Publications by authors named "David La"

87 Publications

The Pediatric Obesity Microbiome and Metabolism Study (POMMS): Methods, Baseline Data, and Early Insights.

Obesity (Silver Spring) 2021 Mar;29(3):569-578

Department of Pediatrics, Duke University, Durham, North Carolina, USA.

Objective: The purpose of this study was to establish a biorepository of clinical, metabolomic, and microbiome samples from adolescents with obesity as they undergo lifestyle modification.

Methods: A total of 223 adolescents aged 10 to 18 years with BMI ≥95th percentile were enrolled, along with 71 healthy weight participants. Clinical data, fasting serum, and fecal samples were collected at repeated intervals over 6 months. Herein, the study design, data collection methods, and interim analysis-including targeted serum metabolite measurements and fecal 16S ribosomal RNA gene amplicon sequencing among adolescents with obesity (n = 27) and healthy weight controls (n = 27)-are presented.

Results: Adolescents with obesity have higher serum alanine aminotransferase, C-reactive protein, and glycated hemoglobin, and they have lower high-density lipoprotein cholesterol when compared with healthy weight controls. Metabolomics revealed differences in branched-chain amino acid-related metabolites. Also observed was a differential abundance of specific microbial taxa and lower species diversity among adolescents with obesity when compared with the healthy weight group.

Conclusions: The Pediatric Metabolism and Microbiome Study (POMMS) biorepository is available as a shared resource. Early findings suggest evidence of a metabolic signature of obesity unique to adolescents, along with confirmation of previously reported findings that describe metabolic and microbiome markers of obesity.
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http://dx.doi.org/10.1002/oby.23081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927749PMC
March 2021

Benefit of a more extended pelvic lymph node dissection among patients undergoing radical prostatectomy for localized prostate cancer: A causal mediation analysis.

Prostate 2021 Apr 18;81(5):286-294. Epub 2021 Feb 18.

Division of Urology, Department of Surgery, Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.

Background: The therapeutic role of extended (ePLND) versus nonextended pelvic lymph node dissection (nePLND) to remove occult micrometastases in men undergoing radical prostatectomy for localized prostate cancer (PC) is conflicting. Therefore, our aim was to quantify the direct effect of ePLND versus nePLND (removal of occult micrometastases), which is not mediated through the detection of nodal disease and potential adjuvant therapy (indirect effect).

Methods: Retrospective, bi-center cohort study of consecutive patients undergoing radical prostatectomy and PLND for PC (January 2006 and December 2016). Patients were followed until April 2018 for the occurrence of either biochemical recurrence or secondary therapy (composite outcome). ePLND was compared to nePLND by unweighted and weighted survival analysis (total effect) as well as by causal mediation analysis (direct and indirect effect).

Results: Positive nodal disease was detected in 71 (7%) out of 1008 patients undergoing radical prostatectomy and PLND for PC (ePLND: 368 [36.5%]; nePLND: 640 [63.5%]). Survival analysis demonstrated results in favor of ePLND (unweighted hazard ratio: 0.77 [95% confidence interval: 0.59-1.01], p = .056; weighted hazard ratio: 0.75 [0.56-0.99], p = .044). The causal mediation analysis confirmed the total effect of 0.77 (0.71-0.82). After disentangling this total effect into an indirect effect (via detection of nodal disease and potential adjuvant therapy) and a direct effect (via removal of occult micrometastases), we identified an even more protective direct effect of 0.69 (0.63-0.75).

Conclusions: Our results not only indicate the utility of ePLND but also that its impact is not restricted to a staging benefit and probably involves a therapeutic benefit mediated through the removal of occult micrometastases.
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http://dx.doi.org/10.1002/pros.24105DOI Listing
April 2021

Naught all zeros in sequence count data are the same.

Comput Struct Biotechnol J 2020 28;18:2789-2798. Epub 2020 Sep 28.

Program in Computational Biology and Bioinformatics, Duke University, Durham, NC 27708, United States.

Genomic studies feature multivariate count data from high-throughput DNA sequencing experiments, which often contain many zero values. These zeros can cause artifacts for statistical analyses and multiple modeling approaches have been developed in response. Here, we apply different zero-handling models to gene-expression and microbiome datasets and show models can disagree substantially in terms of identifying the most differentially expressed sequences. Next, to rationally examine how different zero handling models behave, we developed a conceptual framework outlining four types of processes that may give rise to zero values in sequence count data. Last, we performed simulations to test how zero handling models behave in the presence of these different zero generating processes. Our simulations showed that simple count models are sufficient across multiple processes, even when the true underlying process is unknown. On the other hand, a common zero handling technique known as "zero-inflation" was only suitable under a zero generating process associated with an unlikely set of biological and experimental conditions. In concert, our work here suggests several specific guidelines for developing and choosing state-of-the-art models for analyzing sparse sequence count data.
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http://dx.doi.org/10.1016/j.csbj.2020.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568192PMC
September 2020

Conserved anti-inflammatory effects and sensing of butyrate in zebrafish.

Gut Microbes 2020 11;12(1):1-11

Tuberculosis Research Program at the Centenary Institute, The University of Sydney , Camperdown, Australia.

Short-chain fatty acids (SCFAs) are produced by microbial fermentation of dietary fiber in the gut. Butyrate is a particularly important SCFA with anti-inflammatory properties and is generally present at lower levels in inflammatory diseases associated with gut microbiota dysbiosis in mammals. We aimed to determine if SCFAs are produced by the zebrafish microbiome and if SCFAs exert conserved effects on zebrafish immunity as an example of the non-mammalian vertebrate immune system. We demonstrate that bacterial communities from adult zebrafish intestines synthesize all three main SCFA , although SCFA were below our detectable limits in zebrafish intestines . Immersion in butyrate, but not acetate or propionate, reduced the recruitment of neutrophils and M1-type pro-inflammatory macrophages to wounds. We found conservation of butyrate sensing by neutrophils via orthologs of the () gene. Neutrophils from Hcar1-depleted embryos were no longer responsive to the anti-inflammatory effects of butyrate, while macrophage sensitivity to butyrate was independent of Hcar1. Our data demonstrate conservation of anti-inflammatory butyrate effects and identify the presence of a conserved molecular receptor in fish.
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http://dx.doi.org/10.1080/19490976.2020.1824563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575005PMC
November 2020

Short-Chain Fatty Acid Production by Gut Microbiota from Children with Obesity Differs According to Prebiotic Choice and Bacterial Community Composition.

mBio 2020 08 11;11(4). Epub 2020 Aug 11.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA

Pediatric obesity remains a public health burden and continues to increase in prevalence. The gut microbiota plays a causal role in obesity and is a promising therapeutic target. Specifically, the microbial production of short-chain fatty acids (SCFA) from the fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome. Still, it has not been demonstrated that therapies involving microbiota-targeting carbohydrates, known as prebiotics, will enhance gut bacterial SCFA production in children and adolescents with obesity (age, 10 to 18 years old). Here, we used an system to examine the SCFA production by fecal microbiota from 17 children with obesity when exposed to five different commercially available over-the-counter (OTC) prebiotic supplements. We found microbiota from all 17 patients actively metabolized most prebiotics. Still, supplements varied in their acidogenic potential. Significant interdonor variation also existed in SCFA production, which 16S rRNA sequencing supported as being associated with differences in the host microbiota composition. Last, we found that neither fecal SCFA concentration, microbiota SCFA production capacity, nor markers of obesity positively correlated with one another. Together, these findings suggest the hypothesis that OTC prebiotic supplements may be unequal in their ability to stimulate SCFA production in children and adolescents with obesity and that the most acidogenic prebiotic may differ across individuals. Pediatric obesity remains a major public health problem in the United States, where 17% of children and adolescents are obese, and rates of pediatric "severe obesity" are increasing. Children and adolescents with obesity face higher health risks, and noninvasive therapies for pediatric obesity often have limited success. The human gut microbiome has been implicated in adult obesity, and microbiota-directed therapies can aid weight loss in adults with obesity. However, less is known about the microbiome in pediatric obesity, and microbiota-directed therapies are understudied in children and adolescents. Our research has two important findings: (i) dietary prebiotics (fiber) result in the microbiota from adolescents with obesity producing more SCFA, and (ii) the effectiveness of each prebiotic is donor dependent. Together, these findings suggest that prebiotic supplements could help children and adolescents with obesity, but that these therapies may not be "one size fits all."
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http://dx.doi.org/10.1128/mBio.00914-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439474PMC
August 2020

Predicting Vibrio cholerae Infection and Disease Severity Using Metagenomics in a Prospective Cohort Study.

J Infect Dis 2021 Feb;223(2):342-351

Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington, USA.

Background: Susceptibility to Vibrio cholerae infection is affected by blood group, age, and preexisting immunity, but these factors only partially explain who becomes infected. A recent study used 16S ribosomal RNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution.

Methods: To achieve increased resolution of gut microbial factors associated with V. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera.

Results: Using machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to V. cholerae to identify markers that predict infection and symptoms. Use of metagenomic features improved the precision and accuracy of prediction relative to 16S sequencing. We also predicted disease severity, although with greater uncertainty than our infection prediction. Species within the genera Prevotella and Bifidobacterium predicted protection from infection, and genes involved in iron metabolism were also correlated with protection.

Conclusion: Our results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera.
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http://dx.doi.org/10.1093/infdis/jiaa358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857355PMC
February 2021

Interindividual Variation in Dietary Carbohydrate Metabolism by Gut Bacteria Revealed with Droplet Microfluidic Culture.

mSystems 2020 Jun 30;5(3). Epub 2020 Jun 30.

Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA

Culture and screening of gut bacteria enable testing of microbial function and therapeutic potential. However, the diversity of human gut microbial communities (microbiota) impedes comprehensive experimental studies of individual bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput DNA sequencing to develop a platform for separating and assaying growth of microbiota members in picoliter droplets (MicDrop). MicDrop enabled us to cultivate 2.8 times more bacterial taxa than typical batch culture methods. We then used MicDrop to test whether individuals possess similar abundances of carbohydrate-degrading gut bacteria, using an approach which had previously not been possible due to throughput limitations of traditional bacterial culture techniques. Single MicDrop experiments allowed us to characterize carbohydrate utilization among dozens of gut bacterial taxa from distinct human stool samples. Our aggregate data across nine healthy stool donors revealed that all of the individuals harbored gut bacterial species capable of degrading common dietary polysaccharides. However, the levels of richness and abundance of polysaccharide-degrading species relative to monosaccharide-consuming taxa differed by up to 2.6-fold and 24.7-fold, respectively. Additionally, our unique dataset suggested that gut bacterial taxa may be broadly categorized by whether they can grow on single or multiple polysaccharides, and we found that this lifestyle trait is correlated with how broadly bacterial taxa can be found across individuals. This demonstration shows that it is feasible to measure the function of hundreds of bacterial taxa across multiple fecal samples from different people, which should in turn enable future efforts to design microbiota-directed therapies and yield new insights into microbiota ecology and evolution. Bacterial culture and assay are components of basic microbiological research, drug development, and diagnostic screening. However, community diversity can make it challenging to comprehensively perform experiments involving individual microbiota members. Here, we present a new microfluidic culture platform that makes it feasible to measure the growth and function of microbiota constituents in a single set of experiments. As a proof of concept, we demonstrate how the platform can be used to measure how hundreds of gut bacterial taxa drawn from different people metabolize dietary carbohydrates. Going forward, we expect this microfluidic technique to be adaptable to a range of other microbial assay needs.
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http://dx.doi.org/10.1128/mSystems.00864-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329328PMC
June 2020

Focused ultrasound as a novel strategy for noninvasive gene delivery to retinal Müller glia.

Theranostics 2020 10;10(7):2982-2999. Epub 2020 Feb 10.

Biological Sciences Platform, Sunnybrook Research Institute, Toronto, Ontario, Canada.

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. : As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants . For assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). : We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. : Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.
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http://dx.doi.org/10.7150/thno.42611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053200PMC
February 2020

Novel Immunomodulatory Proteins Generated via Directed Evolution of Variant IgSF Domains.

Front Immunol 2019 21;10:3086. Epub 2020 Jan 21.

Alpine Immune Sciences Inc., Seattle, WA, United States.

Immunoglobulin superfamily member (IgSF) proteins play a significant role in regulating immune responses with surface expression on all immune cell subsets, making the IgSF an attractive family of proteins for therapeutic targeting in human diseases. We have developed a directed evolution platform capable of engineering IgSF domains to increase affinities for cognate ligands and/or introduce binding to non-cognate ligands. Using this scientific platform, ICOSL domains have been derived with enhanced binding to ICOS and with additional high-affinity binding to the non-cognate receptor, CD28. Fc-fusion proteins containing these engineered ICOSL domains significantly attenuate T cell activation and and can inhibit development of inflammatory diseases in mouse models. We also present evidence that engineered ICOSL domains can be formatted to selectively provide costimulatory signals to augment T cell responses. Our scientific platform thus provides a system for developing therapeutic protein candidates with selective biological impact for treatments of a wide array of human disorders including cancer and autoimmune/inflammatory diseases.
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http://dx.doi.org/10.3389/fimmu.2019.03086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985287PMC
November 2020

Preoperative and post-operative psychosocial interventions for bariatric surgery patients: A systematic review.

Obes Rev 2020 04 22;21(4):e12926. Epub 2020 Jan 22.

Department of Psychology, Ryerson University, Toronto, Canada.

Psychosocial interventions are increasingly being utilized to help patients prepare for, and adjust to changes following, bariatric surgery in order to optimize psychosocial adjustment and weight loss. The current systematic review examined the impact of preoperative and post-operative psychosocial interventions with a behavioural and/or cognitive focus on weight, dietary behaviours, eating pathology, lifestyle behaviours, and psychological functioning. A PsycINFO and Medline search of publications was conducted in March 2019. Two authors assessed retrieved titles and abstracts to determine topic relevance and rated the quality of included studies using a validated checklist. Forty-four articles (representing 36 studies) met the study inclusion criteria. The current evidence is strongest for the impact of psychosocial interventions, particularly cognitive behavioural therapy, on eating behaviours (eg, binge eating and emotional eating) and psychological functioning (eg, quality of life, depression, and anxiety). The evidence for the impact of psychosocial interventions on weight loss, dietary behaviours (eg, dietary intake), and lifestyle behaviours (eg, physical activity) is relatively weak and mixed. Psychosocial interventions can improve eating pathology and psychosocial functioning among bariatric patients, and the optimal time to initiate treatment appears to be early in the post-operative period before significant problematic eating behaviours and weight regain occur.
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http://dx.doi.org/10.1111/obr.12926DOI Listing
April 2020

Using DNA Metabarcoding To Evaluate the Plant Component of Human Diets: a Proof of Concept.

mSystems 2019 Oct 8;4(5). Epub 2019 Oct 8.

Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA

Dietary intake is difficult to measure reliably in humans because approaches typically rely on self-reporting, which can be incomplete and biased. In field studies of animals, DNA sequencing-based approaches such as metabarcoding have been developed to characterize diets, but such approaches have not previously been widely applied to humans. Here, we present data derived from sequencing of a chloroplast DNA marker (the P6 loop of the L [UAA] intron) in stool samples collected from 11 individuals consuming both controlled and freely selected diets. The DNA metabarcoding strategy resulted in successful PCR amplification in about 50% of samples, which increased to a 70% success rate in samples from individuals eating a controlled plant-rich diet. Detection of plant taxa among sequenced samples yielded a recall of 0.86 and a precision of 0.55 compared to a written diet record during controlled feeding of plant-based foods. The majority of sequenced plant DNA matched common human food plants, including grains, vegetables, fruits, and herbs prepared both cooked and uncooked. Moreover, DNA metabarcoding data were sufficient to distinguish between baseline and treatment diet arms of the study. Still, the relatively high PCR failure rate and an inability to distinguish some dietary plants at the sequence level using the L-P6 marker suggest that future methodological refinements are necessary. Overall, our results suggest that DNA metabarcoding provides a promising new method for tracking human plant intake and that similar approaches could be used to characterize the animal and fungal components of our omnivorous diets. Current methods for capturing human dietary patterns typically rely on individual recall and as such are subject to the limitations of human memory. DNA sequencing-based approaches, frequently used for profiling nonhuman diets, do not suffer from the same limitations. Here, we used metabarcoding to broadly characterize the plant portion of human diets for the first time. The majority of sequences corresponded to known human foods, including all but one foodstuff included in an experimental plant-rich diet. Metabarcoding could distinguish between experimental diets and matched individual diet records from controlled settings with high accuracy. Because this method is independent of survey language and timing, it could also be applied to geographically and culturally disparate human populations, as well as in retrospective studies involving banked human stool.
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http://dx.doi.org/10.1128/mSystems.00458-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787566PMC
October 2019

Correction to: Dynamic linear models guide design and analysis of microbiota studies within artificial human guts.

Microbiome 2018 11 27;6(1):212. Epub 2018 Nov 27.

Program in Computational Biology and Bioinformatics, Duke University, CIEMAS, Room 2171, 101 Science Drive, Box 3382, Durham, NC, 27708, USA.

AbstractFollowing publication of the original article [1], the authors noticed an error in the presentation of equations in the PDF version.
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http://dx.doi.org/10.1186/s40168-018-0601-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260730PMC
November 2018

Dynamic linear models guide design and analysis of microbiota studies within artificial human guts.

Microbiome 2018 11 12;6(1):202. Epub 2018 Nov 12.

Program in Computational Biology and Bioinformatics, Duke University, CIEMAS, Room 2171, 101 Science Drive, Box 3382, Durham, NC, 27708, USA.

Background: Artificial gut models provide unique opportunities to study human-associated microbiota. Outstanding questions for these models' fundamental biology include the timescales on which microbiota vary and the factors that drive such change. Answering these questions though requires overcoming analytical obstacles like estimating the effects of technical variation on observed microbiota dynamics, as well as the lack of appropriate benchmark datasets.

Results: To address these obstacles, we created a modeling framework based on multinomial logistic-normal dynamic linear models (MALLARDs) and performed dense longitudinal sampling of four replicate artificial human guts over the course of 1 month. The resulting analyses revealed how the ratio of biological variation to technical variation from sample processing depends on sampling frequency. In particular, we find that at hourly sampling frequencies, 76% of observed variation could be ascribed to technical sources, which could also skew the observed covariation between taxa. We also found that the artificial guts demonstrated replicable trajectories even after a recovery from a transient feed disruption. Additionally, we observed irregular sub-daily oscillatory dynamics associated with the bacterial family Enterobacteriaceae within all four replicate vessels.

Conclusions: Our analyses suggest that, beyond variation due to sequence counting, technical variation from sample processing can obscure temporal variation from biological sources in artificial gut studies. Our analyses also supported hypotheses that human gut microbiota fluctuates on sub-daily timescales in the absence of a host and that microbiota can follow replicable trajectories in the presence of environmental driving forces. Finally, multiple aspects of our approach are generalizable and could ultimately be used to facilitate the design and analysis of longitudinal microbiota studies in vivo.
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http://dx.doi.org/10.1186/s40168-018-0584-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233358PMC
November 2018

Microbial nitrogen limitation in the mammalian large intestine.

Nat Microbiol 2018 12 29;3(12):1441-1450. Epub 2018 Oct 29.

Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA.

Resource limitation is a fundamental factor governing the composition and function of ecological communities. However, the role of resource supply in structuring the intestinal microbiome has not been established and represents a challenge for mammals that rely on microbial symbionts for digestion: too little supply might starve the microbiome while too much might starve the host. We present evidence that microbiota occupy a habitat that is limited in total nitrogen supply within the large intestines of 30 mammal species. Lowering dietary protein levels in mice reduced their faecal concentrations of bacteria. A gradient of stoichiometry along the length of the gut was consistent with the hypothesis that intestinal nitrogen limitation results from host absorption of dietary nutrients. Nitrogen availability is also likely to be shaped by host-microbe interactions: levels of host-secreted nitrogen were altered in germ-free mice and when bacterial loads were reduced via experimental antibiotic treatment. Single-cell spectrometry revealed that members of the phylum Bacteroidetes consumed nitrogen in the large intestine more readily than other commensal taxa did. Our findings support a model where nitrogen limitation arises from preferential host use of dietary nutrients. We speculate that this resource limitation could enable hosts to regulate microbial communities in the large intestine. Commensal microbiota may have adapted to nitrogen-limited settings, suggesting one reason why excess dietary protein has been associated with degraded gut-microbial ecosystems.
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http://dx.doi.org/10.1038/s41564-018-0267-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264799PMC
December 2018

Lead Optimization of 5-Aryl Benzimidazolone- and Oxindole-Based AMPA Receptor Modulators Selective for TARP γ-8.

ACS Med Chem Lett 2018 Aug 13;9(8):821-826. Epub 2018 Jul 13.

Janssen Research & Development L.L.C., 3210 Merryfield Row, San Diego, California 92121, United States.

Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead was optimized for potency and brain penetration to provide benzimidazolone , JNJ-55511118.1 Replacement of the benzimidazolone core in with an oxindole mitigated reactive metabolite formation and led to the identification of (GluA1/γ-8 pIC = 9.7). Following oral dosing in rats, demonstrated robust target engagement in hippocampus as assessed by autoradiography (ED = 0.6 mg/kg, plasma EC = 9 ng/mL).
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http://dx.doi.org/10.1021/acsmedchemlett.8b00215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6088354PMC
August 2018

Antibiotic-induced changes in the microbiota disrupt redox dynamics in the gut.

Elife 2018 06 19;7. Epub 2018 Jun 19.

Department of Molecular Genetics and Microbiology, Duke University, Durham, United States.

How host and microbial factors combine to structure gut microbial communities remains incompletely understood. Redox potential is an important environmental feature affected by both host and microbial actions. We assessed how antibiotics, which can impact host and microbial function, change redox state and how this contributes to post-antibiotic succession. We showed gut redox potential increased within hours of an antibiotic dose in mice. Host and microbial functioning changed under treatment, but shifts in redox potentials could be attributed specifically to bacterial suppression in a host-free ex vivo human gut microbiota model. Redox dynamics were linked to blooms of the bacterial family Enterobacteriaceae. Ecological succession to pre-treatment composition was associated with recovery of gut redox, but also required dispersal from unaffected gut communities. As bacterial competition for electron acceptors can be a key ecological factor structuring gut communities, these results support the potential for manipulating gut microbiota through managing bacterial respiration.
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http://dx.doi.org/10.7554/eLife.35987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008055PMC
June 2018

Hamartoma-like lesions in the mouse retina: an animal model of hamartoma tumour syndrome.

Dis Model Mech 2018 05 21;11(5). Epub 2018 May 21.

Biological Sciences Platform, Sunnybrook Research Institute, Room 116, 2075 Bayview Ave, Toronto, ON, M4N 3M5, Canada

hamartoma tumour syndrome (PHTS) is a heterogeneous group of rare, autosomal dominant disorders associated with germline mutations. PHTS patients routinely develop hamartomas, which are benign tissue overgrowths comprised of disorganized 'normal' cells. Efforts to generate PHTS animal models have been largely unsuccessful due to the early lethality of homozygous germline mutations in , together with the lack of hamartoma formation in most conditional mutants generated to date. We report herein a novel PHTS mouse model that reproducibly forms hamartoma-like lesions in the central retina by postnatal day 21. Specifically, we generated a conditional knockout (cKO) using a retinal-specific driver that leads to a nearly complete deletion of in the peripheral retina but produces a mosaic of 'wild-type' and cKO cells centrally. Structural defects were only observed in the mosaic central retina, including in Müller glia and in the outer and inner limiting membranes, suggesting that defective mechanical integrity partly underlies the hamartoma-like pathology. Finally, we used this newly developed model to test whether rapamycin, an mTOR inhibitor that is currently the only PHTS therapy, can block hamartoma growth. When administered in the early postnatal period, prior to hamartoma formation, rapamycin reduces hamartoma size, but also induces new morphological abnormalities in the cKO retinal periphery. In contrast, administration of rapamycin after hamartoma initiation fails to reduce lesion size. We have thus generated and used an animal model of retinal PHTS to show that, although current therapies can reduce hamartoma formation, they might also induce new retinal dysmorphologies.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.031005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992614PMC
May 2018

Human Gut Microbiota Predicts Susceptibility to Vibrio cholerae Infection.

J Infect Dis 2018 07;218(4):645-653

Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts.

Background: Cholera is a public health problem worldwide, and the risk factors for infection are only partially understood.

Methods: We prospectively studied household contacts of patients with cholera to compare those who were infected to those who were not. We constructed predictive machine learning models of susceptibility, using baseline gut microbiota data. We identified bacterial taxa associated with susceptibility to Vibrio cholerae infection and tested these taxa for interactions with V. cholerae in vitro.

Results: We found that machine learning models based on gut microbiota, as well as models based on known clinical and epidemiological risk factors, predicted V. cholerae infection. A predictive gut microbiota of roughly 100 bacterial taxa discriminated between contacts who developed infection and those who did not. Susceptibility to cholera was associated with depleted levels of microbes from the phylum Bacteroidetes. By contrast, a microbe associated with cholera by our modeling framework, Paracoccus aminovorans, promoted the in vitro growth of V. cholerae. Gut microbiota structure, clinical outcome, and age were also linked.

Conclusion: These findings support the hypothesis that abnormal gut microbial communities are a host factor related to V. cholerae susceptibility.
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http://dx.doi.org/10.1093/infdis/jiy192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047457PMC
July 2018

Toward Personalized Control of Human Gut Bacterial Communities.

Authors:
Lawrence A David

mSystems 2018 Mar-Apr;3(2). Epub 2018 Mar 20.

Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA.

A key challenge in microbiology will be developing tools for manipulating human gut bacterial communities. Our ability to predict and control the dynamics of these communities is now in its infancy. To manage human gut microbiota, I am developing methods in three research domains. First, I am refining tools to experimentally study gut microbes at high throughput and in controlled settings. Second, I am adapting "big data" techniques to overcome statistical challenges confronting microbiota modeling. Third, I am testing study designs that can streamline human testing of microbiota manipulations. Assembling these methods creates new challenges, including training scientists who can work across disciplines such as engineering, ecology, and medicine. Nevertheless, I envision that overcoming these obstacles will enable my group to construct platforms that can personalize microbiota treatments, particularly ones based on diet. More broadly, I anticipate that such platforms will have applications across fields such as agriculture, biotechnology, and environmental management.
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http://dx.doi.org/10.1128/mSystems.00165-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881022PMC
March 2018

Plant community and soil conditions individually affect soil microbial community assembly in experimental mesocosms.

Ecol Evol 2018 01 20;8(2):1196-1205. Epub 2017 Dec 20.

Department of Biology Duke University Durham NC USA.

Soils harbor large, diverse microbial communities critical for local and global ecosystem functioning that are controlled by multiple and poorly understood processes. In particular, while there is observational evidence of relationships between both biotic and abiotic conditions and microbial composition and diversity, there have been few experimental tests to determine the relative importance of these two sets of factors at local scales. Here, we report the results of a fully factorial experiment manipulating soil conditions and plant cover on old-field mesocosms across a latitudinal gradient. The largest contributor to beta diversity was site-to-site variation, but, having corrected for that, we observed significant effects of both plant and soil treatments on microbial composition. Separate phyla were associated with each treatment type, and no interactions between soil and plant treatment were observed. Individual soil characteristics and biotic parameters were also associated with overall beta-diversity patterns and phyla abundance. In contrast, soil microbial diversity was only associated with site and not experimental treatment. Overall, plant community treatment explained more variation than soil treatment, a result not previously appreciated because it is difficult to dissociate plant community composition and soil conditions in observational studies across gradients. This work highlights the need for more nuanced, multifactorial experiments in microbial ecology and in particular indicates a greater focus on relationships between plant composition and microbial composition during community assembly.
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http://dx.doi.org/10.1002/ece3.3734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773302PMC
January 2018

Computational design of environmental sensors for the potent opioid fentanyl.

Elife 2017 09 19;6. Epub 2017 Sep 19.

Department of Biochemistry, University of Washington, Seattle, United States.

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.
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http://dx.doi.org/10.7554/eLife.28909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655540PMC
September 2017

An Immunohistochemical Investigation of Renal Phospholipidosis and Toxicity in Rats.

Int J Toxicol 2017 Sep/Oct;36(5):386-394. Epub 2017 Aug 18.

1 Janssen Research and Development, LLC, San Diego, CA, USA.

Immunohistochemical staining for the lysosome-associated membrane protein 2 (LAMP-2) has been proposed previously as an alternative to electron microscopy to identify hepatic phospholipidosis. This study used LAMP-2 immunohistochemistry (IHC) to diagnose phospholipidosis in rats exhibiting renal tubular injury. Rats were administered toreforant, a histamine H4 receptor antagonist by oral gavage at a dose of 3, 10, or 100 mg/kg/d for 6 months. Hematoxylin and eosin staining revealed renal tubular epithelial cell vacuolation, hypertrophy, degeneration, and luminal dilation in the 100 mg/kg/d group animals. Renal tubular injury was confirmed using kidney injury marker 1 (KIM-1) IHC. The involvement of phosopholipidosis in the renal injury was investigated by LAMP-2. Adipophilin IHC was included to differentiate phospholipidosis from lipidosis. Increased LAMP-2 staining was observed in the 100 mg/kg/d group animals when compared to vehicle group animals. Lysosome-associated membrane protein-2 staining was most prominent in the outer stripe of the outer medulla where KIM-1 staining was also most prominent. By contrast, adipophilin staining was not increased. Phospholipidosis was also confirmed by electron microscopy. These data support the use of LAMP-2 IHC as a diagnostic tool and suggest an association between phospholipidosis and the renal tubular injury caused by toreforant.
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http://dx.doi.org/10.1177/1091581817726040DOI Listing
June 2018

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site.

Nat Biotechnol 2017 Jul 12;35(7):667-671. Epub 2017 Jun 12.

Department of Biochemistry, University of Washington, Seattle, Washington, USA.

Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.
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http://dx.doi.org/10.1038/nbt.3907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512607PMC
July 2017

Phylogenetic factorization of compositional data yields lineage-level associations in microbiome datasets.

PeerJ 2017 9;5:e2969. Epub 2017 Feb 9.

Program for Computational Biology and Bioinformatics, Duke University, Durham, NC, United States; Center for Genomic and Computational Biology, Duke University, Durham, NC, United States; Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, United States.

Marker gene sequencing of microbial communities has generated big datasets of microbial relative abundances varying across environmental conditions, sample sites and treatments. These data often come with putative phylogenies, providing unique opportunities to investigate how shared evolutionary history affects microbial abundance patterns. Here, we present a method to identify the phylogenetic factors driving patterns in microbial community composition. We use the method, "phylofactorization," to re-analyze datasets from the human body and soil microbial communities, demonstrating how phylofactorization is a dimensionality-reducing tool, an ordination-visualization tool, and an inferential tool for identifying edges in the phylogeny along which putative functional ecological traits may have arisen.
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http://dx.doi.org/10.7717/peerj.2969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345826PMC
February 2017

Clinical Development of Histamine H Receptor Antagonists.

Handb Exp Pharmacol 2017 ;241:301-320

Janssen Research & Development, LLC, San Diego, CA, 92121, USA.

The discovery of the histamine H receptor (HR) provided a new avenue for the exploration of the physiological role of histamine, as well as providing a new drug target for the development of novel antihistamines. The first step in this process was the identification of selective antagonists to help unravel the pharmacology of the HR relative to other histamine receptors. The discovery of the selective HR antagonist JNJ 7777120 was vital for showing a role for the HR in inflammation and pruritus. While this compound has been very successful as a tool for understanding the function of the receptor, it has drawbacks, including a short in vivo half-life and hypoadrenocorticism toxicity in rats and dogs, that prevented advancing it into clinical studies. Further research let to the discovery of JNJ 39758979, which, similar to JNJ 7777120, was a potent and selective HR antagonist and showed anti-inflammatory and anti-pruritic activity preclinically. JNJ 39758979 advanced into human clinical studies and showed efficacy in reducing experimental pruritus and in patients with atopic dermatitis. However, development of this compound was terminated due to the occurrence of drug-induced agranulocytosis. This was overcome by developing another HR antagonist with a different chemical structure, toreforant, that does not appear to have this side effect. Toreforant has been tested in clinical studies in patients with rheumatoid arthritis, asthma, or psoriasis. In conclusions there have been many HR antagonists reported in the literature, but only a few have been studied in humans underscoring the difficulty in finding ligands with all of the properties necessary for testing in the clinic. Nevertheless, the clinical data to date suggests that HR antagonists can be beneficial in treating atopic dermatitis and pruritus.
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http://dx.doi.org/10.1007/164_2016_130DOI Listing
October 2017

A phylogenetic transform enhances analysis of compositional microbiota data.

Elife 2017 02 15;6. Epub 2017 Feb 15.

Program in Computational Biology and Bioinformatics, Duke University, Durham, United States.

Surveys of microbial communities (microbiota), typically measured as relative abundance of species, have illustrated the importance of these communities in human health and disease. Yet, statistical artifacts commonly plague the analysis of relative abundance data. Here, we introduce the PhILR transform, which incorporates microbial evolutionary models with the isometric log-ratio transform to allow off-the-shelf statistical tools to be safely applied to microbiota surveys. We demonstrate that analyses of community-level structure can be applied to PhILR transformed data with performance on benchmarks rivaling or surpassing standard tools. Additionally, by decomposing distance in the PhILR transformed space, we identified neighboring clades that may have adapted to distinct human body sites. Decomposing variance revealed that covariation of bacterial clades within human body sites increases with phylogenetic relatedness. Together, these findings illustrate how the PhILR transform combines statistical and phylogenetic models to overcome compositional data challenges and enable evolutionary insights relevant to microbial communities.
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http://dx.doi.org/10.7554/eLife.21887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328592PMC
February 2017

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

Methods Mol Biol 2017 ;1529:279-289

Department of Computer Science, Purdue University, West Lafayette, IN, 47907, USA.

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .
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http://dx.doi.org/10.1007/978-1-4939-6637-0_14DOI Listing
January 2018

Erratum to: Host lifestyle affects human microbiota on daily timescales.

Genome Biol 2016 May 31;17(1):117. Epub 2016 May 31.

Computational & Systems Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

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http://dx.doi.org/10.1186/s13059-016-0988-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888531PMC
May 2016

Modification of Ni-Rich FCG NMC and NCA Cathodes by Atomic Layer Deposition: Preventing Surface Phase Transitions for High-Voltage Lithium-Ion Batteries.

Sci Rep 2016 05 26;6:26532. Epub 2016 May 26.

Department of Materials Science and Engineering, University of Michigan Ann Arbor, 2300 Hayward St, Ann Arbor, MI, 48109, USA.

The energy density of current lithium-ion batteries (LIBs) based on layered LiMO2 cathodes (M = Ni, Mn, Co: NMC; M = Ni, Co, Al: NCA) needs to be improved significantly in order to compete with internal combustion engines and allow for widespread implementation of electric vehicles (EVs). In this report, we show that atomic layer deposition (ALD) of titania (TiO2) and alumina (Al2O3) on Ni-rich FCG NMC and NCA active material particles could substantially improve LIB performance and allow for increased upper cutoff voltage (UCV) during charging, which delivers significantly increased specific energy utilization. Our results show that Al2O3 coating improved the NMC cycling performance by 40% and the NCA cycling performance by 34% at 1 C/-1 C with respectively 4.35 V and 4.4 V UCV in 2 Ah pouch cells. High resolution TEM/SAED structural characterization revealed that Al2O3 coatings prevented surface-initiated layered-to-spinel phase transitions in coated materials which were prevalent in uncoated materials. EIS confirmed that Al2O3-coated materials had significantly lower increase in the charge transfer component of impedance during cycling. The ability to mitigate degradation mechanisms for Ni-rich NMC and NCA illustrated in this report provides insight into a method to enable the performance of high-voltage LIBs.
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http://dx.doi.org/10.1038/srep26532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880920PMC
May 2016