Presidential Impoundment of Funds: A Constitutional Crisis

It seems that our maligned Congress, so docile at times in the face of presidential aggrandizement of power, is forging a constitutional crisis. The basic issue involved is whether the President has authority, either constitutional or statutory, to refuse to spend funds appropriated by Congress. The questions pursued in the following pages are basically four: does the President have the (1) statutory, (2) historical, (3) case, or (4) constitutional authority to continue to impound

High-resolution fire danger forecast for Poland based on the Weather Research and Forecasting Model

Due to climate change and associated longer and more frequent droughts, the risk of forest fires increases. To address this, the Institute of Meteorology and Water Management implemented a system for forecasting fire weather in Poland. The Fire Weather Index (FWI) system, developed in Canada, has been adapted to work with meteorological fields derived from the high-resolution (2.5 km) Weather Research and Forecasting (WRF) model. Forecasts are made with 24- and 48-h lead times. The purpose of this work is to present the validation of the implemented system. First, the results of the WRF model were validated using in situ observations from ∼70 synoptic stations. Second, we used the correlation method and Eastaugh\u27s percentile analysis to assess the quality of the FWI index. The data covered the 2019 fire season and were analysed for the whole forest area in Poland. Based on the presented results, it can be concluded that the FWI index (calculated based on the WRF model) has a very high predictive ability of fire risk. However, the results vary by region, distance from human habitats, and size of fire

Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development.

UNLABELLED: Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive

Fluorene and phenanthrene uptake by Pseudomonas putida ATCC 17514: kinetics and physiological aspects

Pseudomonas putida ATCC 17514 was used as a model strain to investigate the characteristics of bacterial growth in the presence of solid fluorene and phenanthrene. Despite the lower water-solubility of phenanthrene, P. putida degraded this polycyclic aromatic hydrocarbon (PAH) at a maximum observed rate of 1.4 ± 0.1 mg Lˉ¹ hˉ¹, higher than the apparent degradation rate of fluorene, 0.8 ± 0.07 mg Lˉ¹ hˉ¹. The role of physiological processes on the biodegradation of these PAHs was analyzed and two different uptake strategies were identified. Zeta potential measurements revealed that phenanthrene-grown cells were slightly more negatively charged (–57.5 ± 4.7 mV) than fluorene-grown cells (–51.6 ± 4.9 mV), but much more negatively charged than glucose-grown cells (–26.8 ± 3.3 mV), suggesting that the PAH substrate induced modifications on the physical properties of bacterial surfaces. Furthermore, protein-to-exopolysaccharide ratios detected during bacterial growth on phenanthrene were typical of biofilms developed under physicochemical stress conditions, caused by the presence of sparingly water-soluble chemicals as the sole carbon and energy source for growth, the maximum value for TP/EPS during growth on phenanthrene (1.9) being lower than the one obtained with fluorene (5.5). Finally, confocal laser microscopy observations using a gfp-labeled derivative strain revealed that, in the presence of phenanthrene, P. putida::gfp cells formed a biofilm on accessible crystal surfaces, whereas in the presence of fluorene the strain grew randomly between the crystal clusters. The results showed that P. putida was able to overcome the lower aqueous solubility of phenanthrene by adhering to the solid PAH throughout the production of extracellular polymeric substances, thus promoting the availability and uptake of such a hydrophobic compound.Fundação para a Ciência e a Tecnologia (FCT) – PRAXISXXI/BD/15944/98.EC Biotech program - BIO4-CT97-2015

Analytical and Clinical Performance of Amyloid-Beta Peptides Measurements in CSF of ADNIGO/2 Participants by an LC–MS/MS Reference Method

BACKGROUND: Cerebrospinal fluid (CSF) amyloid-β1-42 (Aβ42) reliably detects brain amyloidosis based on its high concordance with plaque burden at autopsy and with amyloid positron emission tomography (PET) ligand retention observed in several studies. Low CSF Aβ42 concentrations in normal aging and dementia are associated with the presence of fibrillary Aβ across brain regions detected by amyloid PET imaging. METHODS: An LC-MS/MS reference method for Aβ42, modified by adding Aβ40 and Aβ38 peptides to calibrators, was used to analyze 1445 CSF samples from ADNIGO/2 participants. Seventy runs were completed using 2 different lots of calibrators. For preparation of Aβ42 calibrators and controls spiking solution, reference Aβ42 standard with certified concentration was obtained from EC-JRC-IRMM (Belgium). Aβ40 and Aβ38 standards were purchased from rPeptide. Aβ42 calibrators' accuracy was established using CSF-based Aβ42 Certified Reference Materials (CRM). RESULTS: CRM-adjusted Aβ42 calibrator concentrations were calculated using the regression equation Y (CRM-adjusted) = 0.89X (calibrators) + 32.6. Control samples and CSF pools yielded imprecision ranging from 6.5 to 10.2% (Aβ42) and 2.2 to 7.0% (Aβ40). None of the CSF pools showed statistically significant differences in Aβ42 concentrations across 2 different calibrator lots. Comparison of Aβ42 with Aβ42/Aβ40 showed that the ratio improved concordance with concurrent [18F]-florbetapir PET as a measure of fibrillar Aβ (n = 766) from 81 to 88%. CONCLUSIONS: Long-term performance assessment substantiates our modified LC-MS/MS reference method for 3 Aβ peptides. The improved diagnostic performance of the CSF ratio Aβ42/Aβ40 suggests that Aβ42 and Aβ40 should be measured together and supports the need for an Aβ40 CRM

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrioanguillarum strain 775

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state

A novel pathway producing dimethylsulphide in bacteria is widespread in soil environments

The volatile compound dimethylsulphide (DMS) is important in climate regulation, the sulphur cycle and signalling to higher organisms. Microbial catabolism of the marine osmolyte dimethylsulphoniopropionate (DMSP) is thought to be the major biological process generating DMS. Here we report the discovery and characterisation of the first gene for DMSP-independent DMS production in any bacterium. This gene, mddA, encodes a methyltransferase that methylates methanethiol (MeSH) and generates DMS. MddA functions in many taxonomically diverse bacteria including sediment-dwelling pseudomonads, nitrogen-fixing bradyrhizobia and cyanobacteria, and mycobacteria, including the pathogen Mycobacterium tuberculosis. The mddA gene is present in metagenomes from varied environments, being particularly abundant in soil environments, where it is predicted to occur in up to 76% of bacteria. This novel pathway may significantly contribute to global DMS emissions, especially in terrestrial environments, and could represent a shift from the notion that DMSP is the only significant precursor of DMS

Sleep apnea prevalence in chronic kidney disease - association with total body water and symptoms

Background: Sleep apnea is common and associated with poor outcome in severe chronic kidney disease, but validated screening tools are not available. Our objectives were to determine the prevalence of sleep apnea in this population, to assess the validity of screening for sleep apnea using an ApneaLink device and to investigate the relationship of sleep apnea to; symptoms, spirometry and body water. Methods: Patients with glomerular filtration rate ≤30 mL/min/1.73 m2, whether or not they were receiving haemodialysis, were eligible for enrolment. Participants completed symptom questionnaires, performed an ApneaLink recording and had total body water measured using bioimpedance. This was followed by a multi-channel polysomnography recording which is the gold-standard diagnostic test for sleep apnea. Results: Fifty-seven participants were enrolled and had baseline data collected, of whom only 2 did not have sleep apnea. An apnea hypopnea index ≥30/h was found in 66% of haemodialysis and 54% of non-dialysis participants. A central apnea index ≥5/h was present in 11 patients, with only one dialysis patient having predominantly central sleep apnea. ApneaLink underestimated sleep apnea severity, particularly in the non-dialysis group. Neither total body water corrected for body size, spirometry, subjective sleepiness nor overall symptom scores were associated with sleep apnea severity. Conclusions: This study demonstrates a very high prevalence of severe sleep apnea in patients with chronic kidney disease. Sleep apnea severity was not associated with quality of life or sleepiness scores and was unrelated to total body water corrected for body size. Routine identification of sleep apnea with polysomnography rather than screening is more appropriate in this group due to the high prevalenceThis study was funded by a grant from The Canberra Hospital Private Practice Fund