Rapid Screening for Temperature-Sensitive Alleles in Plants

We developed a simple and fast method to identify temperature-sensitive alleles of essential plant genes. We used primary and tertiary structure information to identify residues in the core of the protein of interest. These residues were mutated and tested for temperature sensitivity, taking advantage of the exceptionally rapid 1-week complementation assay in the moss Physcomitrella patens. As test molecules, we selected the actin-binding proteins profilin and actin-depolymerizing factor, because they are essential and their loss-of-function phenotype can be fully rescued. Screening a small number of candidate mutants, we successfully identified temperature-sensitive alleles of both profilin and actin-depolymerizing factor. Plants harboring these alleles grew well at the permissive temperature of 20°C to 25°C but showed a complete loss of function at the restrictive temperature of 32°C. Notably, the profilin mutation identified in the moss gene can be transferred to profilins from other plant species, also rendering them temperature sensitive. The ability to routinely generate temperature-sensitive alleles of essential plant proteins provides a powerful tool for the study of gene function in plants.Conditional mutants are powerful genetic tools. In yeast, temperature-sensitive mutations have yielded a wealth of information regarding gene function and have aided immensely in the discovery and elucidation of many molecular pathways (Hartwell, 1967; Bonatti et al., 1972; Pringle, 1975; Novick and Botstein, 1985; Johnston et al., 1991; Balasubramanian et al., 1994; Chang et al., 1996, 1997; Iida and Yahara, 1999). In plants, a number of studies have generated temperature-sensitive alleles to study processes ranging from plant morphology to signal transduction (Lane et al., 2001; Whittington et al., 2001; Wiedemeier et al., 2002; Quint et al., 2005; Bannigan et al., 2006, 2007).In addition to temperature-dependent function, conditional expression can be generated in a variety of ways. A common strategy in mouse cells is to incorporate lox-p sites flanking the gene of interest (Sauer and Henderson, 1988; Orban et al., 1992; Vidali et al., 2006). Gene function is conditionally lost by the expression of cre recombinase that fuses the lox-p sites, deleting the intervening sequences. This method and others, such as inducible RNA interference (RNAi; Ketelaar et al., 2004), require long incubation times needed for gene expression and protein depletion. Due to the long time course for these studies, loss-of-function effects can be complicated with the development of the organism. In contrast, temperature-sensitive mutants are potentially fast acting, losing their function in some cases within minutes of exposure to the restrictive conditions (Novick and Botstein, 1985; Pruyne et al., 1998).In most cases, temperature-sensitive mutants are generated randomly and the elucidation of the gene harboring the mutation is uncovered by cloning the mutagenized gene. In plants, this is done by performing a chromosome walk to the mutagenized allele. In yeast, due to the ease of performing complementation, it is also possible to start with a gene of interest, mutagenize that gene, and screen for temperature-sensitive alleles (Shortle et al., 1984; Budd and Campbell, 1987; Mann et al., 1987). In plants, however, this process has not been widely used, presumably due to the time-consuming nature of performing complementation studies in planta.Here, we show that the moss Physcomitrella patens is an ideal plant suited for screening potential temperature-sensitive alleles of a gene of interest. To screen for a temperature-sensitive mutation, loss of the gene of interest must produce a measurable phenotype that can be rescued by reintroduction of the wild-type allele of the gene. We chose two proteins, profilin and actin-depolymerizing factor (ADF)/cofilin, as test molecules. Profilin and ADF are well-characterized actin-binding proteins that are important for cellular growth in plants (Staiger et al., 1994; Ramachandran et al., 2000; Dong et al., 2001; Vidali et al., 2001, 2007; Chen et al., 2002, 2003; McKenna et al., 2004; Augustine et al., 2008). In the moss P. patens, both profilin and ADF are essential for protonemal filament growth. Loss of profilin or ADF results in severely stunted plants, composed of morphologically abnormal cells (Vidali et al., 2007; Augustine et al., 2008). These phenotypes are fully rescued by expression of wild-type profilin or ADF, respectively.Moss has emerged as a facile plant system due to its ability to integrate exogenous DNA molecules by homologous recombination at frequencies enabling gene-targeting studies (Cove et al., 2006). In addition, moss is amenable to transient RNAi (Bezanilla et al., 2003, 2005), which enables the study of terminal phenotypes due to loss of essential genes, something that would not be possible if performing only gene knockout experiments. We have previously demonstrated the ability to knock down essential gene families and obtain quantitative rescue of the knockdown phenotypes (Vidali et al., 2007, 2009; Augustine et al., 2008). We have performed these studies using a rapid transient assay, which enables knock down and complementation studies to be performed within 1 week of transformation (Vidali et al., 2007). This is an extremely rapid assay that is unparalleled in other plant systems. Here, we use this complementation assay to screen for temperature-sensitive alleles of both profilin and ADF. Importantly, we show that the residue that confers temperature sensitivity in moss profilin can also render both Arabidopsis (Arabidopsis thaliana) and lily (Lilium longiflorum) profilins temperature sensitive, demonstrating a wider applicability to this rapid in planta complementation system.     

Hesperidin attenuates inflammation and oxidative damage in pleural exudates and liver of rat model of pleurisy

Objectives: This study investigated the potential anti-inflammatory effect of hesperidin against carrageenan induced pleurisy in rat model.

Methods: Twenty-four adult female Wistar rats (350 - 450g) were grouped as follows: Group I: rats were administered saline solution only (Normal control group); Group II: rats were administered saline solution (NaCl 0.9%) orally and injected with carrageenan (Inflammation control group); Group III: rats were administered hesperidin only (Hesperidin group); Group IV: rats were administered hesperidin orally and intrapleurally injected with 2% carrageenan (Inflammation treated with hesperidin group). The exudate volume, total leukocyte count, reactive oxygen species (ROS), myeloperoxidase (MPO),δ–aminolevulinate dehydratase (δ-ALA-D), catalase (CAT), superoxide dismutase (SOD), activities as well as non-protein thiol group (NPSH) and thiobarbituric acid reactive substances (TBARS) levels were determined.

Results: Pretreatment with hesperidin at a dose of 80 mg/kg orally per day for 21 days, minimized the increase in pleural exudate volume and leucocyte count and modulated the activities of MPO, SOD and CAT, as well as the levels of ROS, NPSH and TBARS in carrageenan-induced rats.

Conclusion: Our results suggest that hesperidin can elicit its anti-inflammatory action by blocking exudate and leukocyte influx into pleural cavity, inhibiting MPO activity and preventing oxidative damage.     

Plasma surfactant protein-D as a diagnostic biomarker for acute respiratory distress syndrome: validation in US and Korean cohorts

Background

Acute respiratory distress syndrome (ARDS) is potentially underrecognized by clinicians. Early recognition and subsequent optimal treatment of patients with ARDS may be facilitated by usage of biomarkers. Surfactant protein D (SP-D), a marker of alveolar epithelial injury, has been proposed as a potentially useful biomarker for diagnosis of ARDS in a few studies. We tried to validate the performance of plasma SP-D levels for diagnosis of ARDS.

Methods

We conducted a retrospective analysis using data from three (two in USA and one in Korea) prospective biobank cohorts involving 407 critically ill patients admitted to medical intensive care unit (ICU). A propensity score matched analysis (patients with versus without ARDS, matched 1:1) was carried out using significant variables from multiple logistic regression. The diagnostic accuracy of plasma SP-D as a diagnostic marker of ARDS was assessed by receiver operating characteristic curve analysis.

Results

Out of the 407 subjects included in this study, 39 (10%) patients fulfilled ARDS criteria. Patients with ARDS had higher SP-D levels in plasma (p?<?0.01) and higher hospital-mortality (p?<?0.001) than those without ARDS. Thirty eight subjects with ARDS (cases) were successfully matched for propensity for ARDS with 38 subjects without ARDS (controls). Plasma levels of SP-D were higher in cases with ARDS compared to their matched controls without ARDS [median 20.8 ng/mL (interquartile range, 12.7–38.4) versus 7.9 (4.1–17.0); p?=?0.001]. The area under the receiver operating characteristic curve for SP-D for the diagnosis of ARDS was 0.71 (95% confidence intervals, 0.60–0.83). A cut-off point of 12.7 ng/mL for SP-D yielded sensitivity of 74% and specificity of 63%.

Conclusions

High levels of SP-D within 48 h after ICU admission might serve as a diagnostic marker for ARDS in patients hospitalized in medical ICU. Further prospective trials are required to validate the diagnostic role of SP-D in ARDS, and if its usefulness is greater in direct than in indirect ARDS, as well as across different strata of severity of ARDS.

     

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users.     

Metabolomic Derangements Are Associated with Mortality in Critically Ill Adult Patients

Objective

To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.

Rationale

Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.

Methods

We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.

Results

We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).

Conclusion

Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.     

Computer-aided X-ray screening for tuberculosis and HIV testing among adults with cough in Malawi (the PROSPECT study): A randomised trial and cost-effectiveness analysis

BackgroundSuboptimal tuberculosis (TB) diagnostics and HIV contribute to the high global burden of TB. We investigated costs and yield from systematic HIV-TB screening, including computer-aided digital chest X-ray (DCXR-CAD).Methods and findingsIn this open, three-arm randomised trial, adults (≥18 years) with cough attending acute primary services in Malawi were randomised (1:1:1) to standard of care (SOC); oral HIV testing (HIV screening) and linkage to care; or HIV testing and linkage to care plus DCXR-CAD with sputum Xpert for high CAD4TBv5 scores (HIV-TB screening). Participants and study staff were not blinded to intervention allocation, but investigator blinding was maintained until final analysis. The primary outcome was time to TB treatment. Secondary outcomes included proportion with same-day TB treatment; prevalence of undiagnosed/untreated bacteriologically confirmed TB on day 56; and undiagnosed/untreated HIV. Analysis was done on an intention-to-treat basis. Cost-effectiveness analysis used a health-provider perspective. Between 15 November 2018 and 27 November 2019, 8,236 were screened for eligibility, with 473, 492, and 497 randomly allocated to SOC, HIV, and HIV-TB screening arms; 53 (11%), 52 (9%), and 47 (9%) were lost to follow-up, respectively. At 56 days, TB treatment had been started in 5 (1.1%) SOC, 8 (1.6%) HIV screening, and 15 (3.0%) HIV-TB screening participants. Median (IQR) time to TB treatment was 11 (6.5 to 38), 6 (1 to 22), and 1 (0 to 3) days (hazard ratio for HIV-TB versus SOC: 2.86, 1.04 to 7.87), with same-day treatment of 0/5 (0%) SOC, 1/8 (12.5%) HIV, and 6/15 (40.0%) HIV-TB screening arm TB patients (p = 0.03). At day 56, 2 SOC (0.5%), 4 HIV (1.0%), and 2 HIV-TB (0.5%) participants had undiagnosed microbiologically confirmed TB. HIV screening reduced the proportion with undiagnosed or untreated HIV from 10 (2.7%) in the SOC arm to 2 (0.5%) in the HIV screening arm (risk ratio [RR]: 0.18, 0.04 to 0.83), and 1 (0.2%) in the HIV-TB screening arm (RR: 0.09, 0.01 to 0.71). Incremental costs were US$3.58 and US$19.92 per participant screened for HIV and HIV-TB; the probability of cost-effectiveness at a US$1,200/quality-adjusted life year (QALY) threshold was 83.9% and 0%. Main limitations were the lower than anticipated prevalence of TB and short participant follow-up period; cost and quality of life benefits of this screening approach may accrue over a longer time horizon.ConclusionsDCXR-CAD with universal HIV screening significantly increased the timeliness and completeness of HIV and TB diagnosis. If implemented at scale, this has potential to rapidly and efficiently improve TB and HIV diagnosis and treatment.Trial registrationclinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03519425","term_id":"NCT03519425"}}NCT03519425.

In a randomised trial, Peter MacPherson and colleagues investigate the costs, timeliness, and completeness of computer-aided X-ray screening for tuberculosis and HIV testing in adults with cough in Malawi.