bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic properties and protein conformational dynamics of the signal transducer HemAT as revealed by time‐resolved stepscan FTIR spectroscopy
Authors: Pinakoulaki Eftychia , Pavlou Andrea , Yoshimura Hideaki , Aono Shigetoshi
Published: June 2021

HemAT is a heme‐based O2 sensor protein that acts as a signal transducer for aerotaxis in Bacillus subtilis. In our work, we have employed time‐resolved step‐scan Fourier transform infrared (TRS2‐FTIR) spectroscopy to investigate the protein structural changes induced by ligand (CO) photodissociation and rebinding that are crucial for understanding the initial events of the intramolecular signal transduction mechanism in HemAT. We have studied the full length and truncated sensor domain HemAT‐CO adducts as well as the Y70F, L92A, T95A and Y133F mutants. Monitoring the rebinding of CO to the heme‐Fe2+ reveals biphasic kinetics for both the full length and truncated sensor domain HemAT. The TRS2‐FTIR experiments demonstrate that Y70 affects the conformational changes that are induced to the protein matrix by CO photodissociation. Moreover, L92 appears to operate as the conformational gate in the migration pathway of photodissociated CO.

This work was funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation (Infrastructure Project ANAVATHMISI/PAGIO/0308/14).
Nicotine regulates FasL, activates Mst‐1, promotes Histone H2B phosphorylation and accelerates neutrophil apoptosis
Authors: Rane Madhavi Jagdish , Kumar Jay , Jin Shunying , Merchant Michael , Ritzenthaler Jeffery , Roman Jesse , Klein Jon
Published: June 2021

An efficient way of resolving inflammation is induction of neutrophil apoptosis. We hypothesized that nicotine accelerates neutrophil apoptosis. Nicotine (3 mM) accelerated human neutrophil apoptosis by enhancing caspase‐3 cleavage in a p38 and ERK dependent manner. LPS‐induced neutrophil survival was significantly inhibited by nicotine. Moreover, □4nAChR antagonist Dihydro‐β‐erythroidine hydrobromide (D□E), inhibited nicotine‐stimulated pERK, pP38 and caspase‐3 cleavage. Caspase‐3 cleavage promoted Mst‐1 cleavage and pSer14‐Histone H2B which correlates with apoptotic chromatin and onset of apoptosis. Mst‐1 cleavage and pSer14‐Histone H2B were dependent upon p38 and ERK. In addition, nicotine induced FasL expression and secretion in neutrophils. Blockade of □4 AChR, p38 and ERK pathways inhibited nicotine‐induced FasL expression. Furthermore, blockade of FasL with anti‐FasL antibody prevented nicotine‐induced caspase‐3 cleavage and neutrophil apoptosis. These results suggest that nicotine stimulates an extrinsic apoptotic cascade in human neutrophils. Thus, FasL/Fas system in neutrophils could serve as a target for further investigation of the cholinergic system in neuroimmunomodulation.

Funding Source: R01AI075212–01 (MJR).
MIX‐cAMP signaling in 3T3‐L1 preadipocyte differentiation
Authors: Gabrielli Matias , Martini Claudia , Romero Damián , del Carmen Vila María
Published: June 2021

Adipogenesis is stimulated in 3T3‐L1 fibroblasts by a combination of insulin, dexamethasone (DEX), and methylisobutylxanthine (MIX). Mitotic clonal expansion (MCE) precedes differentiation of 3T3‐L1 fibroblasts to adipocytes. MIX increases cAMP content, which activates protein kinase A (PKA). However, PKA‐independent cAMP signaling through EPAC (exchange protein activated by cAMP) has also been reported. We found that H89, a PKA inhibitor, blocks MCE but not differentiation of 3T3‐L1 fibroblasts. Differentiation, evaluated by Oil‐Red‐O staining or quantification of triglycerides, did not occur when MIX was not present in the differentiation mixture but it was restored by addition of either dibutyryl‐cAMP (db‐cAMP) or 8‐CPT‐2‐Me‐cAMP. The latter activates EPAC but not PKA signaling. Furthermore, we found that MIX, db‐cAMP or 8‐CPT‐2‐Me‐cAMP increased PPARγ, a transcription factor required for adipocyte differentiation. PI3K‐PKB signaling is known to be required for differentiation. We found that insulin but not MIX, alone or in combination with insulin+DEX, was able to increase PKB phosphorylation. These results suggest that MIX signaling through cAMP‐EPAC, which is independent of PI3K‐PKB signaling, is also required for 3T3‐L1 fibroblasts differentiation to adipocytes.

Supported by National Agency for Scientific and Technological Promotion (Argentina) BID‐PICT: 08–1826.
Stress activated MAPK influence the quality control of misfolded cystic fibrosis transmembrane conductance regulator.
Authors: Hegde Ramanath Narayana , Parashuraman Seetharaman , Iorio Francesco , Di Bernardo Diego , Luini Alberto
Published: June 2021

Cystic fibrosis transmembrane conductance regulator (CFTR) deletion mutation of phenylalanine at position 508 (DF508CFTR) causes 70% Cystic Fibrosis (CF) disease that affects 1 in 2000 live births among Caucasians. This mutation results in misfolding and subsequent retention of the protein in the endoplasmic reticulum (ER) and later removal by ER associated degradation (ERAD). Proteins that are folded in the ER have to pass an obligatory quality control (ERQC) before they can be transported out of the ER to other downstream compartments of the secretory pathway. Here we have used a systems biology based analysis of the mode of action of drugs that have been published to allow the DF508CFTR to cross the ERQC, to arrive at molecular pathways that modulate the ERQC. Our findings here point to a role of stress activated MAPK (SAPK) pathway in the increasing the ERQC threshold that contributes to the ER retention of the already misfolded mutant CFTR. Downregulation of the MAPK by siRNA or inhibition of this pathway using drugs can allow the DF508CFTR to escape the ERQC and reach the plasma membrane.
Identification of novel phosphorylation sites in the mRNA surveillance factor Upf2 from Saccharomyces cerevisiae
Authors: Colon‐Morales Edgardo M. , Lin Qishan , Cajigas Ivan J. , Gonzalez Carlos I.
Published: June 2021

Cells are not exempt from committing errors. One of these errors is the addition of a premature termination codon (PTC) to an mRNA. These PTC‐harboring mRNAs encode potential non‐functional proteins that can lead to disease. Cells rely on the nonsense‐mediated mRNA decay (NMD) pathway to prevent the synthesis of these proteins. NMD is executed by three major proteins: Upf1, Upf2 and Upf3. Evidence has shown that Upf2 is phosphorylated in yeast and mammals but the role of this modification is unclear. The goal of this project is to provide insight on the molecular and biochemical basis of Upf2 phosphorylation and its role in NMD activity. Mass spectrometry analysis of immunopurified Upf2 from S. cerevisiae revealed twelve novel phosphorylation sites (S54, S55, T327, S422, T842, S868, T869, T872, S874, S1021, S1022 and S1023). Residues T842, S868, T869, T872 and S874 are located in the Upf2 acidic domain, previously reported to be phosphorylated. In contrast, S1021, S1022 and S1023 locate in the carboxyl terminal, a region important for NMD activity. Mutations of these residues are being constructed by site‐directed PCR‐mediated mutagenesis to alter the phosphorylated residues and assess their role in NMD. These results suggest that Upf2 phosphorylation is conserved in S. cerevisiae and might play an important role in mRNA surveillance. Supported by U54 CA96297, PRLSAMP HRD‐0601843, and RISE 2R25GM61151.
Activity‐based Proteomic Approaches to Investigate Aging in C. Elegans
Authors: Martell Julianne
Published: June 2021

The elucidation of protein activities dysregulated during the aging process is a vital step toward the discovery of signaling networks and metabolic pathways directly implicated in aging. Genetic studies in the model organism, Caenorhabditis elegans, generated a daf‐2 mutant that has a significantly extended lifespan. The daf‐2‐ gene mutant activates DAF‐16, a transcription factor, which initiates gene expression changes that mediate the life‐extension phenotype of this mutant. Identifying protein‐activity changes that are downstream effects of DAF‐16 activation will illuminate cellular pathways directly implicated in the aging process. We propose to directly monitor protein activity changes using the tools of activity‐based protein profiling. We are focusing our initial efforts on cysteine‐mediated protein activities that encompass a subset of diverse protein classes including proteases, kinases, ubiquitinating proteins, and metabolic enzymes. Combining small‐molecule probes that selectively bind to active members of these enzyme families, together with quantitative mass spectrometrybased proteomics, we are comparing protein activity in daf‐2 and daf‐2/daf‐16 mutant nematodes. Current work is focused on verifying the identity of these dysregulated proteins and understanding the role of these protein activities in slowing down the aging process in daf‐2 nematodes.
Metalloproteomic approach to the determination of calcium, iron and zinc bound to secretory immunoglobulin A in human milk
Authors: Pozzi Carla Mariane Costa , Braga Camila Pereira , Vieira José Cavalcante Souza , Okoshi Marina Politi , de Magalhães Padilha Pedro
Published: June 2021

At this present work, the presence of calcium, iron and zinc bound to secretory immunoglobulin A (sIgA) from human milk was investigated. The sIgA components were separated by two‐dimensional electrophoresis (2D‐PAGE) and they were identified by electrospray ionization mass spectrometry (ESI/MS/MS). A train of eight spots with 5.4<pI<6.5 and a mass of 78 kDa were identified as the Ig alpha‐1 chain constant region. Another spot, with pI of 5.8 and a mass of 100 kDa was identified as transmembrane secretory component, also known as the polymeric immunoglobulin receptor (pIgR). The metal ions in the spots were detected by flame atomic absorption spectrometry (FAAS) after acid mineralization of the spots. It was discovered calcium bound to both pIgR and Ig alpha‐1 constant region, while zinc was found to be bound to the Ig alpha‐1 constant region. The association of a metal ion to a protein (subject of study of metalloproteomics) is important for many reasons and the discoveries of this study can lead to a better understanding of mechanisms of action and additional roles that sIgA and its components have in human milk. Financial support: FAPESP (2011/03291–0).
miR‐10a regulates epithelial‐mesenchymal transition and adhesion and angiogenesis in hepatoma
Authors: Tang Hua
Published: June 2021

MiRNA is involved in the regulation of hepatocellular carcinoma (HCC). Here, we studied the functions of miR‐10a in HCC cells.

miR‐10a promoted cell migration and invasion capacity whereas did not affect cell viability and proliferation in HCC cell lines. However, the intrahepatic metastasis nodules were significantly reduced by miR‐10a in vivo. EphA4 was identified a target gene of miR‐10a. Knockdown of EphA4 promoted cell migration and invasion whereas did not affect cell viability and proliferation. Cell adhesion and vasculogenic minicry assay showed that overexpression of miR‐10a suppressed cell‐matrix adhesion and vessel formation of HCC cells. Blocking expression of miR‐10a or enhanced expression of EphA4 up‐regulated the epithelial marker E‐Cadherin whereas down‐regulate the mesenchymal markers, Vimentin and ICAM‐1.Blocking miR‐10a and up‐regulation of EphA4 enhanced the expression of ¦Â1‐ integrin which mediates cell‐matrix adhesion and MMP‐2.

In summary, miR‐10a can promote EMT process in vitro to affect cell mobility whereas suppress cell‐matrix adhesion and vessel formation ability by regulating ¦Â1‐integrin and MMP‐2 to inhibit the metastasis, and may influence angiogenesis in vivo. All of these will provide new understanding into miR‐10a in the metastasis of HCC.
Identification of regulatory mechanisms and RNA targets of human Pumilio proteins
Authors: VanEtten Jamie , Schagat Trista , Bhasi Ashwini , McEachin Richard , Goldstrohm Aaron
Published: June 2021

Proper gene expression requires a balance between synthesis, modification, and degradation of mRNAs. PUF proteins are a conserved family of RNA binding proteins that regulate specific mRNAs to control many biological processes including development and proliferation. PUFs bind specific sequence elements in the 3′ UTRs of specific mRNAs via an RNA binding domain. PUFs in model organisms repress targets via deadenylation and degradation and/or direct inhibition of translation. The mechanisms by which human PUFs repress their mRNA targets as well as their physiological targets remain unclear. Humans have two related PUFs, PUM1 and PUM2. We found that human PUFs employ a conserved mechanism of repression via direct recruitment of the CCR4‐NOT deadenylase complex to target mRNAs, leading to translational inhibition and accelerated mRNA degradation. Further, PUMs could repress a non‐adenylated mRNA; therefore, PUMs exhibit a second deadenylation independent repression mechanism.

Few specific mRNAs regulated by PUMs have been identified. To better ascertain the roles of human PUMs, we sought to identify mRNAs regulated by them. We depleted PUMs from human cells and measured changes in mRNA expression levels by RNA‐Seq. We identified more than of 200 PUM target RNAs meeting the following criteria: >;1.5‐fold change, >;1 PUM site, and FDR = 0.05. Our results begin to reveal PUM regulated pathways in human networks.

University of Michigan internal grant
Towards background‐free serum analysis
Authors: Danielsson Ake , Markova Natalia , Ohman Johan
Published: June 2021

A fast and automatable sample preparation workflow for the determination of monoclonal antibodies (mAb's) in human blood serum samples using surface plasmon resonance, (SPR) was developed. Using standard sample preparation protocols, we frequently experienced high backgrounds in the analyses. The developed sample preparation workflow comprised two steps. The first step involved affinity capture of the target protein using magnetic beads with Protein G (Protein G Mag Sepharose™). The second step involved buffer exchange using a 96‐well filter plate, PD MultiTrap™G‐25, to a suitable buffer for analysis. To assess the stability of the target protein after sample preparation we used differential scanning calorimetry. The concentration of the target mAb, (Rituximab) in serum samples was determined using a Biacore™ T200 instrument. The results showed a reproducible reduction of the background signals in SPR analysis and an overall target protein recovery above 80%. The developed sample preparation workflow was successfully applied to determine Rituximab in human blood serum samples.
Rapid and efficient derivation of retinal pigment epithelium (RPE) from human pluripotent stem cells
Authors: Pennington Britney Ocean , Buchholz David E. , Hinman Cassidy , Croze Roxanne , Coffey Peter J. , Clegg Dennis O.
Published: June 2021

The death of the retinal pigment epithelium (RPE) induces the leading cause of blindness in the elderly: age‐related macular degeneration (AMD). Human embryonic stem cells (hESCs) have the potential to generate a limitless source of RPE for cellular therapies, but the current methods of both spontaneous and directed differentiation are time consuming and relatively inefficient. This study aims to develop an efficient protocol that derives RPE from hESCs and induced pluripotent stem cells (iPSCs) in 14 days. Applying defined factors at critical points during the differentiation result in ~ 80% of the cells expressing RPE markers at the end of two weeks. This protocol expedites the development of RPE from pluripotent stem cells, which is useful for generating therapeutic cells and investigating maturation of RPE in vitro.
Communicating a Nobel Signal: Exploration of the Heterotrimeric G protein
Authors: Adams Alec , Crowl Madison , Engle Meryl , Jacobson Austin , McClain Amy , Victorine Kasie , Wilkinson Mason , Kessler Eric , Tuel Kelley , Whalen Joseph , Preininger Anita M. , Hamm Heidi E.
Published: June 2021

Cells must sense a host of external signals to be responsive to features in the environment that are vital for survival. Evolutionary pressures have resulted in pathways that translate and amplify such signals into intercellular actions. Visual phototransduction, a model pathway, includes the transmembrane G‐protein‐coupled receptor (GPCR) rhodopsin, activated by light, and the G (guanine‐nucleotide‐binding) protein transducin that communicates to downstream proteins in the photoreceptor. The Blue Valley CAPS 2012–13 SMART Team (Students Modeling a Research Topic) modeled the heterotrimeric G protein transducin (1GOT.pdb) using JMol and their 3D printer. Transducin is composed of a Gα;, containing the GTPase domain, and Gβ; that contains a β‐propeller structure composed of seven similar β‐sheets and an N‐terminal helix that forms a coiled‐coil with one of Gγ two helices. Upon binding of transducin to an activated rhodopsin, GDP is released from Gα. Subsequent GTP binding to Gα causes conformational changes that result in the dissociation and activation of Gα and Gβγ. These separate subunits act upon targets in the downstream cascade resulting in hyperpolarization of the retinal photoreceptor, a sensation of light, and a greater awareness of the environment by the organism. Subsequent hydrolysis of bound GTP to GDP within Gα returns the protein to its inactive state. Supported by a grant from NIH‐SEPA.
Genetic determination of Next Generation sequencing technology error rates within microsatellite sequences
Authors: Allette Kimaada , Eckert Kristin , Hile Suzanne
Published: June 2021

Previous research have shown that microsatellite sequences are underrepresented after massively parallel, Next Generation (NextGen), sequencing approaches compared to the known number detected in the human genome project. The goal of this project is to identify the accuracy and efficiency in reading repeat sequences in both sequencing technologies. To study this mono‐ di‐ and tetra‐nucleotide repeats were inserted in‐frame within the Herpes Simplex Virus1–thymidine kinase gene. Sanger sequencing and selective plating verified correct vector constructions. In addition, we specifically determined the mutation frequencies obtained during NextGen DNA library preparation of the plasmids. The starting background mutational frequencies(MFB) of all plasmids were determined by electroporation of E‐coli and selective plating. The NextGen library preparation steps were mimicked with each of the plasmids. DNA fragments were isolated by gel purification, amplified, and electroporated in E‐coli to determine the MFQ and MFQ+PCR. The degrees of technology‐introduced errors were calculated by comparing MF after amplification to background MF. From this we determined that all microsatellites were susceptible to technology‐introduced errors. The experimental design can be used to compare errors introduced during DNA library preparation. Funding MS Life Cycle NIH Grant GM087474.
SMARTer design of a novel structure‐based ADAM8 inhibitor to combat pancreatic cancer
Authors: Bang JaKyung , Bradford Nora , Harrold Eric , Huang Tianyi , Lu Hancheng , Magliochetti‐Cammarata Tessabella , Nee William , O'Dea Conor , Park Jin Young , Robinson Imogene , Rokous Jack , Sugarman Eli , Tejeda Jeanny , Yan Vincent , Rokous Judy , Bartsch Jörg W.
Published: June 2021

Members of the ADAMs (A Disintegrin And Metalloprotease) family are involved in cell migration, invasion and growth factor signaling, all key processes in cancer. Pancreatic ductal adenocarcinoma (PDAC), marked by massive infiltration of tumor cells into the pancreas and metastasis into liver and lungs, has the highest mortality rate of all solid organ cancers. In PDAC, high ADAM8 levels correlate with decreased survival. In cell membranes, ADAM8 associates with itself to allow autocatalytic activation. ADAM8 then binds to β1‐integrin, a transmembrane receptor that attaches cells to the extracellular matrix (ECM) and allows communication across the membrane. Both interactions cause ECM remodeling via intracellular signaling pathways by phosphorylation of β1‐associated kinases focal adhesion kinase and extracellular signal‐regulated kinase which in turn activate the release of matrix‐metalloproteinases, potent mediators of ECM degradation. The Governor's Academy SMART (Students Modeling A Research Topic) team designed a model using 3D printing technology to highlight structural features, providing functional insight into the mechanism by which ADAM8 causes tumor progression in PDAC. Since its protein‐protein interactions are crucial for its function in tumors, blocking ADAM8 to limit invasive tumor growth is a promising therapeutic strategy. Supported by a grant from NIH‐SEPA 1R25OD010505–01
O‐GlcNAc: The sweet side of protein regulation
Authors: Broderick Bridgette , Fiscus Allie , Patel Nehal , Samms Sarah , Schultz Christine , Seger Hannah , Simmons Jerusha , Wuelzer Jennifer , Dix Randy , Slawson Chad
Published: June 2021

O‐linked N‐acetylglucosamine transferase (OGT) enzymatically regulates many biological processes required by the body to support life, such as: gene transcription, mRNA processing, cytoskeletal solubility, insulin signaling, metabolic function, protein translation, and protein stability. OGT catalyzes the addition of O‐GlcNAc, a post‐translational modification of a single N‐acetylglucosamine, to serine and threonine residues on multiple proteins. OGT is comprised of four domains: two catalytic domains, the tetratricopeptide repeat (TPR) domain, and the phosphatidylinositol 3, 4, 5‐trisphosphate (PIP) binding domain. The TPR domain functions as hinge and latch to fit proteins into the active site, which allows for a broad substrate usage of the enzyme. OGT is a very useful protein, so the protein levels of this enzyme are maintained at a strict equilibrium. When there is an over‐expression of OGT, the process of cell division is skewed and the resulting daughter cells end up with four nuclei. This is called polyploidy. If there is no OGT in the cells, then cell death occurs. Many illnesses present with alterations in the levels of O‐GlcNAc, suggesting that OGT could be a potential drug target to treat chronic diseases. The Olathe North SMART Team (Students Modeling A Research Topic) modeled OGT using 3D printing technology to visualize how the different domains of the protein interact. Funded by a grant from NIH‐SEPA.
Lysophosphatidic Acid Regulates uPAR Expression and Localization
Authors: Ciota Alexandra M , Kobliska Amy , Marsh Sydney , Ellerbroek Shawn M
Published: June 2021

Lysophosphatidic acid (LPA) stimulates in vitro invasion of ovarian epithelial cancer cells through a uPA‐dependent process. We therefore analyzed the effects of LPA on the cell surface uPA receptor (uPAR). While both uPAR mRNA and protein expression was consistent with control after 2 hours of LPA stimulation, LPA strongly stimulated uPAR redistribution into punctate aggregates or to cell junctions, depending on cell line. After 24 hours of stimulation, uPAR mRNA levels increased 4.0±2.3 fold in OVCA 429 cells and were modestly impacted in OVCA 433 cells (1.8±0.5 fold). In accordance, cell surface‐localized uPA activity increased 4.3±2.5 fold (OVCA 429) or 2.1±0.9 fold (OVCA 433) and was accompanied by increased uPAR immunofluorescence staining. At this same time, mRNA levels of uPA and PAI‐1 were not significantly changed. These data provide mechanistic insight into the actions of a lipid that positively correlates with ovarian cancer progression.
Alkaline Phosphatase: IHC Dephosphorylation for Rapid Cancer Detection
Authors: Edwards Ashlyn , Adamson Bennett , Shimmin Bo , Gallagher Colleen , Lantzsch Maddie , Radler Shawn , Seemann Rachel , Stockey Suzanna , Pham Tiffany , Van Scyoc Vanessa , Futch Liane , White Katie
Published: June 2021

According to the American Cancer Society, one third of women and half of men risk developing cancer in the United States. This results in a growing, critical effect on both social and economic aspects of society. Fortunately, researchers are developing simple, accurate tests for cancer detection. Many immunohistochemistry (IHC) assays use alkaline phosphatase (AP) to detect in‐vitro cancers. IHC assays use antibody‐enzyme conjugates that pair with primary antibodies specific to antigens on the surface of cancer cells. As the catalyzing enzyme, AP removes a phosphate from a substrate resulting in a color precipitate localized to cells expressing the specific cancer antigen. AP is a homo‐dimer phosphomonoesterase containing two Zn atoms and one Mg atom in each subunit; one Zn atom and the Mg atom maintain structure, and the other Zn atom works with Ser102 to remove a phosphate. The critical amino acids in AP's active site create the electrostatic environment needed for Ser102 to begin dephosphorylation. AP has many in‐vivo functions and laboratory applications, including calcification of cartilage as well as the synthesis, sequencing, and labeling of DNA. The Ironwood Ridge High School SMART (Students Modeling A Research Topic) Team has modeled AP, using 3D printing technology. By studying AP's structure and function, we are able to visualize its role as a chemical detector. Sponsored by a grant from NIH‐SEPA.
Potential implications of human alpha7 nicotinic acetylcholine receptor genetic variants to the pathogenesis and treatment of HIV‐associated neurocognitive disorders
Authors: Holder‐Viera Mileyshmi , Aponte‐Santiago Nicole Ann , Aviles‐Pagan Emir , Lasalde‐Dominicci Jose Antonio , Baez‐Pagan Carlos Alberto
Published: June 2021

The α7 nicotinic acetylcholine receptor (nAChR), a ligand gated ion channel characterized by its high calcium permeability, was shown to be upregulated in SH‐SY5Y cells exposed to HIV‐1 glycoprotein gp120 leading to Ca2+ overloading and cell death. These results suggest that HIV‐associated neurocognitive disorders (HAND) could be related to increased neuronal death as result of excessive Ca2+ influx through the upregulated α7 nAChR. Furthermore, these results also suggest that α7 nAChR antagonists can be exploited for neuroprotection in HIV‐1 seropositive patients. Genetic variants of the CHRNA7 gene (encodes α7 nAChR) that result in ion channels with increased expression and/or functionality could thus underlie increased risk of HAND. In addition, CHRNA7 genetic variants could also result in receptors with diminished response to antagonists. The hypothesis is that non‐synonymous single nucleotide polymorphisms (SNPs) located in the CHRNA7 gene could result in α7 nAChRs with enhanced functionality and/or expression levels, and with altered responses to drugs aimed at interfering with the excessive Ca2+ flow. Bupropion was studied as a model α7 nAChR antagonist. Herein we present data demonstrating that genetic variants of the CHRNA7 gene may result in α7 nAChRs with altered functionality and response to bupropion. Supported by NIMH grant # P30MH075673–07 and NIMHD grant # 8U54MD007587–03.
Expression of Syntaxin‐2 in Bovine Sperm
Authors: Richardson Leea’ P , McCaskill Shania , Nagdas Subir
Published: June 2021

Syntaxins are membrane integrated Q‐SNARE proteins known to participate in exocytosis. Vesicle docking involves the binding of two plasma membrane proteins, syntaxin and SNAP‐25, to the vesicle membrane protein VAMP to form a stable trimeric core complex; synaptophysin is thought to regulate the formation of this complex. Although the members of Q‐SNARE proteins are characterized in somatic cells, it is not known whether related proteins function in the sperm acrosome reaction. The objective of the present study is to identify and localize syntaxin and synaptophysin in bovine epididymal spermatozoa. Western blots of caput and cauda sperm lysates and plasma membrane fractions stained with anti‐syntaxin and anti‐synaptophysin showed the presence of a 31kDa and 38kDa band in both sperm lysates and plasma membrane fractions, respectively. Indirect immunofluorescence localized syntaxin to the anterior but not equatorial regions of the acrosomal segment. Syntaxin was recovered in the anti‐syntaxin immunoprecipitation pellet. Coimmunoprecipitation analysis will be utilized to identify the syntaxin‐associated protein(s) complex during capacitation and acrosome reaction of bovine spermatozoa. It is assumed that syntaxin‐2 and synaptophysin may be involved in triggering the acrosome reaction through a ligand‐receptor mediated signal transduction pathway. NIH 1SC3GM096875 and FSU‐RISE ‐ STEM Grants.
R61 D,D‐peptidase bound to a Helen‐1 Penicillin Substrate or One “Hel”‐en of an Antibiotic
Authors: Sosa‐Gonzalez Sonia , Cobb Michaun , Osadame Ngozi , Osademe Isioma , Campbell Kyler , Marin Merari , Llamosa Jhordy Rios , Llamosa Brigitte Rios , Jones Kasaundra , Garland Meg , Johnson Carol , Silvaggi Nicholas
Published: June 2021

Although antibiotics like penicillin save lives, antibiotic‐resistant bacteria are a growing issue. According to Purdom (2007), over 70% of infections acquired by hospital patients post‐admission, are resistant to at least one prescribed antibiotic. Penicillin, a β‐lactam antibiotic, treats bacterial infections caused by bacteria producing toxins within a host. Many pathogenic bacteria need a peptidoglycan cell wall for normal functionality. Enzymes in the cell membrane help form this cell wall by cross‐linking peptidoglycan units. β‐lactam antibiotics hinder bacterial cell wall biosynthesis by competing with the peptide substrate for the active site in these enzymes. While not the main enzyme used to produce bacterial cell walls, R61 DD‐peptidase, a cytoplasmic enzyme, is easily crystallized to show bacterial enzyme chemistry. The active site of R61 consists of amino acid residues Ser62, Lys65, Lys159, Arg285, Thr299, and Thr301. The Messmer SMART Team modeled R61 complexed with Helen‐1, a species‐specific β‐lactam, highlighting the functionality and chemistry of the active site amino acids and their interaction with the beta‐lactam. Understanding the structure and function of the active site of penicillin binding proteins, like R61, could lead to new, species‐specific antibiotics that could prevent antibiotic resistance in bacteria. Supported by a grant from NIH‐CTSA UL1RR031973.
Conformational changes of the carbon‐fixing enzyme CODH/ACS revealed by electron microscopy
Authors: Thompson Samuel M. , Brignole Edward J. , Ragsdale Stephen W. , Drennan Catherine L.
Published: June 2021

Carbon monoxide dehydrogenase/acetyl‐CoA synthase (CODH/ACS) is a 310 kDa bifunctional enzyme involved in the anaerobic fixation of atmospheric carbon (CO2) by the acetogenic bacterium Moorella thermoacetica. The CODH subunit reduces CO2 to CO, which transfers through a central hydrophobic tunnel to the active site of ACS. ACS contains a dinickel iron‐sulfur site where CO combines with a methyl group supplied by corrinoid iron‐sulfur protein (CFeSP) and coenzyme A (CoA). Conformational changes of ACS have been hypothesized as the structural mechanism regulating access of methylated‐CFeSP and CoA to the proximal nickel in the active site. However, the conformations of CODH/ACS that have been observed crystallographically are insufficient to accommodate methyltransfer from CFeSP to ACS. To examine the conformations of CODH/ACS we used single‐particle electron microscopy (EM). Because CODH/ACS contains O2 sensitive metallocofactors, we developed a method for preparing negatively stained EM specimens in an anaerobic chamber. The resulting structures of CODH/ACS reveal a dramatic hinge‐like opening of the ACS subunit that can accommodate methyl transfer from CFeSP to CODH/ACS and gives new insight into the catalytic mechanism of this environmentally relevant enzyme.

This work is supported by funding from the NIH. Samuel Thompson is supported by the MIT Undergraduate Research Opportunities Program.
Contribution of neuropilin 1 to increased motility in radiation‐surviving cells
Authors: Tsutsumi Kaori , Chiba Ayaka , Tadaki Yuta , Nakajima Haruka
Published: June 2021

Radiotherapy is an effective approach for the treatment of various types of cancer. However, it has been reported that the malignant capacity of tumor cells is elevated in tumors that survive radiotherapy. In a previous report, we prepared radiation‐surviving tumor cells (IR cells) using the human non‐small cell lung cancer cell line H1299. In preliminary experiments, we found that expression of neuropilin 1 was up‐regulated in IR cells. Here, we investigated the contribution of neuropilin 1 to changes in cell characteristics in these radiation‐surviving cells. Expression levels of neuropilin 1 mRNA were about 1.4‐fold up‐regulated in IR cells, as compared with parental cells. Cell motility was further increased in IR cells than parental cells on cell migration assay, although there were no differences in angiogenic activity on tube formation assay. Furthermore, up‐regulation of cell motility in IR cells was suppressed by pre‐treatment with anti‐neuropilin 1 antibody. Neuropilin 1 was observed at the endosome‐like vesicles in IR cells, on which early endosome autoantigen 1(EEA1), a marker for endosome, was not co‐localized on immunofluorescence. Although further studies are necessary in order to identify other molecules associated with neuropilin 1, neuropilin 1 may be important in controlling the up‐regulation of cell motility in radiation‐surviving tumor cells.
Prion initiation in vivo Requires Limitations in the Proteostasis Network
Authors: Villali Janice , Serio Tricia
Published: June 2021

Neurodegenerative diseases, including Alzheimer's, Huntington's and Parkinson's Diseases and the Transmissible Spongiform Encephalopathies (prion diseases), have been linked to an unusual disease mechanism, in which alternative conformations of cellularly encoded proteins arise and subsequently direct the misfolding of normal conformers of the same protein to a like state. The objective of our research is to determine how a pathway of protein misfolding is initiated in vivo. For these studies, we are exploiting the Sup35/[PSI+] prion system of Saccharomyces cerevisiae. While the mechanism by which Sup35 folding initially switches from the normal ([psi−]) to the misfolded ([PSI+]) pathway is still unknown, this process is dependent on the presence of the misfolded form of another prion protein, Rnq1. Three models were proposed to explain this dependence: Rnq1 acts as a template to stabilize misfolded Sup35, directs the amplification of misfolded Sup35 by the chaperone protein Hsp104, or protects misfolded Sup35 from clearance by Hsp104 in vivo. Each of these models makes unique predictions about the dependence of prion initiation on limiting concentrations of Rnq1, Sup35, and Hsp104. Our data show that Hsp104 has a deleterious effect on [PSI+] formation consistent with Rnq1 acting as a decoy for chaperone‐mediated clearance of protein aggregates. This work was funded by the NIH.
Study on Vitamin A Requirement of Adult Chinese by Isotope Dilution Technique and Vitamin A Intervention Trial
Authors: Wang Zhixu , Li Yonghua , Du Zhen , Shao Zewei , Wu Jieshu , Tang Guangwen
Published: June 2021

The goal of this study is to determine dietary vitamin A requirement for keeping constant vitamin A pool in body in Chinese population. Total 116 healthy adult volunteers (half by genders) were recruited and divided into 4 groups and given vitamin A supplementation with different doses of 0μg/d, 200μg/d, 400μg/d and 600μg/d retinol (by retinyl acetate) for 4 months while they kept low vitamin A diets. Before and after vitamin A intervention, the liver vitamin A storages were determined by two metabolically dynamic experiments with the stable isotope dilution technique using 2H4‐and 2H8‐retinyl acetate. Three 24‐hours dietary recall were carried out once a month to survey background dietary vitamin A intake. The average vitamin A liver storage before and after intervention were (730.75±414.45) μmole and (934.59±786.61) μmole respectively. Average changes of liver vitamin A storage were (203.84±705.49) μmole for all subjects. The linear regression analysis on the data from total 104 subjects (52 males and 52 females) who finished the study showed that the predicted values of dietary vitamin A intake for maintaining total liver vitamin A storage unchanged was about 748.32μg Retinol Equivalents (RE) or 560.79μg Retinol Active Equivalents (RAE) in men, and 599.91μg RE or 472.19μg RAE in women. The values could be used as Estimated Average Requirement (EAR) of vitamin A for adult Chinese.
Dietary intake and major food sources of niacin in adults living in South Korea
Authors: Kim Young‐Nam , Cho Youn‐Ok
Published: June 2021

To estimate niacin intakes and major food sources of Korean adults, three consecutive 24‐hour recalls were collected from 167 healthy adults, aged 20–64y, living in Seoul and metropolitan area, Gumi, and Kwangju, Republic of Korea, during November, 2011 – March, 2012. The mean daily niacin intake was 15.57/5.93 mg NE (mean/SD). There was no significant difference in niacin intake by gender. Foods from animal and plant sources provided 47% and 53%, respectively, of the total niacin intake. The top 10 major dietary sources of niacin were pork, rice, beef, Korean Chinese cabbage kimchi, mandarin, mackerel, rice cake, chicken, yellow croaker, and dried anchovy. As for major dietary sources of niacin, the top 10 foods provided 45% of total niacin intake and the top 30 foods contributed 67% of niacin intake. The Korean adults consuming less the Korean Estimated Average Requirements for niacin was 18% of total subjects and 40% had niacin intakes less than Recommended Nutrient Intakes. Therefore, some of Korean adults in this study had inadequate intake of niacin. Korean adults need to be encouraged to consume niacin‐rich foods such as pork, beef, kimchi, mandarin, and mackerel. Supported by 2012 research fund of National Research Foundation of Korea: 2012R1A1A2A04669745
Chalcones from Angelica keiskei increase insulin receptor (InsR) and insulin receptor substrate‐2 (IRS‐2) mRNA expression in hepatocytes of rats with type 2 diabetes
Authors: LI LEI , ZHONG JINYI
Published: June 2021

Type 2 diabetes mellitus is a chronic metabolic disease characterized by insulin resistance, of which InsR and IRS play very important roles. Chalcones, isolated from Angelica keiskei, have been reported to possess a variety of biological properties including antidiabetes. To study the effect of chalcones from Angelica keiskei on the expression of InsR and IRS‐2 in hepatocytes, the rats with type 2 diabetes were orally given chalcones with 30, 10 and 0 mg/kg.bw per day for 4 wks, respectively. Blood glucose and serum insulin were evaluated by glucose oxidase method and radioimmunoassay, respectively. Reverse transcription polymerase chain reaction (RT‐PCR) was used to determine the mRNA expression of InsR and IRS‐2. Blood glucose and serum insulin of rats with 30 mg/kg.bw chalcones were significantly lower than the diabetes control. Their mRNA expression of InsR and IRS‐2 were significantly higher than the diabetes control. It may conclude that chalcones from Angelica keiskei may up‐regulate the expression levels of InsR and IRS‐2 and improve insulin resistance of rats with type 2 diabetes. [Financial support: The Health Department of Shandong contract#QNW001]
Effects of fetal alcohol exposure on zinc homeostasis in the newborn pulmonary alveolar macrophage
Authors: Konomi Juna , Brown Lou Ann
Published: June 2021

Since zinc is essential for innate and adaptive immunity, zinc deficiency increases susceptibility to infection and magnifies existing infections. To examine the effects of in utero alcohol exposure, C57BL/6 female mice were fed the Lieber DeCarli liquid diet with 25% ethanol derived calories or with 25% maltose dextrin derived calories during preconception and pregnancy. At delivery, alveolar macrophages (AMs) from dams and pups were isolated and treated with FluoZin‐3AM to measure AM zinc and FITC‐labeled inactive Staph. aureus to measure phagocytosis. AMs from pregnant ethanol‐fed dams had lower intracellular zinc (p=0.007) and decreased phagocytic function (p<0.05), when compared to pregnant controls. AMs isolated from pups with fetal ethanol exposure displayed lower zinc levels (p=0.078) and AM function (p=0.041) than unexposed pups. Additionally, protein expression of the zinc transporter, ZnT1, was significantly lower in exposed pups than unexposed pups (p=0.003). Chronic alcohol exposure in adults or chronic in utero exposure decreases pulmonary AM function via decreased zinc availability which may contribute to an increased risk of respiratory infections.

Funding: NIH/NIAAA P50 AA135757(LAB)
Impact of harvest timing on the polyphenol content of black chokeberry (Aronia melanocarpa, cv ‘Viking’) juice.
Authors: Taheri Rod , Durocher Shelley , Brand Mark H , Bolling Bradley Warren
Published: June 2021

Chokeberry (Aronia melanocarpa) is rich in polyphenols with potential nutraceutical value. The objective of this study was to determine the peak polyphenol content of Viking chokeberries over the span of possible harvest dates within a single growing season. Berries were grown in Storrs, CT and randomized into 10 plants/block. At the onset of ripeness, 5 blocks were harvested weekly until the last harvestable date, for 7 weeks. The polyphenol content of pressed juice was measured using UPLC‐MS, and by the 4‐(dimethylamino) cinnamaldehyde (DMAC) and Folin‐Ciocalteu assays. Changes in sugar content were analyzed using a refractometer and quantified in degrees brix. Sugar content rose gradually from 10.5 ± 0 to 14.3 ± 0.3 degree brix between weeks 1 and 7. Anthocyanins increased from 366 ± 3 to 1200 ± 39 μg/mL between weeks 1 and 5 and then declined by 32% by week 7. Proanthocyanidins increased from 214 to 352 μg catechin equivalents/mL between weeks 1 and 7. Hydroxycinnamic acid content declined from 1,250 ± 0 to 835 ± 25 μg/mL between weeks 1 and 7. Total phenol content rose from 4.03 to 5.03 mg gallic acid equivalents/mL between weeks 1 and 7. Thus, the polyphenol content of Viking berry juice was greatest at 7 weeks of ripeness, about 20% higher than week 1. However, maximum juice anthocyanins occurred at 5 weeks of ripeness. Funding provided by the University of Connecticut Diet and Health Initiative.
A randomized, double‐blind, acute crossover, bioavailability study comparing calcium absorption of two calcium formulations in healthy women
Authors: Fouad Gameil T. , Wilson Dale , Guthrie Najla , Baisley Joshua , Pezzullo John C. , Evans Malkanthi
Published: June 2021

Aging of the North American population has increased the incidence of osteoporosis causing concern to health care providers. This study tested the pharmacokinetics of two calcium sources and their relative effects on parathyroid hormone (PTH). Calcium amino acid chelate and calcium carbonate were studied. Fifteen healthy female subjects, ages 25 to 45, having 25(OH) D (vitamin D) levels between 50 nmol/L and 125 nmol/L and a BMI >;20 and ≤35 kg/m2were included. Subjects followed a low calcium diet 7 days prior to and during the clinic test period. Blood samples collected at pre‐dose, 30 minutes, 2, 4, 6, 9, and 12 hours post‐dose were assessed for serum calcium and parathyroid hormone. Subjects administered calcium amino acid chelate had 16.6% higher serum calcium 4 hours post‐dose compared to calcium carbonate. There were no significant differences in serum calcium AUC(0–12h), Cmax, and Tmax between groups. Subjects on calcium amino acid chelate had significantly lower parathyroid hormone levels compared to those on calcium carbonate (P=0.012). The significantly lower values of serum PTH at 4h for calcium amino acid chelate coincided with the maximum concentration of calcium reached in the serum for this formulation. Calcium amino acid chelate produced a significant suppression in serum PTH levels during the test period, suggesting that this formulation was more bioavailable than the calcium carbonate formulation. This study was sponsored by Biotron Laboratories, Inc.
Malvidin and delphinidin exhibit a dose‐dependent effect on cell viability and apoptosis in HT‐29 cells
Authors: Krauss Amy , Fischer Joan
Published: June 2021

Anthocyanidins (ANTH) induce apoptosis in some cancer cell lines, but studies describing the dose‐dependent effects of ANTH on cell populations are limited. The objective of this study was to evaluate the dose‐dependent response of malvidin (MAL) and delphinidin (DEL), ANTH with different chemical structures, on cell viability and apoptosis in HT‐29 cells in comparison with curcumin (CUR), a phenolic compound and a known inducer of apoptosis. Cells were treated with 0, 5, 10, 25, 50, 80, or 100 umol/L MAL or DEL or 40 or 80 umol/L CUR for 48 h at 37°C, 5% CO2, 95% air. Cells were treated with 250 umol/L tert‐butyl hydroperoxide for 2 h as a positive control. A significant ANTH concentration effect on cell proliferation and apoptosis was observed but MAL and DEL had similar effects on these measures. Lower concentrations of DEL (5 and 10 umol/L) and MAL (5 umol/L) increased cell viability by 12–16% compared to control, while higher concentrations of CUR, (80 umol/L), MAL (80 umol/L and 100 umol/L), and DEL (80 umol/L and 100 umol/L) decreased cell viability by 16–44%. Apoptosis was assessed by measuring caspase‐3 activity. Caspase‐3 activity only significantly increased with 80 umol/L MAL and DEL, by a fold increase of about 1.7. This data suggests MAL and DEL have comparable effects on HT‐29 cell viability and concentrations as high as 80 umol/L MAL and DEL are needed to induce apoptosis.
The effect of selective amino acid supplementation on calcium absorption during a low protein diet
Authors: Bihuniak Jessica D , Sullivan Rebecca R , Simpson Christine A , Caseria Donna M , O'Brien Kimberly O , Kerstetter Jane E , Insogna Karl L
Published: June 2021

Increasing dietary protein intake within a physiologic range stimulates intestinal Ca absorption but it is not known if specific amino acids (AA) or dietary protein as a whole is responsible for this effect. We therefore selectively supplemented a low protein (0.7 g/kg) diet with either aromatic (Trp, Phe, His) or dibasic (Arg, Lys) AA to achieve intakes comparable to the content of a high protein diet (2.1 g/kg) and measured Ca absorption. Fourteen young women were randomized to a placebo‐controlled, doubleblind, cross‐over feeding trial in which each subject ingested, in random order, a 6‐day low protein diet supplemented with aromatic AA, dibasic AA, or methylcellulose capsules (control) after a 10‐day adjustment period. All subjects ingested all 3 diets. Intestinal Ca absorption was measured between days 5 and 6 using dual stable Ca isotopes. There was no difference in Ca absorption between the diet supplemented with aromatic AA and the control diet (22.9±2.0% vs.22.3±1.4% respectively, p = 0.322). However, Ca absorption was significantly higher with dibasic AA supplementation (25.2±1.4% vs. 22.3±1.4%, p = 0.047). Individuals with protein intakes below the RDA may benefit from Arg and Lys supplementation to improve Ca economy.