Multi-omics analysis to characterize molecular adaptation of Entamoeba histolytica during serum stress.

Entamoeba histolytica is responsible for dysentery and extraintestinal disease in humans. To establish successful infection, it must generate adaptive response against stress due to host defense mechanisms. We have developed a robust proteomics workflow by combining miniaturized sample preparation, low flow-rate chromatography, and ultra-high sensitivity mass spectrometry, achieving increased proteome coverage, and further integrated proteomics and RNA-seq data to decipher regulation at translational and transcriptional levels. Label-free quantitative proteomics led to identification of 2344 proteins, an improvement over the maximum number identified in E. histolytica proteomic studies. In serum-starved cells, 127 proteins were differentially abundant and were associated with functions including antioxidant activity, cytoskeleton, translation, catalysis, and transport. The virulence factor, Gal/GalNAc-inhibitable lectin subunits, was significantly altered. Integration of transcriptomic and proteomic data revealed that only 30% genes were coordinately regulated at both transcriptional and translational levels. Some highly expressed transcripts did not change in protein abundance. Conversely, genes with no transcriptional change showed enhanced protein abundance, indicating post-transcriptional regulation. This multi-omics approach enables more refined gene expression analysis to understand the adaptive response of E. histolytica during growth stress.

Trends in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Seroprevalence in Massachusetts Estimated from Newborn Screening Specimens.

BACKGROUND: Estimating the cumulative incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for setting public health policies. We leveraged deidentified Massachusetts newborn screening specimens as an accessible, retrospective source of maternal antibodies for estimating statewide seroprevalence in a nontest-seeking population. METHODS: We analyzed 72 117 newborn specimens collected from November 2019 through December 2020, representing 337 towns and cities across Massachusetts. Seroprevalence was estimated for the Massachusetts population after correcting for imperfect test specificity and nonrepresentative sampling using Bayesian multilevel regression and poststratification. RESULTS: Statewide seroprevalence was estimated to be 0.03% (90% credible interval [CI], 0.00-0.11) in November 2019 and rose to 1.47% (90% CI: 1.00-2.13) by May 2020, following sustained SARS-CoV-2 transmission in the spring. Seroprevalence plateaued from May onward, reaching 2.15% (90% CI: 1.56-2.98) in December 2020. Seroprevalence varied substantially by community and was particularly associated with community percent non-Hispanic Black (ß = .024; 90% CI: 0.004-0.044); i.e., a 10% increase in community percent non-Hispanic Black was associated with 27% higher odds of seropositivity. Seroprevalence estimates had good concordance with reported case counts and wastewater surveillance for most of 2020, prior to the resurgence of transmission in winter. CONCLUSIONS: Cumulative incidence of SARS-CoV-2 protective antibody in Massachusetts was low as of December 2020, indicating that a substantial fraction of the population was still susceptible. Maternal seroprevalence data from newborn screening can inform longitudinal trends and identify cities and towns at highest risk, particularly in settings where widespread diagnostic testing is unavailable.

The non-LTR retrotransposons of Entamoeba histolytica: genomic organization and biology.

Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA transposons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8 kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Retrotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8 kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.

Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica.

Ribosome biogenesis, a multi-step process involving transcription, modification, folding and processing of rRNA, is the major consumer of cellular energy. It involves sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. Northern hybridization from different spacer regions depicted the accumulation of unprocessed intermediates during stress. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified ribosome biogenesis factors in E. histolytica. Of the ∼279 Saccharomyces cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had a single copy in E. histolytica. Interestingly E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components compared to S. cerevisiae. Transcriptomic studies revealed the differential regulation of ribosomal factors both in serum starved and RRP6 down-regulation conditions. These included the NEP1 and TSR3 proteins that chemically modify 18S-rRNA. Pre-rRNA precursors accumulate upon downregulation of the latter proteins in S. cerevisiae and humans. These data reveal the major factors that regulate pre-rRNA processing during stress in E. histolytica and provide the first complete repertoire of ribosome biogenesis factors in this early-branching protist.

Transcriptomic analysis of Entamoeba histolytica reveals domain-specific sense strand expression of LINE-encoded ORFs with massive antisense expression of RT domain.

LINEs are retrotransposable elements found in diverse organisms. Their activity is kept in check by several mechanisms, including transcriptional silencing. Here we have analyzed the transcription status of LINE1 copies in the early-branching parasitic protist Entamoeba histolytica. Full-length EhLINE1 encodes ORF1, and ORF2 with reverse transcriptase (RT) and endonuclease (EN) domains. RNA-Seq analysis of EhLINE1 copies (both truncated and full-length) showed unique features. Firstly, although 20/41 transcribed copies were full-length, we failed to detect any full-length transcripts. Rather, sense-strand transcripts mapped to the functional domains- ORF1, RT and EN. Secondly, there was strong antisense transcription specifically from RT domain. No antisense transcripts were seen from ORF1. Antisense RT transcripts did not encode known functional peptides. They could possibly be involved in attenuating translation of RT domain, as we failed to detect ORF2p, whereas ORF1p was detectable. Lack of full-length transcripts and strong antisense RT expression may serve to limit EhLINE1 retrotransposition.

Transcriptomic analysis reveals novel downstream regulatory motifs and highly transcribed virulence factor genes of Entamoeba histolytica.

BACKGROUND: Promoter motifs in Entamoeba histolytica were earlier analysed using microarray data with lower dynamic range of gene expression. Additionally, previous transcriptomic studies did not provide information on the nature of highly transcribed genes, and downstream promoter motifs important for gene expression. To address these issues we generated RNA-Seq data and identified the high and low expressing genes, especially with respect to virulence potential. We analysed sequences both upstream and downstream of start site for important motifs. RESULTS: We used RNA-Seq data to classify genes according to expression levels, which ranged six orders of magnitude. Data were validated by reporter gene expression. Virulence-related genes (except AIG1) were amongst the highly expressed, while some kinases and BspA family genes were poorly expressed. We looked for conserved motifs in sequences upstream and downstream of the initiation codon. Following enrichment by AME we found seven motifs significantly enriched in high expression- and three in low expression-classes. Two of these motifs (M4 and M6) were located downstream of AUG, were exclusively enriched in high expression class, and were mostly found in ribosomal protein, and translation-related genes. Motif deletion resulted in drastic down regulation of reporter gene expression, showing functional relevance. Distribution of core promoter motifs (TATA, GAAC, and Inr) in all genes revealed that genes with downstream motifs were not preferentially associated with TATA-less promoters. We looked at gene expression changes in cells subjected to growth stress by serum starvation, and experimentally validated the data. Genes showing maximum up regulation belonged to the low or medium expression class, and included genes in signalling pathways, lipid metabolism, DNA repair, Myb transcription factors, BspA, and heat shock. Genes showing maximum down regulation belonged to the high or medium expression class. They included genes for signalling factors, actin, Ariel family, and ribosome biogenesis factors. CONCLUSION: Our analysis has added important new information about the E. histolytica transcriptome. We report for the first time two downstream motifs required for gene expression, which could be used for over expression of E. histolytica genes. Most of the virulence-related genes in this parasite are highly expressed in culture.

Regioselectivity of Hydroxyl Radical Reactions with Arenes in Nonaqueous Solutions.

The regioselectivity of hydroxyl radical addition to arenes was studied using a novel analytical method capable of trapping radicals formed after the first elementary step of reaction, without alteration of the product distributions by secondary oxidation processes. Product analyses of these reactions indicate a preference for o- over p-substitution for electron donating groups, with both favored over m-addition. The observed distributions are qualitatively similar to those observed for the addition of other carbon-centered radicals, although the magnitude of the regioselectivity observed is greater for hydroxyl. The data, reproduced by high accuracy CBS-QB3 computational methods, indicate that both polar and radical stabilization effects play a role in the observed regioselectivities. The application and potential limitations of the analytical method used are discussed.

Fluoroquinolone Resistance Mutation Detection Is Equivalent to Culture-Based Drug Sensitivity Testing for Predicting Multidrug-Resistant Tuberculosis Treatment Outcome: A Retrospective Cohort Study.

BACKGROUND: Molecular diagnostics that rapidly and accurately predict fluoroquinolone (FQ) resistance promise to improve treatment outcomes for individuals with multidrug-resistant (MDR) tuberculosis (TB). Mutations in the gyr genes, though, can cause variable levels of in vitro FQ resistance, and some in vitro resistance remains unexplained by gyr mutations alone, but the implications of these discrepancies for treatment outcome are unknown. METHODS: We performed a retrospective cohort study of 172 subjects with MDR/extensively drug-resistant TB subjects and sequenced the full gyrA and gyrB open reading frames in their respective sputum TB isolates. The gyr mutations were classified into 2 categories: a set of mutations that encode high-level FQ resistance and a second set that encodes intermediate resistance levels. We constructed a Cox proportional model to assess the effect of the gyr mutation type on the time to death or treatment failure and compared this with in vitro FQ resistance, controlling for host and treatment factors. RESULTS: Controlling for other host and treatment factors and compared with patients with isolates without gyr resistance mutations, "high-level" gyr mutations significantly predict poor treatment outcomes with a hazard ratio of 2.6 (1.2-5.6). We observed a hazard of death and treatment failure with "intermediate-level" gyr mutations of 1.3 (0.6-3.1), which did not reach statistical significance. The gyr mutations were not different than culture-based FQ drug susceptibility testing in predicting the hazard of death or treatment failure and may be superior. CONCLUSIONS: FQ molecular-based diagnostic tests may better predict treatment response than traditional drug susceptibility testing and open avenues for personalizing TB therapy.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value.

RATIONALE: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. OBJECTIVES: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. METHODS: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. MEASUREMENTS AND MAIN RESULTS: The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. CONCLUSIONS: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Fitness Costs of Drug Resistance Mutations in Multidrug-Resistant Mycobacterium tuberculosis: A Household-Based Case-Control Study.

BACKGROUND: The projected long-term prevalence of multidrug-resistant (MDR) tuberculosis depends upon the relative fitness of MDR Mycobacterium tuberculosis strains, compared with non-MDR strains. While many experimental models have tested the in vitro or in vivo fitness costs of various drug resistance mutations, fewer epidemiologic studies have attempted to validate these experimental findings. METHODS: We performed a case-control study comparing drug resistance-associated mutations from MDR M. tuberculosis strains causing multiple cases in a household to matched MDR strains without evidence of secondary household cases. RESULTS: Eighty-eight multiple-case and 88 single-case household MDR strains were analyzed for 10 specific drug resistance-associated polymorphisms previously associated with fitness effects. We found that the isoniazid-resistant katG Ser315Thr mutation occurred more than twice as frequently in multiple-case households than in single-case households (odds ratio [OR], 2.39; 95% confidence interval [CI], 1.21-4.70), corroborating previous experimental findings. However, strains carrying both the katG Ser315Thr mutation and the rpsL Lys43Arg mutation were less likely to be found in multiple-case households (OR, 0.09; 95% CI, .01-.73), suggesting a negative epistatic interaction which contrasts previous findings. CONCLUSIONS: The case-control design presents a useful approach for assessing in vivo fitness effects of drug resistance mutations.

Gyrase Mutations Are Associated with Variable Levels of Fluoroquinolone Resistance in Mycobacterium tuberculosis.

Molecular diagnostics that rapidly and accurately predict resistance to fluoroquinolone drugs and especially later-generation agents promise to improve treatment outcomes for patients with multidrug-resistant tuberculosis and prevent the spread of disease. Mutations in the gyr genes are known to confer most fluoroquinolone resistance, but knowledge about the effects of gyr mutations on susceptibility to early- versus later-generation fluoroquinolones and about the role of mutation-mutation interactions is limited. Here, we sequenced the full gyrA and gyrB open reading frames in 240 multidrug-resistant and extensively drug-resistant tuberculosis strains and quantified their ofloxacin and moxifloxacin MIC by testing growth at six concentrations for each drug. We constructed a multivariate regression model to assess both the individual mutation effects and interactions on the drug MICs. We found that gyrB mutations contribute to fluoroquinolone resistance both individually and through interactions with gyrA mutations. These effects were statistically significant. In these clinical isolates, several gyrA and gyrB mutations conferred different levels of resistance to ofloxacin and moxifloxacin. Consideration of gyr mutation combinations during the interpretation of molecular test results may improve the accuracy of predicting the fluoroquinolone resistance phenotype. Further, the differential effects of gyr mutations on the activity of early- and later-generation fluoroquinolones requires further investigation and could inform the selection of a fluoroquinolone for treatment.

A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid.

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain was Mycobacterium tuberculosis H37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 µg/ml), rifampin (0.03 to 0.25 µg/ml), ethambutol (0.25 to 2 µg/ml), levofloxacin (0.12 to 1 µg/ml), moxifloxacin (0.06 to 0.5 µg/ml), ofloxacin (0.25 to 2 µg/ml), amikacin (0.25 to 2 µg/ml), kanamycin (0.25 to 2 µg/ml), capreomycin (0.5 to 4 µg/ml), linezolid (0.25 to 2 µg/ml), and clofazimine (0.03 to 0.25 µg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against the M. tuberculosis H37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

A Multilaboratory, Multicountry Study To Determine Bedaquiline MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing.

The aim of this study was to establish standardized drug susceptibility testing (DST) methodologies and reference MIC quality control (QC) ranges for bedaquiline, a diarylquinoline antimycobacterial, used in the treatment of adults with multidrug-resistant tuberculosis. Two tier-2 QC reproducibility studies of bedaquiline DST were conducted in eight laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Agar dilution and broth microdilution methods were evaluated. Mycobacterium tuberculosis H37Rv was used as the QC reference strain. Bedaquiline MIC frequency, mode, and geometric mean were calculated. When resulting data occurred outside predefined CLSI criteria, the entire laboratory data set was excluded. For the agar dilution MIC, a 4-dilution QC range (0.015 to 0.12 µg/ml) centered around the geometric mean included 95.8% (7H10 agar dilution; 204/213 observations with one data set excluded) or 95.9% (7H11 agar dilution; 232/242) of bedaquiline MICs. For the 7H9 broth microdilution MIC, a 3-dilution QC range (0.015 to 0.06 µg/ml) centered around the mode included 98.1% (207/211, with one data set excluded) of bedaquiline MICs. Microbiological equivalence was demonstrated for bedaquiline MICs determined using 7H10 agar and 7H11 agar but not for bedaquiline MICs determined using 7H9 broth and 7H10 agar or 7H9 broth and 7H11 agar. Bedaquiline DST methodologies and MIC QC ranges against the H37Rv M. tuberculosis reference strain have been established: 0.015 to 0.12 µg/ml for the 7H10 and 7H11 agar dilution MICs and 0.015 to 0.06 µg/ml for the 7H9 broth microdilution MIC. These methodologies and QC ranges will be submitted to CLSI and EUCAST to inform future research and provide guidance for routine clinical bedaquiline DST in laboratories worldwide.

Performance of the G4 Xpert® MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings.

BACKGROUND: The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. METHODS: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. RESULTS: Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). CONCLUSIONS: The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

In vitro flowering associated protein changes in Dendrocalamus hamiltonii.

In Dendrocalamus hamiltonii, conversion of vegetative meristem to a floral meristem was successfully achieved on flower induction medium. A total of 128 differentially expressed proteins were evidenced by 2DE in floral meristem protein profiles. Analysis of 103 proteins through PMF revealed change in abundance in the content of 79 proteins, disappearance and new appearance in the content of 7 and 17 proteins, respectively. MS/MS and subsequent homology search identified 65 proteins that were involved in metabolism (22 proteins), regulatory (11 proteins), signaling and transportation (12 proteins), stress (6 proteins), flowering (8 proteins), and unknown functions (6 proteins). The data suggested that change in metabolism related proteins might be providing nutrient resources for floral initiation in D. hamiltonii. Further, interactive effects of various proteins like bHLH145, B-4c transcription factors (heat stress transcription factor), maturase K, MADS box, zinc finger proteins, and scarecrow-like protein 21 (flowering related), a key enzyme of ethylene biosynthesis SAMS (S-adenosylmethionine synthase) and aminocyclopropane-1-carboxylate synthase, improved calcium signaling related proteins (CML36), and change in phytohormone related proteins such as phosphatase proteins (2c3 and 2c55), which are the positive regulators of gibberellic acid and phytochrome regulation related proteins (DASH, LWD1) might be the possible major regulators of floral transition in this bamboo.

A single arabinan chain is attached to the phosphatidylinositol mannosyl core of the major immunomodulatory mycobacterial cell envelope glycoconjugate, lipoarabinomannan.

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.

RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities.

With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.

Impact of emotional intelligence and spiritual intelligence on the caring behavior of nurses: a dimension-level exploratory study among public hospitals in Malaysia.

PURPOSE: The purpose of this research is to study the impact of individual factors such as emotional intelligence (EI) and spiritual intelligence (SI) on the caring behavior of nurses. METHODS: A cross-sectional survey using questionnaire was conducted by sampling 550 nurses working in seven major public hospitals in Malaysia. Data were analyzed using structural equation modeling (SEM). RESULTS: The main findings are: (1) critical existential thinking and transcendental awareness dimensions of SI have significant impacts on assurance of human presence dimension of caring behavior; (2) personal meaning production and conscious state expansion dimensions of SI have significant impacts on perception of emotion and managing own emotions dimensions of EI; and (3) managing own emotions dimension of EI has significant impacts on respectful deference to other and assurance of human presence dimensions of caring behavior of nurses. CONCLUSION: The results can be used to recruit and educate nurses.

Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only.

In vitro flowering--a system for tracking floral organ development in Dendrocalamus hamiltonii Nees et Arn. ex Munro.

Dendrocalamus hamiltonii plants are slender and tall (15-25 m) thereby, rendering tagging, sampling and tracking the development of flowers difficult. Therefore, a reproducible system of in vitro flowering was established for tracking the stages of flower development. MS medium supplemented with 2.22 microM 6-benzylaminopurine, 1.23 microM indole-3-butyric acid and 2% sucrose was optimized as the flower induction medium (FIM) wherein 28 and 42 days were required for the development of gynoecium and androecium, respectively. Six distinct stages of in vitro flower development were identified, and the flowers were comparable with that of in planta sporadic flowers. Pollen viability of the in vitro flowers was higher than those of in planta ones. The in vitro system developed in the present study facilitates easy tracking of different stages of flower development under controlled environmental conditions. It can also be used for medium- or long-term storage of pollens and manipulation of in vitro fertilization.