Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase.

A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C ß-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.

Association of Common and Rare Variants with Alzheimer's Disease in over 13,000 Diverse Individuals with Whole-Genome Sequencing from the Alzheimer's Disease Sequencing Project.

Alzheimer's Disease (AD) is a common disorder of the elderly that is both highly heritable and genetically heterogeneous. Here, we investigated the association between AD and both common variants and aggregates of rare coding and noncoding variants in 13,371 individuals of diverse ancestry with whole genome sequence (WGS) data. Pooled-population analyses identified genetic variants in or near APOE, BIN1, and LINC00320 significantly associated with AD (p < 5×10-8). Population-specific analyses identified a haplotype on chromosome 14 including PSEN1 associated with AD in Hispanics, further supported by aggregate testing of rare coding and noncoding variants in this region. Finally, we observed suggestive associations (p < 5×10-5) of aggregates of rare coding rare variants in ABCA7 among non-Hispanic Whites (p=5.4×10-6), and rare noncoding variants in the promoter of TOMM40 distinct of APOE in pooled-population analyses (p=7.2×10-8). Complementary pooled-population and population-specific analyses offered unique insights into the genetic architecture of AD.

Association between resilience promotion factors during childhood and risk of drug use disorder during adulthood.

Few studies have been conducted on the relationship between "outside-residing" resilience characteristics and the risk of developing drug use disorder later in life. These characteristics include responsive and caring parenting, household routines involving regular family meals and bedtime routines, social support from peers, participation in organized activities, and religious service attendance. We quantified the association between these resilience promotion factors during childhood and the risk of developing criteria for drug use disorder during adulthood using data from a retrospective cohort study of 618 adults born in Massachusetts during 1969-1983, including those with adverse childhood experiences (ACEs). Self-administered questionnaires gathered information on criteria for drug use disorder, ACEs, and family and community resilience promotion factors. Compared to individuals with "low" numbers of resilience promotion factors, 30% (95% CI: 0.5-0.9) and 50% reductions (95% CI: 0.4-0.8) in the risk of developing one or more criteria for drug use disorder were observed among those with "moderate" and "high" numbers of resilience factors, respectively (p value for trend=0.003). Overall, family factors were associated with greater risk reductions than comparable numbers of community factors. Among individuals with ACEs, a "high" number of family factors but not community factors were associated with a reduction in risk (RR:0.6, 95% CI:0.4-1.0 for family factors, RR:1.0, 95% CI:0.5-1.8 for community factors). These results suggest that the risk of developing criteria for drug use disorder decreases in a dose-response fashion according to the number of "outside-residing" resilience promotion factors during childhood, and that family factors are associated with greater risk reductions than community factors, particularly among individuals with ACEs. Coordinated prevention efforts at the family and community level are recommended to reduce the risk of this important societal problem.

Testing the validity and reliability of the Person-Centered Contraceptive Counseling (PCCC) Performance Measure in a Real-World Contraceptive Access Program.

OBJECTIVES: To validate the Person-Centered Contraceptive Counseling (PCCC) patient-reported outcome performance measure and assess for differences by sociodemographic attributes using survey data from a multistate contraceptive access program. STUDY DESIGN: This analysis explored internal reliability and construct validity of the PCCC using survey data from 1413 patients who visited 15 health centers in Washington state and Massachusetts that had partnered with Upstream USA. RESULTS: Multiple psychometric indicators provided evidence of reliability and validity. Significant associations between the highest PCCC rating and conceptually-related survey questions (i.e., experience with bias/coercion and shared decision-making) provided further evidence of construct validity. CONCLUSIONS: Our findings demonstrate that the PCCC is valid and reliable. The results also highlight differences in experience of care by patient-reported race and ethnicity, income level, and language.

Associations of household unmet basic needs and health outcomes among very low birth weight children.

OBJECTIVE: We examined associations of past year household hardships (housing, energy, food, and healthcare hardships) with postnatal growth, developmental risk, health status, and hospitalization among children 0-36 months born with very low birth weight (VLBW) and the extent that these relationships differed by receipt of child supplemental security income (SSI). STUDY DESIGN: We examined cross-sectional data from 695 families. Growth was measured as weight-for-age z-score change. Developmental risk was defined as ≥1 concerns on the "Parents' Evaluation of Developmental Status" screening tool. Child health status was categorized as excellent/good vs. fair/poor. Hospitalizations excluded birth hospitalizations. RESULTS: Compared to children with no household hardships, odds of developmental risk were greater with 1 hardship (aOR 2.0 [1.26, 3.17]) and ≥2 hardships (aOR) 1.85 [1.18, 2.91], and odds of fair/poor child health (aOR) 1.59 [1.02, 2.49] and hospitalizations (aOR) 1.49 [1.00, 2.20] were greater among children with ≥2 hardships. In stratified analysis, associations of hardships and developmental risk were present for households with no child SSI and absent for households with child SSI. CONCLUSION: Household hardships were associated with developmental risk, fair/poor health status, and hospitalizations among VLBW children. Child SSI may be protective against developmental risk among children living in households with hardships.

Prenatal and early childhood exposure to tetrachloroethylene (PCE) and non-medical use of prescription drugs: A retrospective cohort study in Cape Cod, MA.

BACKGROUND: Between 1968 and 1983, public drinking water supplies of Cape Cod, Massachusetts were contaminated with the chlorinated solvent tetrachloroethylene (PCE). We previously found an affinity for risk-taking behaviors, including the use of illicit drugs, following prenatal and early childhood exposure to PCE. Using newly collected data, we investigated the risk of non-medical use of prescription drugs (NMUPD) following prenatal and early childhood PCE exposure. METHODS: Participants were identified from a retrospective cohort study ("Cape Cod Health Study") via cross-matching birth certificates and water system data. The original self-administered questionnaire gathered data on demographics, work and medical history, and alcohol and illicit drug use from 618 individuals (363 exposed and 255 unexposed). The follow-up survey added questions on non-medical use of prescription pain relievers, tranquilizers, stimulants and sedatives. A validated leaching and transport model was used to estimate exposure to PCE exposure in drinking water. RESULTS: There was a wide distribution of cumulative prenatal and early childhood PCE exposure levels (range: 0.04 g-3722.2 g). PCE exposed subjects had a 1.92-fold increase in risk of any non-medical use of prescription drugs [Adjusted RR: 1.92, (95% CI: 1.31, 2.83)]. Furthermore, the association followed a dose-response relationship where the risk of NMUPD was higher for those exposed to PCE levels greater than or equal the median level versus those exposed to levels less than the median [Adjusted RR: 2.05 (95% CI: 1.34, 3.15) vs. 1.83 (95% CI: 1.20, 2.79) (p-value for trend < 0.01)]. Additionally, we found moderate increases in risk by level of non-medical use (any non-medical use, non-medical use of 1 or more categories of prescription drugs, or 2+ categories) as well as by category of drug for pain relivers, stimulants and tranquilizers. CONCLUSION: We found that prenatal and early childhood exposure to PCE was associated with a moderate increase in the risk of NMUPD. Exposed subjects had dose-related increased risks of NMUPD of pain relievers, tranquilizers, and stimulants. This study has a number of limitations and is the first to report this association. Additional longitudinal studies of populations exposed to PCE during early life should be conducted to examine its long-term neurotoxic effects.

Drug use disorder following early life exposure to tetrachloroethylene (PCE)-contaminated drinking water: a retrospective cohort study.

BACKGROUND: Many studies of adults with occupational exposure to solvents such as tetrachloroethylene (PCE) have shown adverse effects on cognition, mood and behavioral problems. Much less is known about neurotoxic effects in early life at lower exposure levels seen in community settings. We recently reported that illicit drug use was more frequent among adults from Cape Cod, Massachusetts who were exposed to PCE-contaminated drinking water during gestation and early childhood than their unexposed counterparts. Using newly collected data from this population-based retrospective cohort study, the current analysis examines whether early life PCE exposure is also associated with drug use disorder over the life course. METHODS: Three-hundred and sixty-three subjects with prenatal and early childhood PCE exposure and 255 unexposed subjects were studied. These individuals (median age: 40-41 years) completed self-administered questionnaires on the eleven established diagnostic criteria for drug use disorder and confounding variables. A validated leaching and transport model was used to estimate exposure to PCE-contaminated water. RESULTS: Overall, 23.3% of subjects reported having at least one criterion for drug use disorder over their lifetime. Early life PCE exposure was associated with a modest increase in the lifetime presence of one or more diagnostic criteria for drug use disorder (adjusted RR: 1.4, 95% CI: 1.0-1.8). Compared to unexposed subjects, PCE-exposed subjects were more likely to report having most diagnostic criteria of drug use disorder, including neglecting major roles due to drug use, physical and psychological problems related to drug use, and giving up activities due to drug use. No dose-response relationships were observed with increasing levels of PCE exposure. CONCLUSIONS: These results suggest that exposure to PCE-contaminated drinking water during early life modestly increases the risk of developing diagnostic criteria for drug use disorder later in life. Because this study has several limitations, these findings should be confirmed in follow-up investigations of other exposed populations with more diverse racial and socioeconomic characteristics.

A γ-Lactam Siderophore Antibiotic Effective against Multidrug-Resistant Gram-Negative Bacilli.

Treatment of multidrug-resistant Gram-negative bacterial pathogens represents a critical clinical need. Here, we report a novel γ-lactam pyrazolidinone that targets penicillin-binding proteins (PBPs) and incorporates a siderophore moiety to facilitate uptake into the periplasm. The MIC values of γ-lactam YU253434, 1, are reported along with the finding that 1 is resistant to hydrolysis by all four classes of ß-lactamases. The druglike characteristics and mouse PK data are described along with the X-ray crystal structure of 1 binding to its target PBP3.

Analytical Representations for Characterizing the Global Aviation Radiation Environment Based on Model and Measurement Databases.

The Nowcast of Atmospheric Ionizing Radiation for Aviation Safety climatological model and the Automated Radiation Measurements for Aerospace Safety (ARMAS) statistical database are presented as polynomial fit equations. Using equations based on altitude, L shell, and geomagnetic conditions an effective dose rate for any location from a galactic cosmic ray (GCR) environment can be calculated. A subset of the ARMAS database is represented by a second polynomial fit equation for the GCR plus probable relativistic energetic particle (REP; Van Allen belt REP) effective dose rates within a narrow band of L shells with altitudinal and geomagnetic dependency. Solar energetic particle events are not considered in this study since our databases do not contain these events. This work supports a suggestion that there may be a REP contribution having an effect at aviation altitudes. The ARMAS database is rich in Western Hemisphere observations for L shells between 1.5 and 5; there have been many cases of enhanced radiation events possibly related to effects from radiation belt particles. Our work identifies that the combined effects of an enhanced radiation environment in this L shell range are typically 15% higher than the GCR background. We also identify applications for the equations representing the Nowcast of Atmospheric Ionizing Radiation for Aviation Safety and ARMAS databases. They include (i) effective dose rate climatology in comparison with measured weather variability and (ii) climatological and statistical weather nowcasting and forecasting. These databases may especially help predict the radiation environment for regional air traffic management, for airport overflight operations, and for air carrier route operations of individual aircraft.

Risk factors for invasive fungal disease in heart transplant recipients.

BACKGROUND: Heart transplant (HT) recipients are at risk for invasive fungal disease (IFD), a morbid and potentially fatal complication. METHODS: We performed a retrospective cohort study to evaluate the incidence and risk factors for IFD in HT recipients from 1995 to 2012 at a single center. IFD cases were classified as proven or probable IFD according to current consensus definitions of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. We calculated IFD incidence rates and used Cox proportional hazards models to determine IFD risk factors. RESULTS: Three hundred sixty patients underwent HT during the study period. The most common indications were dilated (39%) and ischemic (37%) cardiomyopathy. There were 23 (6.4%) cases of proven (21) or probable (2) IFD, for a cumulative incidence rate of 1.23 per 100 person-years (95% CI 0.78 to 1.84). Candida (11) and Aspergillus (5) were the most common etiologic fungi. Thirteen cases (56%) occurred within 3 months of HT, with a 3-month incidence of 3.8% (95% CI 2.2 to 6.4). Delayed chest closure (HR 3.3, 95% CI 1.4 to 7.6, p = 0.01) and the addition of OKT3, anti-thymocyte globulin or daclizumab to standard corticosteroid induction therapy (HR 2.7, 95% CI 1.1 to 6.2, p = 0.02) were independently associated with an increased risk of IFD. CONCLUSIONS: IFD incidence was greatest within the first 3 months post-HT, largely reflecting early surgical-site and nosocomial Candida and Aspergillus infections. Patients receiving additional induction immunosuppression or delayed chest closure were at increased risk for IFD. Peri-transplant anti-fungal prophylaxis should be considered in this subset of HT recipients.

A breath fungal secondary metabolite signature to diagnose invasive aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) remains a leading cause of mortality in immunocompromised patients, in part due to the difficulty of diagnosing this infection. METHODS: Using thermal desorption-gas chromatography/mass spectrometry, we characterized the in vitro volatile metabolite profile of Aspergillus fumigatus, the most common cause of IA, and other pathogenic aspergilli. We prospectively collected breath samples from patients with suspected invasive fungal pneumonia from 2011 to 2013, and assessed whether we could discriminate patients with proven or probable IA from patients without aspergillosis, as determined by European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions, by direct detection of fungal volatile metabolites in these breath samples. RESULTS: The monoterpenes camphene, α- and ß-pinene, and limonene, and the sesquiterpene compounds α- and ß-trans-bergamotene were distinctive volatile metabolites of A. fumigatus in vitro, distinguishing it from other pathogenic aspergilli. Of 64 patients with suspected invasive fungal pneumonia based on host risk factors, clinical symptoms, and radiologic findings, 34 were diagnosed with IA, whereas 30 were ultimately diagnosed with other causes of pneumonia, including other invasive mycoses. Detection of α-trans-bergamotene, ß-trans-bergamotene, a ß-vatirenene-like sesquiterpene, or trans-geranylacetone identified IA patients with 94% sensitivity (95% confidence interval [CI], 81%-98%) and 93% specificity (95% CI, 79%-98%). CONCLUSIONS: In patients with suspected fungal pneumonia, an Aspergillus secondary metabolite signature in breath can identify individuals with IA. These results provide proof-of-concept that direct detection of exogenous fungal metabolites in breath can be used as a novel, noninvasive, pathogen-specific approach to identifying the precise microbial cause of pneumonia.

BK virus disease after allogeneic stem cell transplantation: a cohort analysis.

The clinical epidemiology of BK virus (BKV) disease after allogeneic hematopoietic stem cell transplantation (HSCT) is not well defined. We evaluated 491 patients transplanted from January 2010 to December 2011 at a single transplant center to assess incidence, severity, and risk factors for BKV disease after HSCT. BKV disease was defined as BKV detection in urine by PCR testing in association with genitourinary symptoms without other concurrent genitourinary conditions. BKV disease occurred in 78 patients (15.9%), for an incidence rate of .47/1000 patient-days (95% confidence interval [CI], .37 to .59); BKV disease was considered severe in 27 patients (5.5%). In multivariate Cox modeling, time-dependent acute graft-versus-host disease (aGVHD) grades II to IV (adjusted hazard ratio [aHR] 4.25; 95% CI, 2.51 to 7.21), cord blood HSCT (aHR 2.28; 95% CI, 1.01 to 5.15), post-transplant mycophenolate use (aHR 3.31; 95% CI, 1.83 to 5.99), and high-dose cyclophosphamide conditioning (aHR 2.34, 95% CI 1.45 to 3.77) were significant predictors of BKV disease. Time-dependent aGVHD grades III to IV (aHR 10.5; 95% CI, 4.44 to 25.0) and cord blood HSCT (aHR 5.40; 95% CI, 1.94 to 15.0) were independent risk factors for severe BKV disease. BKV disease is common and is associated with significant and prolonged morbidity after HSCT. Prospective studies are needed to better define the morbidity of post-HSCT BKV disease and inform the design of prophylaxis and treatment trials.

Live attenuated varicella-zoster vaccine in hematopoietic stem cell transplantation recipients.

Hematopoietic stem cell transplantation (HSCT) recipients are at risk for varicella-zoster virus (VZV) reactivation. Vaccination may help restore VZV immunity; however, the available live attenuated VZV vaccine (Zostavax) is contraindicated in immunocompromised hosts. We report our experience with using a single dose of VZV vaccine in 110 adult autologous and allogeneic HSCT recipients who were about 2 years after transplantation, free of graft-versus-host disease, and not receiving immunosuppression. One hundred eight vaccine recipients (98.2%) had no clinically apparent adverse events with a median follow-up period of 9.5 months (interquartile range, 6 to 16; range, 2 to 28). Two vaccine recipients (1.8%) developed a skin rash (one zoster-like rash with associated pain, one varicella-like) within 42 days post-vaccination that resolved with antiviral therapy. We could not confirm if these rashes were due to vaccine (Oka) or wild-type VZV. No other possible cases of VZV reactivation have occurred with about 1178 months of follow-up. Live attenuated zoster vaccine appears generally safe in this population when vaccinated as noted; the overall vaccination risk needs to be weighed against the risk of wild-type VZV disease in this high-risk population.

Coexistence of colonic carcinoma, renal cell carcinoma and gastrointestinal stromal tumour-A case report.

The occurrence of synchronous primary neoplasms remains an issue of great interest to surgeons and oncologists in particular, and the medical field in general. The question of common genetic pathways in the pathogenesis of such neoplasms is always raised when such associations are seen-whether metachronously or synchronously. The possibility of the coexistence of multiple tumours in the same patient must be taken into consideration when preparing patients for operation and a thorough search of the intraperitoneal organs for such coexistence remains important.A case of synchronously resected caecal carcinoma, jejunal gastrointestinal stromal tumour and renal cell carcinoma is presented here, along with a literature review on synchronous tumour resection.

Nonylphenolic compounds in drinking and surface waters downstream of treated textile and pulp and paper effluents: a survey and preliminary assessment of their potential effects on public health and aquatic life.

Eleven drinking water treatment plants, located downstream of textile plants or pulp and paper mills, have been sampled monthly during a year for the analysis of 17 nonylphenol ethoxylates (NP1-17EO) and two nonylphenoxycarboxylic acids (NP1-2EC). At all but one plant, results in the drinking water, for the sum of these 19 substances, range between below detection levels and 6.7 microg/l. Annual means are between 0.02 and 2.8 microg/l. At the other plant, the yearly average concentration is 10.4 microg/l and the monthly maximum is 43.3 microg/l. In the surface (pre-treatment) water, the annual mean concentrations of the 11 plants range between 0.14 and 17.8 microg/l and the recorded instantaneous maximum is 55.3 microg/l. According to Canadian health authorities, drinking water is a negligible route of human exposure to nonylphenolic compounds, even at the highest concentrations found in this study. After transformation of the data into nonylphenol equivalents, about 20% of the surface water samples exceed the Canadian 1 microg/l nonylphenol water quality guideline for the protection of aquatic life. Some results also exceed Québec's 6 microg/l nonylphenol guideline. The efficiency of the plants in removing nonylphenolic compounds from drinking water is highly variable, ranging from 11% to 99%.

ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets.

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.