GABAergic/Glycinergic and Glutamatergic Neurons Mediate Distinct Neurodevelopmental Phenotypes of STXBP1 Encephalopathy.

An increasing number of pathogenic variants in presynaptic proteins involved in the synaptic vesicle cycle are being discovered in neurodevelopmental disorders. The clinical features of these synaptic vesicle cycle disorders are diverse, but the most prevalent phenotypes include intellectual disability, epilepsy, movement disorders, cerebral visual impairment, and psychiatric symptoms ( Verhage and Sørensen, 2020; Bonnycastle et al., 2021; John et al., 2021; Melland et al., 2021). Among this growing list of synaptic vesicle cycle disorders, the most frequent is STXBP1 encephalopathy caused by de novo heterozygous pathogenic variants in syntaxin-binding protein 1 (STXBP1, also known as MUNC18-1; Verhage and Sørensen, 2020; John et al., 2021). STXBP1 is an essential protein for presynaptic neurotransmitter release. Its haploinsufficiency is the main disease mechanism and impairs both excitatory and inhibitory neurotransmitter release. However, the disease pathogenesis and cellular origins of the broad spectrum of neurological phenotypes are poorly understood. Here we generate cell type-specific Stxbp1 haploinsufficient male and female mice and show that Stxbp1 haploinsufficiency in GABAergic/glycinergic neurons causes developmental delay, epilepsy, and motor, cognitive, and psychiatric deficits, recapitulating majority of the phenotypes observed in the constitutive Stxbp1 haploinsufficient mice and STXBP1 encephalopathy. In contrast, Stxbp1 haploinsufficiency in glutamatergic neurons results in a small subset of cognitive and seizure phenotypes distinct from those caused by Stxbp1 haploinsufficiency in GABAergic/glycinergic neurons. Thus, the contrasting roles of excitatory and inhibitory signaling reveal GABAergic/glycinergic dysfunction as a key disease mechanism of STXBP1 encephalopathy and suggest the possibility to selectively modulate disease phenotypes by targeting specific neurotransmitter systems.

Early and exclusive enteral nutrition in infants born very preterm.

OBJECTIVE: To characterise the effects of early and exclusive enteral nutrition with either maternal or donor milk in infants born very preterm (280/7-326/7 weeks of gestation). DESIGN: Parallel-group, unmasked randomised controlled trial. SETTING: Regional, tertiary neonatal intensive care unit. PARTICIPANTS: 102 infants born very preterm between 2021 and 2022 (51 in each group). INTERVENTION: Infants randomised to the intervention group received 60-80 mL/kg/day within the first 36 hours after birth. Infants randomised to the control group received 20-30 mL/kg/day (standard trophic feeding volumes). MAIN OUTCOME MEASURES: The primary outcome was the number of full enteral feeding days (>150 mL/kg/day) in the first 28 days after birth. Secondary outcomes included growth and body composition at the end of the first two postnatal weeks, and length of hospitalisation. RESULTS: The mean birth weight was 1477 g (SD: 334). Half of the infants were male, and 44% were black. Early and exclusive enteral nutrition increased the number of full enteral feeding days (+2; 0-2 days; p=0.004), the fat-free mass-for-age z-scores at postnatal day 14 (+0.5; 0.1-1.0; p=0.02) and the length-for-age z-scores at the time of hospital discharge (+0.6; 0.2-1.0; p=0.002). Hospitalisation costs differed between groups (mean difference favouring the intervention group: -$28 754; -$647 to -$56 861; p=0.04). CONCLUSIONS: In infants born very preterm, early and exclusive enteral nutrition increases the number of full enteral feeding days. This feeding practice may also improve fat-free mass accretion, increase length and reduce hospitalisation costs. TRIAL REGISTRATION NUMBER: NCT04337710.

A decade of progress in juvenile idiopathic athritis treatments and outcomes in Canada: results from ReACCh-Out and the CAPRI registry.

OBJECTIVE: To assess changes in juvenile idiopathic arthritis (JIA) treatments and outcomes in Canada, comparing a 2005-2010 and a 2017-2021 inception cohorts. METHODS: Patients enrolled within three months of diagnosis in the Research in Arthritis in Canadian Children Emphasizing Outcomes (ReACCh-Out) and the Canadian Alliance of Pediatric Rheumatology Investigators Registry (CAPRI) cohorts were included. Cumulative incidences of drug starts and outcome attainment within 70 weeks of diagnosis were compared with Kaplan Meier survival analysis and multivariable Cox regression. RESULTS: The 2005-2010 and 2017-2021 cohorts included 1128 and 721 patients, respectively. JIA category distribution and baseline clinical juvenile idiopathic arthritis disease activity (cJADAS10) scores at enrolment were comparable. By 70 weeks, 6% of patients (95% CI 5, 7) in the 2005-2010 and 26% (23, 30) in the 2017-2021 cohort had started a biologic DMARD (bDMARD), and 43% (40, 47) and 60% (56, 64) had started a conventional DMARD (cDMARD), respectively. Outcome attainment was 64% (61, 67) and 83% (80, 86) for Inactive disease (Wallace criteria), 69% (66, 72) and 84% (81, 87) for minimally active disease (cJADAS10 criteria), 57% (54, 61) and 63% (59, 68) for pain control (<1/10), and 52% (47, 56) and 54% (48, 60) for a good health-related quality of life. CONCLUSION: Although baseline disease characteristics were comparable in the 2005-2010 and 2017-2021 cohorts, cDMARD and bDMARD use increased with a concurrent increase in minimally active and inactive disease. Improvements in parent and patient reported outcomes were smaller than improvements in disease activity.

Early Human Milk Fortification in Infants Born Extremely Preterm: A Randomized Trial.

OBJECTIVES: Enteral nutrition with unfortified human milk during the first 2 postnatal weeks often leads to cumulative protein and energy deficits among preterm infants. Fortified human milk administered soon after birth could increase fat-free mass (FFM) and improve growth in these infants. METHODS: This was a masked, randomized trial. Starting on feeding day 2, extremely preterm infants 28 weeks or younger fed maternal or donor milk were randomized to receive either a diet fortified with a human-based product (intervention group) or a standard, unfortified diet (control group). This practice continued until the feeding day when a standard bovine-based fortifier was ordered. Caregivers were masked. The primary outcome was FFM-for-age z score at 36 weeks of postmenstrual age (PMA). RESULTS: A total of 150 infants were randomized between 2020 and 2022. The mean birth weight was 795±250 g, and the median gestational age was 26 weeks. Eleven infants died during the observation period. The primary outcome was assessed in 105 infants (70%). FFM-for-age z scores did not differ between groups. Length gain velocities from birth to 36 weeks PMA were higher in the intervention group. Declines in head circumference-for-age z score from birth to 36 weeks' PMA were less pronounced in the intervention group. CONCLUSIONS: In infants born extremely preterm, human milk diets fortified soon after birth do not increase FFM accretion at 36 weeks' PMA, but they may increase length gain velocity and reduce declines in head circumference-for-age z scores from birth to 36 weeks' PMA.

Exploring female otolaryngologists' experiences with gender bias and microaggressions: a cross sectional Canadian survey.

BACKGROUND: Gender bias is behavior that shows favoritism towards one gender over another. Microaggressions are defined as subtle, often unconscious, discriminatory, or insulting actions that communicate demeaning or negative attitudes. Our objective was to explore how female otolaryngologists experience gender bias and microaggressions in the workplace. METHODS: Anonymous web-based cross-sectional Canadian survey was distributed to all female otolaryngologists (attendings and trainees) using the Dillman's Tailored Design Method from July to August of 2021. Quantitative survey included demographic data, validated 44-item Sexist Microaggressions Experiences and Stress Scale (MESS) and validated 10-item General Self-efficacy scale (GSES). Statistical analysis included descriptive and bivariate analysis. RESULTS: Sixty out of 200 participants (30% response rate) completed the survey (mean age 37 ± 8.3 years, 55.0% white, 41.7% trainee, 50% fellowship-trained, 50% with children, mean 9.2 ± 7.4 years of practice). Participants scored mild to moderate on the Sexist MESS-Frequency (mean ± standard deviation) 55.8 ± 24.2 (42.3% ± 18.3%), Severity 46.0 ± 23.9 (34.8% ± 18.1%), Total 104.5 ± 43.7 (39.6% ± 16.6%) and high on GSES (32.7 ± 5.7). Sexist MESS score was not associated with age, ethnicity, fellowship-training, having children, years of practice, or GSES. In the sexual objectification domain, trainees had higher frequency (p = 0.04), severity (p = 0.02) and total MESS (p = 0.02) scores than attendings. CONCLUSIONS: This was the first multicenter, Canada-wide study exploring how female otolaryngologists experience gender bias and microaggressions in the workplace. Female otolaryngologists experience mild to moderate gender bias, but have high self-efficacy to manage this issue. Trainees had more severe and frequent microaggressions than attendings in the sexual objectification domain. Future efforts should help develop strategies for all otolaryngologists to manage these experiences, and thereby improve the culture of inclusiveness and diversity in our specialty.

Total Fluid Administration and Weight Loss during the First 2 Weeks in Infants Randomized to Early Enteral Feeding after Extremely Preterm Birth.

BACKGROUND: Randomized trials have not reported the effects of the early progression of feeding volumes on fluid balance and neurodevelopment among infants born extremely preterm (≤28 weeks). METHOD: Fluid, electrolyte, and neurodevelopment data of 60 extremely preterm infants randomly assigned to receive either 1 (early feeding group) or 4 days (late feeding group) of trophic feeding volumes at 20-24 mL/kg/day were analyzed. RESULTS: Infants randomized to the early feeding group received less parenteral fluids, generated lower urine volumes, and had less excessive weight loss during the first 14 days after birth. The 7-point difference in cognitive scores and the 0.5 difference in weight-for-age z-scores favoring the early feeding group did not reach statistical significance. CONCLUSIONS: In extremely preterm infants, early enteral feeding is associated with less total fluid administration and with less excessive weight loss during the first 2 weeks after birth. These short-term effects could have long-lasting benefits.

Introducing immunohistochemistry to the molecular biology laboratory.

Widely used in research laboratories, immunohistochemistry (IHC) is a transferable skill that prepares undergraduate students for a variety of careers in the biomedical field. We have developed an inquiry-based learning IHC laboratory exercise, which introduces students to the theory, procedure, and data interpretation of antibody staining. Students are tasked with performing IHC using an "unknown" antibody and then asked to identify the cells or molecular structures within the nervous systems specific for that unknown antibody. In two lab sessions, students are exposed to handling of delicate brain slices, fluorescent microscopy, and data analysis using the Allen Brain Atlas (ABA), an online freely accessible database of mRNA transcript expression patterns in the brain. Here, we present guidelines for easy implementation in the classroom and assess learning gains achieved by the students upon completion of the IHC laboratory module. Students clearly displayed an increase in knowledge in data interpretation, procedural knowledge, and theory surrounding IHC. Thus, this module works as an inquiry-based learning based method to introduce IHC principles to undergraduate students.

Prevalence of blaCTX-M Genes in Gram-Negative Bloodstream Isolates across 66 Hospitals in the United States.

Understanding bacterial species at greatest risk for harboring blaCTX-M genes is necessary to guide antibiotic treatment. We identified the species-specific prevalence of blaCTX-M genes in Gram-negative clinical isolates from the United States. Twenty-four microbiology laboratories representing 66 hospitals using the GenMark Dx ePlex blood culture identification Gram-negative (BCID-GN) panel extracted blood culture results from April 2019 to July 2020. The BCID-GN panel includes 21 Gram-negative targets. Along with identifying blaCTX-M genes, it detects major carbapenemase gene families. A total of 4,209 Gram-negative blood cultures were included. blaCTX-M genes were identified in 462 (11%) specimens. The species-specific prevalence of blaCTX-M genes was as follows: Escherichia coli (16%), Klebsiella pneumoniae (14%), Klebsiella oxytoca (6%), Salmonella spp. (6%), Acinetobacter baumannii (5%), Enterobacter species (3%), Proteus mirabilis (2%), Serratia marcescens (0.6%), and Pseudomonas aeruginosa (0.5%). blaCTX-M prevalence was 26%, 24%, and 22% among participating hospitals in the District of Columbia, New York, and Florida, respectively. Carbapenemase genes were identified in 61 (2%) organisms with the following distribution: blaKPC (59%), blaVIM (16%), blaOXA (10%), blaNDM (8%), and blaIMP (7%). The species-specific prevalence of carbapenemase genes was as follows: A. baumannii (5%), K. pneumoniae (3%), P. mirabilis (3%), Enterobacter species (3%), Citrobacter spp. (3%), P. aeruginosa (2%), E. coli (<1%), K. oxytoca (<1%), and S. marcescens (<1%). Approximately 11% of Gram-negative organisms in our US cohort contain blaCTX-M genes. blaCTX-M genes remain uncommon in organisms beyond E. coli, K. pneumoniae, and K. oxytoca Future molecular diagnostic panels would benefit from the inclusion of plasmid-mediated ampC and SHV and TEM extended-spectrum beta-lactamase (ESBL) targets.

HIV incidence during breastfeeding and mother-to-child transmission in Cape Town, South Africa.

: Despite widespread concerns about HIV incidence in pregnant and postpartum women, there are few data from Africa on HIV acquisition in breastfeeding and subsequent mother-to-child transmission. We measured HIV incidence in a prospective cohort of 413 peripartum and breastfeeding women who tested HIV-negative during pregnancy. In 377 woman-years accrued postpartum (median duration of follow-up, 1 year), there were seven women infected after delivery (postpartum incidence, 1.86/100 person-years; 95% confidence interval 0.88-3.89) with transmission to 2/7 (28%) infants.

Growth trajectories of breastfed HIV-exposed uninfected and HIV-unexposed children under conditions of universal maternal antiretroviral therapy: a prospective study.

BACKGROUND: Over 1 million HIV-exposed uninfected (HEU) children are born in sub-Saharan Africa annually. Little data exist on the risk of impaired growth in this population under current policies of universal maternal antiretroviral therapy (ART) with breastfeeding. We aimed to study the growth of breastfed HEU children born to women who initiated ART during pregnancy and compare their growth with that of breastfed HIV-unexposed (HU) children drawn from the same community. METHODS: A prospective cohort of HIV-uninfected and HIV-infected pregnant women, who were initiating ART, were enrolled at their first antenatal care visit in a primary care centre in Gugulethu, Cape Town, South Africa. HIV infected women were participants of the Maternal Child Health Antiretroviral Therapy (MCH-ART) study, and HIV-uninfected pregnant women were participants in the HIV-Unexposed-Uninfected (HU2) study. All women were followed up during pregnancy, through delivery, to the early postnatal visit, which was scheduled for the first week after birth. At this visit, eligible breastfeeding mother-child pairs were recruited for continuation of postnatal follow-up until approximately age 12 months. Child anthropometry was measured at around 6 weeks, and every 3 months from month 3 to month 12. Weight-for-age (WAZ), length-for-age (LAZ), weight-for-length (WLZ), head circumference-for-age, and body-mass index-for-age Z scores were compared between HEU and HU children longitudinally using mixed effects linear regression. At 12 months, proportions of HEU and HU children with moderate or severe malnutrition were compared cross-sectionally using logistic regression. MCH-ART is registered with ClinicalTrials.gov, number NCT01933477. FINDINGS: Between June, 2013, and April, 2016, 884 breastfeeding mothers and their newborn babies (HEU, n=471; HU, n=413) were enrolled into postnatal follow-up. Excluding 12 children who tested HIV positive during follow-up, 461 HEU and 411 HU children attended 4511 study visits in total, with a median of 6 visits (IQR 5-6) per child. Birth characteristics were similar (overall, 94 [11%] of 872 preterm [<37 weeks] and 90 [10%] small-for-gestational age [birthweight <10th percentile]). Median duration of breastfeeding was shorter among HEU than HU children (3·9 months [IQR 1·4-12·0] vs 9·0 months [IQR 3·0-12·0]). Although WAZ scores increased over time in both groups, HEU children had consistently lower mean WAZ scores than HU children (overall ß -0·34, 95% CI -0·47 to -0·21). LAZ scores decreased in both groups after 9 months. At 12 months, HEU children had lower mean LAZ scores than HU children (ß -0·43, -0·61 to -0·25), with a higher proportion of children stunted (LAZ score <-2: 35 [10%] of 342 HEU vs 14 [4%] of 342 HU children; odds ratio [OR] 2·67, 95% CI 1·41 to 5·06). Simultaneously, overweight (WLZ score >2) was common in both groups of children at 12 months (54 [16%] of 342 HEU vs 60 [18%] of 340 HU children; OR 0·87, 95% CI 0·58 to 1·31). INTERPRETATION: Compared with HU children, HEU children have small deficits in early growth trajectories under policies of universal maternal ART and breastfeeding. Large proportions of both HEU and HU children were overweight by 12 months, indicating substantial risks for early onset obesity among South African children. Although the longer-term metabolic effects of ART exposure in the context of childhood obesity warrants further investigation, addressing childhood obesity should be an urgent public health priority in this setting. FUNDING: Eunice Kennedy Shriver National Institute of Child Health and Human Development, Elizabeth Glaser Pediatric AIDS Foundation, South African Medical Research Council, and the Fogarty Foundation.

Point-of-Care Diagnosis of Acute Myocardial Infarction in Central Vietnam: International Exchange, Needs Assessment, and Spatial Care Paths.

OBJECTIVES: Objectives were to (a) advance point-of-care (POC) education, international exchange, and culture; (b) report needs assessment survey results from Thua Thien Hue Province, Central Vietnam; (c) determine diagnostic capabilities in regional health care districts of the small-world network of Hue University Medical Center; and (d) recommend Spatial Care Paths that accelerate the care of acute myocardial infarction (AMI) patients. METHODS: We organized progressively focused, intensive, and interactive lectures, workshops, and investigative teamwork over a 2-year period. We surveyed hospital staff in person to determine the status of diagnostic testing at 15 hospitals in 7 districts. Questions focused on cardiac rapid response, prediabetes/diabetes, infectious diseases, and other serious challenges, including epidemic preparedness. RESULTS: Educational exchange revealed a nationwide shortage of POC coordinators. Throughout the province, ambulances transfer patients primarily between hospitals, rarely picking up from homes. No helicopter rescue was available. Ambulance travel times from distant sites to referral hospitals were excessive, longer in costal and mountainous areas. Most hospitals (92.3%) used electrocardiogram and creatine phosphokinase-MB isoenzyme to diagnose AMI. Cardiac troponin I/T testing was performed only at large referral hospitals. CONCLUSIONS: Central Vietnam must improve rapid diagnosis and treatment of AMI patients. Early upstream POC cardiac troponin testing on Spatial Care Paths will expedite transfers directly to hospitals capable of intervening, improving outcomes following coronary occlusion. Point-of-care coordinator certification and financial support will enhance standards of care cost-effectively. Training young physicians pivots on high-value evidence-based learning when POC cardiac troponin T/cardiac troponin I biomarkers are in place for rapid decision making, especially in emergency rooms.

Neurodevelopment of breastfed HIV-exposed uninfected and HIV-unexposed children in South Africa.

OBJECTIVES: To assess neurodevelopment of breastfed HIV-exposed uninfected (HEU) and breastfed HIV-unexposed children in the context of universal maternal antiretroviral therapy (ART). DESIGN: Prospective study with antenatal enrolment and follow-up of breastfeeding HEU and HIV-unexposed mother-infant pairs through 12-18 months postpartum. SETTING: Peri-urban community, Cape Town, South Africa. PARTICIPANTS: HEU (n = 215) and HIV-unexposed (n = 306) children. MAIN OUTCOME MEASURES: Cognitive, motor and language development at median 13 (interquartile range 12-14) months of age: continuous and dichotomous Bayley Scales of Infant and Toddler Development Third Edition (delay defined as composite score <85). RESULTS: Incidence of preterm delivery (<37 weeks) was similar among HEU and HIV-unexposed children (11 vs. 9%, P = 0.31; median gestation 39 weeks); 48% were boys. Median breastfeeding duration was shorter among HEU vs. HIV-unexposed children (6 vs. 10 months). All HIV-infected mothers initiated lifelong ART (tenofovir-emtricitabine-efavirenz) antenatally. HEU (vs. HIV-unexposed) children had higher odds of cognitive delay [odds ratio (OR) 2.28 (95% confidence interval (CI) 1.13-4.60)] and motor delay [OR 2.10 (95% CI 1.03-4.28)], but not language delay, in crude and adjusted analysis. Preterm delivery modified this relationship for motor development: compared with term HIV-unexposed children, term HEU children had similar odds of delay, preterm HIV-unexposed children had five-fold increased odds of delay (adjusted OR 4.73, 95% CI 1.32; 16.91) and preterm HEU children, 16-fold increased odds of delay (adjusted OR 16.35, 95% CI 5.19; 51.54). CONCLUSION: Young HEU children may be at increased risk for cognitive and motor delay despite universal maternal ART and breastfeeding; those born preterm may be particularly vulnerable.

Ambient temperature and risk of cardiovascular events at labor and delivery: A case-crossover study.

BACKGROUND: Extreme ambient temperatures are linked to cardiac events in the general population, but this relationship is unclear among pregnant women. We estimated the associations and attributable risk between ambient temperature and the risk of cardiovascular event at labor/delivery, and investigated whether these associations vary by maternal race/ethnicity. METHODS: We identified 680 women with singleton deliveries affected by cardiovascular events across 12 US sites (2002-2008). Average daily temperature during the week before, delivery day, and each of the seven days before delivery was estimated for each woman. In a case-crossover analysis, exposures during these hazard periods were compared to two control periods before and after delivery using conditional logistic regression adjusted for other environmental factors. RESULTS: During the cold season (October-April), 1°C lower during the week prior to delivery was associated with a 4% (95% CI: 1-7%) increased risk of having a labor/delivery affected by cardiovascular events including cardiac arrest and stroke. During the warm season (May-September), 1°C higher during the week prior was associated with a 7% (95% CI: 3-12%) increased risk. These risks translated to 13.4 and 23.9 excess events per 100,000 singleton deliveries during the cold and warm season, respectively. During the warm season, the risks were more pronounced on days closer to delivery and Black women appeared to be more susceptible to the same temperature increase. CONCLUSION: Small changes in temperature appear to affect the risk of having cardiovascular events at labor/delivery. Black women had a differentially higher warm season risk. These findings merit further investigation.

Clinical outcomes of HIV-exposed, HIV-uninfected children in sub-Saharan Africa.

OBJECTIVE: HIV-exposed but HIV-uninfected (HEU) children are widely considered at increased risk of mortality and morbidity. Recent advances in prevention of mother-to-child HIV transmission (PMTCT) strategies, incorporating life-long universal maternal antiretroviral therapy (ART, "Option B+") with extended breastfeeding, may improve HEU child health substantially. We critically reviewed reports of mortality/morbidity among HEU and HIV-unexposed (HU) children in sub-Saharan Africa. METHODS: We searched Medline, EMBASE, CINAHL, PsycINFO, Academic Search Premier, Global Health & Psychosocial Instruments databases, conference abstracts, and reference lists for longitudinal studies from sub-Saharan Africa reporting mortality and clinical morbidity among HIV-uninfected children aged ≤10 years, by maternal HIV status. Studies were appraised by Newcastle-Ottawa Scale and ACROBAT-NRSI. Due to substantial heterogeneity of study designs, populations and results (I(2) = 75%), data were not synthesised. RESULTS: We included 37 reports (28 studies, 11 164 HEU children); methodological and reporting quality were variable. Most reports came from settings without universal access to maternal ART (n = 35). Results were conflicting, with some studies indicating increased risk of mortality, hospitalisation and/or under-nutrition among HEU children, while others found no evidence of increased risk. In subanalyses, improved maternal health, ART use and breastfeeding were strongly protective for all outcomes. Only 39% (11/28) of studies adjusted for major confounders. Reports from settings using universal maternal ART with breastfeeding (n = 2) found no differences in growth or development but did not report mortality or infectious morbidity. CONCLUSIONS: The existing literature provides little insight into HEU child health under recently adopted PMTCT strategies. There is a need for robust comparative data on HEU and HIV-unexposed child health outcomes under Option B+; optimising breastfeeding practices and increasing maternal use of ART should be urgent public health priorities.

Prevalence of Enamel Markings on Third Molars.

The purpose of this study was to measure the prevalence of enamel markings in routinely extracted third molars. One hundred donated third molars were examined. All had some marking(s). Caries was almost universal; white snowcapping of cusps and ridges was extremely common; pit and valley defects were very common; spots and bands were very common, most were white; horizontal grooves were common; linear enamel hypoplasia, considered to be a true developmental defect, was rare.

Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes.

The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.

TLC plates as a convenient platform for solvent-free reactions.

Solvent-free oxidative couplings of naphthols have been optimized by co-spotting catalysts and substrates directly on silica TLC plates and heating, followed by chromatography, staining, and qualitative visualization.

Reactions of substituted (methylthio)benzylidene Meldrum's acids with secondary alicyclic amines in aqueous DMSO. Evidence for rate-limiting proton transfer.

The replacement of the methylthio group of substituted methylthiobenzylidene Meldrum's acids (2-SMe-Z) by secondary alicyclic amines occurs by a three-step mechanism. The first step is a nucleophilic attachment of the amine to 2-SMe-Z to form a zwitterionic intermediate T(+/-)(A); the second step involves deprotonation of T(+/-)(A) to form T(-)(A); while the third step represents general acid-catalyzed conversion of T(-)(A) to products. At high amine and/or high KOH concentration nucleophilic attachment is rate limiting. At low amine and low KOH concentration the reaction follows a rate law that is characteristic for general base catalysis which, in principle, is consistent with either rate-limiting deprotonation of T(+/-)(A) or rate-limiting conversion of T(-)(A) to products. A detailed structure-reactivity analysis indicates that for the reactions with piperazine, 1-(2-hydroxyethyl)piperazine, and morpholine it is deprotonation of T(+/-)(A) that is rate limiting, while for the reaction with piperidine, conversion of T(-)(A) to products is rate limiting.