bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Determinants in Hinge‐Bend Motions of Neurolysin
Authors: Bibel Brianna , Sigman Jeffrey 
Published: June 2021

Neurolysin (EC 3.4.24.16; NLN) is a zinc (II) metallopeptidase, in the M3 family of enzymes, along with close relatives such as thimet oligopeptidase (TOP). NLN is believed to play a role in diverse physiological processes through cleavage of neuropeptides, including neurotensin and bradykinin. NLN and similar enzymes are thought to operate via a hinge‐clasp mechanism whereby a substrate enters the active site of the open, inactive form and conformational changes in the hinge region lead the enzyme to adopt a closed, active form. These conformational changes likely provide a mechanism for limiting the length of peptides processed by NLN and TOP. This research will use site‐directed mutagenesis followed by spectroscopic and kinetic assays of the engineered enzymes to examine the importance of residues in the clasp region on the mechanism of substrate selectivity and hydrolysis. To this end we have optimized the expression and purification of the cytosolic isoform of mouse NLN – subcloned into a pET28b(+) vector, overexpressed in E. coli, and purified by Ni‐affinity chromatography. We have created homology models of the open and closed conformations of mouse neurolysin; these models suggest that alpha helices 3 and 6, located on either side of the hinge region, play a role in modulating the stability of the closed, active form of the enzyme. Using our homology models, we have analyzed this region and identified several potential stabilizing interactions, including a salt bridge between GLU135 and LYS189, residues highly conserved in NLN and similar enzymes. Analysis of the effect of mutations in the clasp region on enzyme stability and activity are currently underway.Grant Funding SourceThis research was funded by the Robert W. and Beverly J. Summers Scholarship through Saint Mary's College of California's Summer Research Program.
Bioengineered Styrene Monooxygenase: Elucidating the Impact of the N‐Terminus in the Efficiency and Regulation of the Flavin Transfer Reaction
Authors: Okonkwo Nonye , Gassner Geroge 
Published: June 2021

Metabolism of toxic styrene by Pseudomonas putida (S12) bacteria is initiated by a two‐component flavoenzyme system, which consists of the FAD‐specific styrene monooxygenase A (SMOA) and an NADH‐specific styrene monooxygenase B (SMOB), a reductase. Wild‐type SMOB catalyzes the reduction of FAD using a BiBi sequential mechanism and subsequently transfers FAD to the active site of SMOA, which catalyzes the epoxidation of styrene to (S)‐styrene oxide. However, during steady‐state catalysis, we recently discovered that the N‐terminally tagged version, N‐SMOB, employs a double‐displacement mechanism (Kantz, 2005). The efficient delivery of reduced FAD to the SMOA presents a challenge for most two‐component SMO systems and this research aims to further understand the regulation of N‐terminally engineered flavoenzymes.Here we evaluate the impact of an N‐terminal tag on the catalytic mechanisms of SMO. Fluorescence monitored titrations at 4°C determined the Kd value to be ~ 50 nM, an order of magnitude greater than the wild‐type reductase. The increased FAD binding affinity directly affects the steady‐state kinetics at 30°C, as the double‐displacement mechanism with NADH as the leading substrate becomes the preferred method of FAD reduction. Due to these significant changes in flavin binding affinity and catalytic mechanism, we investigated the pre‐steady state and FAD‐transfer kinetics using stopped‐flow fluorescence and absorbance studies. These findings will be presented together with their implications for the engineering of coupled N‐terminally tagged enzymes and N‐terminally linked fusion proteins as potential biocatalysts for the production of essential chiral epoxides.
Size Separation of Large Proteins and Protein Complexes
Authors: Liljedahl Pia , Salomonsson Inger , Graner Therese 
Published: June 2021

The elucidation of the biological function of protein complexes is an area of intense research efforts but isolation and analysis of the complexes is challenging as they can be very large and unstable. Superose™ 6 Increase is a new agarose‐based SEC (size exclusion chromatography) medium with a wide separation range, Mr 5000 to 5 000 000 for globular proteins. The wide separation range allows separation of very large proteins, complexes and membrane proteins both for preparative and analytical purposes under mild, non‐dissociative conditions.Separation of a protein involved in structural coloration on Superdex 200 10/300 resulted in co‐elution of two differently sized protein populations. The wider separation range of Superose 6 Increase 10/300 enabled separation of the two populations. Based on elution position, the size of the separated material was estimated to Mr > 700 000Purification of membrane proteins often require addition of expensive detergents in the buffer to keep the proteins stable and thus low consumption of buffer is important. Purification of a membrane protein complex on Superose 6 Increase 5/150 (3 ml bed volume) resulted in good separation from aggregated material and degradation products.
Substrate Specifity and Quaternary Structure of a Novel Class of Tryptophan Synthases
Authors: Busch Florian , Sterner Reinhard 
Published: June 2021

The analysis of numerous genome sequences uncovered that the TrpB enzyme family IPR023026 is divided into two subfamilies named TrpB1 and TrpB2. The widely distributed TrpB1 enzymes are part of the canonical abba tryptophan synthase complex. They synthesize tryptophan by the condensation of L‐serine with indole, which is provided by the associated TrpA enzyme. TrpB1 and TrpA form a permanent complex. We characterized several TrpB2 enzymes and found that they differ in several respects from TrpB1. First, they synthesize tryptophan by the condensation of O‐phospho‐L‐serine with indole. Second, complex formation between TrpB2 and TrpA is transient and ligand‐dependent. Third, the quaternary structure of TrpB2‐TrpA strikingly differs from those of TrpB1‐TrpA complexes. In summary, our results unravel significant differences between two closely related enzyme subfamilies regarding substrate recognition/specificity and hetero‐oligomerization.Ehrmann, A., Richter, K., Busch, F., Reimann, J., Albers, S.‐V. & Sterner, R. (2010). Ligand‐induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry. Biochemistry 49, 10842‐10853Busch, F., Rajendran, C., Mayans, O. Löffler, P., Merkl, R. & Sterner, R. (2014). TrpB2 enzymes are O‐phospho‐L‐serine dependent tryptophan synthases. <em style=“line‐height: 150%;”>Biochemistry53, 6078‐6083.
Structural‐Function Mechanism of Vibrio cholerae Cytolysin: a b‐barrel Pore‐Forming Toxin
Authors: Rai Anand , Chattopadhyay Kausik 
Published: June 2021

b‐barrel pore‐forming toxins (b‐PFTs) represent an important class of proteins that disrupt membrane permeability of the target cells by forming transmembrane pores. Vibrio cholerae cytolysin (VCC) is a prominent b‐PFT that forms heptameric transmembrane pores in the target eukaryotic cells. Our studies have shown that the membrane binding and pore formation by VCC is governed by multiple regulatory mechanisms. Interaction of VCC with the cell surface glycan(s) appears to modulate the efficacy of the process. Physicochemical conditions like pH also appear to regulate interactions of the toxin with the membranes. It is also evident that VCC may employ distinct structural motifs to mediate interactions with the membrane lipid components. Our study has also explored the sequence of events for the functional pore formation by VCC. We have identified the key amino acid residue(s) in VCC that are crucial to initiate membrane oligomerization. Modification of such residues traps the protein in its membrane‐bound monomeric state that further abrogates oligomerization, membrane insertion and functional transmembrane pore formation. Overall our study provides novel insight regarding the mechanistic aspects of VCC pore‐formation mechanism and also extends our knowledge regarding the structure‐function mechanism of the b‐PFTs family proteins.This work was supported by The Department of Biotechnology, India (grant no. BT/PR13350/BRB/10/751/2009), and by the Indian Institute of Science Education and Research Mohali, India.
Determining The Topology of The Membrane Bound O‐acyltransferase Porcupine
Authors: Sanchez Luis , Fraley Gabriel , Bueno Raymund , Galli Lisa , Burrus Laura 
Published: June 2021

Porcupine (PORCN), a membrane bound O‐acyltransferase (MBOAT) of the endoplasmic reticulum (ER), is essential for Wnt palmitoylation, transport, secretion, and activity. Because all human Wnts require PORCN for their activity, the inhibition of PORCN has become an attractive therapeutic target for the treatment of various cancers displaying an overexpression of Wnt ligands. In order to design and understand how potential PORCN inhibitors work, the topology of PORCN must be determined. Although various bioinformatics algorithms offer numerous predictions, no clear membrane topology of PORCN has emerged. Therefore, we aimed to experimentally establish the membrane topology of PORCN. Preliminary data orient the N and C terminus toward the ER lumen and cytoplasm, respectively. Additional experiments in which consensus glycosylation sites were introduced into the PORCN sequence suggest that the loop containing A134 is localized to the ER lumen. Therefore, we hypothesized that the A134 containing loop is entirely localized to the ER lumen. To test this hypothesis, we expressed two PORCN constructs with FLAG tags inserted at residues 118 and 145 respectively, and a myc tag at the C‐terminus, in COS‐7 cells. Contrary to our hypothesis, immunostaining of COS‐7 cells selectively permeabilized with Triton X‐100 or digitonin revealed that the FLAG tag on both variants was localized to the cytoplasm. These apparently contradictory data suggest that PORCN may exist in multiple conformations. Together, our findings provide structural information about PORCN that can help in the design and understanding of PORCN inhibitors that could be used for the treatment of Wnt driven cancersThis research was made possible by a grant received from the National Institute of Health (NIH).
11β‐HSD1 Mediates Insulin Resistance and Type 2 Diabetes Mellitus through JNK Activation
Authors: Peng Kesong , Liang Guang 
Published: June 2021

BackgroundObesity‐induced insulin resistance is the major determinant of metabolic syndrome, which precedes the development of type 2 diabetes mellitus.11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is considered a potential therapeutic target in the treatment of type 2 diabetes mellitus, while the potential mechanism is not very clearly identified.MethodsIn vitro cultured 3T3‐L1 pre‐adipocytes, prednisone and over‐expressed 11β‐HSD1 were used to induce insulin resistance. Type 2 diabetes mellitus mouse models were treated with 11β‐HSD1 inhibitor PF00195715 and JNK inhibitor C66 by gavage, respectively.ResultsStimulation of prednisone or over‐expressed 11β‐HSD1 significantly disturbed insulin signal pathway, decreased AKT phosphorylation and led to insulin resistance, while these effects were reversed by PF00195715 and C66. Either C66 or DN‐JNK significantly attenuated the 11β‐HSD1 over‐expression induced insulin resistance, indicating a mediated role of JNK. Oral administration with either PF00195715 or C66 in mouse models remarkably relieved insulin resistance and type 2 diabetes mellitus.Conclusion11β‐HSD1 mediates insulin resistance and type 2 diabetes mellitus through JNK activation.
Chronic Sweetener Intake and Oxidative Stress in The Central Nervous System
Authors: Rebolledo Zhenia , Contreras Irazu , Estrada Jose 
Published: June 2021

IntroductionDiseases such as diabetes, obesity and metabolic syndrome are characterized by chronic low‐grade inflammation and increased oxidative stress, which causes changes damage to several tissues, including the central nervous system. Sweeteners are an alternative to sucrose for the treatment of these pathologies, since they provide flavor without the energy content; however, the use of these compounds has been extended to the general population, since they are included in a variety of products for human consumption. Despite being considered safe, there is evidence of significant metabolic alterations after chronic use of large quantities of these substances.ObjectiveTo determine the association between constant intake of commercial sucralose or stevia with an increased production of reactive oxygen species in the central nervous system of mice.Method4 groups of 6 female 8 week‐old BALB/c mice were supplemented with either sucralose, stevia or saccharose in their drinking water, or left unsupplemented (control), for 6 weeks. Subsequently, mice were sacrificed by anesthetic overdose and the nervous tissue was obtained. ROS production in the tissue was determined by the TBARS method.ResultsThe highest production of ROS was observed in the control group (average concentration of MDA 186 mmol/mL). The sucrose (124.11 mmol/mL), sucralose (113 mmol/mL) and stevia (112.3 mmol/mL) samples all had lower ROS concentrations than the unsupplemented control.ConclusionsIn comparison with the sucrose‐supplemented and control groups, supplementation with either sucralose or stevia appear to promote decreased concentrations of ROS in the central nervous system, with the stevia group showing the lowest production.
Toxin‐antitoxin systems and their role(s) in persistence
Authors: Page Rebecca , Peti Wolfgang 
Published: June 2021

During the last decade, efforts to understand the molecular mechanism(s) that underlie bacterial persistence have accelerated substantially. In spite of these efforts, the molecular activities that lead to the persister phenotype remain largely unknown. Persister cells are characterized by an upregulation of genes known as toxin‐antitoxin (TA) pairs, with the toxin mqsR being the most highly upregulated gene in E. coli persisters. During normal cell growth, MqsR and its antitoxin, MqsA, form a non‐toxic complex. However, during stress, MqsA is degraded allowing MqsR to induce growth arrest via mRNA cleavage. We previously reported that the mqsRA TA pair is unique because of the unusual features of MqsA. Now, our most recent work reveals that MqsR also has exclusive properties. Using X‐ray crystallography, mutagenesis, in vitro RNA cleavage assays, EMSAs and ITC, we show that, unlike all other TA systems, the MqsR‐MqsA complex cannot bind DNA. Instead, MqsR functions solely as a de‐repressor, further promoting multidrug tolerance through its ability to disrupt MqsA‐mediated repression of persistence‐related genes. Furthermore, using in vitro studies, we discovered that MqsR is a ‘hybrid’ endoribonuclease (RNase), having characteristics of both RelE‐like and microbial like RNases. Recent work has defined the mechanism of mRNA cleavage and the molecular basis of MqsA neutralization of MqsR activity. Finally, we also discovered a novel Type V TA system that is activated by MqsR, the first TA system has been shown to regulate a second. Collectively, these studies demonstrate that MqsR‐MqsA defines a novel TA system and are ideal targets for developing a new class of antibiotics that inhibit the persister state.This work was supported by an NSF‐CAREER award (MCB0952550) to RP.
Cinnamaldehyde Inhibits Biofilm Formation of MRSA
Authors: Rossi Marco , Heuertz Rita 
Published: June 2021

NIH identified that 80% of chronic infections are caused by biofilm‐producing bacteria. Of concern is biofilm as a contributory component of antimicrobic resistance: identification of anti‐biofilm agents is necessary and timely. Antibacterial properties are associated with many plant‐derived compounds, such as cinnamaldehyde (derived from cinnamon); however, biofilm effects are not reported. Staphylococcus aureus is known for its multi‐drug resistant strains (MRSA) and plant‐based therapy may be appropriate for these antimicrobic resistant bacteria. Clinical MRSA isolates from area hospital laboratories were assessed for cinnamaldehyde effect using a: (i) microplate assay for quantitative spectrophotometric evaluation of crystal violet‐stained biofilm adherent to microwells; and (ii) standard bacterial count assay for colony forming unit (CFU/ml) enumeration. Results indicated that cinnamaldehyde inhibited MRSA biofilm formation in a concentration‐dependent manner with significance (p<0.01) at 50 and 100 uM. Colony counts of MRSA were also significantly (p<0.01) reduced in a concentration‐dependent manner. Taken together, these results indicate that cinnamaldehyde‐induced killing of MRSA is an important mechanism for inhibition of biofilm formation. Clinical relevance of this study is identification of a plant‐derived compound that inhibits MRSA growth and biofilm formation. Given that cinnamaldehyde has no reported human toxicity and is not cost‐prohibitive, its potential impact is in reduction of MRSA morbidity and mortality.Financial support for MWR was received from the DeNardo Education and Research Foundation.
Overweight, Obesity and Cardio‐metabolic Indicators of Peruvian Adolescents Whose Mothers Participated in a Prenatal Zinc Supplementation Trial
Authors: Zavaleta Nelly , Garcia Teresa , Lazarte Fabiola , Caulfield Laura 
Published: June 2021

To characterize the nutritional status and cardio‐metabolic indicators of two groups of adolescents 14 yrs old, born from prenatal supplemented mothers. Methods: We evaluated a group of 86 adolescents from mothers that received iron and folic acid (group A), and 75 from mothers that received iron, folic acid and zinc (Group B).ResultsIn group A, 16 (18.6%) were overweight and 24.4% were obese; in group B, 15 (20%) were overweight and 16% were obese (NS). Among the overweight and obese children (n=64), mean Hb in group A was 13.5 g/dl and 12.8 g/dl in group B (p=0.007), there were no differences on mean serum zinc, ferritin, PCR, triglycerides, HD cholesterol, glucose, fasting insulin, blood pressure, waist circumference. In group A, 16 (43.2 %), 15 (40.5%) and 5 children (13.5%); in Group B, 13 (48.1%), 8 (29.6%) and 3 children (11.1%) had 1, 2 and 3 or more abnormal cardio metabolic indicators (NS). In 53 (82.8%) children had PCR > 5mg/L, in 56 (87.5%) had HDL cholesterol < 40 mg/dl (NS between groups). Results have shown a high prevalence of overweight and obesity in this population, cardio‐metabolic indicators shown a trend to develop metabolic syndrome. Differences were not associated to group of maternal supplementation. Supported by Nestle Foundation.
Associations of Community‐Level Sanitation and Child Stunting in Bengali and Tribal Populations in Bangladesh: A Cross‐Sectional Study
Authors: Sinharoy Sheela , Waid Jillian , Stein Aryeh 
Published: June 2021

ObjectiveTo describe trends and determinants of stunting prevalence among tribal and Bengali children in the Chittagong Hill Tracts of Bangladesh.MethodsWe analyzed surveillance data collected from 2003 – 2006 among 12,006 children aged 6‐23 months.ResultsMean height‐for‐age z‐score (HAZ) was stable for Bengali children (‐1.88 in 2003 to ‐1.90 in 2006) but increased for tribal children (‐1.87 in 2003 to ‐1.68 in 2006). The difference between groups was significant in 2005 (p=0.0017) and 2006 (p=0.0014). In both groups, HAZ was negatively associated with age in months (p=0.001) and distance to health center (p=0.039) and positively associated with mother's height (p=0.012). HAZ was also positively associated with father's schooling (p=0.009) among Bengali children and with consumption of animal source foods (p=0.007) and goat ownership (p=0.045) among tribal children, and negatively associated with village‐level prevalence of open defecation among tribal children (p=0.022). Blinder‐Oaxaca decomposition demonstrated a significant difference in pooled coefficients between the two groups (p=0.003). This was explained primarily by village‐level open defecation, which was significant only for tribal children (p=0.018).ConclusionsVillage‐level prevalence of open defecation is an important explanatory factor for the difference in HAZ between tribal and Bengali populations. Open defecation may also act a proxy for unmeasured factors such as household environmental conditions and food hygiene.Source of Research SupportLaney Graduate School (Emory University) and HKI
Overweight is Prevalent among Cameroonian Women and is Associated with Increased Waist Circumference, Region, and Household Characteristics
Authors: Engle‐Stone Reina , Nankap Martin , Ndjebayi Alex , Friedman Avital , Tarini Ann , Brown Kenneth 
Published: June 2021

We examined the prevalence of overweight and obesity, and associated household and dietary risk factors, in a national sample of Cameroonian women and children, and the change in these values within urban areas after 3 y.In a 2009 national survey (3 strata: South, North, Yaoundé/Douala, 30 clusters/stratum, 10 households/cluster), information on dietary intake, household characteristics, and height/length and weight were obtained from women 15‐49 y and children 12‐59 mo. A 2012 study in Yaoundé/Douala surveyed the same 30 clusters; in addition to the above measures, women's waist and hip circumferences were measured.In 2009, 32% of non‐pregnant women had BMI 蠅 25.0 and 11% of women had BMI 蠅 30. Prevalence of BMI 蠅 25 differed among regions (40% South, 11% North, 48% Yaoundé/Douala), but not between urban and rural areas (36 vs 27%, P=0.11). Greater maternal age, TV ownership, and, in the North, socio‐economic status, were associated with BMI 蠅 25. Weight‐for‐height Z score > 2SD among children was < 4% in all regions. Mean BMI among women in Yaoundé/Douala was 26.0 and 26.9 kg/m2 and BMI 蠅 25 was 48% and 57%, respectively, in 2009 and 2012 (P > 0.1). Overweight/obese women had greater waist‐to‐hip ratios and were more likely to have waist circumference > 88 cm compared to women with BMI < 25 (P < 0.001).The results indicate that overweight/obesity is prevalent among women in some regions of Cameroon. A comprehensive public health strategy is needed to combine chronic disease prevention with efforts to address micronutrient deficiencies and infectious diseases.
Maternal Glycemia and Preterm Birth in the PRECONCEPT Cohort in Vietnam
Authors: Ford Nicole , Nguyen Phuong , Ramakrishnan Usha , Martorell Reynaldo 
Published: June 2021

ObjectiveStudies on gestational glycemia have focused primarily on developed country and predominantly overweight populations. Despite low prevalence of traditional risk factors, Vietnamese women have a high burden of gestational diabetes. Our study examined the role of maternal hyperglycemia in predicting preterm birth in rural Vietnam.MethodsThe PRECONCEPT study recruited women pre‐pregnancy for a randomized micronutrient intervention with prospective data collection through delivery. This analysis is based on a sub‐sample of the cohort with complete information on gestational age and maternal glycemia (N=975). Maternal hyperglycemia was defined as fasting blood glucose 蠅5.1 mmol/L at 23‐28 weeks gestation. Multivariate logistic models assessed the role of maternal hyperglycemia in predicting preterm birth (<37 weeks) and explored predictors of maternal glycemia.ResultsOverall, 46.1% of women had hyperglycemia, and 8.7% had a preterm birth. Maternal hyperglycemia was not associated with preterm birth (RR 0.84; 95% CI 0.53‐1.32). None of the candidate predictors of maternal hyperglycemia, including parity, pre‐pregnancy BMI, gestational weight gain, and physical activity were significant in multivariate analyses (p>.05).ConclusionWhile 46% of women had maternal hyperglycemia, it was not associated with risk of preterm. We were unable to identify risk factors for maternal hyperglycemia, suggesting that universal screening may be required. Future work is needed to examine potential consequences of maternal hyperglycemia for women and their offspring in this population and better define the public health significance of this condition in Vietnam.