Publications by authors named "David B Wilson"

181 Publications

GATA6 modulates the ductular reaction to bile duct ligation.

Hepatol Int 2021 Feb 24;15(1):166-178. Epub 2021 Jan 24.

Department of Pediatrics, St. Louis Children's Hospital, Washington University School of Medicine, St. Louis, MO, USA.

Background: GATA6, a transcription factor expressed in cholangiocytes, has been implicated in the response to liver injury. In biliary atresia, a disease characterized by extrahepatic bile duct obstruction, liver expression of GATA6 increases with pathological bile duct expansion and decreases after successful Kasai portoenterostomy. The aim of this study was to garner genetic evidence that GATA6 is involved in ductular formation/expansion.

Methods: The murine Gata6 gene was conditionally deleted using Alb-cre, a transgene expressed in hepatoblasts (the precursors of hepatocytes and cholangiocytes) and mature hepatocytes. Bile duct ligation (BDL) was used to model biliary obstruction.

Results: Alb-Cre;Gata6 mice were viable and fertile. Cre-mediated recombination of Gata6 in hepatocytes had little impact on cellular structure or function. GATA6 immunoreactivity was retained in a majority of biliary epithelial cells in adult Alb-Cre;Gata6 mice, implying that surviving cholangiocytes were derived from hepatoblasts that had escaped biallelic Cre-mediated recombination. Although GATA6 immunoreactivity was preserved in cholangiocytes, Alb-cre;Gata6 mice had a demonstrable biliary phenotype. A neutrophil-rich infiltrate surrounded newly formed bile ducts in neonatal Alb-Cre;Gata6 mice. Foci of fibrosis/necrosis, presumed to reflect patchy defects in bile duct formation, were observed in the livers of 37% of adult Alb-cre;Gata6 mice and 0% of controls (p < 0.05). Most notably, Alb-cre;Gata6 mice had an altered response to BDL manifest as reduced survival, impaired bile ductule proliferation, increased parenchymal necrosis, reduced fibrosis, and enhanced macrophage accumulation in the portal space.

Conclusions: GATA6 orchestrates intrahepatic biliary remodeling and mitigates liver injury following extrahepatic bile duct obstruction.
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http://dx.doi.org/10.1007/s12072-020-10119-wDOI Listing
February 2021

Enhancing Informal Social Controls to Reduce Crime: Evidence from a Study of Crime Hot Spots.

Prev Sci 2021 Jan 16. Epub 2021 Jan 16.

George Mason University, Fairfax, VA, USA.

There is growing evidence that crime is strongly concentrated in micro-geographic hot spots, a fact that has led to the wide-scale use of hot spots policing programs. Such programs are ordinarily focused on deterrence due to police presence, or other law enforcement interventions at hot spots. However, preliminary basic research studies suggest that informal social controls may also be an important mechanism for crime reduction on high crime streets. Such research has been hindered by a lack of data on social and attitudinal characteristics of residents, and the fact that census information is not available at the micro-geographic level. Our study, conducted in Baltimore, MD, on a sample of 449 residential street segments, overcame these limitations by collecting an average of eight surveys (N = 3738), as well as physical observations, on segments studied. This unique primary data collection allowed us to develop the first direct indicators of collective efficacy at the micro-geographic level, as well as a wide array of indicators of other possible risk and protective factors for crime. Using multilevel negative binomial regression models, we also take into account community-level influences, and oversample crime hot spots to allow for robust comparisons across streets. Our study confirms the importance of opportunity features of streets such as population size and business activity in understanding crime, but also shows that informal social controls, as reflected by collective efficacy, are key for understanding crime on high crime streets. We argue that it is time for police, other city agencies, and NGOs to begin to work together to consider how informal social controls can be used to reduce crime at residential crime hot spots.
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http://dx.doi.org/10.1007/s11121-020-01194-4DOI Listing
January 2021

Chloroquine Triggers Cell Death and Inhibits PARPs in Cell Models of Aggressive Hepatoblastoma.

Front Oncol 2020 17;10:1138. Epub 2020 Jul 17.

Pediatric Research Center, Children's Hospital, Helsinki University Hospital, University of Helsinki, Helsinki, Finland.

Hepatoblastoma (HB) is the most common pediatric liver malignancy. Despite advances in chemotherapeutic regimens and surgical techniques, the survival of patients with advanced HB remains poor, underscoring the need for new therapeutic approaches. Chloroquine (CQ), a drug used to treat malaria and rheumatologic diseases, has been shown to inhibit the growth and survival of various cancer types. We examined the antineoplastic activity of CQ in cell models of aggressive HB. Seven human HB cell models, all derived from chemoresistant tumors, were cultured as spheroids in the presence of relevant concentrations of CQ. Morphology, viability, and induction of apoptosis were assessed after 48 and 96 h of CQ treatment. Metabolomic analysis and RT-qPCR based Death Pathway Finder array were used to elucidate the molecular mechanisms underlying the CQ effect in a 2-dimensional cell culture format. Quantitative western blotting was performed to validate findings at the protein level. CQ had a significant dose and time dependent effect on HB cell viability both in spheroids and in 2-dimensional cell cultures. Following CQ treatment HB spheroids exhibited increased caspase 3/7 activity indicating the induction of apoptotic cell death. Metabolomic profiling demonstrated significant decreases in the concentrations of NAD and aspartate in CQ treated cells. In further investigations, oxidation of NAD decreased as consequence of CQ treatment and NAD/NADH balance shifted toward NADH. Aspartate supplementation rescued cells from CQ induced cell death. Additionally, downregulated expression of PARP1 and PARP2 was observed. CQ treatment inhibits cell survival in cell models of aggressive HB, presumably by perturbing NAD levels, impairing aspartate bioavailability, and inhibiting PARP expression. CQ thus holds potential as a new agent in the management of HB.
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http://dx.doi.org/10.3389/fonc.2020.01138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379510PMC
July 2020

Germline Sequencing Identifies Rare Variants in Finnish Subjects with Familial Germ Cell Tumors.

Appl Clin Genet 2020 30;13:127-137. Epub 2020 Jun 30.

Department of Pediatrics, Division of Hematology and Oncology, Washington University School of Medicine, St. Louis, MO, USA.

Purpose: Pediatric germ cell tumors are rare, representing about 3% of childhood malignancies in children less than 15 years of age, presenting in neonates or adolescents with a greater incidence noted in older adolescents. Aberrations in primordial germ cell proliferation/differentiation can lead to a variety of neoplasms, including teratomas, embryonal carcinoma, choriocarcinoma, and yolk sac tumors.

Patients And Methods: Three Finnish families with varying familial germ cell tumors were identified, and whole-genome sequencing was performed using an Illumina sequencing platform. In total, 22 unique subjects across the three families were sequenced. Family 1 proband (female) was affected by malignant ovarian teratoma, Family 2 proband (female) was affected by sacrococcygeal teratoma with yolk sac tumor in the setting of Cornelia de Lange syndrome, and Family 3 proband (male) was affected by malignant testicular teratoma. Rare variants were identified using an autosomal recessive or de novo model of inheritance.

Results: For family 1 proband (female), an autosomal recessive or de novo model of inheritance identified variants of interest in the following genes: , and . Family 2 proband (female) analysis identified gene variants of interest in the following genes: . Family 3 proband (male) analysis identified the following potential genes: , and .

Conclusion: Leveraging deep pedigrees and next-generation sequencing, rare germline variants were identified that were enriched in three families from Finland with a history of familial germ cell tumors. The data presented support the importance of germline mutations when analyzing complex cancers with a low somatic mutation landscape.
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http://dx.doi.org/10.2147/TACG.S245093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335280PMC
June 2020

Functional Profiling of FSH and Estradiol in Ovarian Granulosa Cell Tumors.

J Endocr Soc 2020 Apr 16;4(4):bvaa034. Epub 2020 Mar 16.

Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERβ) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERβ protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.
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http://dx.doi.org/10.1210/jendso/bvaa034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153750PMC
April 2020

Hb F-Wentzville [γ24(B6)Gly→Glu; : c.74G>A, p.Gly25Glu]: An Unstable γ-Globin Variant Associated with Neonatal Hemolytic Anemia.

Hemoglobin 2020 Jan 27;44(1):67-69. Epub 2020 Jan 27.

Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO, USA.

A novel unstable γ-globin variant, Hb F-Wentzville [γ24(B6)Gly→Glu; : c.74G>A, (p.Gly25Glu)], was identified in a young infant who required a single transfusion of erythrocytes for hemolytic anemia. This is the first reported γ-globin variant affecting the highly conserved glycine residue at helical position B6. In the tertiary structure of hemoglobin (Hb), glycine at B6 is in close proximity to another invariant glycine residue at E8. Prior studies have shown that replacement of the B6 or E8 glycine residues with bulkier amino acids disrupts packing between the B and E helices, resulting in Hb instability. Thus, Hb F-Wentzville is analogous to the following unstable β-globin B6 variants: Hb Savannah (: c.74G>T, p.Gly24Val), Hb Riverdale-Bronx (: c.73G>C, p.Gly24Arg), and Hb Moscva (: c.74G>A, p.Gly24Asp).
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http://dx.doi.org/10.1080/03630269.2020.1716002DOI Listing
January 2020

Differentiation of leukemic blasts is not completely blocked in acute myeloid leukemia.

Proc Natl Acad Sci U S A 2019 12 21;116(49):24593-24599. Epub 2019 Nov 21.

Division of Hematology and Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR 97239;

Hematopoiesis, the formation of blood cells, involves the hierarchical differentiation of immature blast cells into mature, functional cell types and lineages of the immune system. Hematopoietic stem cells precisely regulate self-renewal versus differentiation to balance the production of blood cells and maintenance of the stem cell pool. The canonical view of acute myeloid leukemia (AML) is that it results from a combination of molecular events in a hematopoietic stem cell that block differentiation and drive proliferation. These events result in the accumulation of primitive hematopoietic blast cells in the blood and bone marrow. We used mathematical modeling to determine the impact of varying differentiation rates on myeloblastic accumulation. Our model shows that, instead of the commonly held belief that AML results from a complete block of differentiation of the hematopoietic stem cell, even a slight skewing of the fraction of cells that differentiate would produce an accumulation of blasts. We confirmed this model by interphase fluorescent in situ hybridization (FISH) and sequencing of purified cell populations from patients with AML, which showed that different leukemia-causing molecular abnormalities typically thought to block differentiation were consistently present in mature myeloid cells such as neutrophils and monocytes at similar levels to those in immature myeloid cells. These findings suggest reduced or skewed, rather than blocked, differentiation is responsible for the development of AML. Approaches that restore normal regulation of hematopoiesis could be effective treatment strategies.
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http://dx.doi.org/10.1073/pnas.1904091116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900505PMC
December 2019

Causal effects of education on sexual and reproductive health in low and middle-income countries: A systematic review and meta-analysis.

SSM Popul Health 2019 Aug 20;8:100386. Epub 2019 May 20.

Poverty, Gender and Youth Program, Population Council, New York, NY, USA.

Background: Despite strong theoretical grounding, important gaps in knowledge remain regarding the degree to which there is a causal relationship between education and sexual and reproductive health, as many claims have been made based on associations alone. Understanding the extent to which these relationships are causal is important both to inform investments in education and health, as well as to understand the mechanisms underlying these relationships.

Methods: We conducted a systematic review of the evidence for a causal link between education and sexual and reproductive health (SRH) in low and middle-income countries. Education indicators included exposure to formal schooling and learning. SRH outcomes included: age at first sex, age at first marriage, age at first pregnancy/birth, contraceptive use, fertility, and HIV status and other sexually transmitted infections. When possible, we also conducted meta-analyses to estimate mean effects by outcome, and to understand sources of variation between studies.

Results: We identified 35 papers that met our inclusion criteria. Although many of the studies report evidence of a causal relationship between education and one or more SRH outcomes, estimated effects are often small in magnitude. Our meta-analyses reveal mostly null mean effects, with the exception of small effects of increased grade attainment on lower fertility and HIV positive status. We also found inconsistent evidence supporting mechanisms linking education and SRH.

Conclusions: This review demonstrates that, although investments in schooling may have positive ripple effects for sexual and reproductive health in some circumstances, those effects may not be as large or consistent as expected. Further, our understanding of the circumstances in which schooling is most likely to improve SRH remains somewhat limited. An accurate picture of whether and when improvements in education lead to better health outcomes is essential for the achievement of global development goals.
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http://dx.doi.org/10.1016/j.ssmph.2019.100386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582211PMC
August 2019

Invasive Ductular Reaction: Form and Function.

Am J Pathol 2019 08 14;189(8):1501-1504. Epub 2019 Jun 14.

Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, Missouri; Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

This commentary highlights the article by Clerbaux et al that describes the critical role of invasive ductular reaction in hepatocellular injury.
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http://dx.doi.org/10.1016/j.ajpath.2019.06.002DOI Listing
August 2019

Stereotactic Body Radiation Therapy for the Treatment of Primary Cardiac Angiosarcoma Causing Hemodynamic Instability.

Pract Radiat Oncol 2019 01;9(1):5-8

Department of Radiation Oncology, Washington University School of Medicine in Saint Louis, Saint Louis, Missouri. Electronic address:

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http://dx.doi.org/10.1016/j.prro.2018.07.004DOI Listing
January 2019

Cognitive-behavioural treatment for amphetamine-type stimulants (ATS)-use disorders.

Cochrane Database Syst Rev 2018 12 22;12:CD011315. Epub 2018 Dec 22.

Department of Psychology, Mejiro University, 4-31-1 Naka-Ochiai, Shinjuku-ku, Tokyo, Japan, 161-8539.

Background: Amphetamine-type stimulants (ATS) refer to a group of synthetic stimulants including amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine (MDMA) and related substances. ATS are highly addictive and prolonged use may result in a series of mental and physical symptoms including anxiety, confusion, insomnia, mood disturbances, cognitive impairments, paranoia, hallucinations and delusion.Currently there is no widely accepted treatment for ATS-use disorder. However, cognitive-behavioural treatment (CBT) is the first-choice treatment. The effectiveness of CBT for other substance-use disorders (e.g. alcohol-, opioid- and cocaine-use disorders) has been well documented and as such this basic treatment approach has been applied to the ATS-use disorder.

Objectives: To investigate the efficacy of cognitive-behavioural treatment for people with ATS-use disorder for reducing ATS use compared to other types of psychotherapy, pharmacotherapy, 12-step facilitation, no intervention or treatment as usual.

Search Methods: We identified randomised controlled trials (RCT) and quasi-RCTs comparing CBT for ATS-use disorders with other types of psychotherapy, pharmacotherapy, 12 step facilitation or no intervention. We searched the Cochrane Drugs and Alcohol Group Specialised Register, Cochrane Central Register of Controlled Trials, MEDLINE via PubMed, Embase and five other databases up to July 2018. In addition, we examined reference lists of eligible studies and other systematic reviews. We contacted experts in the field.

Selection Criteria: Eligibility criteria consisted of RCTs and quasi-RCTs comparing CBT versus other types of interventions with adult ATS users (aged 18 years or older) diagnosed by any explicit diagnostic system. Primary outcomes included abstinence rate and other indicators of drug-using behaviours.

Data Collection And Analysis: We used standard methodological procedures expected by Cochrane.

Main Results: Only two studies met the eligibility criteria. Both studies were at low risk of selection bias and reporting bias. In one study, almost half of participants in the intervention group dropped out and this study was at high risk of attrition bias. The studies compared a single session of brief CBT or a web-based CBT to a waiting-list control (total sample size across studies of 129). Results were mixed across the studies. For the single-session brief CBT study, two out of five measures of drug use produced significant results, percentage of abstinent days in 90 days (odds ratio (OR) 0.22, 95% confidence interval (CI) 0.02 to 2.11) and dependence symptoms (standardised mean difference (SMD) -0.59, 95% CI -1.16 to -0.02). Little confidence could be placed in the results from this study give the small sample size (25 participants per group) and corresponding large CIs around the observed effects. For the web-based CBT, there was no significant difference across different outcomes. Neither study reported adverse effects. The meta-analytic mean across these two trials for drug use was not significant (SMD -0.28, 95% CI -0.69 to 0.14). In summary, overall quality of evidence was low and there was insufficient evidence to conclude that CBT is effective, or ineffective, at treating ATS use.

Authors' Conclusions: Currently, there is not enough evidence to establish the efficacy of CBT for ATS-use disorders because of a paucity of high-quality research in this area.
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http://dx.doi.org/10.1002/14651858.CD011315.pub2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516990PMC
December 2018

Lamin B receptor-related disorder is associated with a spectrum of skeletal dysplasia phenotypes.

Bone 2019 03 15;120:354-363. Epub 2018 Nov 15.

Department of Pediatrics, Division of Medical Genetics, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

LBR (Lamin B Receptor) encodes a bifunctional protein important for cholesterol biosynthesis and heterochromatin organization on the inner nuclear membrane. Pathogenic variants in LBR are associated with marked phenotypic variability, ranging from the benign Pelger-Huët anomaly to lethal Greenberg Dysplasia. We performed trio exome sequencing (ES) on two patients with atypical variants of skeletal dysplasia and their unaffected parents. Patient 1 exhibited frontal bossing, mid-face hypoplasia, short stature with rhizomelic limb shortening, and relative macrocephaly at birth. Although remained short, Patient 1 later showed spontaneous improvement in her skeletal findings. Exome sequencing revealed two novel variants in LBR, c.1504C > G (p.Arg502Gly) in exon 12 and c.1748G > T (p.Arg583Leu) in exon 14, which were inherited from her unaffected father and mother, respectively. Sterol analysis revealed an increased level of cholesta‑8,14‑dien‑3β‑ol to 2.9% of total sterols, consistent with a functional deficiency of 3β‑hydroxysterol Δ14‑reductase. Patient 2 presented at birth with short stature and marked rhizomelic limb shortening but later exhibited decreasing severity of shortening of the long bones and improvement in the radiographic skeletal abnormalities although he continued to be significantly short at age 10 years. Exome sequencing revealed that Patient 2 is homozygous for a pathogenic variant c.1534C > T (p.Arg512Trp) in exon 12 of LBR, which was inherited from his unaffected consanguineous parents. This report provides further evidence for a phenotypic spectrum of LBR-associated disorders and expands the genotypic spectrum by describing 3 novel disease-causing variants that have not been previously associated with a disease. Moreover, our data on Patient 1 demonstrate that variants throughout the gene appear to influence both the sterol reductase and nuclear functions of LBR.
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http://dx.doi.org/10.1016/j.bone.2018.11.006DOI Listing
March 2019

Transcription factor GATA4 associates with mesenchymal-like gene expression in human hepatoblastoma cells.

Tumour Biol 2018 Jul;40(7):1010428318785498

1 Pediatric Research Center, Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

GATA4, a transcription factor crucial for early liver development, has been implicated in the pathophysiology of hepatoblastoma, an embryonal tumor of childhood. However, the molecular and phenotypic consequences of GATA4 expression in hepatoblastoma are not fully understood. We surveyed GATA4 expression in 24 hepatoblastomas using RNA in situ hybridization and immunohistochemistry. RNA interference was used to inhibit GATA4 in human HUH6 hepatoblastoma cells, and changes in cell migration were measured with wound healing and transwell assays. RNA microarray hybridization was performed on control and GATA4 knockdown HUH6 cells, and differentially expressed genes were validated by quantitative polymerase chain reaction or immunostaining. Plasmid transfection was used to overexpress GATA4 in primary human hepatocytes and ensuring changes in gene expression were measured by quantitative polymerase chain reaction. We found that GATA4 expression was high in most hepatoblastomas but weak or negligible in normal hepatocytes. GATA4 gene silencing impaired HUH6 cell migration. We identified 106 differentially expressed genes (72 downregulated, 34 upregulated) in knockdown versus control HUH6 cells. GATA4 silencing altered the expression of genes associated with cytoskeleton organization, cell-to-cell adhesion, and extracellular matrix dynamics (e.g. ADD3, AHNAK, DOCK8, RHOU, MSF, IGFBP1, COL4A2). These changes in gene expression reflected a more epithelial (less malignant) phenotype. Consistent with this notion, there was reduced F-actin stress fiber formation in knockdown HUH6 cells. Forced expression of GATA4 in primary human hepatocytes triggered opposite changes in the expression of genes identified by GATA4 silencing in HUH6 cells. In conclusion, GATA4 is highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype of hepatoblastoma cells.
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http://dx.doi.org/10.1177/1010428318785498DOI Listing
July 2018

Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia.

Am J Physiol Gastrointest Liver Physiol 2018 05 1;314(5):G547-G558. Epub 2018 Feb 1.

Pediatric Research Center, Children's Hospital, University of Helsinki and Helsinki University Hospital , Helsinki , Finland.

Biliary atresia (BA), a neonatal liver disease, is characterized by obstruction of extrahepatic bile ducts with subsequent cholestasis, inflammation, and progressive liver fibrosis. To gain insights into the pathophysiology of BA, we focused attention on GATA6, a transcription factor implicated in biliary development. Early in fetal development GATA6 expression is evident in cholangiocytes and hepatocytes, but by late gestation it is extinguished in hepatocytes. Utilizing a unique set of BA liver samples collected before and after successful portoenterostomy (PE), we found that GATA6 expression is markedly upregulated in hepatocytes of patients with BA compared with healthy and cholestatic disease controls. This upregulation is recapitulated in two murine models simulating bile duct obstruction and intrahepatic bile ductule expansion. GATA6 expression in BA livers correlates with two established negative prognostic indicators (age at PE, degree of intrahepatic bile ductule expansion) and decreases after normalization of serum bilirubin by PE. GATA6 expression in BA livers correlates with expression of known regulators of cholangiocyte differentiation ( JAGGED1, HNF1β, and HNF6). These same genes are upregulated after enforced expression of GATA6 in human hepatocyte cell models. In conclusion, GATA6 is a novel marker and a putative driver of hepatocyte-cholangiocyte metaplasia in BA, and its expression in hepatocytes is downregulated after successful PE. NEW & NOTEWORTHY A pathological hallmark in the liver of patients with biliary atresia is ductular reaction, an expansion of new bile ductules that are thought to arise from conversion of mature hepatocytes. Here, we show that transcription factor GATA6 is a marker and potential driver of hepatocyte ductal metaplasia in biliary atresia. Hepatocyte GATA6 expression is elevated in biliary atresia, correlates with bile duct expansion, and decreases after successful portoenterostomy.
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http://dx.doi.org/10.1152/ajpgi.00362.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008062PMC
May 2018

Natural diversity of glycoside hydrolase family 48 exoglucanases: insights from structure.

Biotechnol Biofuels 2017 30;10:274. Epub 2017 Nov 30.

Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401 USA.

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction. Surprisingly, given their importance for these microorganisms, GH48s have intrinsically low cellulolytic activity but even in low ratios synergize greatly with GH9 endoglucanases. In this study, we explore the structural and enzymatic diversity of these enzymes across a wide range of temperature optima. We have crystallized one new GH48 module from in a complex with cellobiose and cellohexaose (GH48). We compare this structure to other known GH48 enzymes in an attempt to understand GH48 structure/function relationships and draw general rules correlating amino acid sequences and secondary structures to thermostability in this GH family.
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http://dx.doi.org/10.1186/s13068-017-0951-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707792PMC
November 2017

Structure of a lytic polysaccharide monooxygenase and mutagenesis of key residues.

Biotechnol Biofuels 2017 30;10:243. Epub 2017 Nov 30.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY USA.

Background: Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity.

Results: In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination.

Conclusions: The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.
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http://dx.doi.org/10.1186/s13068-017-0925-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708082PMC
November 2017

Somatic mutations and clonal hematopoiesis in congenital neutropenia.

Blood 2018 01 1;131(4):408-416. Epub 2017 Nov 1.

Division of Oncology, Department of Internal Medicine.

Severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls ( = NS). Clonal hematopoiesis due to mutations in was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, < .001) or patients with SCN (0/40, < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple mutations. Conversely, clonal hematopoiesis due to mutations of was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand -mutated HSPCs, suggesting that acquisition of mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.
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http://dx.doi.org/10.1182/blood-2017-08-801985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790127PMC
January 2018

Comment on: Acquired monosomy 7 myelodysplastic syndrome in a child with clinical features of dyskeratosis congenita and IMAGe association.

Pediatr Blood Cancer 2018 01 17;65(1). Epub 2017 Aug 17.

Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.

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http://dx.doi.org/10.1002/pbc.26747DOI Listing
January 2018

Probing GATA factor function in mouse Leydig cells via testicular injection of adenoviral vectors.

Reproduction 2017 10 14;154(4):455-467. Epub 2017 Jul 14.

Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, Missouri, USA

Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse and , two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult ; mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (, and ) was reduced, whereas expression of another Leydig cell marker, , was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.Free Finnish abstract: A Finnish translation of this abstract is freely available at http://www.reproduction-online.org/content/154/4/455/suppl/DC2.
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http://dx.doi.org/10.1530/REP-17-0311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589507PMC
October 2017

Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization.

Biochim Biophys Acta Proteins Proteom 2017 Oct 22;1865(10):1237-1245. Epub 2017 Jun 22.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark. Electronic address:

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb, K and Ca ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases.
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http://dx.doi.org/10.1016/j.bbapap.2017.06.015DOI Listing
October 2017

Six-vertex model and Schramm-Loewner evolution.

Phys Rev E 2017 May 30;95(5-1):052146. Epub 2017 May 30.

Microsoft Research, One Microsoft Way, Redmond, Washington 98052, USA.

Square ice is a statistical mechanics model for two-dimensional ice, widely believed to have a conformally invariant scaling limit. We associate a Peano (space-filling) curve to a square ice configuration, and more generally to a so-called six-vertex model configuration, and argue that its scaling limit is a space-filling version of the random fractal curve SLE_{κ}, Schramm-Loewner evolution with parameter κ, where 4<κ≤12+8sqrt[2]. For square ice, κ=12. At the "free-fermion point" of the six-vertex model, κ=8+4sqrt[3]. These unusual values lie outside the classical interval 2≤κ≤8.
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http://dx.doi.org/10.1103/PhysRevE.95.052146DOI Listing
May 2017

Nkx2-5 and Sarcospan genetically interact in the development of the muscular ventricular septum of the heart.

Sci Rep 2017 04 13;7:46438. Epub 2017 Apr 13.

Department of Pediatrics, Washington University School of Medicine, Box 8208 660 South Euclid Avenue, St. Louis, MO, 63110, USA.

The muscular ventricular septum separates the flow of oxygenated and de-oxygenated blood in air-breathing vertebrates. Defects within it, termed muscular ventricular septal defects (VSDs), are common, yet less is known about how they arise than rarer heart defects. Mutations of the cardiac transcription factor NKX2-5 cause cardiac malformations, including muscular VSDs. We describe here a genetic interaction between Nkx2-5 and Sarcospan (Sspn) that affects the risk of muscular VSD in mice. Sspn encodes a protein in the dystrophin-glycoprotein complex. Sspn knockout (Sspn) mice do not have heart defects, but Nkx2-5/Sspn mutants have a higher incidence of muscular VSD than Nkx2-5 mice. Myofibers in the ventricular septum follow a stereotypical pattern that is disrupted around a muscular VSD. Subendocardial myofibers normally run in parallel along the left ventricular outflow tract, but in the Nkx2-5/Sspn mutant they commonly deviate into the septum even in the absence of a muscular VSD. Thus, Nkx2-5 and Sspn act in a pathway that affects the alignment of myofibers during the development of the ventricular septum. The malalignment may be a consequence of a defect in the coalescence of trabeculae into the developing ventricular septum, which has been hypothesized to be the mechanistic basis of muscular VSDs.
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http://dx.doi.org/10.1038/srep46438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390293PMC
April 2017

Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis.

Tumour Biol 2017 Mar;39(3):1010428317695016

1 Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.
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http://dx.doi.org/10.1177/1010428317695016DOI Listing
March 2017

Special issue: Impact of maternal metabolism on newborn heath.

Authors:
David B Wilson

Mol Cell Endocrinol 2016 11;435

Washington University School of Medicine, St. Louis Children's Hospital, USA. Electronic address:

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http://dx.doi.org/10.1016/j.mce.2016.08.038DOI Listing
November 2016

GLI1 progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-like tissue.

Mol Cell Endocrinol 2017 02 29;441:164-175. Epub 2016 Aug 29.

Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO 63110 USA; Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110 USA. Electronic address:

As certain strains of mice age, hyperplastic lesions resembling gonadal tissue accumulate beneath the adrenal capsule. Gonadectomy (GDX) accelerates this heterotopic differentiation, resulting in the formation of wedge-shaped adrenocortical neoplasms that produce sex steroids. Stem/progenitor cells that reside in the adrenal capsule and retain properties of the adrenogonadal primordium are thought to be the source of this heterotopic tissue. Here, we demonstrate that GLI1 progenitors in the adrenal capsule give rise to gonadal-like cells that accumulate in the subcapsular region. A tamoxifen-inducible Cre driver (Gli1-creER) and two reporters (R26R-lacZ, R26R-confetti) were used to track the fate of GLI1 cells in the adrenal glands of B6D2F2 mice, a strain that develops both GDX-induced adrenocortical neoplasms and age-dependent subcapsular cell hyperplasia. In gonadectomized B6D2F2 mice GLI1 progenitors contributed to long-lived adrenal capsule cells and to adrenocortical neoplasms that expressed Gata4 and Foxl2, two prototypical gonadal markers. Pdgfra, a gene expressed in adrenocortical stromal cells, was upregulated in the GDX-induced neoplasms. In aged non-gonadectomized B6D2F2 mice GLI1 progenitors gave rise to patches of subcapsular cell hyperplasia. Treatment with GANT61, a small-molecule GLI antagonist, attenuated the upregulation of gonadal-like markers (Gata4, Amhr2, Foxl2) in response to GDX. These findings support the premise that GLI1 progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic tissue.
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http://dx.doi.org/10.1016/j.mce.2016.08.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5235954PMC
February 2017

Active spanning trees and Schramm-Loewner evolution.

Phys Rev E 2016 06 14;93(6):062121. Epub 2016 Jun 14.

Microsoft Research, Redmond, Washington 98052, USA.

We consider the Peano curve separating a spanning tree from its dual spanning tree on an embedded planar graph, where the tree and dual tree are weighted by y to the number of active edges, and "active" is in the sense of the Tutte polynomial. When the graph is a portion of the square grid approximating a simply connected domain, it is known (y=1 and y=1+sqrt[2]) or believed (1
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http://dx.doi.org/10.1103/PhysRevE.93.062121DOI Listing
June 2016

Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy.

Appl Environ Microbiol 2016 08 15;82(15):4835-4845. Epub 2016 Jul 15.

Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA

Unlabelled: The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.

Importance: The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.
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http://dx.doi.org/10.1128/AEM.01298-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984284PMC
August 2016

GATA4 Regulates Blood-Testis Barrier Function and Lactate Metabolism in Mouse Sertoli Cells.

Endocrinology 2016 06 14;157(6):2416-31. Epub 2016 Mar 14.

Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.

Conditional deletion of Gata4 in Sertoli cells (SCs) of adult mice has been shown to increase permeability of the blood-testis barrier (BTB) and disrupt spermatogenesis. To gain insight into the molecular underpinnings of these phenotypic abnormalities, we assessed the impact of Gata4 gene silencing in cell culture models. Microarray hybridization identified genes dysregulated by siRNA-mediated inhibition of Gata4 in TM4 cells, an immortalized mouse SC line. Differentially expressed genes were validated by quantitative RT-PCR analysis of primary cultures of Gata4(flox/flox) mouse SCs that had been subjected to cre-mediated recombination in vitro. Depletion of GATA4 in TM4 cells and primary SCs was associated with altered expression of genes involved in key facets of BTB maintenance, including tight/adherens junction formation (Tjp1, Cldn12, Vcl, Tnc, Csk) and extracellular matrix reorganization (Lamc1, Col4a1, Col4a5, Mmp10, Mmp23, Timp2). Western blotting and immunocytochemistry demonstrated reduced levels of tight junction protein-1, a prototypical tight junction protein, in GATA4-depleted cells. These changes were accompanied by a loss of morphologically recognizable junctional complexes and a decline in epithelial membrane resistance. Furthermore, Gata4 gene silencing was associated with altered expression of Hk1, Gpi1, Pfkp, Pgam1, Gls2, Pdk3, Pkd4, and Ldhb, genes regulating the production of lactate, a key nutrient that SCs provide to developing germ cells. Comprehensive metabolomic profiling demonstrated impaired lactate production in GATA4-deficient SCs. We conclude that GATA4 plays a pivotal role in the regulation of BTB function and lactate metabolism in mouse SCs.
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http://dx.doi.org/10.1210/en.2015-1927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891789PMC
June 2016

Simulation studies of substrate recognition by the exocellulase CelF from Clostridium cellulolyticum.

Biotechnol Bioeng 2016 07 28;113(7):1433-40. Epub 2016 Jan 28.

Department of Food Science, Cornell University, Ithaca, New York, 14853.

Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, β-d-glucopyranose. Simulations were conducted of the catalytic domain of this enzyme surrounded by a concentrated solution of β-d-glucopyranose, and the full three-dimensional probability distribution for finding sugar molecules adjacent to the enzyme was calculated from the trajectory. A significant probability of finding the sugar stacked against the planar faces of Trp 310 and Trp 312 at the entrance of the active site tunnel was observed. Biotechnol. Bioeng. 2016;113: 1433-1440. © 2015 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.25909DOI Listing
July 2016