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Every year, Hanoi suffers from several episodes (periods with daily concentration of PM 2.5 higher than 50 µg m -3 during at least two consecutive days). These episodes are of health concern because of the high concentration of PM 2.5 and/or PM 0.1 and the presence of PM-bound toxic components, such as, PAHs. In this study, the concentrations of PAHs bound to PM 2.5 and PM 0.1 in night-time and day-time samples during episode and non-episode periods in December 2021 were determined. The concentrations of PAHs bound to PM 2.5 were found to increase significantly from day-time samples of 3.24 ± 0.83 ng m -3 to night-time samples of 10.8 ± 4.45 ng m -3 in episode periods. However, PAHs bound to PM 0.1 increased slightly from day-time samples of 0.58 ± 0.12 ng m -3 to night-time samples of 0.89 ± 0.30 ng m -3 in episode periods. Diagnostic ratios of PAHs indicate that biomass/coal combustion and vehicular emission are the primary sources of PAHs. The incremental lifetime cancer risk was estimated to vary from 8.7E-09 to 2.5E-08 for children and 6.7E-08 to 2.2E-07 for adults, respectively. Accordingly, loss of life expectancy was estimated at 0.11 min and 0.82 min for children and adults, respectively. These findings imply that the carcinogenic impact induced by PAHs via inhalation is negligible during the episode period., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

SignificanceEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process are the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent "pairing" of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study provides observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

A new green method for trace level quantification of four herbicides, glyphosate (GLYP), glufosinate (GLUF), and their main metabolites, aminomethylphosphonic acid (AMPA) and 3-(methyl-phosphinico)-propionic acid (MPPA), was developed. The purification step without any derivatization was conducted by solid-phase extraction using Chelex-100 resin in the Fe (III) form, followed by elution with 5% NH 4 OH. The four analytes were quantified by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. The developed extraction method was validated on five fresh and sea water matrices with mean recoveries ranging from 80.1% to 109.4% (relative standard deviation < 20%). The extraction conditions were evaluated and certified for the high applicability of the extraction method too. The limits of detection (ng/L) in the five water matrices were in ranges 0.70 - 4.0, 2.4 - 3.9, 1.8 - 4.7, and 1.6 - 4.0 for GLYP, AMPA, GLUF, and MPPA, respectively. The method was successfully applied to detect the four compounds in surface waters sampled along the Red River Delta region in July 2019. The highest concentrations were detected at 565, 1,330, 234, and 871 ng/L for GLYP, AMPA, GLUF, and MPPA, respectively. These results showed the potential capacity of this new method for convenient monitoring of herbicides and their metabolites in the diverse natural water system., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1-/- tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis., Competing Interests: The authors have declared that no competing interests exist.

Polycyclic aromatic hydrocarbons (PAHs), the family of organic contaminations, have been shown to have negative effects on human health. However, until now, the comprehension on occurrence, distribution, and risk assessment of human exposure to PAHs has been limited in Vietnam. In this work, a capillary gas chromatography coupled with electron impact ionization tandem mass spectrometry (GC-EI-MS/MS) has been introduced for analysis of 16 PAHs in some particulate matter samples. PAHs have been separated on the TG 5 ms capillary gas chromatographic column and detected by tandem mass spectrometry in multiple reaction monitoring mode. The PAHs in the particulate matter (PM 2.5 and PM 10) samples were extracted by ultrasonic-assisted liquid extraction and cleaned up by an acidic silica gel solid phase extraction. The linearity range of all analyzed PAHs was from 5 to 2000 ng mL -1 with R 2 ≥0.9990. Limit of detection (LOD) of PAHs in particulate matter sample was from 0.001 ng m -3 (Br-Naph) to 0.276 ng m -3 (Fln). The recovery of PAHs was investigated by international proficiency testing samples. The recoveries of PAHs in proficiency testing sample ranged from 79.3% (Chr) to 109.8% (IcdP). The in-house validated GC-EI-MS/MS method was then applied to analysis of some particulate matter samples that were collected in the Hanoi areas. The total concentrations of PAHs in several brands of samples collected from Hanoi were found in the range of 226.3 ng m -3 -706.43 ng m -3 . Among the studied compounds, naphthalene was found at high frequency and ranged from 106.5 ng m -3 to 631.1 ng m -3 . The main distribution of the PAHs in particulate matter samples was two-ring and three-ring compounds., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Nam Vu-Duc et al.)

In this work, twelve heavy metals and arsenic, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn, in a rice sample collected from some areas of Vietnam have been quantified and implemented by using multiple analytical platforms such as ICP-MS, AAS, and mercury analyser. Seventy rice samples collected from the Red River Delta and mining zone activity were analysed. Concentration of heavy metals and arsenic in rice was analysed after appropriated sample digestion using internal or external calibration curves. The mean concentration (mg kg -1 dried weight) of the analysed elements in rice samples decreased on the order of Mn (19.268) > Fe (13.624) > Zn (8.163) > Cu (3.138) > Ni (0.384) > Cr (0.296) > Co (0.279) > As (0.115) > Cd (0.111) > Pb (0.075) > Hg (0.007) > Se (

In this study, an analytical method for the simultaneous determination of 7 major pharmaceutical residues in Vietnam, namely, carbamazepine, ciprofloxacin, ofloxacin, ketoprofen, paracetamol, sulfamethoxazole, and trimethoprim, in surface water and hospital wastewater has been developed. The method includes enrichment and clean-up steps by solid phase extraction using mix-mode cation exchange, followed by identification and quantification using an ultrahigh-performance liquid chromatography and tandem mass spectrometry and employing electrospray ionization (UPLC-ESI-MS/MS). Seven target compounds were separated on the reversed phase column and detected in multiple reaction monitoring (MRM) mode within 6 minutes. The present study also optimized the operating parameters of the mass spectrometer to achieve the highest analytical signals for all target compounds. All characteristic parameters of the analytical method were investigated, including linearity range, limit of detection, limit of quantification, precision, and accuracy. The important parameter in UPLC-ESI-MS/MS, matrix effect, was assessed and implemented via preextraction and postextraction spiking experiments. The overall recoveries of all target compounds were in the ranges from 55% to 109% and 56 % to 115% for surface water and hospital wastewater, respectively. Detection limits for surface water and hospital wastewater were 0.005-0.015  µ g L -1 and 0.014-0.123  µ g L -1 , respectively. The sensitivity of the developed method was allowed for determination of target compounds at trace level in environmental water samples. The in-house validation of the developed method was performed by spiking experiment in both the surface water and hospital wastewater matrix. The method was then applied to analyze several surface water and hospital wastewater samples taken from West Lake and some hospitals in Vietnam, where the level of these pharmaceutical product residues was still missed. Sulfamethoxazole was present at a high detection frequency in both surface water (33% of analyzed samples) and hospital wastewater (81% of analyzed samples) samples., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Bui Van Hoi et al.)

Concentrations of As and other trace elements were measured in groundwater, rice, hair, urine, and blood samples of people consuming As-contaminated groundwater in a village of Ha Nam province, northern Vietnam to understand the recent status of contamination and assess the possible risks of human exposure. Elevated concentrations of As in groundwater were still observed, exceeding the WHO guideline value in most of the tube wells investigated. Significant positive correlations between As concentrations in groundwater and human samples (hair and urine) were observed. Arsenic concentrations in human and hair appeared to be related to the groundwater usage habit, with higher levels found in drinking group than those in the washing group. Significant good correlations were also encountered between cumulative intakes of As, Mn, and Ba through groundwater consumption and hair concentrations. All these results indicate the chronic exposure to As and some other elements such as Mn and Ba. The total intakes of As, Mn, and Ba through rice and groundwater consumption were estimated to be ranged from 80-836, 49.3-1850, and 311-97100  µ g/day, respectively. The daily intakes of As of the study area ranged from 1.6-16.7  µ g/kg body wt./day, mean: 7.15  µ g/kg body wt./day, in which about 85% of the subjects were above the provisional tolerable daily intake proposed by WHO., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Manh Ha Nguyen et al.)

An ultrahigh-performance liquid chromatography in combination with high-resolution mass spectrometry Thermo Q-Extractive Focus Orbitrap MS has been introduced for analysis of multiclass pesticides in vegetable samples collected in Hanoi, Vietnam. Multiclass pesticides were separated on the Thermo Hypersil Gold PFP column utilizing a gradient of the mobile phase consisting of 5 mM ammonium formate, 0.1% formic acid in deionized water, and methanol. The target analytes were detected in the full-scan mode on Thermo Scientific Q-Exactive Focus Orbitrap MS for quantitation at the optimum operating conditions. These conditions included, but not limit to, the resolution of 70000 at the full width at half maximum in both positive and negative mode, mass range from 80 to 1000  m/z , and optimized parameters for the heated electrospray ionization source. The identification of the analytes in real samples was based on retention times, mass to charge ratios, mass accuracies, and MS/MS spectra at the confirmation mode with the inclusion list of target analytes. The mass accuracies of target analytes were from -4.14 ppm (dinotefuran) to 1.42 ppm (cinosulfuron) in the neat solvent and from -3.91 ppm (spinosad D) to 1.29 ppm (cinosulfuron) in the matrix-matched solution. Target analytes in the vegetable-based matrix were extracted by the QuEChERS method. Some critical parameters of the analytical method such as linearity, repeatability, limit of detection, and limit of quantitation have been evaluated and implemented. Excellent LOD and LOQ of the developed method were achieved at the range of 0.04-0.85 and 0.13-2.9  μ g·kg -1 , respectively. Intraday and interday repeatability of the analytical signal (peak area, n =6) of the developed method were below 3% and 10%, correspondingly. The matrix effect, extraction recovery, and overall recovery were fully investigated by spiking experiments. Experimental results demonstrated that the ionization suppression or enhancement was the main contribution on the overall recoveries of target analytes. Finally, the in-house validated method was applied to pesticides screening in vegetables samples in local villages in Hanoi, Vietnam. The concentrations of all target analytes were below limit of quantitation and lower than US-FDA or EU maximum residue levels.

In this work, high-performance liquid chromatography in combination with inductively coupled plasma dynamic reaction cell quadrupole mass spectrometry was introduced and optimized for speciation analysis of five major arsenic species including arsenobetain (AsB), arsenite (As(III)), monomethylarsonic (MMA), dimethylarsenonic acid (DMA), and arsenate (As(V)) in rice samples. Five arsenic compounds were separated on a Hamilton PRP X100 strong anion-exchange column employed with the mobile phase that is compatible with mass spectrometry, containing ammonium carbonate, methanol, and disodium ethylenediaminetetraacetic acid. Arsenic compounds were detected online by inductively coupled plasma dynamic reaction cell quadrupole mass spectrometry utilizing oxygen as the reaction gas at a flow rate of 0.7 mL·min -1 . Five selected arsenic species were baseline separated at the optimum experimental conditions. The excellent LOD and LOQ values of the developed method were achieved in the range of 0.5 to 2.9  μ g·kg -1 and 1.7 to 9.6  μ g·kg -1 for all species of arsenic, respectively. The ionization effect in plasma during chromatographic gradient elution was systematically investigated by using postcolumn injector. Arsenic compounds in rice samples were extracted by diluted nitric acid at elevated temperature. The extraction efficiency and the interconversion of target compounds during sample preparation were also assessed. The full validation of the developed method was performed by using certified reference material, BRC 211, from European Institute of Reference and Standard for speciation analysis. The recovery of all selected arsenic species was in the range of 70 to 135.5%. The validated method was also applied to analyze rice samples collected from some contaminated rice fields. The results showed that As(III), DMA, and As(V) were found in all rice samples. Average concentration (range) of inorganic arsenic and DMA in all rice samples were 130.3 (65.5-228.1) and 32 (8.2-133.01) μ g·kg -1 , respectively. However, total concentration of inorganic arsenic in most of investigated rice samples was below the maximum residual level according to US-FDA and European Union standards.

A high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) as an elemental specific detector was used for the speciation analysis of arsenic compounds in urine and serum samples from Vietnam. Five arsenic species including arsenite (As III ), arsenate (As V ), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) were studied. A gradient elution of ammonium carbonate ((NH 4 ) 2 CO 3 ), ethylenediaminetetraacetic acid disodium salt (Na 2 EDTA), and methanol at pH 9.0 utilizing Hamilton PRP-X100 strong anion-exchange column allowed the chromatographic separation of five arsenic species. In this study, urine and serum samples were prepared by dilution in solvent and protein precipitation by trichloroacetic acid, respectively. The extraction efficiency was greater than 91% for urine matrix, and recoveries from spiked samples were in the range of 94-139% for the arsenic species in human serum. The method limit of detection (MDL) and limit of quantification (MQL), which were calculated by signal to noise ratio, were found to be 0.3-1.5 and 1.0-5.0 ng·mL -1 , respectively. The concentration of arsenic species in 17 pairs of urine and serum samples from Vietnam was also quantified and evaluated. The major species of arsenic in the urine and serum samples were AsB and DMA.

Background: The collapse of the World Trade Center (WTC) on September 11, 2001 released a dust cloud containing numerous environmental contaminants, including polychlorinated dibenzo-para-dioxins and polychlorinated dibenzofurans (PCDD/Fs). PCDD/Fs are toxic and are associated with numerous adverse health outcomes including cancer, diabetes, and impaired reproductive and immunologic function. Prior studies have found adults exposed to the WTC disaster to have elevated levels of PCDD/Fs. This is the first study to assess PCDD/F levels in WTC-exposed children., Methods: This analysis includes 110 participants, a subset of the 2014-2016 WTC Adolescent Health Study, a group of both exposed youths who lived, attended school, or were present in lower Manhattan on 9/11 recruited from the WTC Health Registry (WTCHR) and unexposed youths frequency matched on age, sex, race, ethnicity, and income. Our sample was selected to maximize the contrast in their exposure to dust from the WTC collapse. Questionnaire data, including items about chronic home dust and acute dust cloud exposure, anthropometric measures, and biologic specimens were collected during a clinic visit. Serum PCDD/F concentrations were measured according to a standardized procedure at the New York State Department of Health Organic Analytical Laboratory. We used multivariable linear regression to assess differences in PCCD/Fs between WTCHR and non-WTCHR participants. We also compared mean and median PCDD/F and toxic equivalency (TEQ) concentrations in our cohort to 2003-4 National Health and Nutrition Examination Survey (NHANES) levels for youths age 12-19., Results: Median PCDD/F levels were statistically significantly higher among WTCHR participants compared to non-WTCHR participants for 16 out of 17 congeners. Mean and median TEQ concentrations in WTCHR participants were >7 times those in non-WTCHR participants (72.5 vs. 10.1 and 25. 3 vs. 3.39pg/g lipid, respectively). Among WTCHR participants, median concentrations of several PCDD/Fs were higher than the NHANES 95th percentiles. After controlling for dust cloud exposure, home dust exposure was significantly associated with higher PCDD/F level., Conclusions: Adolescents in lower Manhattan on the day of the WTC attack and exposed to particulate contamination from the WTC collapse had significantly elevated PCDD/F levels >12years later compared to a matched comparison group, driven by chronic home dust exposure rather than acute dust cloud exposure. PCDD/F and TEQ levels substantially exceeded those in similar-aged NHANES participants. Future studies are warranted to explore associations of PCDD/Fs with health and developmental outcomes among individuals exposed to the WTC disaster as children., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Large amounts of various chemical contaminants, including perfluoroalkyl substances (PFASs), were released at the time of the World Trade Center (WTC) disaster. Thousands of children who lived and/or attended school near the disaster site were exposed to these substances but few studies have examined the possible consequences related to these exposures., Objectives: To examine the relationship of PFASs serum levels with cardiometabolic profile in children and adolescents enrolled in the World Trade Center Health Registry (WTCHR) and a matched comparison group., Methods: We evaluated WTCHR enrollees who resided in New York City and were born between September 11, 1993 and September 10, 2001, and a matched comparison group consisting of individuals who were ineligible for WTCHR participation upon distance of their home, school or work from the WTC and lack of participation in rescue and recovery activities. Matching was based on date of birth, sex, race, ethnicity, and income. We assessed exposure to PFASs, as measured by serum levels and association with cardiometabolic profile as measured by arterial wall stiffness, body mass index, insulin resistance, fasting total cholesterol, HDL, LDL and triglycerides., Results: A total of 402 participants completed the study and serum samples were analyzed from 308 participants, 123 in the WTCHR group and 185 in the comparison group. In multivariable regression analysis, after adjusting for relevant confounders, we observed a significant, positive association of perfluorooctanoic acid (PFOA) with triglycerides (beta coefficient=0.14, 95% CI: 0.02, 0.27, 15.1% change), total cholesterol (beta coefficient=0.09, 95% CI: 0.04, 0.14, 9.2% change), and LDL cholesterol (beta coefficient=0.11, 95% CI: 0.03, 0.19, 11.5% change). Perfluorohexanesulfonic acid levels were associated with decreased insulin resistance (beta coefficient=-0.09, 95% CI: -0.18, -0.003, -8.6% change); PFOA and perfluorononanoic acid were associated with increased brachial artery distensibility., Conclusions: This research adds to our knowledge of the physical health impacts in a large group of children exposed to the WTC disaster. Abnormal lipid levels in young adults might be an early marker of atherosclerosis and cardiovascular diseases and our findings highlight the importance of conducting longitudinal studies in this population., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Meiosis is a specialized cellular program required to create haploid gametes from diploid parent cells. Homologous chromosomes pair, synapse, and recombine in a dynamic environment that accommodates gross chromosome reorganization and significant chromosome motion, which are critical for normal chromosome segregation. In Saccharomyces cerevisiae , Ndj1 is a meiotic telomere-associated protein required for physically attaching telomeres to proteins embedded in the nuclear envelope. In this study, we identified additional proteins that act at the nuclear periphery from meiotic cell extracts, including Nup2, a nonessential nucleoporin with a known role in tethering interstitial chromosomal loci to the nuclear pore complex. We found that deleting NUP2 affects meiotic progression and spore viability, and gives increased levels of recombination intermediates and products. We identified a previously uncharacterized 125 aa region of Nup2 that is necessary and sufficient for its meiotic function, thus behaving as a meiotic autonomous region (MAR). Nup2-MAR forms distinct foci on spread meiotic chromosomes, with a subset overlapping with Ndj1 foci. Localization of Nup2-MAR to meiotic chromosomes does not require Ndj1, nor does Ndj1 localization require Nup2, suggesting these proteins function in different pathways, and their interaction is weak or indirect. Instead, several severe synthetic phenotypes are associated with the nup2 Δ ndj1 Δ double mutant, including delayed turnover of recombination joint molecules, and a failure to undergo nuclear divisions without also arresting the meiotic program. These data suggest Nup2 and Ndj1 support partially overlapping functions that promote two different levels of meiotic chromosome organization necessary to withstand a dynamic stage of the eukaryotic life cycle., (Copyright © 2017 by the Genetics Society of America.)

The World Trade Center (WTC) disaster released large amounts of various chemical substances into the environment, including perfluoroalkyl substances (PFASs). Yet, no studies have examined exposures in children living or attending schools near the disaster site. We measured serum PFASs in WTC Health Registry (WTCHR) respondents who were ≤8 years of age on September 11, 2001 and a sociodemographically-matched comparison group. We also examined the relationship of PFASs levels with dust cloud exposure; home dust exposure, and with traumatic exposure, the latter to take into account differences related to possible mental health consequences and associated behavioral problems. Serum samples, collected between 2014 and 2016, were analyzed from 123 WTCHR participants and from 185 participants in the comparison group. In the WTCHR group, median perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) levels were 1.81ng/mL and 3.72ng/mL, respectively. Controlling for sex, caloric intake, race/ethnicity, and date of birth, significant increases among WTCHR participants compared with the matched comparison group were detected for perfluorohexanesulfonate (0.23ng/mL increase or 0.24log unit increase, p=0.006); PFOS (0.86ng/mL increase or 0.16log unit increase, p=0.011); PFOA (0.35ng/mL increase or 0.18log unit increase, p<0.001); perfluorononanoic acid (0.12ng/mL increase or 0.17log unit increase, p=0.003); perfluorodecanoic acid (0.06ng/mL increase or 0.42log unit increase, p<0.001); and perfluoroundecanoic acid (0.03ng/mL increase or 0.32log unit increase, p=0.019). Stronger associations were identified for home dust exposures and traumatic exposures than dust cloud. These findings highlight the importance of conducting longitudinal studies in this population to assess possible cardiometabolic and renal consequences related to these exposures., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions., (Copyright © 2016 Chu and Burgess.)

Monomeric Agaricus meleagris pyranose dehydrogenase (AmPDH) belongs to the glucose-methanol-choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. AmPDH can double oxidize various mono- and oligosaccharides at different positions (C1 to C4). To study the structure/function relationship of selected active-site residues of AmPDH pertaining to substrate (carbohydrate) turnover in more detail, several active-site variants were generated, heterologously expressed in Pichia pastoris, and characterized by biochemical, biophysical and computational means. The crystal structure of AmPDH shows two active-site histidines, both of which could take on the role as the catalytic base in the reductive half-reaction. Steady-state kinetics revealed that His512 is the only catalytic base because H512A showed a reduction in (kcat /KM )glucose by a factor of 10(5) , whereas this catalytic efficiency was reduced by two or three orders of magnitude for His556 variants (H556A, H556N). This was further corroborated by transient-state kinetics, where a comparable decrease in the reductive rate constant was observed for H556A, whereas the rate constant for the oxidative half-reaction (using benzoquinone as substrate) was increased for H556A compared to recombinant wild-type AmPDH. Steady-state kinetics furthermore indicated that Gln392, Tyr510, Val511 and His556 are important for the catalytic efficiency of PDH. Molecular dynamics (MD) simulations and free energy calculations were used to predict d-glucose oxidation sites, which were validated by GC-MS measurements. These simulations also suggest that van der Waals interactions are the main driving force for substrate recognition and binding., (© 2015 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U(13)C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.

Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of sugar phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of sugar phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1-2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6% and an average bias of 1.4%.

A mass spectrometric quantitative assay was developed for the analysis of 10 sugar phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten sugar phosphates in yeast extracts utilizing internal standardization with a fully labeled (13)C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.

In this work, accurate MS-based methods for quantitative profiling of nucleotides, nucleosides, and nucleobases in yeast extracts used as additives in animal feedstuff are presented. Reversed-phase chromatography utilizing a stationary phase compatible with 100% aqueous mobile phases resulted in superior analytical figures of merit than HILIC or ion-pair reversed-phase separation. The novel separation method was combined with both molecular and elemental mass spectrometry. By use of RP-LC-MS-MS, excellent limits of detection <1 μmol L(-1) could be obtained for all the compounds investigated. The elemental speciation analysis approach enabled determination of nucleotides by phosphorus detection. Sensitivity of LC-ICP-MS was 1-2 orders of magnitude lower than that of LC-MS-MS. Quantitative analysis of yeast products using complementary MS detection furnished values in good agreement.

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF-R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1-7 cells) express endogenous gonadotropin-releasing hormone receptors (GnRH-R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1-R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data indicate that GPCR-induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist-induced transactivation of the EGF-R., (Published 2005 Wiley-Liss, Inc.)