Higher-accuracy method for measuring minichromosome stability in Saccharomyces cerevisiae

The minichromosome maintenance assay isfrequently used to characterize mutations genetically that affect the initiation of DNA replication or to decipherfunctional components in autonomously replicating sequences. The assay determines minichromosome loss by measuring the percentage of plasmid-containing cells in cultures after a period of growth in nonselective medium. Here we analyze data acquisition errors that contribute to the low accuracy of the routine versions of the assay. We propose modifications that eliminate errors in the acquisition of two variables and significantly improve the accuracy of the assay.     

Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.     

WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differentiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA‐deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.     

Seasonal Variation of Antifouling Activities of Marine Algae from the Brittany Coast (France)

The antifouling activity of extracts (aqueous, ethanol, and dichloromethane) of 9 marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae has been investigated in relation to season in bimonthly samples from the Bay of Concarneau (France). Of the extracts tested, 48.2% were active against at least one of the fouling organisms, and of these extracts, 31.2% were seasonally active with a peak of activity in summer corresponding to maximal values for water temperature, light intensity, and fouling pressure, and 17% were active throughout the year. This seasonal activity may be adaptive as it coincides with maximal fouling pressure in the Bay of Concarneau. Dichloromethane extracts of Rhodophyceae were the most active in the antifouling assays.     

Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: a guide for prokaryotic and eukaryotic investigation

Many fluorescent techniques are employed to evaluate the viability and activity of microbial cells used in biotechnology. These techniques are sometimes complex and the interpretation of results opened to misunderstanding. Moreover, new developments are constantly proposed especially concerning a more accurate evaluation of the state of the cells including eukaryotic microorganisms. This paper aims at presenting to biotechnologists unfamiliar with fluorescence the principles of these methods and the related possible pitfalls. It focuses on probes of the physical (integrity and fluidity) and energetical (intracellular pH and membrane potential) state of the cell membrane (bacterial and yeast cells) and presents also other probes (nucleic acids, respiration...) and new technical trends. The specificities of Gram-negative bacterial cells are also discussed.     

External validation of the Hospital-patient One-year Mortality Risk (HOMR) model for predicting death within 1 year after hospital admission

Background:

Predicting long-term survival after admission to hospital is helpful for clinical, administrative and research purposes. The Hospital-patient One-year Mortality Risk (HOMR) model was derived and internally validated to predict the risk of death within 1 year after admission. We conducted an external validation of the model in a large multicentre study.

Methods:

We used administrative data for all nonpsychiatric admissions of adult patients to hospitals in the provinces of Ontario (2003–2010) and Alberta (2011–2012), and to the Brigham and Women’s Hospital in Boston (2010–2012) to calculate each patient’s HOMR score at admission. The HOMR score is based on a set of parameters that captures patient demographics, health burden and severity of acute illness. We determined patient status (alive or dead) 1 year after admission using population-based registries.

Results:

The 3 validation cohorts (n = 2 862 996 in Ontario, 210 595 in Alberta and 66 683 in Boston) were distinct from each other and from the derivation cohort. The overall risk of death within 1 year after admission was 8.7% (95% confidence interval [CI] 8.7% to 8.8%). The HOMR score was strongly and significantly associated with risk of death in all populations and was highly discriminative, with a C statistic ranging from 0.89 (95% CI 0.87 to 0.91) to 0.92 (95% CI 0.91 to 0.92). Observed and expected outcome risks were similar (median absolute difference in percent dying in 1 yr 0.3%, interquartile range 0.05%–2.5%).

Interpretation:

The HOMR score, calculated using routinely collected administrative data, accurately predicted the risk of death among adult patients within 1 year after admission to hospital for nonpsychiatric indications. Similar performance was seen when the score was used in geographically and temporally diverse populations. The HOMR model can be used for risk adjustment in analyses of health administrative data to predict long-term survival among hospital patients.The life expectancy of individual patients can be important for both medical decision-making and research. Patients with a short life expectancy may choose to defer preventive treatments, screening interventions or interventional procedures for conditions that are currently asymptomatic. An accurate assessment of risk of death, particularly if that risk is high, could motivate and inform discussions between patients and physicians regarding goals of care. In addition, accurate prognostications are essential for adjusting statistical models that have death as an outcome (or as a competing risk for other outcomes) in both research and administration.We recently derived and internally validated a model that predicts the risk of death from any cause at 1 year after admission to hospital.¹ The Hospital-patient One-year Mortality Risk (HOMR) model consists of covariates whose values are determined at admission using routinely collected health administrative data (Figure 1). These covariates include patient demographics (age, sex and living status); health burden (measured using the Charlson Comorbidity Index score, home oxygen status and the number of visits to emergency departments and admissions to hospital by ambulance in the previous year); and acuity of illness (admission urgency and hospital service, direct admission to an intensive care unit and whether the admission was an urgent readmission to hospital). The latter category was also gauged using the Diagnostic Risk Score, which quantifies risk of death for particular diagnoses beyond that explained by the other covariates (Appendix 1, available at www.cmaj.ca/lookup/suppl/doi:10.1503/cmaj.150209/-/DC1).Open in a separate windowFigure 1:Covariates used to calculate a patient’s Hospital-patient One-year Mortality Risk (HOMR) score at the time of admission to hospital. The Diagnostic Risk Score (Appendix 1) quantifies risk of death for diagnostic groups beyond that explained by the other covariates. Points for the interacting covariates of age and Charlson Comorbidity Index score include the risk of patient age, comorbidity score and their interaction. In contrast, points for living status and admission urgency include the risk of these covariates and their interaction with admissions by ambulance in the previous year; points for the latter covariate are considered separately. See www.cmaj.ca/lookup/suppl/doi:10.1503/cmaj.150209/-/DC1)Discrete values for each covariate are given specific points, which are summed to create the HOMR score (Figure 1). In an internal validation population, the HOMR score accurately predicted the risk of death from any cause within 1 year after admission, with a C statistic of 0.92 and excellent calibration among adult residents of Ontario admitted to hospital for nonpsychiatric indications in 2011.¹Although these statistics are impressive, external validation is required to determine the true usefulness of any statistical model. External validation is necessary to prove that the model’s performance is not idiosyncratic to the patients, physicians, institutions or data systems used to derive and internally test it.^2,3 A prognostic model should remain accurate when retested with different patients (reproducibility), during different periods (historical transportability) and in different locations (geographic transportability).⁴ We conducted an external validation of the HOMR model in a multicentre study that included Canadian and American hospitals.