Rapid detection of Salmonella enterica in leafy greens by a novel DNA microarray-based PathogenDx system.

The diverse matrices pose great challenges for rapid detection of low Salmonella level (<10 CFU) in fresh produce. The applicability of microarray-based PathogenDx system for detecting low contamination of Salmonella Newport from leafy greens was evaluated. A pre-PCR preparation protocol including enrichment in universal pre-enrichment broth for 3 h followed by sample concentration using an InnovaPrep bio-concentrator or 6 h enrichment without a concentration step was used for detecting S. Newport from leafy greens with initial inoculum level at ∼6 CFU/25 g. Among 205 samples tested, 98%, 93%, 76%, and 60% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were tested positive after 3 h of enrichment with sample concentration. After 6 h of enrichment, 100%, 98%, 90%, and 82% of Romaine lettuce, Iceberg lettuce, kale, and spinach samples were positive. The samples were parallelly tested by the FDA bacterial analytical manual (BAM) method and 100% of spiked produce samples were tested positive. The overall analysis time of this methodology was between 8 and 11 h, including all pre-enrichment and concentration steps, in contrast to 4-5 days required for BAM method. The system correctly differentiated all 108 Salmonella strains and 35 non-Salmonella strains used in the study. This novel microarray approach provides a rapid method for detecting Salmonella in leafy greens.

Ongoing risk of bladder cancer among former workers at the last benzidine manufacturing facility in the USA.

OBJECTIVE: To determine whether there is an ongoing risk of developing bladder cancer in a previously studied cohort of workers exposed to both benzidine and dichlorobenzidine or dichlorobenzidine only in the last benzidine manufacturing plant in the USA. METHODS: Workers (n=488) were identified from the quarterly 941 forms the employer was required to submit to the Social Security Administration from 1960 to 1977. Exposures were assigned based on dates worked and known benzidine/dichlorobenzidine production schedules. Incidence, vital status and cause of death were determined through 2014. Analyses were restricted to white men. RESULTS: Bladder cancer incidence and mortality were significantly increased (25 incident cases, standardised incidence ratio (SIR) 2.19, 95% CI 1.42 to 3.23, and 5 deaths, standardised mortality ratio (SMR) 3.79, 95% CI 1.23 to 8.84). There were significant increases in incidence and mortality in those exposed to both benzidine and dichlorobenzidine (SIR 3.11, 95% CI 1.97 to 4.67, SMR 4.10, 95% CI 1.12 to 10.50), but not among workers exposed to dichlorobenzidine only (two incident cases, SIR 0.89, 95% CI 0.11 to 3.23 and one death, SMR 2.90, 95% CI 0.07 to 16.15). Bladder cancer incidence and mortality were increased in individuals with >20 years since last exposure with >5 years worked (six observed, SIR 5.94, 95% CI 2.18 to 12.92 and two deaths, SMR 7.93, 95% CI 0.96 to 28.65). CONCLUSIONS: Incidence and mortality due to bladder cancer increased among workers exposed to benzidine but not among workers exposed only to dichlorobenzidine. The risk of incidence and death from bladder cancer remain elevated more than 20 years after last exposure to benzidine in those who worked >5 years.

Swimming facilities and work-related asthma.

BACKGROUND: Exposure to chlorinated water in swimming facilities may aggravate preexisting asthma or cause new onset asthma. This may be a particular problem for individuals who work and therefore spend prolonged time at swimming facilities. Chloramines formed by the interaction of chlorine-based disinfection products with the nitrogen in water from human sweat, urine and skin cells are the suspected causal agents. METHODS: Cases were reviewed from the state surveillance systems in California (CA), Michigan (MI) and New Jersey (NJ) to identify individuals with confirmed work-related asthma (WRA) attributed to exposures in swimming pools, water parks or hydrotherapy spas. A standardized method was used to confirm cases. RESULTS: A total of 44 confirmed cases of WRA were identified; 17 from 1994 to 2011 in CA, 15 from 1991 to 2012 in MI and 12 from 1990 to 2011 in NJ. A majority (52.2%) of the cases were new onset; 31.8% secondary to an acute exposure incident and 20.4% to repeated exposure. These represented 0.3-1.6% of all confirmed cases of WRA received during these time periods. Maintenance workers (34.9%) and lifeguards (31.8%) were the most common occupations. CONCLUSIONS: Swimming pool workers were identified from three states where the pool environment was either a trigger of preexisting asthma or associated with new onset of WRA. Regulations to require air monitoring and improvements in ventilation are recommended to reduce exposure levels of chloramines, the presumed etiologic agents. Clinical assessment of patients with asthma should include consideration of the effect on respiratory symptoms from exposures in a swimming pool environment.

Silicosis and chronic renal disease.

BACKGROUND: Silica has been associated with end stage kidney disease and kidney dysfunction. METHODS: Calculated glomerular filtration rate, history of kidney disease or chronic dialysis, elevated serum creatinine, and stages of chronic kidney disease among silicotics identified in Michigan's Silicosis Surveillance System from 1987 to 2009 were reviewed to determine the prevalence of kidney disease in confirmed cases of silicosis. RESULTS: Twenty-four percent of 1,072 silicotics had a measure of kidney dysfunction (32.3% if diabetes or hypertension present vs. 20.2% if not). Sixty-nine percent of silicotics had Stage I or greater chronic kidney dysfunction versus 38.8% of the U.S. general population ≥60 years. No association was found between kidney function and measures of silica exposure. CONCLUSIONS: Individuals with silicosis have an increased prevalence of kidney disease. More work to define the pathological changes associated with silica exposure is needed to understand the cause of silica's adverse effect on the kidney.

Independent Study for the Detectx Combined Assay for the Detection of Aspergillus, Salmonella, and STEC (stx1 and/or 2) in Dried Cannabis Flower and Dried Hemp Flower: Level 3 Modification Study 012201.

BACKGROUND: The PathogenDx family of assays uses microarray technology to simultaneously detect the presence of bacterial and fungal pathogens in food products, environmental surfaces, and cannabis products. OBJECTIVE: The Detectx Combined assay was validated for the detection of Aspergillus, (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus), Salmonella, and a broad range of STEC (stx1 and/or 2) species. The validation consisted of two matrix studies in dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol) flower, product consistency, stability, robustness, and inclusivity and exclusivity for two targets: Aspergillus and STEC. METHOD: The PathogenDx Detectx Combined assay was evaluated with 30 replicates in each matrix and confirmed according to the instructions outlined in this study. RESULTS: Results of the validation study met the requirements of AOAC Standard Method Performance Requirement (SMPR®) 2020.002 and 2020.012. In the inclusivity and exclusivity study, all target isolates (Aspergillus and STEC) were correctly detected. For the exclusivity study, 26 out of 30 Aspergillus and 30 out of 30 STEC non-target strains were correctly excluded. In the matrix study, the PathogenDx Detectx Combined assay showed no significant statistical differences between confirmed results for dried hemp and cannabis flower. Robustness testing indicated that small changes to the method parameters did not impact the performance of the assay. Stability and consistency studies verified that the assay's shelf-life claims were appropriate, and manufacturing of the assay was consistent. CONCLUSIONS: The validation study indicated that the PathogenDx DetectX Combined assay was successful in detection of the new target analytes (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus and STEC containing stx1 and/or 2) and could successfully recover these organisms and Salmonella from dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol). HIGHLIGHTS: The PathogenDx DetectX Combined Assay will be the first PTM approved multiplex assay for Aspergillus, E. coli and Salmonella that does not require an enrichment step.

Circulating fibroblast growth factor-23 is associated with increased risk for metachronous colorectal adenoma.

BACKGROUND: Fibroblast growth factor-23 (FGF-23) is a phosphaturic peptide and a key component of an endocrine feedback loop along with the hormonal vitamin D metabolite 1,25(OH)(2)D. Vitamin D has been shown to be inversely related to colorectal neoplasia; therefore, we hypothesized that the effect of FGF-23 on vitamin D metabolite concentrations could have implications for the risk of colorectal neoplasia. MATERIALS AND METHODS: The purpose of this study was to prospectively evaluate the association between circulating concentrations of FGF-23 and the risk of metachronous (recurrent) colorectal adenomas. FGF-23 levels were assessed in 100 male and female participants from the Ursodeoxycholic Acid Trial, 50 of whom had a metachronous colorectal adenoma and 50 who did not. RESULTS: Compared to the lowest tertile of FGF-23, the adjusted odds ratios (95% CIs) for the second and third tertiles were 2.80 (0.94 to 8.31) and 3.41 (1.09 to 10.67), respectively (P-trend=.03). In a linear regression model, there was also a statistically significant inverse relationship between FGF-23 and 1,25(OH)(2)D (ß-coefficient=-1.2; P=.001). In contrast, no statistically significant trend was observed between FGF-23 and 25(OH)D concentrations (ß-coefficient=0.55; P=.10). CONCLUSIONS: The current work presents novel preliminary evidence of a relationship between FGF-23 and the risk for colorectal neoplasia. FGF-23 activity may be mediated through biologic effects on individual serum and colonic 1,25(OH)(2)D levels, or it may be independent from the vitamin D pathway. Further studies in larger populations are necessary for confirmation and expansion of these hypothesis-generating results.

Exposure Assessment Tools for Hypersensitivity Pneumonitis. An Official American Thoracic Society Workshop Report.

This report is based on proceedings from the Exposure Assessment Tools for Hypersensitivity Pneumonitis (HP) Workshop, sponsored by the American Thoracic Society, that took place on May 18, 2019, in Dallas, Texas. The workshop was initiated by members from the Environmental, Occupational, and Population Health and Clinical Problems Assemblies of the American Thoracic Society. Participants included international experts from pulmonary medicine, occupational medicine, radiology, pathology, and exposure science. The meeting objectives were to 1) define currently available tools for exposure assessment in evaluation of HP, 2) describe the evidence base supporting the role for these exposure assessment tools in HP evaluation, 3) identify limitations and barriers to each tool's implementation in clinical practice, 4) determine which exposure assessment tools demonstrate the best performance characteristics and applicability, and 5) identify research needs for improving exposure assessment tools for HP. Specific discussion topics included history-taking and exposure questionnaires, antigen avoidance, environmental assessment, specific inhalational challenge, serum-specific IgG testing, skin testing, lymphocyte proliferation testing, and a multidisciplinary team approach. Priorities for research in this area were identified.

Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design.

Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.

NMR Profiling of Metabolites in Larval and Juvenile Blue Mussels (Mytilus edulis) under Ambient and Low Salinity Conditions.

Blue mussels (Mytilus edulis) are ecologically and economically important marine invertebrates whose populations are at risk from climate change-associated variation in their environment, such as decreased coastal salinity. Blue mussels are osmoconfomers and use components of the metabolome (free amino acids) to help maintain osmotic balance and cellular function during low salinity exposure. However, little is known about the capacity of blue mussels during the planktonic larval stages to regulate metabolites during osmotic stress. Metabolite studies in species such as blue mussels can help improve our understanding of the species' physiology, as well as their capacity to respond to environmental stress. We used 1D ¹H nuclear magnetic resonance (NMR) and 2D total correlation spectroscopy (TOCSY) experiments to describe baseline metabolite pools in larval (veliger and pediveliger stages) and juvenile blue mussels (gill, mantle, and adductor tissues) under ambient conditions and to quantify changes in the abundance of common osmolytes in these stages during low salinity exposure. We found evidence for stage- and tissue-specific differences in the baseline metabolic profiles of blue mussels, which reflect variation in the function and morphology of each larval stage or tissue type of juveniles. These differences impacted the utilization of osmolytes during low salinity exposure, likely stemming from innate physiological variation. This study highlights the importance of foundational metabolomic studies that include multiple tissue types and developmental stages to adequately evaluate organismal responses to stress and better place these findings in a broader physiological context.

Distribution of venous remodeling in exercise-induced pulmonary hemorrhage of horses follows reported blood flow distribution in the equine lung.

Exercise-induced pulmonary hemorrhage (EIPH), which has been reported in humans and a variety of domestic animals following strenuous exercise, is most often documented in racehorses. Remodeling of pulmonary veins (VR) in equine EIPH was recently described, suggesting that it contributes to the pathogenesis of the disease. The cause of VR is unknown. We tested the hypothesis that the development of VR follows pulmonary blood flow distribution, preferentially occurring in the caudodorsal lung region. Furthermore, we hypothesized that VR underpins development of the other lesions of EIPH pathology. The lungs of 10 EIPH-affected horses and 8 controls were randomly sampled for histopathology (2,520 samples) and blindly scored for presence and severity of VR, hemosiderin (H), and interstitial fibrosis (IF). Mean sample score (MSS), mean lesion score, and percent samples with lesions were determined in four dorsal and three ventral lung regions, and the frequency, spatial distribution, and severity of lesions were determined. MSS for VR and H were significantly greater dorsally than ventrally (P < 0.001) and also decreased significantly in the caudocranial direction (P < 0.001). IF decreased only in the caudocranial direction. The percent samples with lesions followed the same distribution as MSS. VR often was accompanied by H; IF never occurred without VR and H. Similarity of the distribution of EIPH lesions and the reported fractal distribution of pulmonary blood flow suggests that VR develops in regions of high blood flow. Further experiments are necessary to determine whether VR is central to the pathogenesis of EIPH.

Oral hydration during growth hormone stimulation with clonidine.

The arginine-clonidine growth hormone (GH) stimulation test causes hypotension, requiring intravenous fluids to stabilize blood pressure (BP) and delaying departure from clinic. We hypothesized that oral hydration during the stimulation test would decrease need for intravenous fluids and shorten clinic stay. Children drank a diet electrolyte drink (10 ml/kg) on arrival to the test, which was repeated after clonidine. Fifteen children (7 girls) were tested without oral hydration, and 23 (6 girls) were tested with oral hydration (age range, 2-15 years). Compared with no oral hydration, intake of >13 ml/kg rarely required intravenous fluids, improved diastolic BP, and permitted discharge at the end of the GH test, with a higher BP.

Dependence of nelfinavir brain uptake on dose and tissue concentrations of the selective P-glycoprotein inhibitor zosuquidar in rats.

Most reverse transcriptase and protease inhibitors used in highly active antiretroviral therapy for treating human immunodeficiency virus (HIV) infections exhibit poor penetration into the brain, raising the concern that the brain may be a sanctuary site for the development of resistant HIV variants. This study explores the relationship between the dose and plasma and brain concentrations of zosuquidar and the effect of this selective P-glycoprotein inhibitor on central nervous system penetration of the HIV protease inhibitor nelfinavir maintained at steady state by intravenous infusions in rats. Nelfinavir was infused (10 mg/kg/h) for up to 10 h with or without concurrent administration of an intravenous bolus dose of 2, 6, or 20 mg/kg zosuquidar given at 4 h. Brain tissue and plasma were analyzed for both drug concentrations. Brain tissue/plasma nelfinavir concentration ratios (uncorrected for the vascular contribution) increased nonlinearly with zosuquidar dose from 0.06 +/- 0.03 in the absence of zosuquidar and 0.09 +/- 0.02 between 2 and 6 h after 2 mg/kg zosuquidar to 0.85 +/- 0.19 after 6 mg/kg and 1.58 +/- 0.67 after 20 mg/kg zosuquidar. Zosuquidar brain tissue/plasma concentration ratios exhibited a similar abrupt increase from 2.8 +/- 0.3 after a 2 mg/kg dose to approximately 15 after the 6 and 20 mg/kg doses. The apparent threshold in the plasma concentration of zosuquidar necessary to produce significant enhancement in brain uptake of nelfinavir appears to be close to the plasma concentrations associated with the maximum tolerated dose reported in the literature after repeated dosing of zosuquidar in patients.

Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering.

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.