bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding the apoptotic potential of heart cells
Authors: Davis Taylor , Del Gaizo Moore Victoria
Published: June 2021

Of all of the organs in the human body, the heart is one of the most vital. Should the heart or the blood vessels around the heart fail to develop properly, congenital heart defect occurs. Apoptosis is a highly controlled and regulated process known to occur throughout embryogenesis and is especially critical in heart development. However, very little is known about the specific apoptotic changes that occur during development. Therefore, our research investigates the molecular mechanisms of heart development. We hypothesize that heart cells isolated from a newly developing heart have greater apoptotic potential than heart cells of a fully developed heart. Utilizing whole hearts dissected from chick embryos, cardiomyocytes and cardiac fibroblasts are isolated from hearts harvested at different developmental stages. Next, these cells are treated with various concentrations of hydrogen peroxide for 2 hours and then stained with Annexin‐V‐FITC. The amount of apoptosis induced, as quantified by flow cytometry, is then used as a measure of susceptibility to apoptosis. Our data indicate that the amount of apoptosis induced by hydrogen peroxide treatment is no different for either cardiomyocytes or cardiac fibroblasts isolated from the same developmental stage. However, cell death for both types of cells at E5, as heart development is initially occurring, is significantly greater as compared to cells at E10, once heart development is complete. These results indicate that once heart development is done, both cardiomyocytes and fibroblasts develop resilience to apoptosis. This is critical, as it is important for heart cells to be susceptible to apoptotic signals throughout development, but also necessary for resistance to apoptosis once formative events have occurred.
Identifying Autopalmitoylation Inhibitors Through Scaffold Ranking
Authors: Hamel Laura , Lenhart Brian , Mitchell David , Giulianotti Marc , Deschenes Robert
Published: June 2021

Protein palmitoylation is a reversible posttranslational lipidation that plays an important role in cellular signaling, protein localization, stability, and aggregation. The dysregulation of palmitoylation has been implicated in a multitude of diseases including cancer, neurological disorders, and infectious diseases. A family of zinc‐finger “zDHHC” enzymes characterized by a conserved Asp‐His‐His‐Cys (DHHC) tetrapeptide sequence mediates palmitoylation. Having a unique inhibitor specific to the zDHHC enzyme(s) could lead to novel disease therapeutics. Despite advances in our understanding of the genetics and enzymology the current best inhibitor of palmitoylation, 2‐bromopalmitic acid, lacks specificity and affinity for zDHHC enzymes. Our lab has developed an assay amenable to high throughput screening, which measures the rate of the first step of the palmitoylation reaction, autopalmitoylation. During autopalmitoylation there is a release of CoASH, which is then coupled to an alpha‐ketoglutarate dehydrogenase reaction to produce fluorescent NADH. The production of NADH occurs at zero‐order kinetics and directly correlates to the reduction of CoA, and thus to the rate of autopalmitoylation. The introduction of an inhibitor of autopalmitoylation to this reaction would cause a reduction in the rate of NADH production. This coupled assay was used to screen for inhibitors of palmitoylation from a unique chemical scaffold library and resulted in the identification of a number of compounds. These results were supported through the use of orthogonal and bio orthogonal autopalmitoylation assays to further elucidate the type of inhibition.
Western Diet Alters Phosphorylation and O‐GlcNAcylation of Proteins Involved in Mouse Heart Metabolic Disease
Authors: Whelan Stephen , Spencer Jean , Heckendorf Christian , Yao Chunxiang , Behring Jessica , Siwick Deborah , Colucci Wilson , Cohen Richard , Bachschmid Markus , Costello Catherine , McComb Mark
Published: June 2021

The Western style diet (high fat and high sucrose) has lead to an epidemic of unfavorable metabolic disorders associated with obesity, diabetes and hyperlipidemia; leading to cardiovascular disease (CVD). Our hypothesis is that poor diet causes unfavorable metabolic conditions due to changes in post‐translational modifications (PTMs) of proteins in heart tissue. Two key PTMs that are perturbed in metabolically diseased tissues are phosphorylation and O‐GlcNAcylation. Both phosphorylation and O‐GlcNAcylation are found on nucleocytoplasmic proteins, regulate one another at the same serine/threonine site or adjacent sites, and are induced by insulin, nutrients (glucose), stress and other factors. Here we apply label‐free proteomics to elucidate potential biomarkers of CVD including both protein changes and changes in post‐translational modifications (PTMs). Heart tissue was from control mice and mice fed a high fat high sucrose diet (HFHS). Label‐free quantification was conducted using Scaffold and Progenesis. Using IPA software revealed a number of cardiovascular disease related proteins with altered levels. Upon further manual inspection many of the key proteins in normal heart function had altered phosphorylation and O‐GlcNAcylation modifications due to the Western diet. Some of these altered PTMs correlated with metabolic disease related mechanisms. NIH‐NHLBI Contract No. HHSN268201000031C and NIH‐GM grant P41 GM104603.
Investigating the Binding Affinity of Nod2 and Soluble Bacterial Cell Wall Dimers
Authors: Genova Lauren , Grimes Catherine
Published: June 2021

The innate immune system is the body's first line of defense against pathogens. The innate immune system is triggered by pathogen associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) such as Toll‐like receptors (TLRs) and Nod‐like receptors (NLRs). This research project focuses on providing a better understanding of how the innate immune system senses and responds to the presence of bacteria. Specifically, our group is interested in the relationship between the nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2), an NLR protein found in the cytosol of mammalian host cells, and muramyl dipeptide (MDP), the smallest bacterial cell wall fragment known to elicit an immunological response. When Nod2 is mutated, the signaling pathway becomes disrupted and uncontrollable inflammation arises, leading to chronic inflammatory bowel disorders such as Crohn's disease. To discover how to better treat these diseases, it is imperative to learn more about how Nod2 and MDP interact, a mechanism which is currently unknown. The Grimes Lab has previously shown that Nod2 binds to MDP in vitro; however, research suggests that a heightened immunological response may be elicited in a host if molecules containing multiple MDP's are used, suggesting multivalency is at play. To test this hypothesis, a variety of novel MDP dimers were synthesized and assessed via in vitro SPR binding assays and cell‐based assays.

This research has been funded by the Howard Hughes Medical Institute.
Recovery and Response of Crohn's Associated Mutants to Bacterial Cell Wall Fragments
Authors: Schaefer Amy , Melnyk James , Mohanan Vishnu , Grimes Catherine
Published: June 2021

The objective of this study is to determine the activity and binding capabilities of Crohn's associated mutant Nod2 protein. Crohn's disease is a debilitating inflammatory bowel disease alleged to be caused by genetic factors and a malfunctioning immune system. A susceptibility gene has been identified on chromosome 16 that is responsible for encoding for nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2). Nod2 is an innate immune receptor that responds to bacterial cell wall fragments to initiate the NF‐κB pathway. Three specific mutations to Nod2 have been implicated in an increased susceptibility to Crohn's disease. These mutations to Nod2 prevent it from recognizing or responding to its proposed ligand, muramyl dipeptide (MDP), a bacterial cell wall fragment. Our lab has shown that the Crohn's‐associated mutants of Nod2 suffer from a stability issue; however, mutants are able to be rescued with the use of a chaperone protein. Upon stabilization of these mutants, activity assays and binding studies can help to elucidate information about where these mutants fail to perform, be it recognizing and binding MDP or signaling NF‐κB activation. Luciferase reporter assays and surface plasmon resonance binding studies were optimized to study Nod2/MDP activity and interaction. In the presence of a chaperone tag, Crohn's mutant activation of NF‐κB signaling was rescued and there was no evidence of diminished binding of the mutants to MDP. This suggests that the root cause of misregulation due to one of these mutations to Nod2 is an issue of protein stability and folding rather than an inability to bind or signal.
Human Single‐chain Antibodies against Polycyclic Aromatic Hydrocarbons.
Authors: Ustinov Valentin , Studennikov Artem , Vavilov Vitaliy , Tyumentseva Marina , Morozova Vera , Tikunova Nina , Glushkov Andrey
Published: June 2021

Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread organic pollutants. In order to select anti‐PAHs antibodies, the conjugate of a benzo[a]pyrene (BP) with BSA (BP‐BSA) was used to screened naïve combinatorial phage library of human scFvs. Seven unique scFvs against BP‐BSA were selected after three rounds of selection. Analysis of the genes encoding the scFvs subdivided them to gene families and subfamilies. Homology with the closest germline ranged from 80.21% to 97.57% for heavy chains and 88.89% to 98.57% for the light chains. Four from seven scFvs amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique nonstructural proteins with low sequence identity among them. The proteomic analysis of all CDRs and the boundaries in the CDR3 formation were carried out. Two of scFvs (T68 and T72) were expressed in E.coli and purified using a nickel resin. The affinity of T68 and T72 was determined by surface plasmon resonance. The KDs values of T68 and T72 to BP‐BSA ​​were 2.93x10‐7 and 1.37x10‐6, respectively, which were close to the previously obtained monoclonal antibodies against BP. The KDs values of T68 to PAHs (BP‐BSA, chrysene‐BSA, pyrene‐BSA, anthracene‐BSA, and benzo[a]anthracene‐BSA) were similar to 10‐7. T72 KDs values were 1.37x10‐6, 2.63x10‐7, 8.22x10‐6, 3.74x10‐6, and 9.42x10‐8 to BP‐BSA, chrysene‐BSA, pyrene‐BSA, anthracene‐BSA, and benzo[a]anthracene‐BSA, respectively. The active centers of T68 and T72 computer built models were different from each other which explained difference in KDs values.
Conserved Residues in TCR CDR Loops and Their Role in Influencing Antigen Recognition
Authors: Blevins Sydney , Baker Brian
Published: June 2021

The Adaptive Immune System is the body's way of identifying and destroying foreign pathogens. This process involves a recognition event utilizing two major proteins: a T cell receptor (TCR) and a Major Histocompatibility Complex (MHC) protein presenting a peptide. The complete mechanism by which a TCR recognizes an antigen (peptide‐ MHC) and proceeds to initiate the signaling cascade to destroy infected cells is still unknown. Much of our work aims to better understand the properties that might influence how a T cell receptor recognizes a peptide‐MHC. One area of interest is the role of conserved TCR residues and how this might influence TCR behavior. Preliminary bioinformatics work has shown that over 80% of TCRs have a conserved histidine residue in the CDR1β loop. Structural observation revealed that the histidine is contacting a surprisingly conserved serine or threonine residue in the CDR3β loop. To explore potential roles this interaction might have, two complementary approaches will be used to examine the effects of this interaction within T cell receptor CDR loops. Surface plasmon resonance will be used for the assessment of non‐additivity between remote CDR loops and fluorescence anisotropy will be used to detect changes in CDR loop flexibility.
Molecular Dynamics Studies of the Role of Protein Flexibility in Immunological Molecular Recognition
Authors: Ayres Cory , Scott Daniel , Baker Brian , Corcelli Steven
Published: June 2021

Activation of the cellular immune response involves the recognition of an antigenic peptide presented by either the class I or class II major histocompatibility complex (MHC) by T‐­cell receptors (TCRs). Because of the large population of potential antigenic peptides in comparison to the number of available TCRs, TCRs must be cross‐­reactive, yet must also maintain a degree of specificity to avoid indiscriminate T cell activation. A commonly implicated factor for TCR specificity and cross reactivity, as well as the current focus for this study, involves the flexibility of TCR complementarity‐­determining region (CDR) loops. This study focuses on the DMF5 TCR, which is specific for epitopes of the MART‐­1 protein, upregulated in the majority of melanomas. Beyond its potential utility in cancer therapy, this particular TCR is of interest because of the apparent rigidity of its CDR loops, which contrasts with what traditionally has been expected of TCR binding loops. Molecular Dynamics simulations of both the unligated and ligated DMF5 TCR were performed in order to assess its flexibility and to gain insight into the roles of mobility in DMF5 binding.
Proinflammatory Environmental Exposures in Asthma
Authors: Cheah Cheryl , Daines Michael , Morgan Wayne
Published: June 2021

Asthma is a chronic inflammatory disease that affects over 35 million people in the United States, and is a leading cause of hospitalization in children. While asthma‐causing allergens like fungi, mites, and insects represent biologically diverse organisms, they have some biological similarities that may underlie their potential to cause asthma. One of these shared biological features is the production of chitin. Studies have shown that chitin impacts lung function through the innate immune system. There are no available chitin assays for assessing environmental samples. Creation of a quantitative assay for chitin is complicated because it is a modified polysaccharide and not a protein. This makes it difficult to make antibodies that will specifically recognize it. Chitin is also poorly soluble in water and many standard solvents, which makes developing adequate standards challenging

After successfully creating a chitin quantification assay with a detection limit of about 20ng/mg dust and validating commercially available assays for endotoxins and lipopolysaccharides, we acquired 387 household dust samples from the Inner‐City Anti‐IgE Therapy for Asthma (ICATA) clinical trial and quantified MAMPS like chitin, endotoxins and lipopolysaccharides in those samples. Quantifying chitin, endotoxins and glucans in household dust samples gave us a better understanding of the microbiome profile in those samples. Overall, this study contributes to our understanding of the mechanisms in human asthma and allergy. It also has the potential to improve existing techniques in the treatment and prevention of a disease that affects millions of people worldwide.
ADP‐ribosylargininyl Activity of Cholix Toxin Is Mediated Through Diffusible Intermediates
Authors: Sung Vicky , Tsai Chia‐Lun
Published: June 2021

OBJECTIVE

We discovered and characterized the ADP‐ribosylargininyl activity of the cholix toxin catalytic fragment, which was first identified as a diphthamide‐dependent ADP‐ribosyltransferase.

METHODS

We utilized genetic approach to construct direct and reverse mutant enzymes and substrates in conjunction with biochemical assays including fluorescence/non‐fluorescence based enzymatic assays, protein/peptide‐2D‐electrophoresis analysis.

RESULTS

Our studies show that the transfer of ADP‐ribose to toxin itself takes place by a predominantly intramolecular mechanism. Multiple arginine residues, located in the proximity of the catalytic site but also at distal sites, can be the ADP‐ribose acceptor in the auto‐reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially modifies arginine residues in proximity to the NAD+ binding pocket. ADP‐ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates, such as oligo arginine peptides and eEF2(H715R) mutant. Auto‐ADP‐ribosylation of cholix toxin appears to have negative regulatory effect on its enzymatic activities. However, at the presence of both endogenous and exogenous substrates, the ADP‐ribosylation of exogenous substrate occurred more efficiently than endogenous substrate.

CONCLUSIONS

The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetime for the cognate oxocarbenium ion. We therefore hypothesize a strained form of NAD+ intermediate generated through an error‐prone mechanism of the enzyme to react with arginine residues in a proximity dependent manner.
Neutron/X‐ray Joint Refinement of The Active and Inactive Form of Helicobacter pylori 5′‐ Methylthioadenosine Nucleosidase
Authors: Banco Michael , Mishra Vidhi , Kovalevsky Andrey , Ronning Donald
Published: June 2021

5′‐Methylthioadenosine nucleosidase (HpMTAN) is an essential Helicobacter pylori enzyme that hydrolyzes N‐ribosidic bonds of a variety of adenosine‐based metabolites. Recently, HpMTAN has become an attractive drug target due to the enzyme's role in the menaquinone pathway that is only specific for H. pylori and Campylobacter. In the proposed catalytic mechanism of HpMTAN, the reaction is initiated by D198 that acts as a general acid to protonate the N7 position of the adenine moiety. The protonation creates a delocalized positive charge in the adenine moiety that promotes stretching of the N‐ribosidic bond to progress the catalytic reaction. Currently, transition state analogs are known to function as powerful inhibitors of MTAN; however, the majority of these compounds exhibit inhibition of the human enzyme 5′‐methylthioadenosine phosphorylase (hMTAP), a homolog to HpMTAN. In this study, we present the first neutron structures of an active and an inactive variant (D198N) of HpMTAN that were co‐crystallized with SAH. The structures were solved using X‐ray crystallography and subjected to joint X‐ray/neutron refinement to allow for direct determination of hydrogen atom positioning throughout the catalytic domain of HpMTAN. The neutron structures confirm the proposed mechanism in which D198 acts as a general acid and illustrates other features of the catalytic reaction. Additionally, key polar interactions are highlighted in the catalytic domain that will aid in structure‐based design of inhibitors that will be selective for HpMTAN and have little to no effect on hMTAP.
Investigating the Catalytic Mechanism of Yeast Cytochrome c Heme Lyase (CCHL) Through Mutation of Highly Conserved Amino Acid Residues
Authors: Kulick Sasha , Junker Matthew , Sanders Carsten
Published: June 2021

Cytochrome c is a vital component in energy transduction pathways, as well as in the process of programmed cell death (apoptosis). As a functional holoform, cytochrome c contains at least one covalently attached heme cofactor. In fungi, metazoans, and some protozoa, heme attachment is catalyzed by the enzyme cytochrome c heme lyase (CCHL), which is also known as holocytochrome c synthase (HCCS). In humans, genetic mutations leading to nonfunctional CCHL are lethal to males and cause microphthalmia with linear skin defects syndrome (MLS) in females. Despite this association with human disease, little is known about the mechanistic and structural properties of CCHL. Here, we used yeast CCHL to carry out site‐directed mutagenesis to evaluate the role of highly conserved residues adjacent to histidine at position 128 (H128), which has been found to be critical for catalytic activity. Generated mutants were co‐expressed with yeast apocytochrome c in an Escherichia coli system, in which successful holocytochrome c formation in vivo is indicated by the development of red colored cells (red phenotype). Cells coexpressing yeast apocytochrome c and CCHL mutants H128N and H128C lost the ability to ligate heme to apoprotein substrates (white phenotype). This ability was retained at varying levels, however, in M124C, V125C, Q126C, 127C, F130C, L131C, and N132C mutants. Absorbance spectroscopy confirmed these findings, revealing characteristic spectra for holocytochrome c in the lysates of red (but not white) colored cells. In some mutants, it has also been observed that the redox behavior of produced holocytochrome c differs from that in wildtype. These mutants are being investigated for potential altered enzyme‐substrate interactions.
Substrate Recognition by the Radical Enzyme Benzylsuccinate Synthase
Authors: Funk Michael , Drennan Catherine
Published: June 2021

Anaerobic bacteria play a crucial role in bioremediation of ecosystems recovering from oil spills and industrial pollution. The first step in the anaerobic degradation of toluene, a major component of gasoline, is addition to fumarate to yield benzylsuccinate by benzylsuccinate synthase (BSS). BSS is a member of glycyl radical enzyme family, which uses free radical chemistry to break and form C‐H and C‐C bonds. In this presentation, we describe the recent X‐ray crystal structures of BSS, and consider how this glycyl radical enzyme controls substrate specificity and affords activation of the otherwise inert toluene molecule.
An Old Cofactor in a New Light: Adenosylcobalamin in Light‐Dependent Gene Regulation
Authors: Jost Marco , Padmanabhan S. , Elíaz‐Arnanz Montserrat , Drennan Catherine
Published: June 2021

Adenosylcobalamin (AdoCbl, coenzyme B12) is one of Nature's most complex enzyme cofactors and enables difficult radical chemistry via cleavage of its covalent cobalt‐carbon bond. In a recently discovered bacterial pathway, however, AdoCbl is used in a completely new function: it is used as a light sensor in light‐dependent gene regulation. Here, AdoCbl binds to the transcription factor CarH to regulate expression of the carotenoid biosynthetic genes. Gene expression is only activated upon light exposure, leading to carotenoid production and protection of the cell from light‐induced damage. This elegant response mechanism to sunlight is found in hundreds of bacterial species.

Here, we present the full structural characterization of the light‐sensing transcription factor CarH. In the dark, AdoCbl mediates formation of a CarH tetramer that positions the DNA‐binding domains on the DNA operator, blocking transcription. Exposure to light cleaves AdoCbl's Co‐C bond, triggering a conformational change that causes tetramer disassembly, dissociation from DNA, and transcription activation. Thus, CarH harnesses the light sensitivity of AdoCbl to drive a light‐dependent gene expression switch. These studies provide fundamental insight into a new mode of light‐dependent gene regulation and provide a new biological role for AdoCbl.

This work is supported by the NIH (CLD), the Ministerio de Ciencia e Innovación–Spain (SP and ME‐A), and a MIT Poitras fellowship (MJ). CLD is a HHMI investigator.
In vitro and In vivo Anti‐hyperglycemia Effect of Novel Amadori Rearrangement Compound, Arginyl‐fructosyl‐galactose
Authors: Choi Hwang‐Yong , Ha Kyoung‐Soo , Lee Se‐Woon , Kang Yu‐Ri , Lee Jung‐Yun , Kim Young‐Ho , Kwon Young‐In
Published: June 2021

Amino acid derivatives such as arginyl‐fructosyl‐galactose (AFL) are formed through Amadori rearrangement, the early step of Maillard reaction from arginine and lactose in daily foods. However, very limited information is available about the effect of AFL on various biological activities. This is the first report of the mode of action and effect of AFL on the type 2 diabetes management related inhibition of postprandial hyperglycemia in vitro and in animal model. The inhibitory activities against a‐glucosidase and porcine pancreatic a‐amylase were investigated in vitro and in vivo animal model. In in vitro study, AFL had a significant inhibition against rat intestinal a‐glucosidase but showed mild inhibitory activity on porcine pancreatic alpha‐amylase. In rat fed on starch or sucrose meals, AFL significantly reduced the postprandial blood glucose increment after starch or sucrose loading in a dose dependent manner. These results indicate that AFL has an anti‐diabetic effect by suppressing carbohydrate absorption from intestine, and thereby reducing the postprandial increase of blood glucose.
Investigation of the evolutionary relationship between Rio2 kinases and the canonical eukaryotic protein kinases using x‐ray crystallography
Authors: Bahmanjah Soheila , LaRonde Nicole
Published: June 2021

Rio proteins are serine kinases that contain few of the conserved residues of typical protein kinases and are thus categorized as atypical protein kinases. Rio proteins are required for the cleavage of 20S rRNA to 18S rRNA.
Canonical protein kinases are very similar to Rio2 proteins but are more active in ATPase and atuophosphorylation activity and they are onlyobserved in archaea. Computational studies have revealed that the arginine residue at position 228 highly affects the activity of canonical protein kinases. This residue in Rio family is replaced by either alanine or glycine. We were interested to study mutation at 228 of Rio2, and have found out how this mutation would affect the structure and activity of the Rio2 family. We have investigated the evolutionary relationship between Rio kinases and the canonical eukaryotic protein kinases. Our observation is that canonical eukaryotic protein kinases have evolved from Rio kinases. We have made several mutations in a key differentiating position of the kinase domain sequence of Ct‐ Rio2, G228, and have collected biochemical data to test the ability of the mutated Rio kinase to autophosphorylate and carry out ATP hydrolysis. We have also co‐crystallized Ct‐Rio2 G228 point mutants with ATP in order to determine the structural differences between the mutant and wild‐type Hence, wild type Ct‐Rio2 and five mutants of Ct‐Rio2 were purified and co‐crystalized with ATP/Mg2+. These mutants include G228A, G228D, G228N, G228R, and G228V and x‐ray diffraction data sets were collected.
Effects of CAII/CAIV double knockout on fertility
Authors: Mannowetz Nadja , Wandernoth Petra , Szczyrba Jaroslaw , Grannemann Laura , Becker Holger , Wolf Anne , Wennemuth Gunther
Published: June 2021

Bicarbonate (HCO3‐) is an essential factor to modulate sperm motility. Female genital tract fluids are rich in HCO3‐ and contribute to an increase of sperm beat frequency thus supporting sperm to reach the site of fertilization. Carbonic anhydrases (CA) catalyze the reversible hydration of CO2 to HCO3‐ and are important candidates in the regulation of both the HCO3‐ homeostasis in sperm and the composition of the male and female genital tract fluids. We show that CAII and CAIV are localized in epithelial cells lining the epididymal duct and that they appear with the onset of puberty. Gene expression analyses with tissue from respective knockout animals reveal an up‐regulation of CAII and CAIV in the different epididymal sections, indicating a compensatory mechanism for the gene defect. In sperm, the presence of CAII is restricted to the principal piece, whereas CAIV is detectable in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an impaired HCO3‐ homeostasis resulting in significantly diminished sperm motility, velocity and response to HCO3‐. Enzyme activity tests show that CAII and CAIV account for nearly 95% of the total CA activity in sperm. We generated CAII/CAIV double knockout animals which display a significant reduction in sperm motility resulting in sub‐fertility. Interestingly, biochemically created CAIV deficient sperm of CAII knockout animals show a lower response to HCO3‐ as compared to sperm from double knockout animals. From these results we conclude that both CAII and CAIV play a key role for a successful fertilization.
Evidence for presence of Focal adhesion kinase and enhanced activity in epithelial and stromal cells of mice cervix during pregnancy
Authors: Gautam Sabrena , Brunson Alicia , Ohashi Takako , Jesmin Subrina , Mowa Chishimba
Published: June 2021

We recently provided the first evidence of a presence of a cluster of mechano‐transducing molecules in the cervix using proteomics analysis, including caveolin‐1 (CAV‐1), talin‐1, vinculin, pro‐filin and others. Here, we use confocal immunofluorescence, Western blot and real time PCR analysis to examine whether a key mechano‐transduction molecule, phosphorylated focal adhesion kinase protein and mRNA (pFAK and FAK mRNA) are expressed in the cervix during pregnancy (days 0, days 14, 15, 16), as well as identify its (pFAK) cellular distribution in mice. pFAK was localized in the epithelial and stromal (fibroblast) cells of the cervix of pregnant mice and its expression and activity increased with advancement in pregnancy but decreased postpartum. The expression was found to be confined to the cell membrane. We conclude that the growing fetus likely induces mechano‐transduction in the cervix and may influence cervical remodeling and most likely the timing of birth. Funding: Office of Student Research, Appalachian State University.
Developing new assays (Fluorosomes) for characterization of drug interactions of ABC multidrug transporters
Authors: Telbisz Agnes , Sarkadi Balazs , Wright George , Bansal Praveen , Melchior Donald
Published: June 2021

ABCB1/Pgp and ABCG2/BCRP multidrug transporters are important in developing multidrug resistance and significantly modify ADME‐Tox parameters of several therapeutic compounds. Regulatory agencies (EMA, FDA) state in their guidelines that drug candidates be tested for in vitro inhibition of Pgp and ABCG2. Only the application of different assays can reveal complex drug interactions of these polyspecific transporters. We investigate these interactions either in a native cell membrane environment or by using purified proteins reconstituted in a liposomal platform. ABC transporters extrude drugs at the expense of ATP hydrolysis, thus drug interactions can be followed in proteoliposomes (PL) by measuring ATPase activity or transport of labeled compounds. Providing appropriate lipid environment for ABCG2 was a key step in establishing new assays. PLs of ABCG2, in contrast to those containing ABCB1, have to contain cholesterol. In addition, phosphatidylethanolamine‐containing E.coli lipids support the ABCG2 activity better than phosphatidylcholine‐based lipids. In our international collaboration we are working on new assays similar to that developed earlier for the Pgp (the Fluorosome‐trans‐pgp assay). This method involves PLs that contain a fluorescent drug sensing probe in the interior of the PL, and measures in real time changes in fluorescence which reflect the Pgp‐ or ABCG2‐mediated transport. With Fluorosome‐trans‐abcg2/pgp assay, and direct liposomal assay observing the inhibition of uptake of fluorescently labeled substrate drugs we examine transporter‐drug interactions. The results are analyzed in correlation with the ATPase activity measurements.
Purification and characterization of a bacterial mating protein
Authors: Bulku Artemisa , Berkmen Melanie
Published: June 2021

Conjugation is the transfer of DNA from a donor to a recipient bacterium through a DNA translocation channel known as a Type IV Secretion System (T4SS). Our work focuses on the T4SS of ICEBs1, the integrative and conjugative element found in Bacillus subtilis. ConE is a component of the ICEBs1 T4SS. ConE is a VirB4‐like ATPase, most likely utilizing the energy generated from ATP hydrolysis for DNA transfer or T4SS assembly. Previously, we fused a hexa‐histidine tag (His6) to ConE for ease of purification via nickel affinity chromatography. However, we found that surprisingly low concentrations of imidazole inadvertently wash His6‐ConE off the nickel resin, preventing us from obtaining a high yield of pure protein. Here, we cloned deca‐histidine tagged ConE (His10‐ConE) and purified it side by side with His6‐ConE. We observed that His10‐ConE can be purified in higher yield and with fewer contaminants than His6‐ConE. After optimization, we conducted a large‐scale purification of His10‐ConE for the purpose of obtaining antigen for production of antibodies to ConE. In the future, the antibodies will be used in western blots, pull‐down assays, and immunofluorescence microscopy to characterize ConE levels, interactions, and localization, respectively. Additionally, we plan on exploring His10‐ConE's ability to interact with DNA using electrophoretic mobility shift assays.
Radical Chemistry in the Human Gut: Discovery of Choline Trimethylamine‐Lyase
Authors: Bodea Smaranda , Balskus Emily
Published: June 2021

The collection of microorganisms that inhabit the human gastrointestinal tract, our gut microbiota, has an extensive set of metabolic capabilities that directly impact human health. Our research seeks to discover and understand the molecular basis of disease‐associated metabolic pathways from the human microbiome. One example under investigation is the catabolism of the essential nutrient choline by anaerobic microbes. Gut bacteria break down choline in an entirely different manner than human cells, cleaving its C–N bond to produce trimethylamine (TMA), which is subsequently oxidized to trimethylamine N‐oxide by liver enzymes. This microbial‐human co‐metabolic pathway has been linked to several diseases, including non‐alcoholic fatty liver disease and atherosclerosis.

Using a genome mining strategy, we recently identified the first gene cluster responsible for anaerobic choline utilization (the cut gene cluster). We verified the functional role of this cluster using both genetic and heterologous expression approaches. These studies revealed that the key C–N bond cleavage step involved in choline breakdown is catalyzed by choline trimethylamine‐lyase (CutC), a new type of glycyl radical enzyme. This reaction is unprecedented chemistry for this enzyme family. We confirmed the activity of the purified choline TMA‐lyase and its activating protein in vitro, observing that CutC is exquisitely specific for choline cleavage. Currently, we are investigating the mechanism of the CutC‐catalyzed reaction, with the ultimate goal of designing small molecule inhibitors for this enzymatic transformation.
Neonatal Antibiotic Exposure Alters Compositional Gut Microbiota Development During the First 6 Months of Life
Authors: Kumar Himanshu , Rautava Samuli , Collado Maria , Borzykh Nikita , Löyttyniemi Eliisa , Isolauri Erika , Salminen Seppo
Published: June 2021

Objective

To investigate the impact of neonatal antibiotic exposure on gut microbiota development in infants born after 36 weeks of gestation

Methods

A case‐control study of 6 neonates exposed to brief (<5 days) empirical antibiotics (penicillin G and gentamicin) during the first 72 hours of life, 6 neonates receiving a 7‐day course of the same antibiotics for documented infection and 24 matched healthy non‐exposed neonates. Gut microbiota composition was assessed at 1 and 6 months of age by qPCR, DGGE and 16S rRNA pyrosequencing

Results

Neonatal antibiotic exposure resulted in a significant decrease in fecal bifidobacteria at 1 month of age (p=0.015), particularly in B. bifidum (p=0.0061). The bacterial richness of infants exposed to antibiotics was significantly lower compared to non‐exposed infants. The principal coordinates analysis (PCoA) formed separate clusters when compared between antibiotic‐exposed and non‐exposed infants at 6 months of age. In particular, an increased frequency of Firmicutes and a decrease in Bacteroidetes was observed in antibiotic‐exposed infants

Conclusions

Aberrant gut microbiota colonization is attributable to antibiotic exposure since it was observed also in antibiotic‐exposed neonates in whom infection was later ruled out. Decreased levels of both bifidobacteria and Bacteroidetes in early infancy have previously been linked to increased risk for non‐communicable disease in later life. Means of accurately detecting bacterial infections in neonates are needed to avoid unnecessary exposure to antibiotics during this vulnerable period

Research support

Functional Foods Forum, University of Turku
Developing an Approach for Using Low Quantities of DNA for HLA typing: A Tool for Determining the Ancestral Link of Disease
Authors: Uaisele Mele , Marquez‐Magana Leticia
Published: June 2021

Molecular genetic research relies on determining the underlying biology of health disparity populations; however, biospecimen donations from these populations are scarce. Factors that contribute to limited biospecimen donation include lack of time, trust and transparency. In fact, often small quantities of biospecimen and/or DNA are available. Therefore, we sought to develop a new approach for using small quantities of DNA to study genetic links between individuals burdened by health disparities. These links can be uncovered by human leukocyte antigen (HLA) typing. Unfortunately, this method requires micrograms of DNA when often only nanograms of DNA are available from health disparity populations. Consequently, we developed a method for amplifying low quantities of genomic DNA for use in HLA typing. Preliminary results suggest that the amplified DNA yields the same HLA types as genomic DNA thus validating our approach. In future work, this approach can be used to study the genetic link between individuals afflicted with a particular health disparity. For example, the high incidence of triple‐negative breast cancer for African American women in the U.S. and for Ghanian women world‐wide suggests an ancestral link for this disease that can be uncovered by HLA typing.
Cell‐fie of a Cancer Cell: Imaging Metastatic Potential of Single Living Tumor Cells with Fluorescent Biosensors
Authors: Midde Krishna , Sun Nina , Mehlawat Priyanka , Husain Hatim , Ghosh Pradipta
Published: June 2021

Recognizing the metastatic potential of solid tumors is of utmost importance to personalize treatment plans for cancer patients. Conventional approaches utilize ensemble studies in which read‐outs from a heterogeneous population of cells are averaged to assess tumor aggressiveness. Such averaging obscures precious information on those individual tumor cells that are a minority within any given tumor, but possess the most ominous characteristics that contribute to their intrinsic metastatic potential, e.g., stem‐ness, EMT/invasiveness, drug resistance). Thus, the quest for the Holy Grail in measuring such ‘potential’ continues. Here we developed FRET‐based biosensors that measure the metastatic potential of single living cancer cells by confocal imaging.

By design, these biosensors measure functional phosphorylation of Gα‐Interacting and Vesicle Associated Protein (GIV) a multimodular signal transducer and a bona‐fide metastasis‐related protein across a variety of solid tumors. These biosensors allow the generation of spatial and temporal signaling profiles of single cells, downstream of multiple receptors, even at a steady‐state. Such single‐cell signaling profiles are reflective of tumor heterogeneity, are unbiased to a given receptor/pathway, and eliminate the need to know which receptors/pathways drive a given tumor. Using these probes we detected those few cells within the primary lung and breast tumors with the highest metastatic potential, which are subsequently selected during metastasis.

These results provide proof‐of‐concept that GIV‐biosensors are effective in monitoring the metastatic potential of single‐cells, in a sensitive and unbiased way, across different tumors.
A microRNA signature of response to erlotinib is impacted by the EMT‐ inducing cytokine TGFβ1
Authors: Krentz Madeline , Astley Rebecca , Black Esther
Published: June 2021

Purpose

We endeavored to understand the contribution of canonical TGFβ signaling on expression of a 13‐gene microRNA (miR) signature of erlotinib response.

Background

We identified a 13‐gene miRNA signature predictive of response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in Non‐Small Cell Lung Cancer (NSCLC) cell lines. Bioinformatic analysis of the signature showed a functional convergence on TGFβ canonical signaling. We hypothesize that TGFβ signaling controls expression of the miR genes comprising an erlotinib response signature in NSCLC.

Methods

Two signature miR genes (miR‐140‐3p and miR‐141) were chosen for study. Promoters regions of both genes harbor putative Smad Binding Elements (SBEs), DNA binding domains for TGFβ‐responsive Smad transcription factors. TGFβ signaling impact on miR‐140 and ‐141 expression was assessed in erlotinib‐resistant (A549) and –sensitive (PC9) NSCLC cells by quantitative real‐time PCR (qRT‐PCR), luciferase reporter assays, and chromatin immunoprecipitation (ChIP).

Results

(1) qRT‐PCR showed endogenous miR 140/141 expression decreased after short term TGFβ1 treatment in both cell lines. After 7 days of treatment, the trend continued. (2) The promoter region of miR‐141, containing 6 SBEs, was cloned into a luciferase reporter vector. The miR‐141 luciferase construct is responsive to TGFβ stimulation like that seen in qRT‐PCR. (3) ChIP experiments will assess specific Smad complexes binding SBE sites.

Conclusion

We expected that canonical TGFβ signaling molecules, Smads, bind to and control miR genes with SBEs in A549 cells. In PC9 cells, some miR genes likely control Smad protein expression while a non‐canonical TGFβ response may regulate signature miR expression

College funds were used.
Downregulation of miR‐155 Contributes to The Anti‐proliferative Effects of The Soy Isoflavone Genistein in Estrogen Receptor Negative Breast Cancer
Authors: De La Parra Columba , Cruz‐Collazo Ailed , Castillo‐Pichardo Linette , Cubano Luis , Redis Roxana , Calin George , Dharmawardhane Suranganie
Published: June 2021

Consumption of the soy isoflavone genistein has been associated with reduced breast cancer risk. We previously reported that dietary genistein inhibits mammary tumor growth and metastasis of estrogen receptor (ER) (‐) cancer cells in mice. The objective of this study was to investigate a role for the oncomir miR‐155 in the anticancer effects of genistein. At low physiologically relevant concentrations, genistein inhibited cell viability, induced apoptosis, and downregulated miR‐155 in the ER (‐) MDA‐MB‐435 and Hs578t breast cancer cells, without significantly affecting the viability or miR‐155 levels of ER (+) MCF‐7 breast cancer cells. In parallel, the pro‐apoptotic and anti‐cell proliferative miR‐155 targets FOXO3, PTEN, casein kinase, and p27 were upregulated in response to genistein in the ER (‐) cells. Ectopic expression of miR‐155 in the MDA‐MB‐435 and Hs578t cells decreased the effects of genistein on cell viability, and abrogated the effects of genistein on apoptosis and expression of pro‐apoptotic genes. We conclude that genistein‐mediated downregulation of miR‐155, a critical suppressor of apoptosis, contributes to the anticancer effects of genistein in ER (‐) breast cancer.
PKCδVIII and Bcl2 Increase Ovarian Cancer Survival via LncRNA NEAT1 Secreted by Obese Adipose Derived Stem Cells
Authors: Patel Niketa , Morris Amanda , Patel Rekha , Carter Gay , Cooper Denise , Murr Michel
Published: June 2021

Ovarian cancer remains the most common cause of death from gynecologic malignancy in US. The impact of obesity on the incidence and treatment of gynecologic cancer is growing in importance. Little is known about the molecular factors obesity imparts to the cancer niche that account for this relationship. Adipose‐derived stem cells (ADSC) can be isolated from stromal vascular fraction of abdominal white adipose tissue. We isolated and differentiated ADSC into adipocytes and previously showed that ADSC derived from lean and obese patients have distinct differences in gene expression. We evaluated the possibility that the secretome of obese ADSC could have paracrine effects on ovarian cells promoting growth and development of cancer cells. We show that NEAT1 levels secreted by ADSC from obese patients (ADSCo) were considerably higher than from normal ADSC both in cells and its secretome. Ovarian cancer high mortality rates are further exacerbated by cisplatin‐resistant cancers. Our data shows cisplatin‐resistant ovarian cells TOV‐112D cells had 5–fold higher expression of the survival protein Bcl2, and 10‐fold higher PKCδVIII, a signaling kinase which upregulates expression of Bcl2. Further, NEAT1 secreted from ADSCo increases Bcl2 and PKCδVIII expression. Cell proliferation assays and migration assays indicated that ADSCo‐CM increase TOV112D tumor cell migration. When ADSC NEAT1 was silenced, tumor cell migration decreased. These results indicate that the deleterious paracrine effects of genetically modified ADSC from obese patients may contribute to the poorer oncologic outcomes of ovarian cancer.

Supported by VA Merit Grant awarded to N.A.P.
Socio‐Ecological Determinants of Type 2 Diabetes among South‐Indians: Influence of Sedentary Behaviours and High Energy Intakes
Authors: Bermudez Odilia , Vanzan Hari , Ravi Sripriya , Thanikachalam Sadagopan , Vasudevan Preethi , Must Aviva , Thanikachalam Mohan
Published: June 2021

Objective

To examine the association between energy intakes (EI) and physical activity (PA) levels with prevalence of type 2 diabetes (T2D) among adults from the state of Tamil Nadu, India

Methods

PURSE‐HIS is an ongoing population‐based study following a sample of 8080 adults, men and women, >20y from rural, semi‐urban and urban areas of Chennai City, Tamil Nadu. Participants were assessed by questionnaire for PA, EI with 24‐hr recalls and prevalence of T2D by history or oral glucose tolerance test. SES was assessed with the Kuppuswamy index.

Results

In the low SES group, 74% of men and women had moderate PA across rural, semi urban and urban areas. High PA was reported by only 5% of the sample with more (11%) of rural men reporting high PA. Women with high PA had lower EI than recommendations with highest deficit in rural women (18%). Men with high PA in rural and semi urban areas had EI deficits of 18% and 8%. Across areas and sexes with low and moderate PA, excess EI was seen. Among men highest excess in EI was seen in urban (40%) and semi urban (36%) men with low PA (p< 0.01). Low PA was associated with highest prevalence of T2D among rural (25%), semi urban (26%) and urban (30%) women. Among men, highest prevalence of T2D (26%) was in the low and moderate PA groups of all three areas. Significant association were seen among low PA, excess EI and prevalence of T2D across sex and regions (p <0.01).

Key findings

Low physical activity and excess energy intake are associated with higher prevalence of T2D in the low socio‐economic groups.