bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of human polymerase ν in E. coli
Authors: Shissler Susannah Claire , Jiang Xiaohua
Published: June 2021

The human genome has a vast repertoire of proteins involved in replication and repair of DNA. Among those proteins, there are 15 total polymerases, and polymerase ν (pol ν) is one of the least studied. It is a member of A‐family polymerases, which are highly conserved. Studies of human polymerase ν have described a significantly higher error rate due to lack of proof‐reading capabilities. Because of this, pol ν is highly regulated within the cell to prevent extraneous mutation. Although pol ν has been shown to participate in translesion synthesis in vitro, its precise function has yet to be determined. Our Research with pol ν will include subcloning the catalytic core into the vector pBG106. The plasmid will be transformed into E. coli cells and the protein will be over‐expressed. Histidine‐tagged pol ν should simplify the separation and purification of the protein by use of a Nickel affinity column.
Replication of G‐quadruplexes by archaeal DNA polymerases
Authors: Eddy Sarah Diane , Maddukuri Leena , Eoff Robert Lawton
Published: June 2021

Evolutionarily retained G‐quadruplex DNA (QDNA) is comprised of Hoogsteen hydrogen bonded guanines that form stable stacked tetrads, which can impede DNA synthesis. QDNA replication is critical to genomic maintenance and stability because the sequences have been conserved in regulatory regions of the genome where they influence transcription and expression of important genes, including tumor suppressors and proto‐oncogenes. Y‐family polymerases (pols) may help bypass these sites. We analyzed the functional activity of the archaeal Y‐family pol Dpo4 against QDNA substrates derived from the c‐MYC promoter. Preliminary extension assays with control non‐QDNA and QDNA substrates demonstrate that Y‐family pols are better able to insert cytosines opposite quadruplex tetrad guanines than Dpo1, a model B‐family pol. Calculated changes in heat capacity (ΔCp°) associated with Y‐family pol‐QDNA complex formation indicate a favorable binding interface, a trend not observed with the model B‐family pol. These results reveal molecular interfaces and functional properties for Y‐family polymerases that are indicative of their suitability for QDNA replication. Supported in part by USPHS R00 GM084460 (R.L.E.).
Antibiotic resistance acquired through a DNA damage‐inducible response in Acinetobacter baumannii
Authors: Norton Matthew D , Godoy Veronica G
Published: June 2021

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen. It can survive desiccation and acquire resistance to multiple antibiotics. Escherichia coli can gain antibiotic resistances as a result of the expression of genes involved in a global response to DNA damage. Therefore, we asked whether A. baumannii does the same through a yet undetermined DNA damage response akin to the E. coli paradigm. Here we present evidence consistent with this hypothesis. We find that recA, which is essential to mount a response in E. coli, and multiple, error‐prone DNA Polymerase V genes are induced upon DNA damage. The regulation of these genes was found to be governed by RecA. Consequently, we find that the frequency of rifampicin resistant mutants is dramatically increased upon DNA damage and desiccation in a RecA‐dependent manner. It appears that the number of error‐prone DNA polymerase genes A. baumannii strains have acquired may directly contribute to mutagenesis. These studies provide evidence for an A. baumannii global and inducible DNA damage response that directly contributes to genomic evolution and acquisition of antibiotic resistance, especially in hospitals where A. baumannii desiccates and survives on equipment.
Extra‐transcriptional effects of RNA polymerase III transcription complex on neighboring RNA polymerase II transcribing genes in Saccharomyces cerevisiae
Authors: Korde Asawari , Rosselot Jessica M. , Donze David
Published: June 2021

RNA polymerase III (Pol III) transcribes tRNAs, 5S RNA and other non‐coding RNAs. Moreover, Pol III transcription complexes (TFIIIC, TFIIIB and Pol III) assembled on DNA can exert extra‐transcriptional effects on chromosome function. Transfer RNA genes (tDNAs) can act as chromatin boundaries in yeast. In S. cerevisiae, a tDNA tV(UAC)D (tVD) lies between divergently transcribed ATG31 and SES1 genes. Higher levels of SES1 transcription relative to ATG31 led us to speculate an insulator function of tVD, that prevents activation of ATG31 transcription by SES1 regulatory elements. RNA analysis of tVD deleted strains revealed increased ATG31 transcripts and surprisingly a longerATG31 transcript. This extended ATG31 mRNA is due to readthrough of the untranslated transcript SUT467 originating near the SES1 promoter. We show that tVD blocks progression of this intergenic transcript, suggesting a unique barrier function. Western blots show reduced translation of ATG31 in tVD deleted strains. The readthrough transcript is also observed in yeast containing mutations in the Pol III machinery and other chromatin proteins associated with tDNAs. Our results indicate that in addition to the boundary activity, Pol III complexes, with other chromatin proteins at tDNAs, can function as barriers to Pol II progression, which may be important in protecting promoters from deleterious effects of cryptic intergenic transcription.
The Ure2 αCap Is Required for GATA Factor Responses to Rapamycin, But Not Nitrogen Limitation
Authors: Tate Jennifer J. , Feller Andre , Georis Isabelle , Dubois Evelyne , Cooper Terrance G.
Published: June 2021

Ure2 is a phosphoprotein and central negative regulator of nitrogen‐responsive Gln3 and Gat1 localization and their abilities to activate transcription. This negative regulation is achieved by forming Ure2‐Gln3 and Ure2‐Gat1 complexes which sequester these GATA factors in the cytoplasm of cells cultured in excess nitrogen. Ure2 itself is a dimer, the monomer of which consists of two core domains and a flexible protruding αcap. Here, we show that alterations in this αcap abolish rapamycin‐ (Rap‐) elicited nuclear Gln3 and, to a more limited extent, Gat1 localization but have little effect on the Gln3 and Gat1 responses to nitrogen limitation. Rap is a specific inhibitor of the global regulator, TorC1. Ure2 dephosphorylation is also stimulus‐specific, occurring after Rap treatment, but only minimally if at all in nitrogen‐limited cells. In contrast to Gln3, Rap‐elicited Ure2 dephosphorylation is independent of Sit4 and PP2A. Our data suggest that distinct regions of Ure2 are associated with the receipt and/or implementation of signals calling for cessation of GATA factor sequestration in the cytoplasm. This in turn is more consistent with the existence of distinct pathways for TorC1‐ and nitrogen limitation‐dependent control of GATA factor localization and function than it is with these stimuli representing sequential steps in a single regulatory pathway. Supported by: NIH GM‐35642–23, COCOF, FRFC 2.4547.11.
Insights into splicing: structure of the yeast U1 snRNP
Authors: van der Feltz Clarisse , Grigorieff Nikolaus , Krummel Daniel Pomeranz
Published: June 2021

Precursor‐mRNA splicing is catalyzed by a large and dynamic macromolecular assemblage termed the spliceosome. Integral to spliceosome function are five RNA‐protein complexes termed uridine‐rich small nuclear ribonucleoprotein particles, the U1, U2, U4, U5 and U6 snRNPs. An early and significant regulatory step in assembly of the spliceosome and splice‐site selection is U1 snRNP recognition of the junction between the 5′ exon and intron, a 5′ splice‐site. This pivotal event will act to: (1) trigger spliceosome assembly; (2) demarcate a 5′ splice‐site; and (3) contribute to spliceosome fidelity. In metazoans, U1 snRNP 5′ splice‐site selection is aided by transiently associated proteins known as alternative splicing factors that function to guide U1 snRNP to specific 5′ splice‐site(s). In contrast, Saccharomyces cerevisae has very few protein coding genes with more than a single intron. S. cerevisae U1 snRNP is an 18‐subunit, ~800 kDa assembly that has as integral subunits proteins that are alternative splicing factors in higher eukaryotes. We report the structure of a native S. cerevisae U1 snRNP determined by electron microscopy and present a working model that provides insight into the function of its protein subunits.

Funding provided by the National Science Foundation; Howard Hughes Medical Institute
Molecular dynamics study of the stability of the sarcin/ricin domain of RNA
Authors: Bruist Michael Frederik , Cavanaugh Cassandre
Published: June 2021

The importance of noncodoing RNAs is being appreciated more. These participate in regulation of all aspects of nucleic acids biology and also have structural and catalytic roles. Noncoding RNAs are assembled from motifs, A‐form helices and unstructured single strands. We are using molecular dynamics (MD) to understand the basis of stability of RNA motifs and their junctions to other motifs and helices.

The stability of the sarcin/ricin domain (SRD) RNA motif is being studied in a dumbbell RNA in which a G/C‐rich and a A/U‐rich A‐form helix capped with a tetraloop flank the SRD domain. RNA melting is being observed using adaptively biased and replica exchange methods to explore the free energy profile as the dumbbell melts. We have found that more conformations can be explored more rapidly when the negative charges of the RNA are not completely neutralized by the cations. Artificial cations with less than unit charge allow multiple runs with different degree of neutralization. We are comparing the conformations explored under these conditions with those seen in a standard net neutralized system.
Conformational stability of loop E in the Potato Spindle Tuber Viroid (PSTVd)
Authors: Salvo Renee M , Bruist Michael F , Baumstark Tilman
Published: June 2021

Viroids are small autonomously replicating plant pathogens consisting of non‐encapsidated, circular, single‐stranded RNA. Viroid RNA does not code for proteins; therefore their primary function is to interact with host proteins or other RNAs to carry out replication and pathogenesis. The potato spindle tuber viroid (PSTVd) is composed of 359 nucleotides and adopts a rod‐like secondary structure. The viroid has a central conserved region that contains a loop E motif. During various stages of viroid replication, the loop E motif takes on different folds, even though the sequence of the monomer remains unchanged. The fold of the loop E sequence dictates the cleavage and ligation that create the final viroid circle. Little direct evidence of what stabilizes these folds has been found. We show the contribution of component base pairs to stability using susceptibility to chemical probes of wild‐type and mutated loop E sequences.
Expression of recombinant human neutrophil Cathepsin G in Pichia pastoris
Authors: Perry Evan Thomas , Smith Eliot , Johnson David
Published: June 2021

Cathespin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG has dual specificity for chymotrypsin‐like and tryspin‐like substrates. CatG is a poorly understood enzyme and is currently only commercially available as mature enzyme purified from human sources. The yeast Pichia pastoris is being used to express CatG to study its dual specificity and its C‐terminal processing. The full length (C‐terminus present) human CatG amino acid sequence was modified to remove one glycosylation site and eight dibasic sites to avoid potential cleavage by yeast kexin protease. The construct was engineered to have an N‐terminal 6‐His‐cytochrome B5 (CytB5) heme binding fusion domain linked to the modified human CatG by an enterokinase cleavage site for activation. The amino acid sequence was used to generate a codon‐optimized gene that was placed in the pPICzα secretion vector. After transforming Pichia pastoris strain X‐33, 48 Zeocin‐resistant clones were screened for relative levels of CatG activity. Recombinant CatG has been partially purified from fermentation media by nickel affinity chromatography and its activity has been confirmed by assays using synthetic substrates. Supported by a Student Faculty Collaborative Grant from the ETSU Honors College and ETSU Office of Research and Sponsored Programs and by NHLB grant R15HL091770.
Extracellular Thimet Oligopeptidase is carried by cell membrane microvesicles of prostate cancer cells
Authors: Liu Yu , Bruce Lisa A. , Wolfson Adele J.
Published: June 2021

Peptidases often generate bioactive peptides that regulate and mediate intercellular communication. Although processing of peptide precursors is initiated intracellularly, additional modifications by peptidases are often completed extracellularly. Plasma membrane microvesicles (MVs or microparticles) can carry membrane‐associated molecules, including proteins, when released from their parental cells. Thimet oligopeptidase (TOP) is a biologically significant peptidase that is known to be secreted to the extracellular space. Since TOP has been localized on the external face of the cell plasma membrane, we asked whether TOP might be carried by cellular MVs into the extracellular space, and whether this process is influenced by steroid hormones. DU145 androgen‐sensitive prostate cancer cells were treated with dihydrotestosterone (DHT). The cell culture supernatant was subjected to ultracentrifugation to pellet MVs. Western blot analysis of the homogenized MV sample revealed that TOP protein is, indeed, carried by the MVs of both DHT‐treated and control cells. MVs isolated from PC3 androgen‐sensitive prostate cancer cells treated with a calcium ionophore also carry TOP. Thus, our studies strongly suggest that, in addition to the soluble form, extracellular TOP also exists in MVs. This is a novel form of the extracellular peptidase that may play important pathophysiological roles.
Isolation and Characterization of Novel Folic Acid Degrading Enzymes
Authors: Vekasy Shelley , Watkins Linette , Cherry Elyssa
Published: June 2021

Folic acid functions in multiple life critical metabolic pathways, and is an important coenzyme that aids in redox processing and transfer of one‐carbon units, which involves molecules including nucleotides as well as amino acids. This allows organisms to properly metabolize food sources. Fifteen soil samples gathered from the central Texas area were collected and screened against various antibiotics and vitamins for growth in sterile single carbon source media. Two bacterial strains of organisms were able to grow in media with only folic acid as their only carbon source. These cells were grown, lysed, and the folic acid degrading enzyme was purified from the cells using affinity chromatography on a folate‐sepharose column. A pathway of folic acid degradation is studied, and the folic acid degrading enzyme is characterized.
Deciphering the Role of Sucrose Phosphate Synthase in Root Nodules of Medicago sativa (alfalfa)
Authors: Grimes Martha Martinez , Ortega Jose Luis , Sengupta‐Gopalan Champa
Published: June 2021

Leguminous plants, like alfalfa, can form a symbiotic relationship with a N fixing bacteria that can result in the formation root nodules. They have the ability to sustain the N fixing bacteria by providing optimal conditions for the bacterially encoded enzyme, nitrogenase, to reduce N to ammonia. The ammonia is then assimilated and exported to the rest of the plant. The plant provides the bacteria in the nodules with a source of C and energy in the form of sucrose in exchange for the fixed N. Sucrose Phosphate Synthase (SPS) catalyzes the penultimate step in sucrose biosynthesis and plays a key regulatory role in C partitioning in the pathway. Preliminary work form our lab has shown one isoform to have high SPS activity in the nodules, SPSA. To address the issue of what might be role of SPSA gene product in the nodules, we are using both immunolocalization and site of promoter function to localize the site of expression of SPSA in the nodules in context of N fixation, ammonia assimilation, and C metabolism. We are also using a genetic engineering approach to modulate the expression of SPS genes in a cell specific manner to monitor any changes in the function of the nodules.
Identification and biochemical characterization of the PP1‐binding protein: Consortin
Authors: Lin Yu Shan , Chen Chun yu , Wang Tzu Fan , Lai Ning Sheng , Huang Hsien bin
Published: June 2021

Protein phosphatase 1 (PP1), one of the major serine/threonine protein phosphatases in eukaryotic cells, controls various key biological pathways, including protein synthesis, carbohydrate metabolism, muscle contraction, cell cycle and neuron transmission, et al. The catalytic subunit of PP1 is in vivo associated with different regulatory subunits to form a variety of holoenzymes. These regulatory subunits target the enzyme to specific subcellular compartments and regulate the functions of its substrates. We have conducted the search for the new PP1‐binding proteins of heart cells by yeast two‐hybrid screen, and identified several candidate proteins. One of them is consortin, a connexin trafficking protein. Consortin was found to associate with PP1α in the co‐immunoprecipitation, and GST pull‐down assay. Mutation at the PP1‐binding motif of consortin disrupted binding with PP1. Immunofluorescence microscopic analysis revealed that the PP1/consortin complex was conspicuously localized at ER and Golgi. The recombinant consortin protein can inhibit PP1 with the IC50 of 3±0.2 nM, but fail to inhibit PP2A. Taken together, our results suggested that consortin is a PP1 binding protein and is capable of modulating PP1 activity.
Peptide‐Based Probes to Monitor Disparte Cysteine‐Mediated Protein Activities
Authors: Pace Nicholas , Weerapana Eranthie
Published: June 2021

Cysteine residues facilitate many important protein functions including metal binding, structural stability, redox‐activity, metal‐binding, and catalysis. Small‐molecule cysteine‐reactive chemical probes have been utilized as a means to modulate cysteine‐mediated protein activity. Specifically, we synthesized a small‐molecule library consisting of variable peptide sequences coupled to moderate cysteine‐reactive electrophiles: sulfonate esters, Michael acceptors, and haloacetamides. The peptide basis of these probes was intended to exploit the inherent structural diversity found within amino acids. A number of library members were identified that selectively target a single enzyme class. These peptides show potential in elucidating enzymatic mechanisms to potentially aid in future inhibitor design. Other library members moderately labeled multiple enzyme classes, and thus appeared suitable a tool for the design of a competitive activity‐based proteomic approach for identifying metal‐binding cysteines. Lastly, library members were distinguished by an ability to inhibit a single enzyme class within select populations of cells, specifically healthy vs apoptotic cells. Together, these studies illustrate the utility of peptides to serve as a valuable scaffold in the expansion of activity‐based probes for cysteines participating in disparate functions across diverse enzyme classes.
Preparation and high resolution analysis of a Fab fragment
Authors: Danielsson Ake , Widehammar Jens , Rodrigo Gustav
Published: June 2021

In this work a Fab fragment was produced by papain cleavage of a purified monoclonal antibody (Mab) followed by purification on MabSelect™ Sure™ and Capto™ L media (protein A and protein L, respectively, immobilized onto high flow agarose). Fractions from the different steps were analyzed by gel filtration (GF) to follow purification progress. The process started with Mab being purified from feed on MabSelect Sure and thereafter the Mab was digested by papain overnight at 37 degrees into Fab‐ and Fcfragments. Cleavage was interrupted with the inhibitor antipain and the digest was applied to a column containing MabSelect Sure. Fc and partially digested Mab bind to the chromatography medium whereas the Fab fragment elutes in the flow through together with other contaminants such as papain. The flow through fraction was applied to Capto L medium and the Fab fragment was thus further purified. The purity of the Fab fragment was confirmed by analysis on new high resolution GF media. This new GF media has the resolution power to separate both Mab from aggregates and Fab fragments from Fc fragments.
Building a new anatomy program in Israel: Second year update
Authors: Karasik David , Weingarten Michael , Luder Anthony , Barash Alon
Published: June 2021

There is a growing shortage of doctors in Israel. This led to two decisions: first to open a new medical school and second to initiate studies with a 4‐year graduate‐entry curriculum. The Bar Ilan University (BIU) Faculty of Medicine opened in October 2011 in Northern Israel. This new school enabled us to implement a new anatomy curriculum, using novel but proven teaching methods. In the Functional Anatomy course we integrated the instruction of anatomical knowledge with clinical exposure. The 1st‐year block included the thorax, abdomen, pelvis, head and neck. Each topic had the same structure: we began with basic macro‐ and micro‐anatomy lectures, followed by radiology lectures and dissection laboratories. A special focus was on ultrasound and computer‐based imaging laboratories in which the students practiced structure identification guided by a radiologist. To integrate all learned material and develop clinical thinking, the next phase comprised clinical lectures, followed by case‐based learning. Surgical residents performed basic medical procedures in the dissection room, while ENT doctors applied real‐time laryngoscopy on student volunteers. Among the unique challenges we faced (a) a heterogeneous student body, which required some extra time to synchronize the basics of embryology and physiology, and (b) the necessity to emphasize Jewish ethical values as a fundamental part of BIU's philosophy.
High Throughput In Situ Transfection of Primary and iPSC‐derived Cells
Authors: Pihl Johan , Dalen Jennie Svensson , Aspengren Sara , Karlsson Mattias
Published: June 2021

Primary cells in culture are typically considered to be a highly relevant in vitro models. They can be cultivated and differentiated in vitro, but are typically difficult to transfect using conventional methods. In this work, we've developed a system, Cellaxess Elektra, and methods for high‐throughput in‐vitro transfections of primary and iPSC‐derived cells at any developmental stage with virtually no impact on cell viability or morphology. The platform utilizes a capillary electroporation concept that can be used for introduction of different types of molecules, ranging from small molecules, peptides, siRNAs, and plasmids, to adherent primary and stem cell derived cultures in 384‐well plates. The focused electrical field delivered by the platform minimizes toxicity and joule heating, The platform is truly enabling, and has the capability to transfect primary cells from a variety of sources, e.g. rat mouse & iPSC‐derived human neurons, primary human immune cells and many more cell types with excellent viability and a completely retained morphology. Data from cDNA and RNAi experiments are presented for different types of cultures and applications.
Modeling the Regulation of the Kinase Domain of EGFR
Authors: Aguero‐Sinclair Michael , Barasch Julia , Borenstein Zoe , Cohen Samantha , Foltz‐Navab Shazy , Katz Olivia , Polycarpe Francesca , Vallimarescu Ben , Hackett Jennifer , Shan Yibing
Published: June 2021

Cell division is a tightly regulated process, and if not controlled, may lead to the formation of tumors. Epidermal growth factor receptor (EGFR) is a transmembrane receptor that causes cell division in a wide range of cell types in response to several different growth factors. Mutations that activate EGFR in the absence of growth factors can contribute to cancer. The Dalton School SMART Team (Students Modeling A Research Topic) modeled EGFR using 3D printing technology. EGFR is a kinase that phosphorylates other proteins to turn them on or off. EGFR consists of an extracellular ligand‐binding domain, an anchoring intramembrane segment, a juxtamembrane connector segment, and a phosphorylating intracellular kinase domain. Generally, EGFR remains in an inactive form and becomes active when a ligand growth factor binds to the extracellular domain. Ligand binding changes the shape of the intracellular kinase domain, including the active site, turning it on. Active EGFR initiates a signaling pathway that causes cell growth and division. However, mutations in EGFR, including deletion of the extracellular domain or kinase domain point mutations, can activate EGFR in the absence of a growth factor, which may cause the formation of tumors because cells grow and divide uncontrollably. Therefore, EGFR is a potential target for cancer therapy. Supported by a grant from the Camille and Henry Dreyfus Foundation.
Salivary antigen‐5/CAP family members are Cu2+‐dependent antioxidant enzymes which negatively modulate platelet function
Authors: Assumpcao Teresa , Ma Dongying , Schwarz Alexandra , Reiter Karine , Santana Jaime M. , Andersen John F. , Ribeiro Jose M. C. , Nardone Glenn , Yu Lee L. , Francischetti Ivo M. B.
Published: June 2021

The function of the antigen‐5/CAP family of salivary gland (SG) proteins ubiquitously found in bloodsucking animals has remained elusive for decades. Antigen 5 members from the hematophagous bugs Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation by low doses of collagen (<1 μg/mL) but no other agonists. This inhibitory profile resembles the effects of antioxidants (e.g., superoxide dismutase [SOD]) in platelet function. Accordingly, our results demonstrate that DMAV blunts the luminescence signal of O2− generated by phorbol myristate acetate (PMA)‐stimulated neutrophils. Moreover, Cu2+‐DMAV was found to inhibit cytochrome c reduction by O2− generated by the xanthine/xanthine oxidase implying that it exhibits antioxidant activity. Mechanistically, ICP‐MS and fluorescence spectroscopy revealed that DMAV, like SOD, interacts with Cu2+ which provides redox potential for catalytic removal of O2−. Notably, SG of D. maxima with native DMAV contain Cu2+ and display metal‐dependent antioxidant properties. Antigen‐5/CAP emerges as a novel family of Cu2+‐dependent antioxidant enzymes that negatively modulate platelet aggregation by a unique salivary mechanism.
Receptor Mimicry by Human Antibody F045–092 Inhibits Influenza H3N2
Authors: Considine Camille , Palakodeti Samhita , Handmacher Matt , Sagart Arman , Eherenfeldt Hannah , Moerder Susan , Lee Peter
Published: June 2021

According to the CDC, influenza is the 9th leading cause of death in the US, killing over 50,000 people in 2011. H3N2, an influenza A serotype, is important for current research due to its increasing circulation in humans this season. To combat infections, flu vaccines are administered each year; however, due to continual mutations of the viral proteins, a vaccine offers little protection if the match between it and the dominant circulating strain is poor. Hemagglutinin (HA), the major glycoprotein on the viral surface, binds to host cell sialic acid receptors and initiates viral infection; thus blocking this viral‐host interaction prevents infection. F045–092, a broadly‐neutralizing antibody, targets the receptor binding site of HA and uses remarkable receptor mimicry to recognize all H3 strains from 1968 to 2011. The antibody binds primarily through a long HCDR3 loop that inserts into the HA receptor binding pocket. In addition, the antibody extends its breadth of neutralization using avidity, as the bivalent IgG has higher apparent affinity than the monovalent Fab. This broadly‐neutralizing antibody will potentially enable and inform the development of a pan‐H3 vaccine and small molecule therapeutics. Using 3D printing technology, the Francis Parker SMART (Students Modeling A Research Topic) Team modeled the antibody F045–092 in complex with HA. Supported by a grant from TSRI and UCSD.
From the origin of Life to the present: A novel method for teaching introductory biology
Authors: Gaiser J. Christopher , Roberts Michael Foster
Published: June 2021

Introductory biology courses usually consist of a survey of biological concepts presented in a compartmentalized fashion. Very often this approach focuses on levels of organization: molecules, cells, organisms, etc. We find this approach limited in at least two significant ways: 1) Often evolution and ecology are taught as distinct sections near the end of textbooks, which implies that they are only significant for the multicellular organisms covered near the end of the course. 2) This compartmentalized approach makes it difficult for students to develop an integrated understanding of biology. Here we describe a novel method of delivering a year‐long introductory biology sequence. Our approach surveys the origin of biological processes, cells, and multicellular organisms in the historical sequence in which they actually occurred. Thus, the course begins with the chemistry of the early earth and moves to the origins of metabolism and the appearance of RNA as a catalyst and a hereditary molecule. This leads naturally into the first cells and the structural and metabolic characteristics of prokaryotes. The next step in the course is the endosymbiotic events that led to the eukaryotes, allowing us to explore for a second time cellular mechanisms such as replication, transcription, translation, and metabolism, now in eukaryotic cells. After discussion of sexual reproduction and phylogenetic analysis, we explore the diversity of single‐celled and then multicellular eukaryotes, their physiological processes, and their interactions with the environment. Throughout each of these stages we emphasize how ecological niche and natural selection are the twin drivers of evolution on earth. We find that this method allows students to more effectively connect the vast amount of information presented in an introductory survey course. We present here our initial successes and challenges with this approach.
The interplay between substrate and allosteric effectors of the photosynthetic C4 isoenzyme of phosphoenolpyruvate carboxylase from monocot and dicot plants
Authors: Guemez‐Toro Rodrigo , Muñoz‐Clares Rosario A.
Published: June 2021

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible formation of oxaloacetate and Pi from phosphoenolpyruvate (PEP) and bicarbonate. This reaction constitutes the first step in the assimilation pathway of atmospheric CO2 in C4 plants, such as Zea mays, monocot, and Amaranthus hypochondriacus, dicot. The maize and amaranth photosynthetic PEPC isoenzymes (ZmPEPC‐C4 and AhPEPC‐C4 respectively) are activated by glucose‐6‐phosphate (Glc6P) and inhibited by malate and aspartate, but only ZmPEPC‐C4 is regulated by glycine (Gly), which is its main activator under physiological conditions. In this work we have explored the interplay between substrate and allosteric effectors in the two enzymes by initial velocity studies. At pH 7.3, 0.4 mM free Mg2+, and 0.1 mM bicarbonate, we found that: 1) The affinities for PEP and malate of AhPEPC‐C4, measured as Ks and Ki respectively, are higher than those of ZmPEPC‐C4, which result in similar I50 values for malate in both enzymes at physiological PEP concentrations. 2) Glc6P is an atypical activator because, unexpectedly, increases the inhibition by malate in both enzymes. 3) As expected, Gly counteracts the inhibition of ZmPEPC‐C4 by malate, which indicates the physiological relevance of the regulation by Gly of this enzyme. Why the activation by Gly of AhPEPC‐C4 is physiologically unnecessary remains to be explained.

Supported by PAPIIT‐UNAM IN216911 grant.
Detection and Characterization of R‐loops at the Murine Immunoglobulin S α Region
Authors: Huang Feng‐Ting , Kao Yu‐Pu , Hsieh Wen‐Chuan , Hung Shu‐Ting , Huang Chiun‐Wei , Lieber Michael
Published: June 2021

IgA is the most abundant antibody in mammals. However, the mechanism of its class switching is still not clear. The formation of the R‐loops has been proposed to play a crucial role during mammalian class switch recombination. Hence, we investigate if R‐loops exist at Sα. In addition, the correlation between R‐loops and IgA class switching is also been investigated. Here, we are the first to provide a systematic evaluation of R‐loops at Sα (IgA) in vitro and in situ in CH12F3–2A cells, which is a unique cell model system for class switch recombination for its consistent switching to IgA upon stimulation. R‐loops form at Sα, despite variable G density and relatively few GGGG clusters in Sα. The short distance from the promoter to Sα may compensate for the less robust R‐loop‐forming factors at Sα relative to other switch regions. R‐loops have distinct features specific to Sα. Therefore, R‐loops at the Sα region further support R‐loop formation as a general feature of all stimulated switch regions. Furthermore, the IgA class switching is altered in the deficiency of R‐loops‐related enzymes in CH12F3–2A cells.
Structural and functional basis for substrate specificity and catalysis of levan fructotransferase
Authors: Park Jinseo , Kim Myung‐Il , Park Young‐Don , Shin Inchul , Cha Jaeho , Kim Chul Ho , Rhee Sangkee
Published: June 2021

Levan is β‐2,6‐linked fructan and serves as reserve carbohydrate in some plants and microorganisms. This fructan is usually utilized by enzymes such as glycoside hydrolase (GH) for producing a fructose. Levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate‐specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form and complex with sucrose and levanbiose. Despite the structural similarity with members of the GH32 family, LFTase contains the‐1 and ‐2 subsites for a difructosaccharide in an active site. This feature is unique among GH32 proteins and is facilitated by small side‐chain residues in the loop region of a catalytic domain, which is conserved in the LFTase family. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements. This work was supported by a grant from Next‐Generation BioGreen21 Program Plant Molecular Breeding Center, the Rural Development Administration, Republic of Korea.
Specificity of Alcohol Dehydrogenases Among Various Substrates
Authors: Puente Roberto Airan , Watkins Linette , Correa Jaime
Published: June 2021

The use of enzymes in organic transformations has increased dramatically over the last 20 years. A common industrial reaction that could benefit from an enzyme is the oxidation of alcohols to aldehydes. Alcohol dehydrogenase is one enzyme that oxidizes alcohols to form aldehydes. The specificity of alcohol dehydrogenase enzymes varies greatly. The kinetic activity of alcohol dehydrogenase enzymes from different sources was measured with a range of model substrates of increasing chain length and branching. The rate of the reaction was measured by monitoring the rate of reduction of NAD+ to NADH that occurs during the formation of aldehyde from the alcohol, using absorbance at 340nm. The results revealed specificity patterns dependent on length of carbon group chain length, presence of other functional groups and solvent composition. The specificity varied dramatically depending on the source of the alcohol dehydrogenase.
Basis of Prokaryotic Selectivity in the Antibiotic Paromomycin
Authors: Puri Simar , Kalsi Satvir , Singh Ricky , Chand Neil , Singh Vipul , Park Sangwoo , Aguwa Chi , Ali Moid , Dhingra Anshul , Zhu Justin , Gill Griffin , Sportiello Mike , Tuinstra Robbyn , Dey Madhusudan
Published: June 2021

Ribosomes are responsible for protein synthesis and are a major target of antibiotics. While translation is a universally conserved cellular process, the ability of drugs to target prokaryotic ribosomes depends on subtle variations from eukaryotic ribosomes. The ribosome is composed of ribosomal RNA (rRNA) and protein. The small ribosomal subunit, called 30s in prokaryotes, contains 21 proteins and one rRNA (16S) and the large subunit, called 50S, contains 31 proteins and two rRNAs (23S and 5S). Recent crystal structures reveal that the rRNAs adopt a 3D fold generating (I) decoding center for codon‐anticodon recognition, (II) a peptidyl‐transfer center (PTC) for a peptide bond formation and (III) an exit tunnel through which the nascent protein emerges. Paromomycin, used in the treatment of intestinal infections, inhibits prokaryotic ribosomes at the decoding site. Paromomycin physically restructures helix H44 of the 16S rRNA, preventing proper rotation of A1492 and A1493 during anticodon:codon recognition, decreasing tRNA selection accuracy in prokaryotic ribosomes. However, paromomycin fails to affect eukaryotes due to an A to G transition at position 1408. The Brookfield Academy SMART Team (Students Modeling A Research Topic) modeled a prokaryotic ribosome, highlighting nucleotides responsible for the prokaryotic specificity of paromomycin. (Supported by a grant from the NIH‐CTSA UL1RR031973)
Devastating Degradations: Hya and PLA's “Inflammous Plot”
Authors: Ross Khadiyah , Lam Emily , Graham Eric , Moore Melshae , Wofford Ayanna , Pharr Carmen , Johnson‐Anthony Chasity , Stubbs Tamica , Colton Shannon
Published: June 2021

According to the CDC, 90–100 Americans stung by bees annually die due to anaphylactic shock. Using 3D printing technology, the Berry Academy SMART Team (Students Modeling a Research Topic) modeled two proteins in bee venom: hyaluronidase (Hya), a TIM barrel structured enzyme, and bee venom phospholipase (PLA2). Hya and PLA2 work in tandem to spread bee venom by degrading the extracellular matrix (ECM) and disrupting cellular membranes, respectively. Hya begins degradation by binding to and cleaving hyaluronic acid (HA), a glycosaminoglycan made of repeating disaccharide (GlcNac and GlcA) units in the ECM that facilitate tissue stabilization. Arg116 and Arg224 guide HA to the active site, where HA binds to Ser304, Ser303, Tyr227, and Asp111. Hya cleaves glycosidic bonds within HA, mediated by Glu113, thus degrading the ECM and allowing PLA2 to access to membrane's lipids. Once exposed, PLA2 mediates the hydrolysis of phospholipids by attacking the sn‐2 acyl‐bond of phospholipids through polarization of sn‐2 to sequestrate catalytic H2O (W5) and abridgment of W5 to W6 by His34, which then is neutralized by Asp35 and Asp64. This cleavage reaction releases archindonate products, which incite inflammatory responses, leading to the pain and swelling associated with bee stings. Understanding how these proteins function may help to improve anti‐venom medicines. Supported by a grant from the NIH‐CTSA UL1RR031973.
Active learning in the medical histology laboratory engages students and faculty, improving performance
Authors: Childs Gwen V. , Stanley Laura C. , Burns E Robert , Drew Paul D , Kane Cynthia J , Syed Mohsin M , Reeves Melanie M , Davies David Lewis
Published: June 2021

Students at UAMS have traditionally studied histology with microscopes and the computer program, Histology Time, that encouraged independent study, but limited faculty‐student interactions. A major curriculum reconstruction offered the faculty an opportunity to convert most lecture and laboratory periods to interactive learning laboratories. Students were given homework assignments and mini‐lectures (25–35 min), which prepared them to apply the material. During class, the students worked in teams, reviewing clinical cases and applying histological knowledge to answer questions. Motivated by the clinical relevance, students came to class better prepared and eager to complete point‐getting exercises. The quality of the faculty interactions also improved learning, as faculty helped students with problem‐solving exercises, rather than spoon‐feed answers. Moreover, class attendance approached an unprecedented 100%. Using the same question bank, each unit exam showed improved class performance, especially in units teaching musculoskeletal, cardiovascular and respiratory systems, (increased by 4, 5.5, and 9.8 percentage points, respectively). No students were in academic jeopardy, compared with 5–8% of previous classes, which also shows that this approach facilitates learning for those having difficulties with the material. Supported by the Dept. of Neurobiology and Developmental Sciences.
Physicochemical and release properties of retinol‐loaded zein chitosan nanoparticles
Authors: Kim Bum‐Keun , Kim Jong‐Chan , Kim Myung‐Ho , Park Chan‐Eun , Lee Eun‐Ji
Published: June 2021

In this study, one of the hydrophobic vitamins, retinol was successfully encapsulated into zein chitosan nanoparticles. EF‐TEM study revealed the spherical shape with smooth surface of nanoparticles. The particle size of retinol‐loaded zein nanoparticles without chitosan coating was around 6 nm; however, particle size increased to around 400 nm after coating with chitosan. The encapsulation efficiencies slightly increased from 66.2% to 72.8% as zein concentration increased. The kinetic release profile of retinol from nanoparticles can be described as a two‐step biphasic process, i.e., an initial burst effect followed by subsequent slower release. These results show that processing parameters, such as concentration of zein and chitosan, molecular weight of chitosan, and zein/chitosan weight ratios, and so on, could be important factors to affect the properties of zein‐chitosan nanoparticles.
Increasing dietary protein selectively upregulates the DMT1 isoform that contains an iron responsive element
Authors: Thomas Carrie , Yao Gang‐Qing , O'Brien Kimberly , Kerstetter Jane , Insogna Karl
Published: June 2021

Intestinal iron absorption is a critical regulator of whole body iron homeostasis. The Divalent Metal Transporter 1 (DMT1) is a nonheme iron importer found on the brush border membrane of enterocytes. Its genetic absence in experimental animals leads to markedly impaired intestinal iron absorption. Recent data from our laboratory demonstrate that increasing dietary protein in rats enhances intestinal iron absorption by 50%, and that this is associated with a 3.8‐fold induction in DMT1 mRNA expression. To determine whether dietary protein affects the expression of the isoform of DMT1 that includes an iron responsive element (IRE), isoform‐specific qPCR was performed using RNA isolated from the duodenum of rats consuming either a 20% (control) or 40% (high) protein diet for 1 wk. Rats on the 40% protein diet exhibited significantly greater DMT1A‐IRE mRNA expression compared to animals consuming the 20% protein diet (fold change: 2.8; p<0.01), while no significant change was observed in the level of expression of the DMT1A isoform that does not include an IRE. These findings indicate that duodenal DMT1A‐IRE is selectively and significantly upregulated in response to increasing dietary protein.
Exclusive breastfeeding to six months: Results from a randomised controlled trial including lactation consultant support
Authors: Cameron Sonya Lynne , Taylor Rachael W , Gray Andrew R , Taylor Barry J , Heath Anne‐Louise M
Published: June 2021

To investigate the impact of lactation support on exclusive breastfeeding (EBF) to 6 months, 804 mother‐infant pairs were randomised to 4 equal‐sized groups: 1) breastfeeding education and support, 2) infant sleep education, 3) both interventions, or 4) usual care. Certified lactation consultants (LC) delivered intervention sessions (ante‐natal, 1 week, 4 months postpartum) and were available on request to 6 months postpartum. Data on breastfeeding status and age at introduction of formula, other liquids, or complementary foods were collected at monthly interviews from 3 to 27 weeks postpartum. Exclusive breastfeeding was: “since birth, the infant had no food or drink, not even water, except breast milk or prescribed medicines”.

At 6 months, 764 (93%) participants had known EBF status with 7.2% exclusively breastfeeding. No group differences in EBF rates were observed at 6 weeks, 4 months or 5.5 months (New Zealand recommendation is “around” 6 months) but marginal evidence of differences was found at 6 months of age (Wald test p=0.038). After adjusting for multiple comparisons, these differences were no longer statistically significant. Providing an LC at this intensity did not consistently improve EBF rates to 6 months of age in this highly educated sample. Future analysis will determine the effect of an LC on duration of any breastfeeding and timing of introduction of complementary foods.

Funding source:

Health Research Council of New Zealand.
Relationship between household environmental exposures and child stunting in India
Authors: Bondre Ameya P , Rogers Beatrice Lorge
Published: June 2021

CARE phased over its 10 yr nutrition program to Gov't of India in 2009; a survey of mothers of children 6–23 mo (n‐3455) was conducted in 4 CARE program states 2 yrs later. This study investigates the relationship between environmental exposures and child stunting in 2011. Relationships between binary predictors (having a pucca [cement, no dirt floor] house, latrine or improved water source in the house) and stunting (HAZ LE ‐2 SD) were assessed through logistic and linear regression adjusted for clustering and for caste, tribal status, supplementary food receipt, feeding practices, distance from Anganwadi (child care) center, child age, state. For 4 states combined, not having a pucca house, latrine or improved water was associated with stunting. The association was significant for pucca house (OR: .81, CI: 0.67 – 0.98). Among states, most associations were negative but inconsistently significant. By linear regression, pucca house, improved water, and latrine significantly increased HAZ (by .22, .18 and .24 SD respectively), but R2 were low (.06). Latrine and improved water increased from 2009 and 2011 but is low (24%; 35%); stunting is high and unchanged (39.9% in 2009; 40% in 2011).

Further longitudinal studies could establish these as causal associations, to better guide programs to reduce stunting, so that they also focus on improved water, sanitation and hygiene.

Support: USAID FFP with FANTAIII Project, FHI360
Anti‐Diabetic Potential of Flaxseed Lignan Secoisolariciresinol Diglucoside in Diet‐Induced Obese Mice
Authors: Wang Yanwen , Fofana Bourlaye , Roy Moumita
Published: June 2021

The current study was to determine the anti‐diabetic potential of semi‐purified (~ 60% pure) flaxseed SDG lignan in obese mice. Sixty male C57BL/6J mice were fed a high‐fat diet to induce insulin resistance and 11 male C57BL/6J mice fed a low‐fat diet were used as the normal control. After 12 weeks, mice on the high‐fat diet were treated by oral gavage with 0, 10, 100, and 1,000 mg/kg/d of flaxseed SDG lignan, respectively, for 6 weeks. The two control groups were gavaged with the vehicle. The results showed that oral administration of SDG lignan lowered body weight and improved blood glucose levels. SDG lignan treatment significantly lowered insulin levels and improved insulin tolerance. The treatment of SDG lignan also improved oral glucose tolerance. Moreover, the serum total cholesterol and free fatty acids were lowered due to SDG lignan treatment. The results suggest that flaxseed SDG lignan is a promising natural product for improving or preventing obesity‐related insulin resistance. Further experiments are underway to determine the mechanisms of action. (Supported by Genome Canada/TUFGEN grant 6003321).