bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational Analysis of the Dimer and Decamer Interface Residues in the Yeast Peroxiredoxin Tsa1
Authors: Loberg Matthew , Buchan John , Allan Kristin , Spencer Matthew , West James
Published: June 2021

Oxidative damage of biological macromolecules is associated with a number of disease pathologies and the aging process. To defend against oxidative injury caused by peroxides, organisms express numerous peroxidases, including a family of conserved peroxiredoxins. Of the five peroxiredoxins found in baker's yeast, four of these proteins (Tsa1, Tsa2, Ahp1, and Prx1) are targets of the protein cross‐linker divinyl sulfone (DVSF) in treated yeast cells. While previous work has implicated the redox‐active cysteine residues in Tsa1 as targets of DVSF, the cysteine residues are brought into close proximity via dimeric quaternary structure. Therefore, we are investigating the quaternary structures formed by Tsa1, seeking to understand what amino acid residues are critical for the formation of dimers and higher order structures. To this end, we have performed a structure‐guided mutational analysis of conserved residues at the interfaces in Tsa1. Specifically, we have focused our studies on two putative ion‐pairing regions at the dimer interface and potential aromatic interactions at the decamer interface. Mutations at these sites in Tsa1 disrupt the quaternary structures formed, as measured by protein cross‐linking and native PAGE analysis. Moreover, the mutations studied compromise Tsa1′s ability to defend against oxidative insults and carry out other functions in cells.
The Effect of P6 Dual Orientation on Anti‐P6 Bactericidal Activity in Nontypable Haemophilus influenzae
Authors: Morrow Kasey , Kumar Sanjana , Pichichero Michael , Michel Lea
Published: June 2021

Nontypable Haemophilus influenzae (NTHi) bacteria cause ear and respiratory tract infections that can lead to short and long‐term health effects in patients and high healthcare costs for treatment. The current treatment for NTHi infection is antibiotics, which is controversial due to the emergence of antibiotic‐resistant bacteria. For these reasons, there is an urgent need for a vaccine against NTHi. The outer membrane lipoprotein P6 is currently a leading a vaccine candidate for NTHi. We have shown that a subpopulation of P6 is surface exposed in several strains of NTHi. However, the majority of P6 is not surface exposed. Our current study uses bactericidal activity assays to determine the effect of minimal P6 surface exposure on its vulnerability to bactericidal antibodies. Good vaccine candidates should be susceptible to bactericidal antibodies. Our preliminary results have shown that only a small percentage of surface exposed P6 is necessary to elicit P6‐mediated killing.
Extracellular Substrate Stiffness Alters Macrophage Morphology and Function
Authors: Gruber Erika , Sinha Siddhartha , Ehrlich Emma , Leifer Cynthia
Published: June 2021

Macrophages participate in immunity, tissue repair, and tissue homeostasis, and also mediate pathologic conditions including atherosclerosis and cancer. Depending on microenvironmental cues, macrophages may adopt a pro‐inflammatory or anti‐inflammatory phenotype. Tissue stiffness influences the behavior and differentiation of cells, yet little is known about how the physical extracellular environment influences macrophage function. To study the role of substrate stiffness in determining macrophage phenotype, we cultured murine macrophages on tunable polyacrylamide gels that approximate different tissue stiffnesses. Macrophages on rigid surfaces had larger surface area and reduced circularity compared to macrophages on compliant gels. On rigid surfaces, phagocytosis was increased, but lipid uptake was decreased. Cytokine secretion differed in macrophages grown on compliant versus rigid surfaces. Differences in response to the toll‐like receptor ligands CpG oligonucleotide and lipopolysaccharide were also noted. The distribution of vinculin, a component of mature focal adhesions that mediate mechanosensing, was punctate in macrophages grown on glass and diffuse in macrophages grown on compliant gels, suggesting a fundamental difference in the quantity and/or character of the focal adhesions. Together, our studies demonstrate that the mechanical properties of the extracellular environment have a direct impact on macrophage behavior and function. Furthermore, these findings have important implications for a variety of pathologic conditions that involve both macrophages and altered tissue stiffness such as fibrosis, atherosclerosis, and neoplasia.

The studies were supported by NIH T32 Comparative Medicine Training Grant.
NMR plasma metabolome discriminates subjects with dengue infection from non‐specific febrile infection
Authors: El‐Bacha Tatiana , Struchiner Claudio , Almeida Fabio , Da Poian Andrea
Published: June 2021

Brazil has a very high incidence of dengue. In 2013, approximately 1,500,000 cases of dengue infection, which presents no specific treatments,and over 200 deaths were reported. We aim to investigate plasma metabolome of subjects presenting symptoms of dengue fever (DF; mild), dengue hemorrhagic fever (DHF; severe), and subjects with non‐specific infection (NS). Samples were obtained in a cohort with dengue patients in Recife, Brazil. Plasma metabolomic analyzes were carried out by 1H Nuclear Magnetic Resonance. Using multivariate analysis, plasma metabolome from NS was discriminated from DF and DHF subjects. Additionally, DF plasma metabolome presenting secondary Dengue infection varies during the course infection. Candidate metabolites that contributed to the discriminatory pattern include β‐hydroxybutirate, citrate, succinate, tyrosine and valine. Discriminating NS from dengue infection through plasma NMR‐based metabolomics indicates that these analyzes could provide insights into the underlying molecular mechanisms of dengue pathogenesis. Moreover, following plasma metabolome during the course of dengue infection could help discriminating individuals at risk to develop severe infection and to predict the disease outcome.
The Interaction of the LL‐37 Peptide with CpG DNA Oligonucleotides
Authors: Avelsgard Irmamarie , Craig Maria
Published: June 2021

LL‐37, a member of the cathelicidin family of innate immune peptides, forms complexes with extracellular self‐DNA and aids in the transport of DNA across cell membranes. LL‐37/DNA complex formation and DNA internalization by immune cells activates a series of events that lead to an autoimmune response. We have used electrophoretic mobility shift assays (EMSA) and western blotting to examine the details of complex formation between LL‐37 and 24‐bp DNA fragments containing CpG sequences. We observe by EMSA that increasing amounts of CpG DNA are shifted as the concentration of LL‐37 increases. Quantitative analysis of DNA bands in the EMSA gels has allowed us to construct binding curves for the formation of LL‐37/DNA complexes. The curves indicate that a strong cooperative effect in the binding process leads to tight binding with low apparent dissociation constants for CpG DNA above an LL‐37 concentration of about 20 µM. Complementary experiments in a THP‐1 monocytic cell model investigate the uptake of LL‐37 and LL‐37/DNA complexes.
Biophysical Characterization of TCR Variants with Reengineered Specificity and Affinity
Authors: Singh Nishant , Pierce Brian , Weng Zhiping , Baker Brian
Published: June 2021

Autologous cytotoxic T‐cells, CD8+ T‐cells, recognize and target certain cancer cells through an interaction between T‐cell receptors (TCR) and antigen bound major histocompatibility complex (MHC complex) on the target cells. This phenomenon has led to advent of adoptive immunotherapy for melanoma. In this therapy, T‐cells from the patient are genetically engineered to express TCRs that respond specifically to melanoma associated antigens and then reintroduced into patients. The first set of clinical trials examining this approach in humans utilized DMF4 and DMF5 TCRs, and showed that DMF4 TCR led to 13% tumor regression, whereas DMF5 TCR led to 30% tumor regression. Upon biophysical characterization it was shown that the improvement in immunogenicity from DMF5 is due to its higher affinity towards the cognate melanoma antigen. This made higher affinity TCRs therapeutically relevant. Traditionally higher affinity TCRs have been generated via in vitro selection techniques. However, in vitro selection methods have inherent limitations. Most notably, gaining peptide independent TCR binding. Structure guided design (SGD) provides an alternative technique for developing therapeutically relevant TCRs. It is an in silico method that combines structural and biophysical information with a modeling software to generate high affinity TCR variants, allowing for highly controlled manipulation of affinity and specificity. While SGD of TCRs is promising, it is in early stages of development and requires further optimization. Structural and biophysical characterization of TCR variants that have high affinity or re‐engineered specificity will help improve TCR SGD.
Immunoglobulin Knockout Chickens Lack Detectable Circulating IgA in Response to Keyhole Limpet Hemocyanin Vaccination
Authors: Bolek Kevin , Klasing Kirk
Published: June 2021

Immunoglobulin knockout chickens have a unique potential for use as a research tool. One could examine the contribution of immunoglobulins to pathogen resistance or further characterize the nutritional implications of mounting an immune response. Researchers have attempted to examine the effects of eliminating antibody production in chickens by ablation of the bursa of Fabricius. These techniques cause a loss of patency of the cloaca causing leakage of rectal contents. This results in continuous diarrhea and complications with dehydration and hygiene. Thus, knockout chickens are a superior model because the bursa is maintained, which eliminates the hygienic complications, while also eliminating immunoglobulin production. We examined chickens that were either heterozygous or homozygous for a knockout of the immunoglobulin heavy chain J gene segment. It was previously shown that heterozygotes but not homozygotes produce circulating IgY and IgM in response to vaccination, but IgA was not characterized. The goal of the present study was to affirm that IgA producing cells are also absent in the homozygotes. At 4 weeks of age, birds were injected intramuscularly with 300µg of keyhole limpet hemocyanin mixed 1:1 with complete Freund's adjuvant. A booster was administered at 5 and 6 weeks of age using incomplete Freund's adjuvant. Serum was collected via wing vein puncture at 6 and 8 weeks of age for IgA detection using a sandwich ELISA. It was found that circulating IgA was present in heterozygotes, but homozygotes had no detectable IgA concentrations. In conjunction with previous research showing homozygotes also lack circulating IgY and IgM, these results confirm that homozygotes lack all major classes of circulating immunoglobulin known in chickens.
In Vitro Modeling of Extracellular Trap Release and Association with LL‐37 Peptide in THP‐1‐Derived Macrophages
Authors: Stone Sophia , Craig Maria
Published: June 2021

Leukocyte‐derived extracellular traps (ETs) are DNA webs coated with histones and antimicrobial peptides that ensnare and kill bacteria in a phagocytosis‐independent manner. The cell death pathway resulting in ET release (ETosis) has been studied extensively in neutrophils, but it is now known to occur in other immune cell types via similar mechanisms. Antimicrobial peptide LL‐37 is a potent DNA‐binding peptide that facilitates ETosis in neutrophils and protects neutrophil ETs (NETs) from degradation by bacterial nucleases. Here, quantification of extracellular DNA was performed using immunocytochemistry and fluorimetric analysis to study immunogenic species that may activate ETosis in THP‐1 monocyte‐derived macrophages. We will also investigate a potential role for LL‐37 in the formation and antimicrobial activity of monocyte/macrophage ETs (METs). Shared ETotic mechanisms exist between multiple leukocyte lineages, but cooperative targeting of diverse microbial species via specialized mechanisms could contribute to broad‐spectrum innate immune defense, as well as inflammatory tissue damage in human diseases.

This work was supported by a grant from the TriBeta Research Scholarship Foundation.
Investigation of the Radical SAM Enzyme CDG Synthase
Authors: Bruender Nathan , Dowling Daniel , Drennan Catherine , Bandarian Vahe
Published: June 2021

7‐Deazapurine containing secondary metabolites and hypermodified tRNA bases are produced in a variety of marine and terrestrial organisms. Although these 7‐deazapurines vary in both their structure and biological roles, they share a common precursor, 7‐cyano‐7‐deazaguanine (preQ0), which is derived from GTP through the action of four enzymes. CDG synthase (QueE) catalyzes the penultimate step, which generates the 7‐deazapurine moiety. QueE is a member of the radical S‐adenosyl‐L‐methionine (SAM) superfamily and catalyzes a complex radical‐mediated ring contraction to convert 6‐carboxy‐5,6,7,8‐tetrahydropterin (CPH4) into 7‐carboxydeazaguanine (CDG). This paper will discuss the structure function relationships that are emerging from studies of this fascinating transformation. This work was supported by NIH grant GM72623 to VB.
SIRT5 Mutants Reveal Insights into Sirtuin Regulation
Authors: Yu Junru , Mueller Dustin , Singh Raushan , Haldar Manas , Sanku Mallik , Srivastava D.K.
Published: June 2021

Sirtuins are emerging as key regulators for metabolism and aging. These regulations were primarily attributed to sirtuin's NAD+ dependent deacetylase activity. Among the seven human sirtuins, SIRT5 is the only one that also catalyzes desuccinylation. It is becoming clear that sirtuins have evolved to adapt different regulatory mechanisms of their activities. However, detailed studies on substrate specificity, isozyme selectivity, and inhibition mechanisms of sirtuins are still lacking. Two amino acids—Y102 and R105—are responsible for SIRT5′s desuccinylation activity. Therefore, we made single and double muatations on the two residues and compared their steady‐state kinetic features with those of wild type SIRT5 and SIRT1. We noticed that Y102 and R105 account partially for the differential substrate specificity and drug isoform selectivity of SIRT5, implying that sirtuin activity is also regulated by the dynamic flexibility in the protein structures. Our inhibition analysis showed that nicotinamide inhibited SIRT5′s desuccinylation activity by competing with NAD+; while it inhibited SIRT1′s and SIRT5′s deacetylation activity via a combination of two pathways—competing with NAD+ and base‐exchange. Combining it with our bisubstrate kinetic analysis, we conclude that the binding of succinylated substrate creates a tighter binding site for both NAD+ and nicotinamide, generating alternative nicotinamide regulations of SIRT5. Our research studies provide insight into Sirtuin regulation andthe rationale designing of sirtuin isoform selective inhibitors.
Towards Optimization of a General RNA Labeling Deoxyribozyme: Characterization of a Unimolecular Deoxyribozyme
Authors: Ashton Matthew , Carrocci Tucker , Hoebartner Claudia , Hoskins Aaron
Published: June 2021

Using fluorescence microscopy as a tool to study biological systems has been proven to be useful in both experiments in cells and in vitro. However, current methods for preparing fluorescent RNAs for use in vitro fluorescence assays are challenging and inefficient. Recently, we have shown that deoxyribozymes (DNAzymes) can be used to site‐specifically label RNA molecules with fluorescent GMP moieties. We believe that this method will prove generally useful for incorporation of fluorescent labels into different sites within an RNA molecule for use in biochemical studies. We propose that the annealing of two RNA molecules (the substrate RNA and a “helper” RNA) to the DNAzyme results in formation of a ternary complex that brings key catalytic sites within close proximity in order to facilitate GMP transfer. Initial experiments suggested that this ternary complex is not stable during native PAGE analysis. I am currently studying a unimolecular RNA/DNA hybrid in which the substrate RNA, the helper RNA, and the DNA are all part of a single oligonucleotide. Our goal is to use this simplified construct to characterize the folding and reaction pathways for DNAzyme catalysis and RNA labeling. These experiments will lead to development of next generation deoxyribozymes with improved function. This work was supported by a Beckman Young Investigators award to AAH and a Hilldale Undergraduate Research Fellowship to MA.
Study of Kinetic Mechanism of Flavin‐Dependent Thymidylate Synthase from Thermotoga Maritima
Authors: Sapra Aparna , Palfey Bruce
Published: June 2021

Thymidylate, an essential DNA nucleotide, is synthesized de novo by all organisms. The enzyme thymidylate synthase catalyses the last committed step of this de novo pathway, which is the reductive methylation of 2′‐deoxyuridine‐5′‐monophosphate (dUMP) to produce 2′‐deoxythymidine‐5′‐monophosphate (dTMP). Most organism including humans, rely on classical thymidylate synthase. Recently the identification of many bacteria and human pathogens lacking this enzyme led to the discovery of flavin‐dependent thymidylate synthase (FDTS) encoded by the thyX gene. The two classes of enzyme share no structural or sequence homology and while the mechanism of classical enzyme has been studied over decades and is well established, the mechanism of thyX is still not clear. Overall, thyX receives reducing equivalents from NADPH and uses its flavin prosthetic group for redox chemistry and methylenetetrahydrofolate (CH2H4folate) as the methylene donor to produce dTMP with the concomitant release of tetrahydrofolate. Many different mechanisms have been proposed for the oxidative half‐reaction of this enzyme. One of the major questions involving its catalytic mechanisms is the activation of the pyrimidine substrate. Here we tested the feasibility of a recently published mechanism by synthesizing one of the proposed intermediates. We also present kinetic and spectroscopic data that provides more details into the kinetic mechanism of this flavoenzyme.
Dramatic Conformational Flexibility of Carbon Monoxide Dehydrogenase/Acetyl‐CoA Synthase Revealed by Electron Microscopy
Authors: Brignole Edward , Wittenborn Elizabeth , Thompson Samuel , Ragsdale Stephen , Drennan Catherine
Published: June 2021

Carbon monoxide dehydrogenase/acetyl‐CoA synthase (CODH/ACS) is a 310‐kDa bifunctional enzyme involved in anaerobic fixation of atmospheric carbon (CO2) by acetogenic bacteria. The CODH subunit reduces CO2 to CO, which passes through a 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 transfers the acetyl product to coenzyme A (CoA). Structures of CODH/ACS previously observed by X‐ray crystallography indicate that ACS must open to accommodate methyl transfer from CFeSP, but the full extent of that motion was not visualized. To examine the conformational flexibility of CODH/ACS, we performed single‐particle electron microscopy (EM) on anaerobically prepared samples. The resulting structures reveal never before seen conformations of the ACS subunit, suggesting that dramatic movements are involved in the methyl transfer from CFeSP to the ACS subunit of CODH/ACS.
Diacyltransferase Activity and Chain Length Specificity of Mtb PapA5 in Synthesis of Alkyl Beta‐Diol Lipids
Authors: Touchette Megan , Bommineni Gopal , Delle Bovi Richard , Gadbery John , Metz Thomas , Martin Dwight , Sampson Nicole , Miller Todd , Tonge Peter , Seeliger Jessica
Published: June 2021

Phthiocerol dimycocerosate (PDIM) is a Mycobacterium tuberculosis (Mtb) outer membrane, wax ester lipid implicated in Mtb virulence. This work provides a better understanding of how PDIM biosynthesis occurs in Mtb by studying PapA5, an acyltransferase responsible for esterifying phthiocerol with mycocerosic acid. We show using a series of synthetic substrates that PapA5 has a preference for longer alkyl chains and under novel biphasic reaction conditions is capable of catalyzing two successive acylation reactions, consistent with its proposed role in modifying both hydroxyls on phthiocerol. The introduction of bulky amino acids in the putative substrate‐binding pockets of PapA5 yielded partial or complete inhibition of product formation helping to confirm substrate‐binding channel assignment. Furthermore, the introduction of a salt bridge or a disulfide bond to constrain a hypothesized flexible helix led to partial inhibition of product. These data support a role for conformational flexibility in ligand binding and/or product release. Finally, in vitro phosphorylation of PapA5 did not alter activity and the modified residues were localized to a disordered loop suggesting PapA5 phosphorylation may regulate protein interactions.
Seeing Is Believing: Visualizing Hydrolysis and Inhibition of an Epoxide Hydrolase Virulence Factor
Authors: Hvorecny Kelli , Bahl Christopher , Bomberger Jennifer , Moreau‐Marquis Sophie , Kitamura Seiya , Morisseau Christophe , Hammock Bruce , Stanton Bruce , Madden Dean
Published: June 2021

Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the US, results in poor mucociliary clearance and increased lung colonization by opportunistic pathogens, such as Pseudomonas aeruginosa (Pa). Pa drives a decrease in levels of the ion channel CFTR, which regulates the fluid balance at the air‐liquid interface of the lungs for proper mucociliary clearance. Pa acts on CFTR through the secretion of an epoxide hydrolase, CFTR Inhibitory Factor (Cif). Here we show that Pa decreases mucociliary transport in vitro. Deletion of the Cif gene from Pa prevents this decrease. Cystic fibrosis patients, a group chronically infected with Pa, harbor serum antibodies to Cif, indicating that Pa secretes Cif in situ. Further work ties Cif's hydrolase activity to the decrease of CFTR and thus to the observed mucociliary transport defect. To gain an understanding of Cif's substrate selectivity and enzyme kinetics, we employed hydrolase null mutants and small molecule inhibitors. Utilizing a combination of X‐ray crystallography and fluorogenic reporter assays, we can describe Cif's steric requirements for substrates and the cross talk occurring between the monomers of the dimer during hydrolysis and inhibition. This work provides a rare look into the enzymatic intermediates of a bacterial toxin, and produces structural insights key to the development of second‐generation inhibitors with the potential to act as a complement to antibiotic and disease therapies

Funding: NIH and CFF
Incorporation of Azide Functionality into Recombinant Protein via Recombinant, Enzymatic, and Chemical Approaches for Bio‐Orthogonal Modification
Authors: Wang Lin , Jiang Rui , Wang Lin , Sun Xue‐Long
Published: June 2021

Introducing unique functional group into proteins is an attractive approach for site‐specific protein modification applications. In this study, we systemically investigated four different strategies to introduce azide functionality into recombinant thrombomodulin (TM), site‐specifically by recombinant, chemical, and enzymatic methods at either N‐terminus or C‐terminus for its bio‐orthogonal modification application. The first method is to express near C‐terminus azido‐TM by unnatural amino acid expression. The second method is to incorporate azide molecule into the C‐terminus of recombinant TM via sortase‐mediated ligation (SML). The third method is to add azide molecule to the N‐terminal amine of recombinant TM via amidation chemistry, and the fourth method is to introduce azide to recombinant TMvia a tyrosine selective three‐component Mannich reaction. The azido‐TMderivatives obtained in all four methods were successfully applied for site‐specific modification and immobilization of recombinant TM through copper‐free click chemistry with upholding their protein C activation activities. In comparison, incorporating azide molecule into the C‐terminus of recombinant TM via SML affords highest overall yield since the key protein expression step uses all natural amino acids. Also, singly site modification and immobilization facilitate the highest TM activity. Overall, the reported site‐specific azide incorporation strategies can be widely used in chemical biology and biomedical research and applications.
Yeast Env9 is a Conserved Oxidoreductase Involved in Lipid Droplet Biogenesis
Authors: Siddiqah Ikha , Manandhar Surya , Gharakhanian Editte
Published: June 2021

Interactions between organelles and trafficking of cellular materials are finely regulated to maintain cellular integrity. The complexity of such events is highlighted in the endomembrane system, which is comprised of the endoplasmic reticulum (ER), Golgi apparatus, late endosomes, lysosomes, lipid droplets, and plasma membrane. As endomembrane system regulation are conserved between yeast and human, yeast serves as an ideal model for its study. Yeast ENV9 is a novel gene involved in vesicular trafficking. Previous characterization by our lab revealed that ENV9 deletion leads to defects in a lysosomal enzyme processing, increase in vesiculated vacuoles, and caffeine sensitivity. Our bioinformatic studies show that ENV9 is conserved among eukaryotes and exhibits similarity to Retinol Dehydrogenase 12 (RDH12) involved in redox reactions in visual cycle. Mutations of RDH12 have been linked to Leber Congenital Amaurosis. Using microscopic and biochemical approaches, we show localization of Env9 to lipid droplets. Here we report that ENV9 plays a role in lipid droplet morphology by positively regulating lipid droplet expansion and carbon stress‐induced lipid droplet fragmentation. Lastly, Env9 displays reductase activity towards sterol synthesis substrate 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG CoA) and toxic aldehyde 4‐Hydroxynonenal (4‐HNE). Point mutations in the conserved residues in the active (N146L) and cofactor‐binding sites (G23‐24A) of Env9 abolish its reductase activity, as reported in other retinol dehydrogenases. Thus, we report a novel oxidoreductase involved in lipid droplet morphology. ENV9 may serve as a model to study lipid droplet expansion, organelle interactions in endomembrane system, and retinal disease‐associated mutations. (NIH‐AREA #R15 GM85794‐02)
Dopamine and 5HT Transporter Amino And Carboxy Termini Contribute To Substrate Affinity And Psychostimulant Activity
Authors: Sweeney Carolyn , Holy Marion , Sitte Harald , Melikian Haley
Published: June 2021

Determining how psychoactive drugs interact with their molecular targets is essential for understanding psychostimulant abuse and pharmacotherapies. The SLC6 carriers, DAT and SERT, are the primary targets for both addictive and therapeutic psychostimulants. Recent SLC6 structures have shed light on these molecular interactions. However, these structures lack much of the carriers' N‐ and C‐termini, raising questions regarding their potential contributions to the transport mechanism. To address this question, we generated DAT/SERT chimeras that exchange the N‐, C‐, or both termini, and tested their effect on substrate uptake, ligand binding and efflux kinetics. The SERT N‐terminus decreased DAT's affinity for DA and AMPH affinity two‐fold, whereas substituting the SERT C‐terminus had no effect on DAT substrate affinities. Moreover, replacing both DAT intracellular termini with SERT domains rescued DAT substrate affinity. In contrast, substituting the DAT N‐terminus had no effect on SERT substrate affinities, whereas the DAT C‐terminus decreased SERT substrate affinities and this was not rescued when both DAT termini were substituted onto SERT. Chimeric DAT and SERT protein affinities for cocaine were unaffected by interchanging domains, suggesting that DAT and SERT tail domains contribute in substrate translocation but not ligand binding. Ongoing studies test whether these intracellular domains contribute to AMPH‐stimulated substrate efflux and catalytic turnover rates and should shed considerable light onto the differential contribution of these domains to the DA vs. 5HT transport processes.
Distinct Intracellular Trafficking Patterns of Host IgG by Herpes Virus Fc‐Receptors
Authors: Ndjamen Blaise , Bjorkman Pamela
Published: June 2021

Members of both alpha and beta herpes viruses affects 50–98% of people around the world. They cause severe symptoms in congenitally infected newborns, a lifelong latent infection that is lethal in immuno‐compromised individuals, and are associated with several types of cancer. Human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV‐1) viruses express proteins (HCMV gp68 and gp34; HSV‐1 gE‐gI) that function as Fcγ receptors (FcγRs) by binding to the Fc regions of human IgG. In addition to binding free IgG, these viral FcγRs can bind to IgG complexed with an antigen to form an antibody bipolar bridged (ABB) complex. The effects of Fcγ binding by these viral molecules are poorly understood. Although HCMV gp68 and HSV‐1 gE‐gI have an overlapping binding site on Fc, the finding that the gp68/Fc interaction is stable at pH values between 5.6 and 8.1 but that gE‐gI binds only at neutral or basic pH suggests distinct pH‐based downstream events after IgG is internalized via receptor‐mediated endocytosis into intracellular compartments. Here we developed a cell‐based in vitromodel system and performed 4D‐confocal fluorescence imaging to define the fates of ABB complexes formed by the two types of viral FcγRs. We found that alpha (HSV‐1) and beta (HCMV) herpes virus FcγRs displayed distinct intracellular trafficking patterns to target internalized ligands: HSV‐1 gE‐gI dissociates from its IgG‐antigen ligand in acidic endosomal compartments and recycles back to the cell surface (Ndjamen et al., 2014), whereas HCMV FcγRs (gp68) are transported together with IgG‐antigen complexes to lysosomes for degradation. In both cases, anti‐viral IgGs and their viral targets are selectively degraded, a potential immune evasion strategy allowing herpes viruses to escape from IgG‐mediated immune responses.
Genotyping of a p73 Dinucleotide Polymorphism in Human Cancer Cell Lines
Authors: Cordova Ricardo , Hampton Kaia , Kim Dongwha , Park Jong , Carastro L Michael
Published: June 2021

The p73 gene is a member of the p53 tumor suppressor family. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14‐to‐A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and DNp73 isoforms, respectively. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI =0.31‐0.99] for aggressive prostate cancer (PCa) and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. We hypothesize the presence of the p73 DNP allele causes altered p73 promoter usage, ultimately resulting in increased TAp73/DNp73 protein isoform ratios, which could explain the observed decreased risk for PCa aggressiveness. Our ultimate goal is to assess the potential of p73 DNP as a biomarker for lower aggressive PCa risk. Therefore, our initial goal in this study was to determine the p73 DNP genotype in four human cancer cell lines (DU 145, MDA‐MB‐468, PC‐3 and SW48) not yet characterized for p73 DNP status. We previously characterized the p73 DNP genotype of three human cancer cell lines that we used as genotyping controls: Caco‐2 (homozygous polymorphic), NCI‐H1299 (homozygous wild type), and HepG2 (heterozygous). These seven cell lines were cultured and genomic DNA was isolated and quantitated. Cell line genomic DNA samples were used in TaqMan Real Time‐PCR allelic discrimination assays to determine the p73 DNP genotypes. Our data conclusively determined that DU 145, MDA‐MB‐468, and SW48 were p73 DNP homozygous wild type, and PC‐3 was p73 DNP heterozygous.
Effects of Lung Carcinogens on PPARgamma Activity in A549 Cell Line
Authors: Carrasco Nestor , Tennis Meredith , Nield Lori
Published: June 2021

PPARgamma (PPARG) is a transcription factor that associates with retinoic acid receptor to enhance transcription of genes regulating pathways of adipogenesis, macrophage programming, growth, and inflammation. PPARg agonists are effective at preventing lung cancer in mice and cause regression of human pre‐neoplastic lung lesions. However, the mechanisms by which PPARg prevents lung cancer progression are not completely understood. Herein, we examined the effects of endogenous and exogenous PPAR agonists on expression of PPARg‐regulated genes (E‐cadherin and Ptgs2) in human lung cancer A549 cells. We treated cells with the endogenous PPARg agonist 15‐PGJ2and exogenous agonists pioglitazone (Pio) and iloprost. We also tested acrolein (component of cigarette smoke) and 4‐hydroxynonenol (4‐HNE; increased in the lungs of smokers). These molecules are chemically related to 15‐Deoxy‐Δ12,14‐prostaglandin J2 (15‐PGJ2), and 4‐HNE has been shown to enhance PPARg activity. We found that Pio, iloprost, and 15‐PGJ2increased expression of E‐cadherin at 24h and 48h of exposure. HPGD expression was increased at 24 h with Pio, iloprost, 15‐Deoxy‐Δ12,14‐prostaglandin J2, and 4‐HNE, but returned to baseline by 48 h. NFκB protein is down‐regulated in the presence of PPARg, but its gene expression increased with Pio, iloprost, 15‐Deoxy‐Δ12,14‐prostaglandin J2, and acrolein exposure at 24 h, indicating that decreased protein leads to increased transcription. The peak of agonist‐induced expression effects occurs by 24 h. Increases in expression of the Pio off‐target regulated gene HPGD with agonists other than Pio indicates that regulation of these genes is more complicated than was previously thought.
What's up with catch‐up? No evidence of population‐level catch‐up growth in children under 5 years of age when using height‐for‐age difference (HAD) instead of height‐for‐age z‐scores (HAZ)
Authors: Leroy Jef , Ruel Marie , Habicht Jean‐Pierre , Frongillo Edward
Published: June 2021

Studies conducted in past decades showed little or no population‐level linear catch‐up growth (defined as reductions in absolute height deficit) in children after 2 y of age. Recent studies, however, have reported catch‐up growth in groups of children using height‐for‐age z‐scores (HAZ). We assessed whether population‐level catch‐up growth is found when height‐for‐age difference (HAD; child's height compared to standard in cm) is used instead of HAZ.

Using data from Demographic and Health Surveys (DHS), Young Lives (YL), and the Consortium on Health‐Oriented Research in Transitional Societies (COHORTS), we compared changes in HAD and HAZ in populations of children between 2 and 5 y. We show that HAZ is inappropriate to measure changes in linear growth as populations of children age because they are constructed using SDs from cross‐sectional data, which increase with age. Using HAD, we find not only an absence of population‐level catch‐up growth, but a continued deterioration reflected in a decrease in mean HAD between 2 and 5 y; HAZ shows either no change (DHS) or an improvement in mean HAZ (YL, COHORT) as groups of children age.

We show no evidence of population‐level catch‐up in children between 2 to 5 y when using HAD. The continued widening of the height deficit after 2 y does not challenge the current focus on the first 1,000 days, but raises questions regarding prevention of continued deterioration and the potential of children to benefit from nutrition interventions after 2 y of age.

Funded by the CGIAR Research Program on Agriculture for Nutrition and Health (A4NH), led by IFPRI.
Water, Sanitation and Hygiene (WASH) and Breastfeeding Contribute to Child Stunting in Rural Malawi
Authors: Ruel‐Bergeron J , Christian P , Hurley K , Bou Monclus M , Phuka J , Wu L , Klemm R , West K
Published: June 2021

Evidence linking water, sanitation and hygiene (WASH) to stunting has been mixed but constitutes a possibly important contributor to chronic undernutrition in low and middle income countries. Using cross‐sectional baseline data of an impact evaluation of a nutrition program targeted at reducing maternal and child undernutrition, we assessed the extent to which optimal WASH practices contribute to reduced odds of stunting (length‐for‐age z‐score <‐2 SD) among children 6 to 24 months of age living in two rural districts of Malawi. We used multivariable logistic regression with robust standard error estimation to measure the relationship between stunting and a composite of six WASH indicators (measured as an unweighted score ranging from 0‐11, with scores of 0, 1 or 2 assigned for levels of optimal water source and treatment, sanitation facility and cleanliness, handwashing with soap, and safe disposal of child feces), controlling for potential health, dietary, and socioeconomic confounders. 53% of children were stunted in this population. The median (IQR) of the WASH score was 5.5 (4‐6). A high WASH score (蠅8) (OR=0.71, 95% CI 0.51‐1.00) and breastfeeding 蠅10 times per day in the last 7 days (OR=0.50, 95% CI 0.27‐0.92) were associated with a reduced odds of stunting in an adjusted analysis; diarrhea, child age, sex, and maternal education, were all significantly associated with stunting in this population. Integration of WASH and infant and young child feeding interventions in nutrition programs may contribute to reductions in stunting in Malawi. Funded by the Children's Investment Fund Foundation, UK.
Association between Fish and Seafood Consumption and DHA Levels in Cord Blood and Breast Milk in Taiwan
Authors: SU YU‐TING , WANG SHU‐LI , CHEN HSIAO‐YEN , SUN PEI‐YI , HUANG MENG‐CHUAN
Published: June 2021

Docosahexaenoic acid (DHA; C22:6n3), a long chain polyunsaturated fatty acids (LCPUFA), has positive effects in neural and visual function development in early life. It is important for developing fetus to obtain DHA through preferential placental transfer during prenatal period and later via breast milk. We aimed to evaluate the correlations between third trimester maternal intake of fish and seafood products and n‐3 LCPUFA in cord blood and breast milk. Further, we assessed the relationship between cord blood‐ and breast milk n‐3 LCPUFA. Using multivariate analysis, freshwater and marine fish intakes significantly correlated with cord blood DHA (p<0.05), and marginally associated with breast milk DHA (freshwater: p=0.065; marine: p=0.057). Additionally, processed fish products correlated with breast milk DHA (p=0.022), and shellfish with cord blood DHA (p=0.015). Furthermore, DHA (p=0.063) and n‐3 LCPUFA (p=0.029) levels in cord blood and breast milk were positively associated. Our results support that beneficial effects of consuming seafood and marine products may increase DHA transfer during pre‐ and postnatal period. Therefore, seafood and marine products consumption should be encouraged during pregnancy and lactation.
Factors Associated with Subclinical Mastitis (SCM) Among Indigenous Mothers in the Western Highlands of Guatemala
Authors: Wren Hilary , Solomons Noel , Scott Marilyn , Koski Kristine
Published: June 2021

Background

SCM is an asymptomatic inflammatory condition of the lactating breast where a breast milk ratio of Na/K>0.6 is the current indicator. Our objective was to determine maternal and infant factors associated with an increased or decreased likelihood of SCM.

Methods

Breast milk samples were collected from 105 lactating Mam‐Mayan mothers with infants < 6 mo. Inductively Coupled Plasma Mass Spectrometry measured Na and K. A structured in‐depth questionnaire for independent factors including socio‐demographic characteristics, maternal and infant health status, breastfeeding (category, initiation, feeding frequency), cultural practices and beliefs, and maternal physical activity that might be associated with SCM was administered to mothers; anthropometry of both mother and infant was measured.

Results

Fourteen‐percent had a breast milk Na/K ratio >0.6. SCM was associated with maternal age (OR=1.1, p=0.011), parity (OR=1.3, p=0.011), walking (OR=1.7, p=0.008), breastfeeding frequency (OR=1.1, p=0.024), and infant weight‐for‐age (WAZ) score (OR=0.57, p=0.049)

Conclusion

Results suggest that interventions to decrease the prevalence of SCM should be targeted to older mothers with high parity. Interestingly, breastfeeding frequency and walking were associated with SCM whereas cultural practices and beliefs were not in our population. Our findings were consistent with previous research where maternal SCM was associated with decreased infant weight.

Funding

McGill University International Mobility Travel Award
Using Videoconferencing for Lactation Consultation: An Online Survey of Acceptance among a Sample of Mothers in the United States
Authors: Habibi MF , Springer CM , Spence ML , Hansen‐Petrik MB , Fouts HN , Kavanagh KF
Published: June 2021

The objective of this study was to assess the relationship of acceptance of remote lactation consultation using videoconferencing (VC) with maternal learning styles and demographic factors among mothers of infants 蠄16 weeks old who also reported breastfeeding initiation. This cross‐sectional research design used an online survey methodology with mothers recruited from 26 randomly selected states. To date, 78 mothers have completed the survey, including responding to statements about acceptance of technology (using a 7‐point Likert scale), and completing a learning styles assessment. Preliminary analysis indicated mothers were 29.6 years old and their infants were 10.7 weeks old, on average. The sample was predominantly white (92.3%), and well‐educated (65.4% with an advanced degree) with reported incomes above 185% of the Federal Poverty Level (79.5%). Nearly half were primiparous (46.2%). Mothers generally accepted the use of VC (mean responses were all significantly greater than 4.0). No significant relationships were detected between acceptance of use of VC and any maternal demographic or learning style. Mothers in this sample appear to be relatively unconcerned about physically revealing themselves or their infants via VC, but acceptance of its use was significantly negatively related to concerns about maintaining their home as a private space. To conclude, preliminary analysis indicates general acceptance of this technology medium for lactation consultation, but providers should be aware of concerns related to home privacy. Funding Source: The University of Tennessee, Professional Development Award.
Insulin Resistance, Thyroid‐Related Hormones, and Duration of Postpartum Amenorrhea in Lactating Women
Authors: McGuire Michelle , Williams Janet , McGuire Mark
Published: June 2021

Although the mechanisms regulating fertility during the postpartum period are not well understood, maternal nutritional status and suckling behaviors have been shown to be related to duration of postpartum amenorrhea (PPA). Some data also suggest that insulin resistance may play a role. The goal of this study was to test the hypothesis that women who experience relatively shorter periods of PPA are more resistant to insulin compared to those who have longer periods of PPA. We also wished to explore the possibility that thyroid function might be related to duration of PPA. To test these hypotheses, we documented baseline serum insulin, glucose, insulin‐like glucose factor‐I (IGF‐I), triiodothyronine (T3), thyroxine (T4), and thyroid‐stimulating hormone (TSH) as well as their response to an oral glucose tolerance test (OGTT) in healthy, lactating women (n = 17). OGTT were administered at 12, 16, 20, and 24 wk postpartum, and women were divided into earlier and later ovulation groups: ovulation 蠄 25 wk (EO, n = 10) and ovulation > 25 wk (LO, n = 7). There was no interaction between ovulation group and time postpartum on any variable measured. However, there was a main effect (P < 0.05) of ovulation group on serum insulin concentrations before and after OGTT, such that EO women had higher levels than did LO women. There was also an effect (P < 0.05) of ovulation group on change in serum T3 due to OGTT, such that the EO group experienced less of a change than did the LO group. In summary, although reproductive capacity in the postpartum period may be affected by insulin and T3 concentrations, insulin resistance does not appear to play a role.
Spatiotemporal Profile of the Human Milk Proteome across Populations and Lactation Stages: GEHM Study Insights
Authors: Zhang Qiang , Lohe Kimberly , Cundiff Judy , Maria Sarah , Phillips Shay , McMahon Robert , Davidson Barbara , Morrow Ardythe
Published: June 2021

Improved understanding of temporal and geographical differences in the human milk proteome may provide novel insights into changes in milk composition and function across the globe during breastfeeding. We have compared milk proteomes among three populations (Shanghai, China, Mexico City, Mexico and Cincinnati, United States; n = 9 for each population) at two different stages of lactation (4 and 26 weeks), with all samples collected as part of the Global Exploration of Human Milk (GEHM) study. At each of the two lactation stages, the profiles of milk proteins are largely similar among the three populations. Differences were found to be greater with respect to lactation stage as opposed to population. Interestingly, proteins with significant abundance changes over time show patterns of regulation that are broadly similar among populations. Nevertheless, at each of the two lactation stages, immunoglobulins IgA2, IgG1, IgG2 and IgM are present at higher levels in milk from China and Mexico compared to those from the U.S. with fold changes between China and U.S. samples of 2.0, 1.6, 2.3 and 2.1, respectively, and fold changes between Mexico and U.S. samples of1.8, 1.3, 1.1 and 2.0, respectively. Similar observations can be made for the antimicrobial protein lactoferrin. Our findings suggest broad congruency in milk protein composition among the three populations. This study also suggests that a number of host‐defense proteins may be more abundant in human milk from developing countries. This research was supported by Mead Johnson Pediatric Nutrition Institute.
Determining the Feasibility and Acceptability of a Campus Dairy Campaign Designed to Increase College Students' Awareness of the Importance of Dairy Calcium
Authors: Rose Angela , Seger Jamie , Gillis Brooke , Kennel Julie , Gunther Carolyn
Published: June 2021

Calcium in many young adults is below the recommended intake. The objective of this study was to test feasibility and acceptability of a 32‐week campus dairy campaign in promoting awareness of the importance of calcium intake among the target audience of college students. Rooted in the Social‐Ecological Model and aligned with USDA MyPlate guidelines, tailored messages and a campaign logo were developed. Campaign pages were created for social media outlets and print materials featuring student ‘celebrities' were displayed across campus. Booths were hosted at campus events and included interactive games, student ‘celebrity’ guests, educational pamphlets, and give‐aways. Students were invited to complete online surveys at three time points throughout the campaign to assess awareness of campaign and the most effective aspects of campaign. Demographic data were also collected. Two thousand one hundred and four students completed the initial survey (5% response rate). One‐third of respondents were aware of the campaign, primarily through campus events (64%) and posters/flyers (45%). Students ranked the promotional items distributed at events and the posters/flyers as the most influential parts of the campaign. Students preferred Facebook posts that contained nutrition information concerning dairy (64%) and simple recipes to incorporate dairy into their diet (36%). A campaign designed to promote dairy calcium has potential to engage its target audience of college students. Future research will focus on the extent to which engagement in the campaign influences attitudes and behaviors concerning dairy consumption.