Publications by authors named "R W Flick"

206 Publications

Development of omics biomarkers for estrogen exposure using mRNA, miRNA and piRNAs.

Aquat Toxicol 2021 Mar 12;235:105807. Epub 2021 Mar 12.

US Environmental Protection Agency, Office of Research and Development, 26 W. Martin Luther King Dr., Cincinnati, OH 45268, United States. Electronic address:

The number of chemicals requiring risk evaluation exceeds our capacity to provide the underlying data using traditional methodology. This has led to an increased focus on the development of novel approach methodologies. This work aimed to expand the panel of gene expression-based biomarkers to include responses to estrogens, to identify training strategies that maximize the range of applicable concentrations, and to evaluate the potential for two classes of small non-coding RNAs (sncRNAs), microRNA (miRNA) and piwi-interacting RNA (piRNA), as biomarkers. To this end larval Pimephales promelas (96 hpf +/- 1h) were exposed to 5 concentrations of 17α- ethinylestradiol (0.12, 1.25, 2.5, 5.0, 10.0 ng/L) for 48 h. For mRNA-based biomarker development, RNA-seq was conducted across all concentrations. For sncRNA biomarkers, small RNA libraries were prepared only for the control and 10.0 ng/L EE2 treatment. In order to develop an mRNA classifier that remained accurate over the range of exposure concentrations, three different training strategies were employed that focused on 10 ng/L, 2.5 ng/L or a combination of both. Classifiers were tested against an independent test set of individuals exposed to the same concentrations used in training and subsequently against concentrations not included in model training. Both random forest (RF) and logistic regression with elastic net regularizations (glmnet) models trained on 10 ng/L EE2 performed poorly when applied to lower concentrations. RF models trained with either the 2.5 ng/L or combination (2.5 + 10 ng/L) treatments were able to accurately discriminate exposed vs. non-exposed across all but the lowest concentrations. glmnet models were unable to accurately classify below 5 ng/L. With the exception of the 10 ng/L treatment, few mRNA differentially expressed genes (DEG) were observed, however, there was marked overlap of DEGs across treatments. Overlapping DEGs have well established linkages to estrogen and several of the 81 DEGs identified in the 10 ng/L treatment have been previously utilized as estrogenic biomarkers (vitellogenin, estrogen receptor-β). Following multiple test correction, no sncRNAs were found to be differentially expressed, however, both miRNA and piRNA classifiers were able to accurately discriminate control and 10 ng/L exposed organisms with AUCs of 0.83 and 1.0 respectively. We have developed a highly discriminative estrogenic mRNA biomarker that is accurate over a range of concentrations likely to be found in real-world exposures. We have demonstrated that both miRNA and piRNA are responsive to estrogenic exposure, suggesting the need to further investigate their regulatory roles in the estrogenic response.
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http://dx.doi.org/10.1016/j.aquatox.2021.105807DOI Listing
March 2021

Effect of upper respiratory infection on anaesthesia induced atelectasis in paediatric patients.

Sci Rep 2021 Mar 16;11(1):5981. Epub 2021 Mar 16.

Department of Anaesthesiology and Pain Medicine, Anaesthesia and Pain Research Institute, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.

Upper respiratory tract infection (URI) symptoms are known to increase perioperative respiratory adverse events (PRAEs) in children undergoing general anaesthesia. General anaesthesia per se also induces atelectasis, which may worsen with URIs and yield detrimental outcomes. However, the influence of URI symptoms on anaesthesia-induced atelectasis in children has not been investigated. This study aimed to demonstrate whether current URI symptoms induce aggravation of perioperative atelectasis in children. Overall, 270 children aged 6 months to 6 years undergoing surgery were prospectively recruited. URI severity was scored using a questionnaire and the degree of atelectasis was defined by sonographic findings showing juxtapleural consolidation and B-lines. The correlation between severity of URI and degree of atelectasis was analysed by multiple linear regression. Overall, 256 children were finally analysed. Most children had only one or two mild symptoms of URI, which were not associated with the atelectasis score across the entire cohort. However, PRAE occurrences showed significant correspondence with the URI severity (odds ratio 1.36, 95% confidence interval 1.10-1.67, p = 0.004). In conclusion, mild URI symptoms did not exacerbate anaesthesia-induced atelectasis, though the presence and severity of URI were correlated with PRAEs in children.Trial registration: Clinicaltrials.gov (NCT03355547).
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http://dx.doi.org/10.1038/s41598-021-85378-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966804PMC
March 2021

DNA methylation and expression of estrogen receptor alpha in fathead minnows exposed to 17α-ethynylestradiol.

Aquat Toxicol 2021 Apr 23;233:105788. Epub 2021 Feb 23.

US Environmental Protection Agency, Office of Research and Development, Cincinnati, OH, 45268, United States. Electronic address:

The gene expression response thought to underlie the negative apical effects resulting from estrogen exposure have been thoroughly described in fish. Although epigenetics are believed to play a critical role translating environmental exposures into the development of adverse apical effects, they remain poorly characterized in fish species. This study investigated alterations of DNA methylation of estrogen receptor alpha (esr1) in brain and liver tissues from 8 to 10 month old male fathead minnows (Pimephales promelas) after a 2d exposure to either 2.5 ng/L or 10 ng/L 17α-ethynylestradiol (EE2). Changes in the patterns of methylation were evaluated using targeted deep sequencing of bisulfite treated DNA in the 5' region of esr1. Methylation and gene expression were assessed at 2d of exposure and after a 7 and 14d depuration period. After 2d EE2 exposure, males exhibited significant demethylation in the 5' upstream region of esr1 in liver tissue, which was inversely correlated to gene expression. This methylation pattern reflected what was seen in females. No gene body methylation (GBM) was observed for liver of exposed males. Differential methylation was observed for a single upstream CpG site in the liver after the 14d depuration. A less pronounced methylation response was observed in the upstream region in brain tissue, however, several CpGs were necessarily excluded from the analysis. In contrast to the liver, a significant GBM response was observed across the entire gene body, which was sustained until at least 7d post-exposure. No differential expression was observed in the brain, limiting functional interpretation of methylation changes. The identification of EE2-dependent changes in methylation levels strongly suggests the importance of epigenetic mechanisms as a mediator of the organismal response to environmental exposures and the need for further characterization of the epigenome. Further, differential methylation following depuration indicates estrogenic effects persist well after the active exposure, which has implications for the risk posed by repeated exposures..
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http://dx.doi.org/10.1016/j.aquatox.2021.105788DOI Listing
April 2021

Hypotension and adverse neurodevelopmental outcomes among children with multiple exposures to general anesthesia: Subanalysis of the Mayo Anesthesia Safety in Kids (MASK) Study.

Paediatr Anaesth 2021 Mar 4;31(3):282-289. Epub 2021 Jan 4.

Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, Rochester, MN, USA.

Background: The potential adverse effects of exposures to general anesthesia on the developing human brain remain controversial. It has been hypothesized that hypotension accompanying anesthesia could be contributory. We hypothesized that among children exposed to multiple anesthetics prior to age 3, children developing adverse neurodevelopmental outcomes would be more likely to have intraoperative hypotension.

Methods: Two previously published study cohorts were utilized for analysis: the retrospective and prospective Mayo Anesthesia Safety in Kids cohorts. The two lowest consecutive systolic blood pressure measurements were abstracted and standardized by calculating a z-score for noninvasive blood pressure reference ranges for children. The lowest systolic blood pressure z-score (continuous variable) and intraoperative hypotension (lowest systolic blood pressure z-score <-1.0) were used to assess the association of intraoperative hypotension with the incidence of learning disabilities or attention-deficit/hyperactivity disorder(retrospective cohort) and factor scores/cluster membership (prospective cohort).

Results: One hunderd and sixteen and 206 children with multiple exposures to general anesthesia were analyzed in the retrospective and prospective cohorts with mean lowest systolic blood pressure z-scores -0.26 (SD 1.02) and -0.62 (SD 1.10), respectively. There was no overall association of the lowest z-score or hypotension with learning disabilities or attention-deficit/hyperactivity disorder in the retrospective cohort. In the prospective cohort, there was no overall association of the lowest systolic blood pressure or hypotension with factor scores or cluster membership.

Conclusions: We did not find evidence to support the hypothesis that, among children exposed to multiple anesthetics prior to age 3, children developing adverse neurodevelopmental outcomes would be more likely to have intraoperative hypotension compared with those who did not.
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http://dx.doi.org/10.1111/pan.14106DOI Listing
March 2021

Global transcriptomic profiling of microcystin-LR or -RR treated hepatocytes (HepaRG).

Toxicon X 2020 Dec 7;8:100060. Epub 2020 Oct 7.

U.S. Environmental Protection Agency, Office of Research and Development, Research Triangle Park, NC, 27709, USA.

The canonical mode of action (MOA) of microcystins (MC) is the inhibition of protein phosphatases, but complete characterization of toxicity pathways is lacking. The existence of over 200 MC congeners complicates risk estimates worldwide. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or -RR for 2 h. Significant reduction in cell survival was observed in LR1000 and LR100 treatments whereas no acute toxicity was observed in any MR-RR treatment. RNA-seq was performed on all treatments of MC-LR and -RR. Differentially expressed genes and pathways associated with oxidative and endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) were highly enriched by both congeners as were inflammatory pathways. Genes associated with both apoptotic and inflammatory pathways were enriched in LR1000. We present a model of MC toxicity that immediately causes oxidative stress and leads to ER stress and the activation of the UPR. Differential activation of the three arms of the UPR and the kinetics of JNK activation ultimately determine whether cell survival or apoptosis is favored. Extracellular exosomes were enrichment of by both congeners, suggesting a previously unidentified mechanism for MC-dependent extracellular signaling. The complement system was enriched only in MC-RR treatments, suggesting congener-specific differences in cellular effects. This study provided an unbiased snapshot of the early systemic hepatocyte response to MC-LR and MC-RR congeners and may explain differences in toxicity among MC congeners.
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http://dx.doi.org/10.1016/j.toxcx.2020.100060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670210PMC
December 2020

Lignin-oxidizing activity of bacterial laccases characterized using soluble substrates and polymeric lignin.

J Biotechnol 2021 Jan 10;325:128-137. Epub 2020 Nov 10.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada; Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, LL57 2UW, UK. Electronic address:

Efficient biotransformation of lignin requires the activity of different oxidative enzymes. In this work, 19 bacterial multi-copper oxidases were screened for oxidase activity against 19 soluble substrates and revealed the highest activity in the laccase CotA (BSU0630) from Bacillus subtilis. Structure-based site-directed mutagenesis of CotA identified four conserved residues (His419, Cys492, His497, and Met502) as critical for activity against 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). Greatly reduced oxidase activity was found in the CotA mutant proteins E213A, N214A, C229A, N264A, E298A, T415A, R416A, Q468A, and T480A. We also designed a lignin-agarose plate screen for detecting oxidase activity of purified proteins against polymeric lignin, which confirmed the results obtained with ABTS and identified three mutant variants with increased activity toward kraft lignin (E213A, T415A, and T260A). X-ray photoelectron spectroscopy analysis of low sulfonate kraft lignin after incubation with CotA revealed a reduction in the content of CC/CC bonds and increase in CO/CO bonds. Product analyses using mass spectrometry, liquid chromatography, and bright-field microscopy revealed an increased polymerization state of reaction products suggesting that formation of radical intermediates was followed by radical coupling. Our results provide further insights into the mechanisms of lignin oxidation by laccases.
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http://dx.doi.org/10.1016/j.jbiotec.2020.11.007DOI Listing
January 2021

Utility of medical record diagnostic codes to ascertain attention-deficit/hyperactivity disorder and learning disabilities in populations of children.

BMC Pediatr 2020 11 7;20(1):510. Epub 2020 Nov 7.

Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 200 1st St SW, Rochester, MN, 55905, USA.

Background: To develop and evaluate machine learning algorithms to ascertain attention-deficit/hyperactivity (ADHD) and learning disability (LD) using diagnostic codes in the medical record.

Method: Diagnoses of ADHD and LD were confirmed in cohorts of children in Olmsted County of Minnesota based on validated research criteria. Models to predict ADHD and LD were developed using ICD-9 codes in a derivation cohort of 1057 children before evaluated in a validation cohort of 536 children.

Results: The ENET-MIN model using selected ICD-9 codes at prior probability of 0.25 has a sensitivity of 0.76, PPV of 0.85, specificity of 0.98, and NPV of 0.97 in the validation cohort. However, it does not offer significant advantage over a model using a single ICD-9 code of 314.X, which shows sensitivity of 0.81, PPV of 0.83, specificity of 0.98, and NPV of 0.97. None of the models developed for LD performed well in the validation cohort.

Conclusions: It is feasible to utilize diagnostic codes to ascertain cases of ADHD in a population of children. Machine learning approaches do not have advantage compared with simply using a single family of diagnostic codes for ADHD. The use of medical record diagnostic codes is not feasible to ascertain LD.
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http://dx.doi.org/10.1186/s12887-020-02411-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648408PMC
November 2020

Association Between Behavioral and Learning Outcomes and Single Exposures to Procedures Requiring General Anesthesia Before Age 3: Secondary Analysis of Data From Olmsted County, MN.

Anesth Analg 2020 Sep 7. Epub 2020 Sep 7.

From the Departments of Anesthesiology and Perioperative Medicine.

Background: Two prior population-based (children born in Olmsted County, MN), retrospective cohort studies both found that multiple exposures to anesthesia before age 3 were associated with a significant increase in the frequency of attention-deficit hyperactivity disorder (ADHD) and learning disabilities (LD) later in life. The primary purpose of this secondary analysis of these data was to test the hypothesis that a single exposure to anesthesia before age 3 was associated with an increased risk of ADHD. We also examined the association of single exposures with LD and the need for individualized educational plans as secondary outcomes.

Methods: This analysis includes 5339 children who were unexposed to general anesthesia before age 3 (4876 born from 1976 to 1982 and 463 born from 1996 to 2000), and 1054 children who had a single exposure to anesthesia before age 3 (481 born from 1976 to 1982 and 573 born from 1996 to 2000). The primary outcome of interest was ADHD. Secondary outcomes included LD (reading, mathematics, and written language) and the need for individualized educational programs (speech/language and emotion/behavior). To compare the incidence of each outcome between those who were unexposed and singly exposed to anesthesia before the age of 3 years, an inverse probability of treatment weighted proportional hazards model was used.

Results: For children not exposed to anesthesia, the estimated cumulative frequency (95% confidence interval [CI]) of ADHD at age 18 was 7.3% (95% CI, 6.5-8.1) and 13.0% (95% CI, 10.1-16.8) for the 1976-1982 and 1996-2000 cohorts, respectively. For children exposed to a single anesthetic before age 3, the cumulative frequency of ADHD was 8.1% (95% CI, 5.3-12.4) and 17.6% (95% CI, 14.0-21.9) for the 1976-1982 and 1996-2000 cohorts, respectively. In weighted analyses, single exposures were not significantly associated with an increased frequency of ADHD (hazard ratio [HR], 1.21; 95% CI, 0.91-1.60; P= .184). Single exposures were also not associated with an increased frequency of any LD (HR, 0.98; 95% CI, 0.78-1.23), or the need for individualized education plans.

Conclusions: This analysis did not find evidence that single exposures to procedures requiring general anesthesia, before age 3, are associated with an increased risk of developing ADHD, LD, or the need for individualized educational plans in later life.
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http://dx.doi.org/10.1213/ANE.0000000000005180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936987PMC
September 2020

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution.

Biotechnol Biofuels 2020 29;13:114. Epub 2020 Jun 29.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5 Canada.

Background: Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability.

Results: Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium using error-prone PCR-based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in , thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo.

Conclusion: This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.
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http://dx.doi.org/10.1186/s13068-020-01753-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325246PMC
June 2020

Biocatalytic and FMN Prenylation and (De)carboxylase Activation.

ACS Chem Biol 2020 07 7;15(7):1874-1882. Epub 2020 Jul 7.

Department of Chemical Engineering and Applied Sciences, University of Toronto, Toronto, Ontario M5S 3E5, Canada.

Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for and FMN prenylation and UbiD activation under aerobic conditions. The one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from and protocatechuic acid decarboxylase ENC0058 from . The system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in cells under aerobic conditions in the presence of prenol. The and FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.
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http://dx.doi.org/10.1021/acschembio.0c00136DOI Listing
July 2020

Evidence for extensive anaerobic dechlorination and transformation of the pesticide chlordecone (C10Cl10O) by indigenous microbes in microcosms from Guadeloupe soil.

PLoS One 2020 13;15(4):e0231219. Epub 2020 Apr 13.

Département de Chimie, Laboratory COVACHIMM2E, Université des Antilles, Pointe à Pitre Cedex, Guadeloupe (FWI), France.

The historic use of chlordecone (C10Cl10O) as a pesticide to control banana weevil infestations has resulted in pollution of large land areas in the French West Indies. Although currently banned, chlordecone persists because it adsorbs strongly to soil and its complex bis-homocubane structure is stable, particularly under aerobic conditions. Abiotic chemical transformation catalyzed by reduced vitamin B12 has been shown to break down chlordecone by opening the cage structure to produce C9 polychloroindenes. More recently these C9 polychloroindenes were also observed as products of anaerobic microbiological transformation. To investigate the anaerobic biotransformation of chlordecone by microbes native to the French West Indies, microcosms were constructed anaerobically from chlordecone impacted Guadeloupe soil and sludge to mimic natural attenuation and eletron donor-stimulated reductive dechlorination. Original microcosms and transfers were incubated over a period of 8 years, during which they were repeatedly amended with chlordecone and electron donor (ethanol and acetone). Using LC-MS, chlordecone and degradation products were detected in all the biologically active microcosms. Observed products included monohydro-, dihydro- and trihydrochlordecone derivatives (C10Cl10-nO2Hn; n = 1,2,3), as well as "open cage" C9 polychloroindene compounds (C9Cl5-nH3+n n = 0,1,2) and C10 carboxylated polychloroindene derivatives (C10Cl4-nO2H4+n, n = 0-3). Products with as many as 9 chlorine atoms removed were detected. These products were not observed in sterile (poisoned) microcosms. Chlordecone concentrations decreased in active microcosms as concentrations of products increased, indicating that anaerobic dechlorination processes have occurred. The data enabled a crude estimation of partitioning coefficients between soil and water, showing that carboxylated intermediates sorb poorly and as a consequence may be flushed away, while polychlorinated indenes sorb strongly to soil. Microbial community analysis in microcosms revealed enrichment of anaerobic fermenting and acetogenic microbes possibly involved in anaerobic chlordecone biotransformation. It thus should be possible to stimuilate anaerobic dechlorination through donor amendment to contaminated soils, particularly as some metabolites (in particular pentachloroindene) were already detected in field samples as a result of intrinsic processes. Extensive dechlorination in the microcosms, with evidence for up to 9 Cl atoms removed from the parent molecule is game-changing, giving hope to the possibility of using bioremediation to reduce the impact of CLD contamination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231219PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153859PMC
July 2020

Adoption of COVID-19 triage strategies for low-income settings.

Lancet Respir Med 2020 04 11;8(4):e22. Epub 2020 Mar 11.

Global Health Security Department, Infectious Disease Institute, Makerere University, Kampala 22418, Uganda.

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http://dx.doi.org/10.1016/S2213-2600(20)30114-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103952PMC
April 2020

The Video intervention to Inspire Treatment Adherence for Life (VITAL Start): protocol for a multisite randomized controlled trial of a brief video-based intervention to improve antiretroviral adherence and retention among HIV-infected pregnant women in Malawi.

Trials 2020 Feb 19;21(1):207. Epub 2020 Feb 19.

ICAP at Columbia, Mailman School of Public Health and Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Background: Improving maternal antiretroviral therapy (ART) retention and adherence is a critical challenge facing prevention of mother-to-child transmission (PMTCT) of HIV programs. There is an urgent need for evidence-based, cost-effective, and scalable interventions to improve maternal adherence and retention that can be feasibly implemented in overburdened health systems. Brief video-based interventions are a promising but underutilized approach to this crisis. We describe a trial protocol to evaluate the effectiveness and implementation of a standardized educational video-based intervention targeting HIV-infected pregnant women that seeks to optimize their ART retention and adherence by providing a VITAL Start (Video intervention to Inspire Treatment Adherence for Life) before committing to lifelong ART.

Methods: This study is a multisite parallel group, randomized controlled trial assessing the effectiveness of a brief facility-based video intervention to optimize retention and adherence to ART among pregnant women living with HIV in Malawi. A total of 892 pregnant women living with HIV and not yet on ART will be randomized to standard-of-care pre-ART counseling or VITAL Start. The primary outcome is a composite of retention and adherence (viral load < 1000 copies/ml) 12 months after starting ART. Secondary outcomes include assessments of behavioral adherence (self-reported adherence, pharmacy refill, and tenofovir diphosphate concentration), psychosocial impact, and resource utilization. We will also examine the implementation of VITAL Start via surveys and qualitative interviews with patients, partners, and health care workers and conduct cost-effectiveness analyses.

Discussion: This is a robust evaluation of an innovative facility-based video intervention for pregnant women living with HIV, with the potential to improve maternal and infant outcomes.

Trial Registration: ClinicalTrials.gov, NCT03654898. Registered on 31 August 2018.
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http://dx.doi.org/10.1186/s13063-020-4131-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031891PMC
February 2020

One-Pot Biocatalytic Transformation of Adipic Acid to 6-Aminocaproic Acid and 1,6-Hexamethylenediamine Using Carboxylic Acid Reductases and Transaminases.

J Am Chem Soc 2020 01 7;142(2):1038-1048. Epub 2020 Jan 7.

Department of Chemical Engineering and Applied Chemistry , University of Toronto , Toronto , Ontario M5S 3E5 , Canada.

Production of platform chemicals from renewable feedstocks is becoming increasingly important due to concerns on environmental contamination, climate change, and depletion of fossil fuels. Adipic acid (AA), 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD) are key precursors for nylon synthesis, which are currently produced primarily from petroleum-based feedstocks. In recent years, the biosynthesis of adipic acid from renewable feedstocks has been demonstrated using both bacterial and yeast cells. Here we report the biocatalytic conversion/transformation of AA to 6-ACA and HMD by carboxylic acid reductases (CARs) and transaminases (TAs), which involves two rounds (cascades) of reduction/amination reactions (AA → 6-ACA → HMD). Using purified wild type CARs and TAs supplemented with cofactor regenerating systems for ATP, NADPH, and amine donor, we established a one-pot enzyme cascade catalyzing up to 95% conversion of AA to 6-ACA. To increase the cascade activity for the transformation of 6-ACA to HMD, we determined the crystal structure of the CAR substrate-binding domain in complex with AMP and succinate and engineered three mutant CARs with enhanced activity against 6-ACA. In combination with TAs, the CAR L342E protein showed 50-75% conversion of 6-ACA to HMD. For the transformation of AA to HMD (via 6-ACA), the wild type CAR was combined with the L342E variant and two different TAs resulting in up to 30% conversion to HMD and 70% to 6-ACA. Our results highlight the suitability of CARs and TAs for several rounds of reduction/amination reactions in one-pot cascade systems and their potential for the biobased synthesis of terminal amines.
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http://dx.doi.org/10.1021/jacs.9b11761DOI Listing
January 2020

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of ()-1,3-butanediol.

J Biol Chem 2020 01 5;295(2):597-609. Epub 2019 Dec 5.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada; Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor LL57 2UW, United Kingdom. Electronic address:

Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of ()-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from , TM1559 from , and DeoC from The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for and 1,3BDO biosynthesis.
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http://dx.doi.org/10.1074/jbc.RA119.011363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956516PMC
January 2020

Sixteen years of bathymetry and waves at San Diego beaches.

Sci Data 2019 08 29;6(1):161. Epub 2019 Aug 29.

Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92037, USA.

Sustained, quantitative observations of nearshore waves and sand levels are essential for testing beach evolution models, but comprehensive datasets are relatively rare. We document beach profiles and concurrent waves monitored at three southern California beaches during 2001-2016. The beaches include offshore reefs, lagoon mouths, hard substrates, and cobble and sandy (medium-grained) sediments. The data span two energetic El Niño winters and four beach nourishments. Quarterly surveys of 165 total cross-shore transects (all sites) at 100 m alongshore spacing were made from the backbeach to 8 m depth. Monthly surveys of the subaerial beach were obtained at alongshore-oriented transects. The resulting dataset consists of (1) raw sand elevation data, (2) gridded elevations, (3) interpolated elevation maps with error estimates, (4) beach widths, subaerial and total sand volumes, (5) locations of hard substrate and beach nourishments, (6) water levels from a NOAA tide gauge (7) wave conditions from a buoy-driven regional wave model, and (8) time periods and reaches with alongshore uniform bathymetry, suitable for testing 1-dimensional beach profile change models.
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http://dx.doi.org/10.1038/s41597-019-0167-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715754PMC
August 2019

Accumulation of soluble menaquinones MK-7 in honey coincides with death of spp. present in honey.

Food Chem X 2019 Mar 20;1:100008. Epub 2019 Feb 20.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, BioZone, 200 Collage Street, Toronto, Ontario M5S 3E5, Canada.

Long-chain menaquinones (MK) are of bacterial origin. We investigated the possibility that MKs observed in honey are also the products of bacteria present in honey. The bacterial composition of honey was analyzed using culture-dependent methods. 16S rRNA gene sequencing and MALDI-TOF showed prevalence of the members of and groups The dominant menaquinones in both bacteria and honey were menaquinones MK-7 and MK-8 as indicated by UHPLC-ESI-MS/MS coupled to quadrupole orbitrap. The EICs showed alignment of mass ions of MK-7 and MK-8 from culture supernatants with that of honey. The unique MS/MS fragmentation pattern indicated that fragment ions were arising from the same menaquinone present in both samples. During growth, the accumulation of MK-7 in supernatants occurred in a stationary phase and coincided with cell death. These novel findings suggest that the soluble MKs in honey originate from shedding of cell membranes of dead vegetative cells.
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http://dx.doi.org/10.1016/j.fochx.2019.100008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694848PMC
March 2019

Mechanisms of pharmaceutical and personal care product removal in algae-based wastewater treatment systems.

Sci Total Environ 2019 Dec 5;695:133772. Epub 2019 Aug 5.

Department of Civil & Mineral Engineering, University of Toronto, 35 St. George Street, Toronto, Ontario M5S 1A4, Canada; Department of Chemical Engineering & Applied Chemistry, 200 College Street, Toronto, Ontario M5S 3E5, Canada. Electronic address:

The widespread distribution of pharmaceuticals and personal care products (PPCPs), particularly in the built environment, has led to increased concern about their effects on both human and ecosystem health. In this research, we investigated the role of algae species Scenedesmus obliquus and Chlorella vulgaris in governing PPCP transfer and transformation mechanisms in algae-containing environments. Lab-scale algal bioreactors were created under various conditions of light, water matrix, and sterilization method to isolate and elucidate reaction mechanisms affecting carbamazepine, ibuprofen, gemfibrozil, and triclosan. The parent compounds and their potential transformation products were analyzed in both the water and algae phases. The results showed that ibuprofen was primarily biotransformed due to synergistic relationships between the algae and the bacteria. Ibuprofen biotransformation products tentatively identified as hydroxy-ibuprofen, carboxy-ibuprofen, and 4-isobutylcatechol were detected in several samples. In all the reactors exposed to light, triclosan underwent both phototransformation and biotransformation. Triclosan biotransformation took place in Scenedesmus obliquus, as demonstrated by the presence of triclosan-O-sulfate in the algae extracts. No evidence of significant carbamazepine and gemfibrozil transfer or transformation was observed under the experimental conditions tested. These results suggest that microalgal-bacterial consortia can facilitate PPCP transformation in algae-based passive water treatment systems.
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http://dx.doi.org/10.1016/j.scitotenv.2019.133772DOI Listing
December 2019

VITAL Start: Video-Based Intervention to Inspire Treatment Adherence for Life-Pilot of a Novel Video-Based Approach to HIV Counseling for Pregnant Women Living with HIV.

AIDS Behav 2019 Nov;23(11):3140-3151

ICAP at Columbia, Mailman School of Public Health and Vagelos College of Physicians & Surgeons, Columbia University, New York, USA.

We developed and piloted a video-based intervention targeting HIV-positive pregnant women to optimize antiretroviral therapy (ART) retention and adherence by providing a VITAL Start (Video-intervention to Inspire Treatment Adherence for Life) before ART. VITAL Start (VS) was grounded in behavior-determinant models and developed through an iterative multi-stakeholder process. Of 306 pregnant women eligible for ART, 160 were randomized to standard of care (SOC), 146 to VS and followed for one-month. Of those assigned to VS, 100% completed video-viewing; 96.5% reported they would recommend VS. Of 11 health workers interviewed, 82% preferred VS over SOC; 91% found VS more time-efficient. Compared to SOC, VS group had greater change in HIV/ART knowledge (p < 0.01), trend towards being more likely to start ART (p = 0.07), and better self-reported adherence (p = 0.02). There were no significant group differences in 1-month retention and pharmacy pill count. VITAL Start was highly acceptable, feasible, with promising benefits to ART adherence.
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http://dx.doi.org/10.1007/s10461-019-02634-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803103PMC
November 2019

Site-directed mutagenesis and stability of the carboxylic acid reductase MAB4714 from Mycobacterium abscessus.

J Biotechnol 2019 Sep 2;303:72-79. Epub 2019 Aug 2.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada; Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, Gwynedd LL57 2UW, UK. Electronic address:

Carboxylic acid reductases (CARs) catalyze ATP- and NADPH-dependent reduction of carboxylic acids to corresponding aldehydes. Although successful applications of these enzymes for the bioconversion of monocarboxylic acids have already been reported, their applicability for the reduction of dicarboxylic acids is not well understood. Here, we explored the possibility of engineering CARs for enhanced activity toward succinic acid for potential applications in 1,4-butanediol production. Structural models of the carboxylate-binding pocket of the CAR enzyme MAB4714 from Mycobacterium abscessus suggested that its reactivity toward succinic acid could be enhanced by reducing the pocket volume. Using site-directed mutagenesis, we introduced larger side chains into the MAB4714 carboxylate binding pocket and compared the activity of 16 mutant proteins against cinnamic and succinic acids. These experiments revealed that, although the reaction rates remain low, the replacement of Leu284 or Thr285 with Trp increased activity toward succinic acid more than two times. The T285E mutant protein also showed increased activity toward succinic acid, but it was lower than that of T285W. The mutated residues of MAB4714 are located on the flexible loop covering the carboxylate-binding pocket, which appears to contribute to substrate preference of CARs. Thus, reductase activity of CARs against succinic acid can be improved by introducing large side chains into the carboxylate-binding pocket. We also discovered that alanine replacement of the conserved Ser713 in the CAR phosphopantetheine attachment site resulted in complete degradation of the full-length protein into separate A and R domains, suggesting that CAR phosphopantetheinylation is important for its stability in solution.
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http://dx.doi.org/10.1016/j.jbiotec.2019.07.009DOI Listing
September 2019

Multigene Biomarkers of Pyrethroid Exposure: Exploratory Experiments.

Environ Toxicol Chem 2019 11 3;38(11):2436-2446. Epub 2019 Oct 3.

Office of Research and Development, National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio, USA.

We describe initial development of microarray-based assays for detecting 4 pyrethroid pesticides (bifenthrin, cypermethrin, esfenvalerate, and permethrin) in water. To facilitate comparison of transcriptional responses with gross apical responses, we estimated concentration-mortality curves for these pyrethroids using flow-through exposures of newly hatched Daphnia magna, Pimephales promelas adults, and 24 h posthatch P. promelas. Median lethal concentration (LC50) estimates were below most reported values, perhaps attributable to the use of flow-through exposures or of measured rather than nominal concentrations. Microarray analysis of whole P. promelas larvae and brains from exposed P. promelas adults showed that assays using either tissue type can detect these pyrethroids at concentrations below LC50 values reported for between 72 and 96% of aquatic species, depending on the pesticide. These estimates are conservative because they correspond to the lowest concentrations tested. This suggests that the simpler and less expensive whole-larval assay provides adequate sensitivity for screening contexts where acute aquatic lethality is observed, but the responsible agent is not known. Gene set analysis (GSA) highlighted several Gene Ontology (GO) terms consistent with known pyrethroid action, but the implications of other GO terms are less clear. Exploration of the sensitivity of results to changes in data processing suggests robustness of the detection assay results, but GSA results were sensitive to methodological variations. Environ Toxicol Chem 2019;38:2436-2446. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/etc.4552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836324PMC
November 2019

Alliance, Trust, and Loss: Experiences of Patients Cared for by Students in a Longitudinal Integrated Clerkship.

Acad Med 2019 11;94(11):1806-1813

R.J. Flick is a resident physician, Osler Medical Residency Training Program, Johns Hopkins Hospital, Baltimore, Maryland; ORCID: https://orcid.org/0000-0002-8155-6398. C. Felder-Heim is a resident physician, Family and Community Medicine Residency Program, University of California, San Francisco, San Francisco, California. J. Gong is assistant professor, Department of Family Medicine, University of Colorado School of Medicine, Aurora, Colorado; ORCID: https://orcid.org/0000-0001-7530-8358. J. Corral is associate professor, Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado; ORCID: https://orcid.org/0000-0001-8576-6192. K. Kalata is a fourth-year medical student, University of Colorado School of Medicine, Aurora, Colorado. A. Marin is a fourth-year medical student, University of Colorado School of Medicine, Aurora, Colorado. J.E. Adams is director, Denver Health Longitudinal Integrated Clerkship, assistant dean of clinical curriculum, and associate professor, Department of Internal Medicine, University of Colorado School of Medicine, Aurora, Colorado, and Denver Health and Hospital Authority, Denver, Colorado; ORCID: https://orcid.org/0000-0002-5433-8600.

Purpose: The longitudinal integrated clerkship (LIC) model, which allows medical students to participate in comprehensive care of a panel of patients over time, is rapidly expanding because of recognized benefits to students and faculty. This study aimed to determine how LIC student contact affected patients' experiences and self-described health outcomes.

Method: This qualitative case study used semistructured patient interviews to understand the impact of LIC learners at the University of Colorado School of Medicine on patients at Denver Health. Patients with at least 3 encounters with an LIC student and over age 18 were selected. Thirty patients were invited to participate in 2016-2017; 14 (47%) completed interviews before the thematic analysis reached saturation. Four researchers independently analyzed interview transcripts and reached consensus on emergent categories and themes.

Results: Six broad themes were identified: beginnings of a relationship, caring demonstrated by student, growing to trust student, reaching a therapeutic alliance, improvement of patient outcomes due to student involvement, and a sense of loss after students completed the LIC program.

Conclusions: Patients deeply valued the therapeutic alliances built with LIC students involved in their care over time. These alliances led to improved patient experience, mitigation of perceived health system failures, and subjective improvement in health outcomes. Patients described a sense of loss at the end of the LIC when students were no longer involved in their care. Curricula that support students building longitudinal therapeutic relationships with their patients are an opportunity to improve patient experience while promoting students' professional development.
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http://dx.doi.org/10.1097/ACM.0000000000002812DOI Listing
November 2019

Routine laboratory measures of heparin anticoagulation for children on extracorporeal membrane oxygenation: Systematic review and meta-analysis.

Thromb Res 2019 Jul 7;179:132-139. Epub 2019 May 7.

Mayo Clinic, Evidence-based Practice Center, Mayo Clinic Robert D. and Patricia E. Kern Center for the Science of Health Care Delivery, Rochester, MN, USA.

Objective: Specific protocols for anticoagulation for children on ECMO vary across institutions, with most using a continuous infusion of unfractionated heparin. The goal of this study is to aid clinician's decision on the best measure of heparin anticoagulation test; which would be the one that correlates well with heparin activity and helps in predicting hemorrhagic and thrombotic complications.

Data Sources: A comprehensive search of MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, and Scopus was conducted from each database's inception to 07/13/2018.

Study Selection: Studies evaluating children (<18 years) treated with ECMO and evaluating ACT, aPTT, TEG and Anti-Xa in any language were included.

Data Extraction: Two reviewers selected and appraised studies independently, and abstracted data.

Results: We included 19 studies (759 patients, mean age 19.8 months). Meta-analysis showed strong correlation between heparin dosing and anti-Xa. Additionally, there was not a strong correlation between laboratory tests and complications (hemorrhagic and thrombosis), or mortality.

Conclusion: Based on current evidence, Anti-Xa is the only laboratory test that shows strong correlation with heparin infusion dose and seems like the most suitable test for monitoring of anticoagulation with heparin in children on ECMO.
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http://dx.doi.org/10.1016/j.thromres.2019.05.006DOI Listing
July 2019

Prenylated FMN: Biosynthesis, purification, and Fdc1 activation.

Methods Enzymol 2019 11;620:469-488. Epub 2019 Apr 11.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada; Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, United Kingdom. Electronic address:

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.
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http://dx.doi.org/10.1016/bs.mie.2019.03.021DOI Listing
February 2020

The HIV diagnostic assistant: early findings from a novel HIV testing cadre in Malawi.

AIDS 2019 06;33(7):1215-1224

Baylor College of Medicine Children's Foundation, Malawi, Lilongwe, Malawi.

Objectives: In 2015, Malawi piloted the HIV diagnostic assistant (HDA), a cadre of lay health workers focused primarily on HIV testing services. Our objective is to measure the effect of HDA deployment on country-level HIV testing measures.

Design: Interrupted time series analysis of routinely collected data to assess immediate change in absolute numbers and longitudinal changes in trends.

Methods: Data from all HDA sites were divided into two periods: predeployment (October 2013 to June 2015) and postdeployment (July 2015 to December 2017). Monthly rates of several key HIV testing measures were evaluated: HIV testing, including all tests done, new positives, and confirmatory testing. Syphilis testing at antenatal clinic (ANC) and early infant diagnosis were also assessed.

Findings: The number of patients tested for HIV per month increased after HDA deployment across all sex, age, and testing subgroups. The number of tests immediately increased by 35 588 (P = 0.031), and the postintervention trend was significantly greater than the preintervention slope (+3442 per month, P = 0.001). Of 7.4 million patients tested for HIV in the postdeployment period, 2.6 million (34%) were attributable to the intervention. The proportion of new positives receiving confirmatory tests increased from 28% preintervention to 98% postintervention (P < 0.0001). Syphilis testing rates at ANC improved, with 98% of all tests attributable to HDA deployment. The number and proportion of infants receiving DNA-PCR testing at 2 months experienced significant trend increases (P < 0.0001).

Interpretation: HDA deployment is associated with significant increases in total HIV testing, identification of new positives, confirmatory testing, syphilis testing at ANC, and early infant diagnosis testing.
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http://dx.doi.org/10.1097/QAD.0000000000002159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511419PMC
June 2019

Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress.

J Biol Chem 2019 05 17;294(22):9007-9015. Epub 2019 Apr 17.

From the Departments of Laboratory Medicine and Pathobiology and

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic , which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.
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http://dx.doi.org/10.1074/jbc.RA119.007997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552437PMC
May 2019

Patterns of neuropsychological changes after general anaesthesia in young children: secondary analysis of the Mayo Anesthesia Safety in Kids study.

Br J Anaesth 2019 May;122(5):671-681

Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, Rochester, MN, USA. Electronic address:

Background: We hypothesised that exposure to multiple, but not single, procedures requiring general anaesthesia before age 3 yr is associated with a specific pattern of deficits in processing speed and fine motor skills.

Methods: A secondary analysis (using factor and cluster analyses) of data from the Mayo Anesthesia Safety in Kids study was conducted, in which unexposed, singly exposed, and multiply exposed children born in Olmsted County, MN, USA from 1994 to 2007 were sampled using a propensity-guided approach and underwent neuropsychological testing at ages 8-12 or 15-20 yr.

Results: In the factor analysis, the data were well fit to a five factor model. For subjects multiply (but not singly) exposed to anaesthesia, a factor reflecting motor skills, visual-motor integration, and processing speed was significantly lower [standardised difference of -0.35 (95% confidence interval {CI} -0.57 to -0.13)] compared with unexposed subjects. No other factor was associated with exposure. Three groups were identified in the cluster analysis, with 106 subjects (10.6%) in Cluster A (lowest performance in most tests), 557 (55.9%) in Cluster B, and 334 (33.5%) in Cluster C (highest performance in most tests). The odds of multiply exposed children belonging to Cluster A was 2.83 (95% CI: 1.49-5.35; P=0.001) compared with belonging to Cluster B; there was no other significant association between exposure status and cluster membership.

Conclusions: Multiple, but not single, exposures to procedures requiring general anaesthesia before age 3 yr are associated with a specific pattern of deficits in neuropsychological tests. Factors predicting which children develop the most pronounced deficits remain unknown.
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http://dx.doi.org/10.1016/j.bja.2019.01.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549043PMC
May 2019

Performance on the Operant Test Battery in young children exposed to procedures requiring general anaesthesia: the MASK study.

Br J Anaesth 2019 Apr 4;122(4):470-479. Epub 2019 Feb 4.

Department of Anesthesiology and Perioperative Medicine, USA.

Background: It is not known whether the neurotoxicity produced by anaesthetics administered to young animals can also occur in children. Exposure of infant macaques to ketamine impairs performance in selected domains of the Operant Test Battery (OTB), which can also be administered to children. This study determined whether a similar pattern of results on the OTB is found in children exposed to procedures requiring general anaesthesia before age 3 yr.

Methods: We analysed data from the Mayo Anesthesia Safety in Kids (MASK) study, in which unexposed, singly-exposed, and multiply-exposed children born in Olmsted County, MN, USA, from 1994 to 2007 were sampled using a propensity-guided approach and prospectively underwent OTB testing at ages 8-12 or 15-20 yr, using five tasks that generated 15 OTB test scores.

Results: In primary analysis, none of the OTB test scores depended upon anaesthesia exposure status when corrected for multiple comparisons. Cluster analysis identified four clusters of subjects, with cluster membership determined by relative performance on the OTB tasks. There was no evidence of association between exposure status and cluster membership. Exploratory factor analysis showed that the OTB scores loaded onto four factors. The score for one factor was significantly less in multiply-exposed children (mean standardised difference -0.28 [95% confidence interval, -0.55 to -0.01; P=0.04]), but significance did not survive a sensitivity analysis accounting for outlying values.

Conclusions: These findings provide little evidence to support the hypothesis that children exposed to procedures requiring anaesthesia show deficits on OTB tasks that are similar to those observed in non-human primates.
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http://dx.doi.org/10.1016/j.bja.2018.12.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435940PMC
April 2019

Characterization of the Fundulus heteroclitus embryo transcriptional response and development of a gene expression-based fingerprint of exposure for the alternative flame retardant, TBPH (bis (2-ethylhexyl)-tetrabromophthalate).

Environ Pollut 2019 Apr 10;247:696-705. Epub 2019 Jan 10.

U.S. EPA Office of Research and Development, National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH, 45268, USA. Electronic address:

Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (n = 16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.
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http://dx.doi.org/10.1016/j.envpol.2019.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495336PMC
April 2019

An interspecies malate-pyruvate shuttle reconciles redox imbalance in an anaerobic microbial community.

ISME J 2019 04 3;13(4):1042-1055. Epub 2019 Jan 3.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 3E5, Canada.

Microbes in ecosystems often develop coordinated metabolic interactions. Therefore, understanding metabolic interdependencies between microbes is critical to deciphering ecosystem function. In this study, we sought to deconstruct metabolic interdependencies in organohalide-respiring consortium ACT-3 containing Dehalobacter restrictus using a combination of metabolic modeling and experimental validation. D. restrictus possesses a complete set of genes for amino acid biosynthesis yet when grown in isolation requires amino acid supplementation. We reconciled this discrepancy using flux balance analysis considering cofactor availability, enzyme promiscuity, and shared protein expression patterns for several D. restrictus strains. Experimentally, C incorporation assays, growth assays, and metabolite analysis of D. restrictus strain PER-K23 cultures were performed to validate the model predictions. The model resolved that the amino acid dependency of D. restrictus resulted from restricted NADPH regeneration and predicted that malate supplementation would replenish intracellular NADPH. Interestingly, we observed unexpected export of pyruvate and glutamate in parallel to malate consumption in strain PER-K23 cultures. Further experimental analysis using the ACT-3 transfer cultures suggested the occurrence of an interspecies malate-pyruvate shuttle reconciling a redox imbalance, reminiscent of the mitochondrial malate shunt pathway in eukaryotic cells. Altogether, this study suggests that redox imbalance and metabolic complementarity are important driving forces for metabolite exchange in anaerobic microbial communities.
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http://dx.doi.org/10.1038/s41396-018-0333-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461761PMC
April 2019