bluuebunny/crossref_metadata_2025 huggingface dataset:
Heavy metals and polyaromatic hydrocarbon contaminants in smoked and fresh Clarias gariepinus from a cultured system in Lagos, Nigeria (959.5)
Authors: Osuala Fidelia , Igwo – Ezikpe Miriam , Emmanuel Babatunde
Published: June 2021

This study investigated the level of heavy metals and poly ‐ aromatic hydrocarbon (PAH) concentrations in ten randomly selected life fresh and smoked African cat fish, Clarias gariepinus from a cultured system in Lagos, Nigeria using FAO / SIDA, 1983 method for heavy metals analysis while PAH was analysed using gas chromatograph ‐ mass spectrometry (GC‐MS). Heavy metals and PAHs were detected in both samples at varying concentration, with higher concentration of cadmium, lead and zinc in smoked fish though not significantly (P > 0.05) different from that in fresh fish whereas iron and copper were insignificantly (P > 0.05) higher in fresh fish. Naphthalene, fluorene, phenanthrene, anthracene and benzo (g,h,i) perylene were higher in the smoked fishes though naphthalene and anthracene were significantly (P < 0.05) higher in smoked fishes than fresh fishes. The maximum concentration of lead was 0.0492 mg/kg which is higher than the EPA safety limit (0.001 mg/kg) while 2‐methylnaphthalene concentration was highest in the smoked fishes with a mean value of 4.13 mg/kg. The statistical analysis using one way ANOVA showed that there was significant (P < 0.05) differences in the heavy metals and PAH content in the fresh and smoked fish. This study suggests that smoked fish have higher concentrations of heavy metals and poly ‐ aromatic hydrocarbons which might pose health risk to humans when consumed.
Molecular mechanism studies of mammalian sterile20‐like protein kinase 3 in mitochondrial dynamics (961.2)
Authors: Lin Wei , Yuan Chiun‐Jye
Published: June 2021

Mammalian sterile20‐like protein kinase 3 (Mst3) is a serine/threonine protein kinase that plays roles in various biological functions, including cell migration, cell cycle regulation and apoptosis. In preliminary studies we have shown that Mst3 may play a role in mediating the activity of mitochondrial OXPHOS system. Mitochondrion is a highly dynamic organelle that frequently proceeds fusion and fission to maintain its function and to adapt the metabolic needs of the cells. Dys‐regulation of mitochondrial morphology usually associates with many diseases, including diabetes, neurodegenerative diseases and cancer. However, how Mst3 regulates the mitochondrial function and dynamics remains unknown so far. In this study we found that the mitochondria in HeLa cells with selective knockdown of Mst3 exhibited more fragmented morphology compared to that of HeLa cells. Further studies shown that the proteins controlling the mitochondrial fusion and fission were altered by the selective knockdown of Mst3. These results suggest that Mst3 may not only play a role in regulating mitochondrial OXPHOS system, but also mediating mitochondrial morphological dynamics. Studies are undergoing to further elucidate the molecular mechanism underlying Mst3‐mediated mitochondrial function and dynsmics.
Insight into the lysosomal storage disorder cystinosis: genetic interactions with ERS1 in Saccharomyces cerevisiae (963.1)
Authors: Rickel Kirby , Simpkins Jessica , Vitiello Seasson
Published: June 2021

Cystinosis is an inherited pediatric lysosomal storage disorder caused by loss‐of‐function mutations in the gene CTNS, which encodes the Cystinosin protein. While the causative gene and primary biochemical defect have been identified, it is still unclear exactly which pathways contribute to the cell death that is observed in cystinotic patients. To investigate the implications of the loss of Cystinosin, we have employed the power of the model organism Saccharomyces cerevisiae, a model organism for higher eukaryotes. Yeast is an organism that is amenable to genetic manipulation and has a fully sequenced and highly annotated genome. With the understanding that the yeast protein Ers1p is the ortholog of human Cystinosin, the overall aim of this research was to better understand the role of Ers1p in yeast by identifying genes that interact with the cognate gene ERS1. Using data derived during a functional genomics screen, we propose to investigate genes that exhibit increased or decreased expression in the absence of ERS1. Our findings will not only contribute to our understanding of cystinosis, but also to primordial pathways in yeast and other eukaryotes.

Grant Funding Source: Supported by NIH Grant Number 2 P20 RR016479
Non‐telomerase telomere maintenance in human cells (97.2)
Authors: Reddel Roger , Conomos Dimitri , Henson Jeremy , Napier Christine , Neumann Axel , Pickett Hilda
Published: June 2021

Although it has recently been demonstrated that telomeres can be lengthened by a non‐telomerase mechanism (i.e., Alternative Lengthening of Telomeres; ALT) in normal mouse tissues, we have learnt most about ALT in mammalian cells from the study of in vitro‐immortalized and tumor‐derived human cell lines. Apart from its intrinsic biological interest, it is important to understand ALT because ~10% of human cancers utilize it to avoid senescence, and some of the tumor types where ALT is common, such as soft tissue sarcomas and astrocytic brain tumors, are currently difficult to treat. Loss of normal ATRX or DAXX function contributes to, but is insufficient for the upregulation of ALT in many cancers and cell lines. An increased non‐canonical telomeric repeat sequence content may also foster increased levels of ALT activity by decreasing shelterin binding sites. ALT involves synthesis of new telomeric DNA via recombination‐mediated copying of existing telomeric sequences. The template may be the DNA of another telomere or a more proximal region of the telomere that is being lengthened. There is a tight correlation between the level of C‐circles, i.e., extrachromosomal circles of partially double‐stranded telomeric DNA with an intact C‐rich strand and an incomplete G‐rich strand, and ALT activity. We are therefore using a C‐circle assay to identify genes/ proteins involved in the ALT mechanism.
Identification and characterization of novel granzyme in fish (971.1)
Authors: Matsuura Yuta , Yabu Takeshi , Shibasaki Yasuhiro , Shiba Hajime , Moritomo Tadaaki , Nakanishi Teruyuki
Published: June 2021

Granzymes are serine proteases released by cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Granzymes induce apoptosis within virus‐infected and transformed cells. Information on the function of fish granzymes is limited, although granzyme‐like genes have been reported in several fishes. Here, we report the identification and characterization of a granzyme in ginbuna crucian carp, Carassius auratus langsdorfii, focusing on substrate specificity and gene expression after allo‐sensitization and bacterial infection.

A full‐length granzyme cDNA, termed gcGranzyme was identified from ginbuna crucian carp. gcGranzyme molecule has trypsin‐like catalytic triad: His66, Asp109, and Ser201 residues, and active center: Ser201 residue. Phylogenetic analysis of deduced amino acids sequence revealed that gcGranzyme formed a cluster with human and mouse granzyme B (separate from the other members of the granzyme). gcGranzyme was secreted from the HeLa cells transfected with gcGranzyme as in mammals. Enzyme assay using recombinant gcGranzyme indicated that the enzyme hydrolyzed Bz‐Arg‐4Methyl‐Coumaryl‐7‐amide, suggesting that the gcGranzyme was different from mammalian granzyme B in substrate specificity.

To investigate the role of gcGranzyme in cell‐mediated immunity in fish, we analyzed the gcGranzyme mRNA expression in allo‐sensitized fish. Expression level of gcGranzyme mRNA from CD8+ T cells was greatly elevated by allo‐sensitization. Furthermore, gcGranzyme mRNA expression in CD8+ T cells was also enhanced by the in vivo infection with intracellular bacteria, Edwardsiella tarda.

These results suggest that newly found gcGranzyme is a novel secretory serine protease involved in cell‐mediated immunity in fish with similar structure to human granzyme B but different substrate specificity.
Re‐evaluation of desmethyl, desamino‐PateamineA targeting of eukaryotic translation initiation factor 4F (975.2)
Authors: Kommaraju Sai Shilpa , Aulicino Julieta , Li Jing , Zhu Mingzhao , Romo Daniel , Low Woon‐Kai
Published: June 2021

The marine natural product PateamineA (PatA) and the simplified analog desmethyl, desamino PateamineA (DMDAPatA) have cytotoxicity selective for rapidly proliferating cells by inhibition of cap‐dependent translation initiation through binding to eIF4A (eukaryotic initiation factor 4A). eIF4A is a member of eIF4F which includes eIF4E (cap‐binding protein) and eIF4G (scaffolding protein). eIF4A is a DEAD‐box helicase that unwinds secondary structure in the 5ʹ‐untranslated region (5ʹ‐UTR) of mRNA allowing for recruitment of the 43S pre‐initiation complex containing the small ribosomal subunit. The small ribosomal subunit then “scans” the 5ʹ‐UTR in search of the AUG start codon which triggers translation. Previously we had proposed a model where binding by PatA/DMDAPatA to eIF4A disrupts the integrity of the eIF4F complex by directly perturbing the protein‐protein interactions between eIF4A and eIF4G causing an inhibition of eIF4A‐ and hence eIF4F‐dependent translation initiation. However, more detailed structural studies have suggested that the eIF4A‐eIF4G interaction is not directly disrupted by PatA. We have re‐examined the integrity of eIF4F complexes in the presence of DMDAPatA, and our preliminary results suggest the potential dependency of prior binding of eIF4E to eIF4G along with eIF4E cap‐binding for disruption of the eIF4A‐eIF4G interaction by DMDAPatA.

Grant Funding Source: Supported by St. John's University
Kinetics and molecular dynamic modeling of a potent and selective marine natural product inhibitor of human cathepsin L (975.3)
Authors: Miller Bailey , Friedman Aaron , Choi Hyukjae , McCammon J. , Hook Vivian , Gerwick William
Published: June 2021

Protease inhibitors are an important drug target class due to their wide and varied roles in both physiological and pathological states. In particular, the human cysteine cathepsin proteases have been implicated in the progression and metastasis of cancer, participation in the development of neurodegenerative diseases, and are important processing enzymes for the production of multiple neuropeptides. Past marine natural products screening efforts have successfully yielded potent and selective inhibitors of proteases, hence, marine cyanobacterial extracts were assessed for inhibitory activity to human cathepsin L. Gallinamide A, a depsipeptide secondary metabolite isolated from a collection of filamentous marine cyanobacteria, was identified as a hit structure with potent activity. With 30 minutes of preincubation, gallinamide A displayed an IC50 of 5.0 nM, and kinetic analysis demonstrated an inhibition constant of ki = 9009 ± 262 M‐1 s‐1. A preincubation‐dilution experiment revealed an irreversible mode of inhibition, and comparative IC50 values display a 28‐ to 331‐ fold greater selectivity toward cathepsin L than closely related human cysteine cathepsins V or B. Gallinamide A shows no inhibitory activity against the exopeptidase cathepsin H, further exemplifying its selectivity. Molecular docking and molecular dynamics simulations were used to determine the pose of gallinamide in the active site of cathepsin L. These data resulted in the identification of a pose characterized by high stability, a consistent hydrogen bond network, and the reactive Michael acceptor enone functional group of gallinamide A positioned near the active site cysteine of the protease, leading to a proposed mechanism of covalent inhibition. These data reveal and characterize the novel activity of gallinamide A as a potent inhibitor of human cathepsin L.

Grant Funding Source: Supported by NIH T32GM007752, NIH FIC U01 TW006634, and NIH R01MH077305
Effect of a combined fraction of protease inhibitor and lectins on hematopoietic system in rats (975.7)
Authors: Moreno‐Celis Ulisses , Ferriz‐Martinez Roberto , Andrade‐Portillo Veronica , Lopez‐Martinez Josue , Aranda‐Vargas Paola , Noriega‐Giron Hector , Guerrero‐Carrillo Maria De Jesus , Blanco‐Labra Alejandro , Rodriguez‐Mendez Adriana Jheny , Garcia‐Gasca Teresa
Published: June 2021

Studies from our group have shown that lectins and protease inhibitors (PIs ) of Tepary beans have individual effects on colon cancer cells. In order to know the in vivo effects of a combined fraction of lectins and protease inhibitor (LIP‐60), alterations in the hematopoietic system were determined. Sprague Dawley rats of 3 weeks old were administered intragastrically with LIP‐60 (100 mg/kg body weight) for 28 days. Blood samples were obtained on 0, 1, 3, 9, 14, 19 and 24 days, animals were sacrificed at day 30 and spleen, thymus and femur bone marrow were obtained for histopathological analysis. Body weight and food consumption decreased were observed respect to control group. Complete blood count showed decrease in lymphocytes while granulocytes increased, suggesting allergic process. Histopathological analyzes showed reduced white pulp of the spleen, related with an increase in the release of mature leukocytes. The results suggest that LIP‐60 provoke allergy‐like response.

Grant Funding Source: CONACYT FOMIX (QRO‐2011‐C02‐175340)
Investigation of potential topoisomerase I inhibitors from indigenous natural products in southwestern region of Nigeria (975.8)
Authors: Magbagbeola Olubunmi , James Ayorinde , Odesanmi Selina , Samuel Titilola , Oshodi Temitope
Published: June 2021

Polyphenolic compounds are important secondary metabolites that may serve as anti‐tumour agents by binding the nuclear enzymes topoisomerase I thereby affecting DNA function and cell survival. Polyphenols from sixteen hypothesized antitumour plants in the south western region of Nigeria were screened for topoisomerase I inhibition in comparison to camptothecin.

Partially purified bulky polyphenols from the selected plants (Costus afer, Piper guineense, Andrographis paniculata, Securidaca longipedunculata, Olax subsorpiodea, Vernonia amygdalina, Ocimum gratissimum, Annona muricata, Bryocarpus coccineus, Combretum racemosum, Citrus aurantifolia, Amaranthus virides, Catharantus roseus, Garcinia kola, Zanthoxylum zanthoxyloides) from different GPS locations were incubated at 10 µg/ml with 0.5 ug supercoiled pBR322 and 1 U topoisomerase I enzyme at 37oC for 2hrs and reaction was terminated using a stop buffer containing 3% SDS, 60 mM EDTA, 50% glycerol, 0.25% bromphenol blue. Reaction products were determined by electrophoresis on 1% agarose gel in TAE (Tris‐acetate‐EDTA) running buffer at 6.5 V/cm for 2 hrs.

Conclusively, significant (P<0.05) purification of bulky polyphenols from the aqueous extract of each study plant was achieved by our newly rapid and inexpensive modified method of adsorption chromatography of polyphenols. Polyphenols from the aqueous extract of Annona muricata plant showed promise to contain topoisomerase I inhibitors by inhibiting the relaxation of supercoiled DNA pBR322.
Development of selective inhibitors against homoserine transacetylase ő a novel target for drug development (976.2)
Authors: Thangavelu Bharani , Pavlovsky Alexander , Viola Ronald
Published: June 2021

Homoserine transacetylase (HTA) lies at a branch point in the aspartate metabolic pathway which leads to the biosynthesis of the essential amino acids methionine and lysine in plants, bacteria and fungi. The branch leading to the synthesis of methionine and the essential methyl transfer cofactor S‐adenosylmethionine (AdoMet) starts with the acetylation of homoserine to form O‐acetylhomoserine catalyzed by HTA. AdoMet is also the substrate for a group of enzymes that synthesize acylhomoserine lactones, virulence signaling molecules in Gram‐negative bacteria. Since the aspartate pathway is not found in humans, but is necessary for bacterial survival, the reaction catalyzed by HTA is a critical control point for cell growth and viability and is a potential target for the development of new antibiotics. We have cloned and purified homoserine transacetylases from the Gram‐positive bacteria Staphylococcus aureus, Gram‐negative bacteria Pseudomonas aeruginosa and fungi Candida albicans. A sensitive coupled‐enzyme activity assay had been developed to determine the kinetic properties of each enzyme form. A customized fragment library has been screened against these enzymes and initial hits have been identified with inhibition constants (Ki) values in the low millimolar range. Docking studies with published HTA structures are being used to guide the design and development of potent inhibitors. Initial crystallization of these new enzyme forms have produced diffraction quality crystals that are being optimized for data collection. The long term goal of this project is to develop drugs that selectively target this enzyme and block the methionine biosynthetic pathway. This work is supported by funding from the NIH (AI077720).

Grant Funding Source: NIH (AI077720)
Controlling low rates of terminal cell differentiation through noise and ultra‐high feedback (981.1)
Authors: Teruel Mary , Ahrends Robert , Ota Asuka , Kovary Kyle , Park Byung Ouk
Published: June 2021

Mammalian tissue size is maintained by slow replacement of aging cells. For adipocytes, key regulators of glucose and lipid metabolism, the renewal rate is only 10% per year. Here we use computational modeling, quantitative mass spectrometry (1) and single‐cell microscopy (2) to show that expression noise acts within a network of more than six positive feedbacks to permit pre‐adipocytes to differentiate at very low rates. This solves two fundamental opposing requirements: high cell‐to‐cell signal variability generates very low differentiation rates while low signal variability keeps cells irreversibly locked in the differentiated state. Thus, the challenge of how higher eukaryotes control low rates of irreversible cell fate decisions can be solved by a balancing act between expression noise and ultra‐high feedback connectivity.

(1) Abell E,…, Teruel MN (2011). Proc Natl Acad Sci U S A. Aug 30; 108(35):14485‐90.

(2) Park BO,…, Teruel MN. (2012). Cell Reports 2012 Oct 25. PMID: 23063366.
Antigens with the potential to differentiate between acute and chronic phases of Schistosoma mansoni (984.2)
Authors: Moraes Vanessa , Siqueira Jaqueline Maria , Coelho Paulo Marcos , Grenfell Rafaella
Published: June 2021

Schistosomiasis mansoni is a serious problem of public health in the world and the improvement and development of new diagnostics tests is necessary to the control of disease. Our group performed a study in different areas in southeast of Brazil, with acute and chronic patients. The samples of serum and feces of patients were collected and submitted to Kato‐Katz and ELISA to diagnostic. Positive patients were treated with praziquantel and monitored after treatment. Western blotting analysis using cercariae, adult worms and eggs antigens was applied to these samples for screening of immunoreactive fractions. Some fractions were more immnunoreactive to specific phases and others decreased the intensity after treatment. Five fractions were selected to compose a diagnostic kit: two with 18 and 25 kDa to detected acute phase and one with 22 kDa to chronic phase, all of cercariae newly transformed antigens; one with 50 kDa to detected acute and chronic phase and two with 17 and 20 kDa to detected the post‐treatment phase, all of egg antigens. These fractions will be indentified and recombinant proteins produced to confection of an indirect diagnostic based in lateral flow methodology. This assay is fast, simple, requires less equipment, and is an accurate tool of screening for low resource‐regions.

Grant Funding Source: Supported by Capes, CNPq, Fiocruz, PDTIS (Brazil), Fiotec, UFSJ
A comparative survey of the impact of NHE1 phosphorylation on pHi and cell motility (994.1)
Authors: Zamit Andrea , Strong Molly , Hovde Moriah , Wallert Mark , Provost Joseph
Published: June 2021

The sodium hydrogen exchanger isoform one (NHE1) exchanges an intracellular proton for an extracellular sodium ion playing a key role in the regulation of pH and directed cell motility. The regulation of NHE1 function is complex, involving multiple protein‐protein interactions and several phosphorylation sites. While most of the kinase sites have been identified the role of each phosphorylation event has not been defined in the context of the other phosphorylation events. There are many challenges involved in gaining a comprehensive understanding of the role of the different phosphorylation events. In part, these challenges come from the fact that data has been collected from multiple cell types being stimulated by a wide array of agonists. The key phosphorylation sites on NHE1 include: RhoA associated kinase (Rock), ribosomal S‐6 kinase (Rsk), protein kinase B (AKT) and extracellular‐signal regulated kinase (Erk). We have created cell lines expressing two distinct mutations at each Ser/Thr phosphorylation site. The first mutation changes the Ser/Thr to Ala removing the ability of a kinase to phosphorylate that site. The second changes a Ser/Thr to Asp to mimic the phosphorylation event. To screen the roles of each of these phosphorylation sites, changes in intracellular pHi and cell motility will be determined in cell lines stably expressing each NHE1 mutant. Our goal is to evaluate the relative impact of each phosphorylation site on NHE1 function in a comparative comprehensive manner
Direct uptake of copper from plasma ceruloplasmin by cells that do and do not express CTR1 (996.2)
Authors: Vargas Rebecca , Ramos Danny , Mar David , Ishida Michaella , Lee Kyoung , Linder Maria , Montgomery Aaron
Published: June 2021

The main Cu‐binding protein of blood plasma, ceruloplasmin (Cp), has mainly been studied for its role in mediating cellular iron efflux but also has other functions, and as shown by i.v. infusion of 67Cu‐Cp, its Cu enters most major organs. To determine whether it delivers Cu directly to cells and the mechanisms involved, we incubated purified radio‐copper‐labeled and unlabeled Cp with cells and measured rates of Cu uptake and accumulation. Uptake was demonstrated for human mammary epithelial cell monolayers (PMC42 cells) with tight junctions, and with mouse embryonic fibroblasts expressing/not expressing Ctr1. Cp‐Cu was internalized. It could not be washed from the cell surface even with acidic buffers, and was found in the cytosol. Inhibitors of endocytosis did not impair uptake. Cu deficient cells exposed to Cp increased their cellular Cu contents; and holo‐Cp was converted to apo‐Cp during cell treatment, with no loss of total Cp protein. Uptake was almost the same in cells not expressing Ctr1. Excess ionic Cu(II) and Cu(I) virtually eliminated Cp‐Cu uptake. The presence of Cu(II) and Fe(III) reductase activities on the cell surface was verified. We conclude that Cp‐Cu delivers Cu to cells by a process involving cell surface reduction of Cp‐Cu(II), release of apo‐Cp, and uptake of the resulting Cu(I) by Ctr1 and an additional as yet unidentified Cu(I) transporter.

Grant Funding Source: Supported by PHS Grant R15 GM100464
Investigating the domain motions of an asymmetric ABC transporter (997.1)
Authors: Tieleman Peter , Corradi Valentina , Singh Gurpreet , Seeger Markus
Published: June 2021

ATP‐binding cassette (ABC) transporters transport lipids, sterols, peptides, toxins, nutrients, drugs and ions across membranes and consist of two transmembrane domains (TMDs) and two nucleotide‐binding domains (NBDs).

Asymmetric ABC transporters feature specific mutations in one of the two ATP‐binding sites (the degenerate site), resulting in a reduced ATPase activity compared to the non‐mutated site (the consensus site). It has been proposed that in asymmetric ABC transporters ATP remains bound at the degenerate site, while ATP‐binding and ‐hydrolysis at the consensus site drive the transport cycle.

Here, we investigate the effects of ATP‐binding and domains' motions of the heterodimeric transporter TM287/288 (Hohl et al., Nat. Struct. Mol. Biol. 2012). We have performed molecular dynamics (MD) simulations, for a total simulation time of ca. 10 microseconds, on the structure of TM287/288 in different nucleotide‐bound states. Our results show that in the absence of ATP at the consensus site, the simulation systems with and without ATP at the degenerate site do not completely dissociate at the NBD level, but reach an intermediate state in which contacts between the NBDs are still observed.
Identification of essential residues in the extracellular region of the epithelial sodium channel (997.4)
Authors: Berman Chance , Booth Rachell
Published: June 2021

The epithelial sodium channel (ENaC) is a membrane bound protein responsible for regulating the reabsorption of sodium in the distal nephron of the kidneys. Genetic mutations in the intracellular regions of ENaC are known to cause diseases such as Liddle’s syndrome and Pseudohypoaldosteronism type 1, severe hypertension or hypotension, respectively. In an effort to identify critical residues in the extracellular region of αENaC that are necessary for proper function, error prone polymerase chain reaction (EP‐PCR) was employed to generate random mutations in the extracellular loop followed by expression in yeast. A serial dilution pronging assay was used to monitor the salt sensitivity, an induced phenotype, in yeast expressing various αENaCs. Mutant αENaCs that altered yeast growth were sequenced and expression was confirmed by western blotting. Mutations effecting αENaC function included mutant 125, Y334H; mutant 135, F306L; and mutant 137, R202A. Future studies are aimed at understanding the role of these residues in ENaC function.

Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David) and Welch Foundation Departmental Grant AI‐0045
A comparative analysis of mitral valve changes in different age groups by histochemical, immunohistochemical and ultrastructural study (LB1)
Authors: Sahni Daisy , Saini Narbada , Saikia Uma
Published: June 2021

Objectives: Mitral valve prolapse (MVP) is an increasingly prevalent clinical problem which poses a surgical and diagnostic challenge for cardiac surgeons and cardiologists respectively. The commonest MVP etiologies are age‐related degenerative (60%), rheumatic (12%) and functional (25%). Previous studies have described the degenerative changes in various pathological conditions of mitral valve such as rheumatic heart disease, mitral regurgitation and mitral valve prolapse. However there is paucity of literature regarding histomorphological details of age‐related degenerative changes in normal human mitral valve leaflets. Thus we planned to analyze the histomorphological features and ultrastructural changes in mitral valve along with the distribution of proteoglycans in mitral valve leaflets with ageing.

Methods: Tissues from 60 formalin fixed human hearts including 30 males and 30 females were taken for histomorphology and immunohistochemistry and for ultrastructural analysis in 15 of them.

Results: Among 60 mitral valves, mild fibrosis was present in 4 cases (40%) in 2nd decade, 8 cases (80%) in 3rd decade, 4 cases (40%) in 4th and 5th decades, and 6 cases (6%) in 6th decade and only 1 case (10%) in 7th decade. Majority of the mitral valves (90%) in 7th decade showed moderate fibrosis, however severe fibrosis was found only in 10% cases in 6th decade. The MPS material deposition was mild in 60%, 20% and 10% cases in 2nd, 3rd and 5th decades respectively whereas it was moderate to severe in 4th to 7th decades, being maximum in 7th decade (90%). Immunohistochemistry(IHC) showed moderate and severe decorin positivity in 18/60 (30%) and 1/60 (1.66%) cases respectively whereas mild and moderate biglycan positivity was seen in 34/60 (56.66%) and 8/60 (13.33%) cases respectively. Ultrastructurally, collagen fibres were fragmented with abundant glycogen granules, numerous inclusions of MPS material, and fibroblasts with increasing age.

Conclusion: The present study highlighted that fibrosis and MPS accumulation in mitral valve leaflets increases with advancing age along with the increased expression of decorin and biglycan.

Key words: Mitral valve, aging, fibrosis, immunohistochemistry, ultrastructure

Grant Funding Source: Indian Council of Medical Research
Understanding The Magic Touch (LB101)
Authors: Garb Ashley , Barbeau Delaney , Ihrig Daniel , Henke Noah , Opheim Allison , Hansen Brandon , Respalje Lindsay , Schroeder Mitchell , Garb Jodie , Weyer Andy , Zappia Katherine
Published: June 2021

The Institute of Medicine estimates $635 billion dollars are spent annually on people with chronic pain conditions. One debilitating symptom of these conditions is hypersensitivity to touch, where daily activities can be painful. Few therapeutics to ameliorate mechanical hypersensitivity exist because the mammalian ion channels that sense touch are poorly understood. The mechanosensitive channel of large conductance (MscL) is an ion channel in Mycobacterium tuberculosis which allows bacteria to respond to mechanical stimuli by electrochemical response, regulating membrane ion flow. Research shows structural changes in MscL causes the protein to open, allowing ions into the cell. Key amino acids include hydrophobic residues I14 and V21, creating a constriction at the cytoplasmic surface. R98, K99, K100, E102 and E104 are possibly a ligand binding site, potentially participating in the ion conduction pathway. Residues at the N‐terminus of MscL, K3, F5, E7 and F8, may play a role in sensing membrane stretch. The Laconia SMART (Students Modeling A Research Topic) Team used 3D printing technology to model MscL. Understanding the structure‐function relationships of the MscL channel protein may lead to better comprehension of how human mechanosensitive ion channels, like the Transient Receptor Potential Ankyrin 1, work and lead to a cure for hypersensitivity to touch.

Grant Funding Source: Supported by a grant from NIH‐CTSA.
Beta‐2 adrenergic receptor: a SMART team modeling project (LB106)
Authors: Ellingwood Lexy Ellingwood , Sanders Mitchell , Sanders Katie Sanders , Vinod Anjan , Wang James , Wang John , Link Tina , Zhu Mengyuan Zhu
Published: June 2021

Asthma is a condition that affects millions. In response to allergens, the smooth muscles surrounding the lungs undergo bronchoconstriction which reduces the amount of air accessible. Bronchoconstriction can be countered by bronchodilation which results from the side effects of Beta‐2 adrenergic receptor (b2AR) antagonists. B2AR is a type of G protein coupled receptors (GPCRs) that bind to a drug like salmeterol and induce a conformational change that activates signal transduction in the cell that results in the relaxation of smooth muscle in the lungs. In 2012, Brian Kobilka and Bob Lefkowitz received a Nobel prize for their biochemical analysis and structural determination of b2AR. Kobilka used the T4 lysozyme to stabilize the protein and create a clearer view of the protein. The critical residues for agonist binding are Phe290, Asp113, Ser207, Ser203, Asn312 and Phe193. The Walton SMART Team (Students Modeling A Research Topic) modeled the b2AR using 3D printing technology demonstrating the interaction profile between the high affinity agonist BI‐167107 that stabilizes the active state of b2AR.

Grant Funding Source: Supported by grants from the NIH‐SEPA and NIH‐CTSA.
Akt1 signaling in dopamine‐dependent behaviors (LB116)
Authors: Lo Yu‐Hsiang , Franke Thomas
Published: June 2021

Background: One biochemical change acquired following induction of depression‐like behavior and exposure to drugs of abuse is decreased phosphorylation of the Akt1 serine‐threonine kinase.

Methods: To examine the relevance of intact Akt signaling, we used Akt1‐mutant mice as genetic model. By combining biochemical, pharmacological, genetic and behavioral approaches, we examined if altered Akt signal transduction affects behavioral responses in experimental paradigms related to mesolimbic/nigrostriatal dopamine function.

Results: Loss‐of‐function Akt1‐mutant mice exhibited a phenotype of increased susceptibility in the learned helplessness paradigm. In this experimental model for acquired behavioral depression, the exposure to inescapable electric foot shocks (IES) disrupts the subject’s escape to subsequent noxious stimuli. While no significant differences were observed between sham‐treated control mice depending on genotypes, IES‐treated Akt1‐mutant mice showed increased latencies of crossing and a higher number of escape failures when compared to IES‐treated wild‐type mice. In further support of the importance of intact Akt signaling in dopamine‐dependent behavioral responsiveness, Akt1‐mutant mice displayed enhanced responses to acute cocaine, diminished sensitization to chronic cocaine exposure and altered conditional place preference for cocaine.

Western blotting and immunohistochemical analyses after IES training revealed genotype‐dependent changes in Akt phosphorylation and regulation of its substrates following IES training. These biochemical changes phenocopied differences in behavioral outputs when comparing wild‐type and Akt1‐mutant mice.

Discussion: Several mechanisms have been proposed to explain increased excitability of DA neurons after drug exposure. One that is increasingly attractive involves dephosphorylation and inhibition of the serine/threonine kinase Akt. Reduced Akt kinase activity is frequently observed in psychiatric disorders with high comorbidity for substance use. By exploring Akt1‐dependent stress responses in the mesolimbic system using genetic models, we gain novel insights into the role of Akt signaling in regulating critical brain circuitry involved in motivation, decision‐making and reward.

Grant Funding Source: Supported by NIH DA032280
Physical and Functional Interactions of the E. coli Monothiol Glutaredoxin GrxD Suggest a Role in FeS Apoprotein Maturation (LB132)
Authors: Saini Avneesh , Boutigny Sylvain , Baidoo Edward E.K. , Yeung Natasha , Keasling Jay D. , Butland Gareth
Published: June 2021

The biosynthesis of iron sulfur (FeS) clusters is a highly coordinated process enabled by multi‐protein systems such as Isc and Suf in E. coli. Additional accessory factors including the monothiol glutaredoxin, GrxD, are located outside of these defined systems. GrxD has been proposed to function as a shuttle protein acting to transfer FeS clusters between scaffold and carrier proteins, although specific functional role remain unknown. We have previously linked GrxD in FeS client protein interactions with the radical SAM enzyme MiaB. To address these different hypotheses, we conducted an extensive survey for physical interaction of GrxD with a comprehensive set of FeS cluster biosynthesis factors including cysteine desulfurase components (IscS, SufSE), FeS scaffold (IscU, SufBC2D) , carrier proteins (IscA SufA, ErpA, NfuA), HscA, HscB and various FeS apoproteins. We have employed various methods, including affinity co‐purification, analytical gel filtration and surface plasmon resonance (SPR) to obtain interaction data. To date we have observed no physical interaction between GrxD and any FeS biosynthesis factor except the SufBC2D scaffold and the SufA carrier protein. With SufA a reproducible but extremely weak association is observed by SPR alone (KD >> 700 µM). In contrast we have observed specific interactions between GrxD and two radical SAM enzymes, MiaB (KD =12 µM) and BioB (KD =34 µM). These data strongly indicate a role for GrxD in FeS protein maturation or repair, working downstream of the Suf system

Grant Funding Source: NIH award GM088196 to G.B
Understanding epoxide hydrolase regiospecificity: towards the discovery and design of highly selective biocatalysts (LB133)
Authors: Horsman Geoff , Aliwalas Mark
Published: June 2021

The ever‐increasing quantity of genomic data continues to reveal a great deal about microbial natural product biosynthesis and has enabled new molecule and biocatalyst discovery. However, predicting chemical structures from gene sequences remains challenging because even enzymes with high sequence similarity may catalyze unexpectedly different reactions. This information gap between genes and chemical structure can be narrowed by careful comparison of related biosynthetic enzymes. For example, epoxide hydrolases (EHs) of very high sequence similarity from related enediyne biosynthetic pathways can have different regiospecificities, resulting in opposite stereochemical configuration in the products. Specifically, “inverting” EHs hydrolyze an (S)‐epoxide substrate to generate an (R)‐vicinal diol while “retaining” EHs yield an (S)‐vicinal diol. Intriguingly, the inverting EHs possess only one of the two canonical Tyr residues present in EHs. Biochemical characterization of several EH mutants suggests that Tyr substitution may direct EH regioselectivity. Indeed, genome mining has identified additional enediyne biosynthesis‐associated EHs that further the predictive utility of the Tyr substitution, thereby setting the stage for understanding EH regioselectivity.
Biological Nitrile Reduction ‐ Structure and Mechanism (LB136)
Authors: Swairjo Manal , Kiani Maryam , Chikwana Vimbai , Lee Bobby , Iwata‐Reuyl Dirk
Published: June 2021

The enzyme QueF catalyzes the NADPH‐dependent reduction of the nitrile group of 7‐cyano‐7‐deazaguanine (preQ0) to 7‐aminomethyl‐7‐deazaguanine (preQ1) in the biosynthesis pathway to the tRNA modified nucleoside Queuosine. The only nitrile reductase known in biology, QueF is a potential new candidate for industrial biocatalysis of nitrile transformations to amines. QueF belongs to the tunneling fold (T‐fold) structural superfamily built around a small ββααββ protein domain that oligomerizes to form a β2nαn barrel and facilitates interfacial catalysis of a variety of chemical reactions. In recent years, several crystal structures have been determined of unimodular and bimodular QueF enzymes, and of active site mutants and complexes with substrate and cofactor. Here we present crystal structures and biochemical and transient kinetic data that elucidate the mechanism of biological nitrile reduction and provide insight into the molecular evolution of T‐fold mediated interfacial catalysis. Structures of substrate‐bound QueF demonstrate that formation of a thioimide covalent adduct is a key step in the mechanism of reaction and that preQ0 binding, not thioimide formation, induces closure of the active site. A strictly conserved active site disulfide bridge suggests possible enzyme regulation by allosteric disulfide. We propose a mechanism for the QueF‐catalyzed reaction.

Grant Funding Source: Sponsored by the National Institutes of Health and the National Science Foundation
Characterization of beta‐glucosidase from Physarum polycephalum (LB141)
Authors: Oh‐date Ayaka , Minami Yoshiko
Published: June 2021

Physarum polycephalum, which is one of the true slime molds, has a unique life cycle that includes plasmodia, sclerotia, spores, and amoebae. Furthermore, the plasmodia cultured in liquid medium form a small lump called microplasmodia. The microplasmodia secrete a large amount of slime that includes the various proteins such as beta‐glucosidase: intracellular soluble, membrane‐bound, and secretory enzymes. Previously, we had reported that the membrane‐bound beta‐glucosidase BglM1, which has a molecular mass of 130 kDa, has a unique primary structure. The structure of BglM1 consists mainly of two regions: the N‐terminal region, which contains a typical glycosyl hydrolase family 3 domains and the C‐terminal region, which has a unique sequence that is unrelated to hydrolytic reactions. Notably, the C‐terminal region of BglM1 contains two calx‐beta motifs known as calcium binding sites, an RGD sequence, and a transmembrane region. In this study, we focus on the characteristics of a secretory enzyme that may be a membrane‐free type of BglM1.

The molecular mass of the secretory enzyme purified in the presence of Ca ion and absence of Ca ion is 130 and 95 kDa, respectively. The 130kDa molecules react with antibodies that are specific for the BglM1 N‐terminal and C‐terminal regions. However, the 95kDa molecules are not detected by the C‐terminal antibody. These results indicate that in the absence of Ca ion, the 130kDa molecules are unstable and are degraded to 95kDa molecules. The kinetics data obtained from the 95 and 130kDa molecules showed that the calx region plays an important role on the stability and function of the enzyme. Further, to clarify the physiological functions of BglM1 with a calx region and of the secretory form, we assessed their mRNA expression during the Physarum cell cycle.
Crystal structure of trehalose synthase from Deinococcus radiodurans reveals an active conformation (LB142)
Authors: WANG Yun‐lin , Liaw Shwu‐Huey
Published: June 2021

Trehalose, a non‐reducing disaccharide, displays exceptional stability and has been used in the food, cosmetic, medical and biotechnological industries. Trehalose synthase catalyzes a simple conversion of maltose into trehalose and hence has great potential in industrial application. However, the disadvantages of low thermo‐stability and the side product of glucose by the hydrolytic activity are needed to be improved. TS belongs to the glycoside hydrolase family 13 (GH13), which represents the largest family of glycoside hydrolases, transferases and isomerases. Here we have determined a crystal structure of TS from Deinococcus radiodurans (DrTS), which consists of a catalytic (β/α)8 barrel, a subdomain B, a C‐terminal β domain and two TS‐unique subdomains (S7 and S8). The C‐terminal domain and S8 contribute the majority of the dimeric interface. The active‐site architecture of DrTS is virtually identical to those of several glycoside hydrolase 13 members, suggesting of an active conformation. However, the recently published TS structures from Mycobacterium smegmatis and M. tuberculosis display an inactive conformation because the S7 loop blocks the substrate‐binding site. A detailed comparison revealed open/closed conformations with distinct S7 structures and different inter‐domain orientations. Such conformational changes through subdomains during enzyme catalysis are also observed in other GH13 members. Based on the structural comparison, particularly with sucrose isomerase and sucrose hydrolase, several mutants are generated and characterized. Hopefully, the residues involved in isomerase/hydrolase activities will be identified.
Is knocking out nagZ the answer to routine production of O‐GlcNAc‐modified proteins in E. coli? (LB143)
Authors: Macnaughtan Megan
Published: June 2021

The overall goal of this research is to develop a robust co‐expression system to produce large quantities of O‐beta‐N‐acetylglucosamine (O‐GlcNAc)‐modified proteins using E. coli. It has been shown by several groups that O‐GlcNAc transferase (OGT) can be expressed in E. coli and can modify co‐expressed substrate proteins in vivo. The O‐GlcNAc‐modified protein yields from these co‐expressions are typically low, on the order of nanograms to micrograms, even though E. coli is routinely used to produce milligrams of unmodified protein. We have discovered that an endogenous beta‐N‐acetylglucosaminidase (NagZ) sabotages O‐GlcNAc‐modified protein yields by cleaving O‐GlcNAc from modified proteins in vivo. A nagZ knockout strain of E. coli was made, and co‐expression of human OGT with two substrate proteins, human cAMP responsive element‐binding protein (CREB1) and Abelson tyrosine‐kinase 2 (ABL2), show a temporal production of O‐GlcNAc‐modified protein. The current results do not lead to a definitive conclusion, but to more questions about E. coli and OGT.
Sialic acid‐like molecules in motility, biofilm formation, and systemic pathogenesis by the seafood‐borne pathogen Vibrio vulnificus (LB144)
Authors: Lubin Jean Bernard , Lewis Warren , Gilbert Nicole , Boyd Ethna , Lewis Amanda
Published: June 2021

Sialic acids are found on all vertebrate cell surfaces. Dozens of pathogens have evolved a variety of ways to mask themselves with sialic acids, which can promote bloodstream survival of pathogens via multiple mechanisms. Many other bacterial pathogens synthesize sialic acid‐like molecules to decorate lipopolysaccharides or flagella; however, the role(s) of sialic acid‐like molecules in the bloodstream have not been investigated. Vibrio vulnificus is a seafood‐borne pathogen and a leading cause of food‐related death in the U.S. because of its ability to quickly access the host bloodstream. However, virtually nothing is known about how this organism persists in the systemic vasculature. We previously showed that many clinical isolates of Vibrio vulnificus express high levels of sialic acid‐like molecules (di‐N‐acetylated nonulosonic acids or NulOs). Here we elucidate the genetic basis of NulO biosynthesis in V. vulnificus and, using an isogenic in‐frame deletion of the putative NulO synthase gene nab2, we demonstrate a vital role for these molecules in systemic virulence of V. vulnificus. The data show that V. vulnificus LPS are adorned with NulO residues and that expression of these molecules is required for optimal biofilm formation, motility and flagellar expression. Competition experiments in a mouse model of septecemia revealed a striking 500‐fold survival advantage of the WT versus nab2 strain. In summary, these data demonstrate several biological functions of sialic acid‐like molecules in V. vulnificus and provide the first molecular explanation for how members of this species cause life‐threatening systemic infections.
VPA inhibits myo‐inositol phosphate synthase activity by inhibition of Gsk3 (LB150)
Authors: Yu Wenxi , Greenberg Miriam
Published: June 2021

Valproic acid (VPA) has been used for decades for the treatment of bipolar disorder. Yet, the therapeutic mechanism of VPA is not understood, hindering the development of more effective drugs. VPA causes inositol depletion in the human brain cells and in yeast cells. A similar degree of inositol depletion was observed in the yeast gsk3Δ mutant, in which the four glycogen synthase kinase 3 (GSK3) homologs were deleted including MCK1, MRK1, MDS1 and YGK3. VPA was also shown to inhibit Gsk3 in mammalian cells. These findings suggest that Gsk3 may play a role in inositol metabolism, and that VPA may cause inositol depletion by inhibiting Gsk3. The rate limiting enzyme in yeast inositol de novo synthesis is myo‐inositol phosphate synthase (MIPS). VPA treatment has been shown to cause a 50% decrease in MIPS activity. However, VPA did not inhibit MIPS in gsk3Δ cells, suggesting that VPA‐induced inositol depletion occurs by inhibition of Gsk3, leading to decreased MIPS activity. Of the four GSK3 homologs, we show that MCK1 is required for optimal inositol synthesis. mck1Δ is the only GSK3 single mutant that exhibited multiple features of inositol depletion, including inositol‐dependent growth, decreased intracellular inositol and decreased MIPS activity. VPA‐induced inhibition of MIPS was not observed in mck1Δ, indicating that Mck1 is required by VPA to inhibit MIPS. To verify that MCK1 is the sole GSK3 gene involved in regulating inositol metabolism, a triple mutant (mrk1Δmds1Δygk3Δ) was constructed. The triple mutant exhibited normal growth in inositol‐free media and increased intracellular inositol compared to WT, indicating that inositol depletion observed in gsk3Δ does not result from deletion of MRK1, MDS1 or YGK3. In conclusion, MCK1 is the yeast GSK3 homolog required for optimal inositol synthesis and VPA‐induced inhibition of MIPS is Mck1 dependent.

Grant Funding Source: Supported by NIH‐DK081367 & Wayne State University Enhancement Award for Doctoral Student Travel