hortorum pv. pelargonii strains, typically the main causal agent of bacterial leaf spot on geranium. Clade III also contained strains isolated from zebra plant (Aphelandra squarrosa, family Acanthaceae), which is a newly recognized host for X. codiaei and X. axonopodis pv. poinsettiicola. Xanthomonas leaf spot is a serious threat to poinsettia production that can be caused by several Xanthomonas spp. that can infect different ornamental plant hosts. It is imperative that growers maintain a strict sanitation program because reservoirs of inoculum can occur on a number of ornamental hosts.

First Report of Atypical Xanthomonas euvesicatoria Strains Causing Bacterial Spot of Tomato in Nigeria.

Bacterial spot (BS) is an important disease of tomato in Nigeria (2). Although a xanthomonad was isolated from tomato in Nigeria and characterized using phenotypic and pathogenicity tests, the bacterium was not characterized genetically to confirm the species. To determine the species associated with BS, leaves were collected in fields in northwestern Nigeria from tomato plants showing typical BS symptoms, which consisted of dark, irregular-shaped brown leaf spots that coalesced, resulting in a blighted appearance. Isolations from individual lesions were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated from 14 lesions and all were determined to be amylolytic (3). To determine the races of these strains, bacterial suspensions of the tomato strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The tomato strains elicited hypersensitive reactions (HRs) on the four pepper differential lines and an HR on the tomato genotype FL 216, which contains the R gene Xv3, but elicited susceptible reactions on the tomato genotypes Hawaii 7998 and Bonny Best. These reactions are typical of X. perforans tomato race 3 strains (5). Multilocus sequence analysis (MLSA) of six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze four representative strains (1) (GenBank Accession Nos. KJ938581 to KJ938584, KJ938588 to KJ938591, KJ938595 to KJ938598, KJ938602 to KJ938605, KJ938629 to KJ938632, and KJ938636 to KJ938639, respectively). A partial sequence of hrpB2 was also made since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence of this gene (3) (KJ938609 to KJ938621 and KJ938628). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the National Center for Biotechnology Information (NCBI) database ( www.ncbi.nlm.nih.gov ) using the MUSCLE tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the strains grouped most closely with the X. euvesicatoria reference strain 85-10 but more distantly to X. perforans. The hrpB2 sequence, which is highly conserved for each Xanthomonas species pathogenic on tomato (4), was sequenced from the tomato strains. These sequences were identical to the hrpB2 sequence from X. perforans strains but different from X. euvesicatoria. Although BS is common in Nigeria, to our knowledge, this represents a unique group of X. euvesicatoria strains from tomato that are identical to X. perforans based on pathogenic reactions on tomato and pepper and hrpB2 sequence identity but are more closely related to X. euvesicatoria based on the six housekeeping gene sequences. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) E. U. Opara and F. J. Odibo. J. Mol. Genet. 1:35, 2009. (3) J. B. Jones et al. Syst. Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (5) R. E. Stall et al. Annu. Rev. Phytopathol. 47:265, 2009.

First Report of Xanthomonas euvesicatoria Causing Bacterial Spot Disease in Pepper in Northwestern Nigeria.

Bacterial spot (BS) has been reported as an important disease on pepper in Nigeria (4). Xanthomonas campestris pv. vesicatoria was identified as the causal agent using phenotypic and pathogenicity tests; however, X. campestris pv. vesicatoria is a synonym for two genetically distinct groups that have been elevated to the species X. euvesicatoria and X. vesicatoria (2). Furthermore, the latter two species and X. gardneri cause similar diseases on pepper (2). In order to determine the species associated with BS on pepper, leaves with irregular, dark brown lesions were collected from pepper plants in fields from northwestern Nigeria, and isolations were made on nutrient agar (NA). Yellow, mucoid colonies typical of Xanthomonas were isolated. Six strains isolated from pepper were determined to be non-amylolytic. For race determinations, bacterial suspensions of the pepper strains, derived from 24-h cultures grown on NA at 28°C, were adjusted to 108 CFU/ml and infiltrated into leaves of tomato and pepper differential genotypes (5). The six pepper strains elicited HRs on the tomato differential genotypes. The strains produced a susceptible reaction on all pepper differentials and were designated as pepper race 6 (5). Multilocus sequence analysis (MLSA) using six housekeeping genes (fusA, lacF, gyrB, gltA, gapA, and lepA) was used to further analyze the strains (1) (GenBank Accession Nos. KJ938585 to KJ938587, KJ938592 to KJ938594, KJ938599 to KJ938601, KJ938606 to KJ938608, KJ938633 to KJ938635, and KJ938640 to KJ938642). A partial sequence of hrpB2 was also sequenced since the four Xanthomonas species associated with BS can be differentiated based on sequence divergence (3) (KJ938622 to KJ938627). The housekeeping gene sequences were aligned along with other Xanthomonas sequences imported from the NCBI database using muscle tool from MEGA software, 5.2.2. Maximum likelihood phylogenetic trees constructed for the six housekeeping gene sequences individually and in concatenation revealed that the Nigerian pepper strains were identical to the X. euvesicatoria reference strain 85-10. Although BS is common in Nigeria, to our knowledge, this represents the first report for this pepper pathogen in Nigeria. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (3) J. B. Jones et al. System Appl. Microbiol. 27:755, 2004. (4) A. Obradovic et al. Eur. J. Plant Pathol. 88:736, 2004. (2) E. U. Opara and F. J. Odibo. J. Mol. Gen. 1:35, 2009. (5) R. E. Stall et al. Ann. Rev. Phytopathol. 47:265, 2009.

Tobacco and alcohol: the relation to pulmonary tuberculosis in household contacts.

Tuberculosis is transmitted commonly by droplet nuclei and facilitated by weak immune system. Lowered immunity may be associated with cigarette smoking, tobacco chewing and alcohol consumption. The co-relationship between these all factors to TB should be explored. This study aims to detect the hidden household contacts (HC) cases early and to examine the relative contribution of tobacco and alcohol use to the risk of TB. Across-sectional study was in Dharan among HCs. From June 2009 to May 2010, 184 index cases with sputum smear positive for AFB and their 802 HCs were included. Three sputum specimens were collected from each HCs and examined microscopically for AFB detection. AFB were detected in sputum of 13 (1.6%) HCs. The association between habits (alcohol user and smoking) and TB was found except with chewing tobacco user (P > 0.05). The risk of contact TB was 4 and 8 times greater in smoker (OR = 3.94 95% CI = 1.26-12.26, P < 0.05) and alcoholic (OR = 8.23 95% CI = 2.71-24.98, P < 0.05) HCs respectively. This study has revealed smoking and alcohols as the risk factors for tuberculosis. Effective campaign to discourage use of alcohol and tobacco, and awareness programme about the mode of transmission of TB are needed in community.

Mucositis does not lead to false-positivity of the Platelia Aspergillus enzyme-linked immunosorbent assay.

We are reporting a study evaluating the crossover of antigens reacting in Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) from faeces to vessels during mucositis as a possible cause of false-positivity of this test. In our series of 102 episodes of different grades of mucositis, we found strong reactivity of faeces in the PA ELISA test irrespective of the grade of mucositis, the percentage of oral food intake or the presence of total parenteral nutrition. However, none of the patients included in the study were positive in the serum (when the criterion of two samples with cut-off index of positivity [IP] > 0.5 was used).

Antibiotic susceptibility of group A Streptococcus isolated from throat swab culture of school children in Pokhara, Nepal.

Group A Streptococcus (GAS) or Streptococcus pyogenes is estimated to be present in 5.0-15.0% of norma individual in the respiratory tract, vagina, skin and anus without any sign of disease. This study was carried out to find out the rate of asymptomatic throat carriage of S. pyogenes and antibiotic susceptibility of the isolates in school children of Pokhara, Western Nepal. A total of 487 randomly selected children younger than 16 years were included in the study. Throat swabs collected were subjected to 5.0% Sheep blood agar supplemented with crystal violet (CVBA).GAS was identified by a-haemolytic colonies, bacitracin sensitivity, cotrimoxazole resistivity, catalase negativity and PYR positivity. Antibiotic susceptibility test was performed on Muller Hinton agar (MHA) containing 5% sheep blood by modified Kirby-Bauer disc diffusion method. Out of total 487 throat swabs, GAS was isolated in 9.2% (n = 45). Among the isolates, 46.6% (n = 21) were from male children where as 53.4% (n = 24) from female children. There was no significant sex difference in colonization of GAS (p > 0.05). Out of 45 isolates, 100.0% isolates were sensitive to antibiotic penicillin-G and amoxycillin where as 15.6%, 6.6%, and 2.2% isolates were resistant to antibiotic erythromycin, tetracycline and azithromycin respectively.