bluuebunny/crossref_metadata_2025_split huggingface dataset:
The genetic basis of mitochondrial DNA depletion syndrome (958.2)
Authors: Venkataraman Chantel , Field Martha , Stover Patrick 
Published: June 2021

Mutations in DGUOK, a gene encoding an enzyme in the mitochondrial nucleotide synthesis salvage pathway, and MPV17, a gene encoding an inner mitochondrial membrane protein of unknown function, have been shown to cause the hepatocerebral form of mitochondrial DNA depletion syndrome. MPV17 has been postulated to function as a nucleotide transporter. Transient HeLa cell culture models using siRNA and stable HeLa cell culture models using shRNA were created. The models facilitated studies addressing the effects of nucleotide availability on mtDNA copy number. It was observed that mtDNA copy number modestly decreased in MPV17 siRNA cells cultured in nucleotide replete medium. Supplementation with nucleotides individually indicates that pyrimidine availability increases mtDNA copy number in MPV17 siRNA‐treated cells as much as 10‐fold. Further experiments to determine the quality of the mtDNA through determination of uracil content are currently being conducted.Grant Funding Source: Supported by NIH R37DK58144
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
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
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, ultrastructureGrant Funding Source: Indian Council of Medical Research