bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing CDK12 and CDK13 Activity in Transcription and mRNA Biogenesis in Mouse Embryonic Stem Cells
Authors: Dubbury Sara , Sharp Phillip
Published: June 2021

The RNA Polymerase II (RNAPII) C‐terminal domain (CTD) heptapeptide repeat (YSPTSPS) is dynamically phosphorylated during transcription. Serine 2 phosphorylation of the RNAPII CTD (Ser2P) occurs upon the transition to productively elongating RNAPII and extends through the gene body. Ser2P recruits mRNA processing machinery, histone modifying enzymes, chromatin remodelers, and transcription elongation factors directly to the transcription machinery consequently coupling transcription and mRNA processing. Recently, two paralogous kinases in mammals, CDK12 and CDK13, were identified as members of the family of cyclin‐dependent kinases responsible for Ser2P. Additionally, CDK12 has been identified as a tumor suppressor in several tumor types. However, the function of each kinase in regulating transcription and mRNA biogenesis remains unclear.

Here, we report the establishment of a genetic system to interrogate CDK12 and CDK13 activity. CDK12 or CDK13 knockouts were generated in mouse embryonic stem cells using CRISPR/Cas9 genome editing. The rescue of knockout cells with an inducible CDK12 or CDK13 epitope‐tagged expression system permits high‐confidence genomic characterization. Currently RNA sequencing (RNA‐seq) and Chromatin Immunoprecipitation Sequencing (ChIP‐seq) with antibodies specific for phosphorylated and total RNAPII, CDK12, and CDK13 are being performed in knockout and rescued cells. The derivation of this cell system and progress in CDK12 and CDK13 characterization will be presented.

NIH RO1‐CA133404 to P.A. Sharp, NIH Pre‐Doctoral Training Grant T32GM007287 to MIT Biology Dept., and MIT School of Science Fellowship in Cancer Research Award to S. Dubbury supported this work.
Post­translational Modifications Promote Formation of SOD1 Oligomers With Potential Toxicity in ALS
Authors: Dokholyan Nikolay , Redler Rachel , Proctor Elizabeth , Ding Feng , Caplow Michael
Published: June 2021

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) causes a subset of cases of familial amyotrophic lateral sclerosis (FALS) through structural destabilization of SOD1, which leads to misfolding and aggregation. The non‐heritable (sporadic) nature of most cases of ALS and the late onset of symptoms, even for patients expressing disease‐linked mutant proteins throughout life, suggest a significant role of environmental factor(s) in ALS etiology. We have previously found that SOD1 from human tissue is extensively post‐translationally modified, with the most prevalent modifications being glutathionylation at Cys‐111 and phosphorylation at Thr‐2. We further found that glutathionylation significantly destabilizes SOD1WT dimers, increasing the equilibrium dissociation constant approximately 1,000‐fold, such that its stability is comparable to the A4V FALS‐causative mutation. SOD1A4V is further destabilized by glutathionylation. Using computational structural modeling, we show that glutathionylation has distinct effects on different ALS‐linked SOD1 mutants, which correspond to changes in composition of the dimer interface. Glutathionylation also stabilizes an intermediate misfolded species in which the b‐barrel core of each monomer is “loosened”. These findings suggest a mode by which the cellular environment impacts the propensity of SOD1 to adopt toxic misfolded conformations in ALS.
Effect of Glucocerebrosidase and Its Substrate, Glucosylceramide on α‐Synuclein Aggregation and Degradation
Authors: McGlinchey Ryan , Lee Jennifer
Published: June 2021

α‐Synuclein (α‐syn), is a presynaptic protein widely known for its amyloid formation properties and its involvement in Parkinson disease (PD) and Lewy body disorders. Recent genetic studies point to a correlation between mutations in the gene GBA1, which encodes for glucocerebrosidase (GCase), a lysosomal hydrolase, and the development of PD. The deficiency of GCase is the cause of a lysosomal storage disorder, Gaucher disease (GD), where glucosylceramide (GlcCer), the substrate of GCase accumulates. Recently, we discovered a physical interaction exists between α‐syn and GCase under lysosomal‐like conditions. Since α‐syn is mostly degraded in the lysosome, this protein‐protein interaction offers potential insight into the connection between PD and GD. In this study, we explored the effect of this interaction on α‐syn aggregation. Initial data suggest that α‐syn aggregation is accelerated in the presence of low amounts of GCase, but inhibited with higher amounts of enzyme. We have begun to investigate the effect GlcCer has on α‐syn turnover by lysosomal cathepsin proteases in order to determine whether the observed inverse relationship between GCase and α‐syn levels are associated with impaired α‐syn degradation in the presence of increased GlcCer.
Mitochondrial Ribosome Profiling Reveals Rapid and Dynamic Translation Regulation During Yeast Mitochondrial Biogenesis
Authors: Couvillion Mary , Shipkovenska Gergana , Tye Blake , Churchman L Stirling
Published: June 2021

Regulation of mitochondrial gene expression is essential to prevent accumulation of harmful intermediates at respiratory chain reaction centers that cause aging phenotypes and disease. Translation in mitochondria is carried out by dedicated ribosomes (mitoribosomes) that are distinct from cytoplasmic ribosomes. The mitochondrial translational response to environmental and developmental changes is currently unexplored because tools have not been developed for quantitative, time‐resolved studies in vivo. Genomic approaches have typically overlooked the mitochondrial genome either through experimental design or through biased bioinformatics approaches. We have developed mitoribosome profiling, an approach to determine quantitative, genome‐wide measures of mitochondrial translation at codon resolution. Applying mitoribosome profiling to study gene expression during mitochondrial biogenesis in S. cerevisiae, we find that translation occurs in the mitochondria even under conditions in which the translation products do not accumulate, indicating that the relative contributions of protein synthesis and turnover change depending on environment. As mitochondria differentiate when cells shift from glycolytic to oxidative metabolism, mitoribosomes are redistributed across mRNAs to regulate expression in a precise temporal pattern. Respiratory complexes are unique in the cell as their proteins are encoded by two separate genomes. To understand at a global level how mitochondrial and nuclear protein synthesis and turnover are coordinated we are now quantifying cytoplasmic translation using ribosome profiling, as well as the cellular proteome using tandem mass tags, during mitochondrial biogenesis.
Molecular Basis for Substrate and Inhibitor Interactions with the Glycine Transporter, GlyT2
Authors: Vandenberg Robert , Carland Jane , Edington Ashleigh , Ryan Renae
Published: June 2021

The glycine transporter GlyT2 regulates synaptic glycine concentrations to control inhibitory neurotransmission. We aim to understand how glycine and GlyT2 inhibitors interact with the transporter. We have used the crystal structure of the leucine transporter, LeuT, to better understand the structural basis for GlyT2 function. Mutations of residues in GlyT2, that correspond to the leucine binding site of LeuT, change substrate selectivity of the transporter. For example, W482F changes GlyT2 from being selective for glycine to a promiscuous transporter that allows transport of Alanine, Valine, Leucine, Cysteine, Serine, Tyrosine and Phenylalanine. These results suggest that this site forms the primary substrate binding site and other secondary sites are not required for determining substrate selectivity.

ALX1393 is a GlyT2 inhibitor that in analgesic in animal models of pain. Inhibition of GlyT2 by ALX1393 causes a reduction in maximal rate of glycine transport and also increases the K0.5 for glycine demonstrating that it has characteristics of both a competitive and non‐competitive inhibitor. 7 mutations, that alter substrate selectivity of GlyT2, cause 4‐20 fold reductions in potency of ALX1393 inhibition confirming the overlap in substrate and inhibitor binding sites. The outward facing conformation of LeuT contains an aqueous accessible vestibule, which has been proposed to form a low affinity binding site for non‐competitive inhibitors. We introduced a variety of mutations in this vestibule that may have the capacity to interact with ligands, but none of the 10 mutations caused any change in the potency or selectivity for glycine or ALX1393 inhibition. We conclude that the external vestibule does not contribute to the inhibitor or substrate binding sites.
Comparative Proteomics of Amylolytic Strains of Bacillus subtilis and Bacillus licheniformis
Authors: Adeyanju Muinat , Adebawo Olugbenga
Published: June 2021

Bacillus is known to be one of the most important sources of proteins with industrial applications. Not much is known about the local strains of this genus in Nigeria. This study aimed at investigating the comparative proteomics of the intracellular and extracellular proteins from B.subtilis and B. licheniformis to identify proteins of industrial importance. B. subtilis and B.licheniformis strains earlier isolated were reactivated and used in this study. The organisms were incubated at 37oC and 50oC. The intracellular and extracellular proteins were extracted from the cells. The proteomes were resolved using one dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and two dimensional differential gel electrophoresis. The proteins were further analysed using Decyder 2D version 6.0 software and the selected spots were subjected to Trypsin digestion. The peptide‐fragments were eluted into Linear Trapped Quadrupole Mass Spectrometer (LTQ‐MS/MS) and the RAW files obtained were searched against the database of the B. subtilis and B. licheniformis fasta files. The results obtained revealed that B. subtilis at 37oC secreted alpha‐amylase and YmcB protein extracellularly while the prominent intracellular proteins were bacitracin synthetase and glutathione peroxidase. In contrast, the prominent extracellular proteins secreted by B. licheniformis at 37oC were leucine aminopeptidase precursor and bacitracin transport ATP binding proteins while the intracellular proteins at 37oC were homoserine‐O‐succinyl transferase and bacitracin transport ATP binding protein. B. licheniformis grew at 50oC and produced many proteins. It was concluded that B. licheniformis was a more versatile producer of industrial proteins.
A Scoring Function for Motif Template Alignments with ProMOL
Authors: Madha Shariq , Kovuri Venkata Aditya , Bryan Michael , Craig Paul , Bernstein Herbert
Published: June 2021

ProMOL is a 3D motif alignment plugin for PyMol, the molecular visualization tool. The tool compares the structures of proteins against a library of known motifs to attempt to assign function to the protein. By using a scoring function to compare the results, it is possible to better discriminate between true and false positives. The scoring function uses a 3‐dimensional matrix to score how likely residues are to be a certain distance from the each other. This matrix was developed using empirical data retrieved from multiple pdb files determining the frequencies of residues distances. It was shown using discriminant analysis that the scoring function was able to accurately rank the obtained matches found by ProMOL. Funding was provided by RIT, Dowling College, and NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1, and 3R15GM078077‐02S2.
Müller Cells Regulate Notch Signaling And Retinal Angiogenesis Via The Soluble Epoxide Hydrolase‐Dependent Generation Of 19,20‐Dihydroxydocosapentaenoic Acid
Authors: Fleming Ingrid , Hu Jiong , Popp Rüdiger , Frömel Timo
Published: June 2021

Soluble epoxide hydrolase (sEH) is a promising pharmacological target based on its enzymatic function of metabolizing potent bioactive substrates, epoxyeicosatrienoic acids (EETs), epoxydocosapentaenoic acids (EDPs) and other lipid epoxides to their corresponding diols. Despite many studies characterized the proangiogenic and anti‐inflammatory effect of EETs, the biological function of sEH in angiogenesis in vivo remains unclear. We found that the retinal angiogenesis was significant delayed in sEH‐/‐ mice and was associated with reduced tip cell numbers and filopodia. This phenotype was associated with the induction of the Notch‐dependent transcription factors Hes1 and Hey1 and attenuated endothelial cell proliferation. sEH was mainly expressed in Müller glia cells and Müller glia cell specific sEH knockout mice displayed delayed retinal angiogenesis similar to sEH‐/‐ mice. Lipid profile analyses revealed a significant decrease of the sEH metabolite 19,20‐dihydroxydocosapentaenoic acid (DHDP) in sEH‐/‐ retinas compared to WT littermates. 19,20‐DHDP suppressed Notch signaling via inhibition of γ‐secretase in vitro, as well as rescued retinal vasculature defects in a Notch overactivating mutant (Fbxw7iEC) in vivo. Moreover, intravitreal injection of 19,20‐DHDP significant increased tip cell and filopodia number as well as primary vessel network density in sEH‐/‐ mice retina. Our data demonstrated that Müller glia cells are involved in the development of the superficial retinal vasculature by secreting 19,20‐DHDP which inhibits γ‐secretase mediated Notch signaling.
Effect of oxidized linoleic acid on plasma apoC 3 levels in C57BL mice
Authors: Ainsworth Gregory , Mahini Halleh , Alaimo Hope , Jamil Naseeha , Garelnabi Mahdi
Published: June 2021

Background

We have previously shown that oxidized linoleic acid significantly lower plasma triglyceride (TG) levels as compared to animals fed oleic acid, the changes was associated with increased APOA5 and acetyl‐CoA oxidase genes expression in the mice. The focus of the current study is to determine the mechanisms and plasma and tissue metabolites associated with the plasma triglycerides decreased levels.

Study Design

We fed C57BL6 normal male mice 4 groups (N=10 each) 4 different types of mouse diet formulations as follows: normal mouse chow (P), normal chow supplemented with 18 mg/day linoleic acid (C), oxidized linoleic acid 9 mg (A) or 18 mg /animal/day) for 2 months. Mice were sacrificed; blood and tissues (liver, adipose, kidney, and heart) were collected. We measured the plasma lipids, performed Western Blot for the Apo5, ApoC3 and related proteins expression.

Results and Conclusion

This is a going study, we currently completed the plasma APoC3 measurements; as anticipated our data have shown a significant dose dependent decreases in plasma ApC3 on mice fed low (P<0.01) and high (P<0.003) dose oxidized linoleic acid compared to the control group on linoleic acid. These finding indicate for the first time that intake of oxidized linoleic acid decreases plasma ApoC3 levels and modulatesplasma TG..
The Interplay between Cholesterol and Vitamin D Biosynthesis
Authors: Prabhu Anika , Luu Winnie , Sharpe Laura , Brown Andrew
Published: June 2021

Cholesterol is dangerous to human health in excess, but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to balance cholesterol levels. 7‐dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis, acting on 7‐dehydrocholesterol (7DHC) to produce cholesterol. In the absence of DHCR7, a lack of cholesterol production leads to the devastating developmental disorder, Smith‐Lemli‐Opitz Syndrome (SLOS).

We examined human DHCR7 at the transcriptional level to characterize its promoter and identified two sterol regulatory elements (SREs) that work cooperatively to activate DHCR7 expression [Prabhu et al. BBA Lipids 1841 (2014) 1431‐9]. This method of cooperativity ensures that there is high demand for cholesterol before resources are committed to the energetically expensive process of cholesterol synthesis. Although this is similar to other cholesterol synthetic enzymes we have studied, DHCR7 differs at the post‐translational level.

We found that unlike other enzymes involved in the final stages of cholesterol synthesis (e.g. DHCR24), DHCR7 is rapidly turned over. This degradation is induced by the cholesterol product itself, resulting in decreased DHCR7 protein levels and activity, resulting in 7DHC accumulation. As 7DHC is a precursor to both cholesterol and Vitamin D, we have also shown that in conditions of high cholesterol, DHCR7 degradation is associated with increased Vitamin D production. Investigating these unique modes of regulation of DHCR7 is important for further understanding SLOS etiology, and the interplay between cholesterol and Vitamin D biosynthesis.

This work is supported by funding from the National Health and Medical Research Council (NHMRC).
Acrolein Exacerbates HAART–Induced Apoptotic Death in Hepatocytes by Enhancing Transcriptionally Permissive Epigenetic Modifications at FasL Promoter
Authors: Ghare Smita , Donde Hridgandh , Joshi‐Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

Highly active antiretroviral therapy (HAART) ‐ associated hepatotoxicity is becoming a major clinical problem leading to morbidity, mortality and treatment discontinuation in HIV patient. Environmental/dietary factors are known to significantly impact general health as well as therapeutic outcomes. Acrolein is one such common dietary and environmental pollutant and is also generated endogenously by cellular metabolism. The present study examines the hepatotoxic interactions of acrolein with HAART drug, azidothymidine (AZT).

Human hepatoma cells (HepG2) as well as rat primary hepatocytes were used to examine the cytotoxic effects of acrolein (ACR) and AZT treatment either individually or in combination (AZT+ACR). Data showed that acrolein enhances expression of FasL gene, leading to increased apoptosis in AZT treated cells. Chromatin immunoprecipitation (ChIP) analysis revealed that ACR+AZT induced enhanced FasL transcription was mediated by increased in promoter histone H3K9 acetylation allowing recruitment of NFkB and RNA Polymerase II at the FasL promoter. Notably, acrolein scavenger, hydralazine significantly attenuated acrolein induced transcriptionally permissive modifications at the FasL promoter preventing FasL expression and hepatocyte cell death. Overall, the data suggest that environmental and/or dietary exposure to acrolein can induce chromatin modifications and further exacerbate the hepatotoxic effects of HAART medication affecting treatment options for HIV patients. This work was supported by NIH grants.
Role of mTOR and ERK in PMT‐induced Cell Proliferation and Survivin Upregulation
Authors: Oubrahim Hammou , Bacsik David , Chock P
Published: June 2021

Bacterial virulence factors that interfere with cell signaling and result in disruption of normal cell division and apoptosis could facilitate cancer metastasis via unknown mechanisms. Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage‐independent growth for certain type of cells. These properties suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections. We have shown that rPMT induces protein and ATP synthesis, cell migration and proliferation in serum‐starved Swiss 3T3 cells by hijacking cellular signal transduction pathways via deamidation of heterotrimeric G‐proteins and leads to a sustained activation of mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, which in part, leads to cell proliferation and migration. Furthermore, survivin , a subunit of chromosomal passengers complex also known as inhibitor of apoptosis protein, was upregulated in PMT treated 3T3 cells. Normal survivin level is undetectable in differentiated tissue but highly expressed in most human tumors. PMT induced upregulation of survivin is mediated by activating both ERK and mTOR signaling pathways, at least at the transcriptional level.
Nuclear localization and stabilization of GPS2 depend on arginine methylation and interaction with the cofactor TBL1
Authors: Huang Jiawen , Johnson Holly , Chan Michelle , Lentucci Claudia , Cardamone Maria , Perissi Valentina
Published: June 2021

G‐protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of different metabolic organs. First identified as part of the N‐CoR corepressor complex, GPS2 has been shown to be an important cofactor in the nucleus for mediating transcriptional regulation and meiotic recombination. In addition, we recently reported that GPS2 plays a key non‐transcriptional role in the cytosol where it regulates the pro‐inflammatory tumor necrosis factor alpha (TNFα) pathway by directly interacting with the ubiquitin conjugating enzyme Ubc13 and inhibiting its enzymatic activity. While these observations suggest that GPS2 localization is an important determinant of its molecular function, a clear understanding of GPS2 differential targeting to specific cellular locations is still missing.

Here we show that GPS2 nuclear localization is tightly regulated by a fine balance between stabilization and protein degradation, with the interaction between GPS2 and the exchange cofactor TBL1 being required to protect GPS2 from proteasomal degradation and to mediate its nuclear localization. Moreover, our data data indicate that arginine methylation on R323 by PRMT6 plays a critical role in regulating GPS2 stability. Thus, our findings together further support the idea that posttranslational modifications, including ubiquitination and arginine methylation, are necessary to regulate GPS2 molecular function in vivo.
Understanding the Mechanisms that Lead to Dynamic Stress‐Induced O‐GlcNAcylation; Identification of the Stress‐Dependent Interactome of the O‐GlcNAcase
Authors: Groves Jennifer , Maduka Austin , O'Meally Robert , Cole Robert , Zachara Natasha
Published: June 2021

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification of intracellular proteins thought to regulate protein function in a manner analogous to phosphorylation. The addition and removal of O‐GlcNAc is catalyzed by the O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA), respectively. O‐GlcNAc is induced in response to many forms of cellular stress and injury in ex vivo, in vivo, and in vitro models. Suggesting that O‐GlcNAc regulates cell survival decisions, elevation of O‐GlcNAc levels prior to or after injury is protective. However, little is known about how the cell communicates with OGT and OGA during times of stress to regulate pro‐survival signaling networks. One mechanism by which these enzymes may be regulated is via protein‐protein interactions that alter their expression, activity, and/or targeting. The goal of this study is to define the stress‐dependent interactome of OGA using the BioID technique. We have fused full‐length OGA to the N‐ or C‐terminus of a mutant E. coli biotin ligase, which will promiscuously biotinylate interacting partners in vivo. These fusion proteins express and localize properly in U2OS cells, possess hexosaminidase activity, and biotinylate proteins differentially in response to oxidative stress. Numerous stress‐dependent binding partners of OGA were identified by SILAC‐based proteomics including calcium/calmodulin‐dependent kinase II, fatty acid synthase, and filamin A. These data provide the framework for future physiological studies that will provide mechanistic insight into how OGA is regulated during stress and contributes to O‐GlcNAc‐mediated cell survival.
Biological Significance of Dual Oriented Nontypeable Haemophilus influenzae Vaccine Candidate P6
Authors: Reulbach Casey , Shaul Jeffrey , Pichichero Michael , Michel Lea
Published: June 2021

Nontypeable Haemophilus influenzae (NTHi) are pathogenic bacteria and the cause such diseases as pneumonia, bacteremia, meningitis, and sepsis in adults. NTHi is also linked to 30‐35% of the roughly 25 million annual cases (in The United States) of acute otitis media (ear infections) in children. Vaccines against encapsulated strains of Hi have proven effective, but no vaccines are available in the US to protect against NTHi. The 16‐kDa outer membrane lipoprotein P6 has been shown to be nearly homologous in all tested NTHi strains, making it one of the leading vaccine candidates for NTHi. However, it was recently demonstrated, using flow cytometry, confocal microscopy, and other biochemical methods, that P6 exhibits dual orientation in the outer membrane of NTHi. A small percentage of the P6 population faces out of the cell while a much larger percentage faces in toward the periplasm. However, these studies were only performed on a single strain of NTHi, which was cultured in a laboratory under aerobic conditions. In order to gain insight into P6′s in vivo orientation(s), similar studies were performed on multiple clinically‐relevant strains of NTHi and on NTHi cultured under different physiologically‐relevant conditions. Results suggest that P6 localization does not vary between strains, but localization does change in response to varying culture conditions.
Identification of the Antimicrobial Peptide LL‐37 as a Ligand for Integrin αMβ2 (Mac‐1, CD11b/CD18) Reveals its Role in Macrophage Phagocytosis
Authors: Lishko Valeryi , Moreno Benjamin , Podolnikova Nataly , Ugarova Tatiana
Published: June 2021

The cathelicidin peptide LL‐37 has numerous immune‐modulating effects and has been implicated in a bactericidal effect. However, the mechanisms by which LL‐37 mediates these activities remain unclear. Our recent elucidation of the recognition specificity of integrin Mac‐1, a major receptor on the surface of myeloid cells, indicated that LL‐37 contains several putative Mac‐1 recognition motifs and could potentially interact with the receptor. Using a peptide library spanning the sequence of LL‐37, we show that the αMI‐domain of Mac‐1 bound several overlapping LL‐37‐derived peptides. The full‐length peptide and its C‐terminal derivative (18‐37) supported strong adhesion of various Mac‐1‐expressing cells, including human U937 monocytic cells, murine IC‐21 macrophages and HEK293 cells transfected with Mac‐1. The cell adhesion to LL‐37 was partially inhibited by mAbs against the αM and β2 integrin subunits, and completely blocked when anti‐Mac‐1 antibodies were combined with heparin, suggesting that cell surface proteoglycans act cooperatively with integrin Mac‐1. Coating both Gram‐negative and Gram‐positive bacteria with LL‐37 significantly enhanced their phagocytosis by macrophages, and this process was blocked by a combination of anti‐Mac‐1 mAbs and heparin. Phagocytosis by murine peritoneal macrophages of LL‐37‐coated latex beads, a model of foreign surfaces, was several fold higher than that of untreated beads. Furthermore, LL‐37 potentiated phagocytosis by wild‐type, but not Mac‐1‐deficient macrophages. These results identify LL‐37 as a ligand for integrin Mac‐1 and opsonin, revealing a new aspect of LL‐37 action.

NIH Support: NIH HL 63199
Mechanism of Action of OXA‐58 β‐Lactamase
Authors: Golemi‐Kotra Dasantila , Gill Preet , Wilson Derek
Published: June 2021

Carbapenems such as imipenem and meropenem are β‐lactam antibiotics with a wide spectrum of activity, initially designed to overcome β‐lactam resistance due to their stability toward most β‐lactamases. However, as a result of selective pressure these carbapenems have succumbed to the hydrolytic power of β‐lactamases. These enzymes hydrolyze the β‐lactam bond of these antibiotics. They are classified in four classes based on their primary sequences, classes A, B, C and D. Classes A, C and D are serine‐active site β‐lactamases and class B β‐lactamases are metallo‐enzymes. The progressively increasing ability of all four classes of β‐lactamases to hydrolyze carbapenems has been documented. Among them, Carbapenem‐hydrolyzing Class D β‐lactamases (CHDLs) are directly associated with outbreaks of carbapenem‐resistant Acinetobacter baumannii around the world and have emerged as a major problem in treatment of multidrug resistant A. baumannii, for which carbapenems have been the drug of choice. We have undertaken a structural study using H/D exchange and a kinetic study using a number of OXA‐58 mutants to delineate the mechanism of hydrolysis of imipenem by OXA‐58. Our studies show that OXA‐58 employs enzyme dynamics to gain specificity toward imipenem hydrolysis.
B12 in a New Light: Queuosine tRNA Modification
Authors: Dowling Daniel , Miles Zachary , Köhrer Caroline , Bandarian Vahe , Drennan Catherine
Published: June 2021

The tRNA modification queuosine (Q) was first discovered in the wobble position of His, Tyr, Asp, and Asn anticodons over 40 years ago, yet only recently was the final biosynthetic enzyme responsible for Q‐base maturation identified. Even more perplexing was the apparent dependence on cobalamin (B12) and reducing equivalents. Here, we present structural insight into how epoxyqueuosine reductase uses B12 in a new light to react with nucleic acid molecules and expands the exciting family of B12‐dependent enzymes. CLD is an HHMI investigator, and this work was supported by NIH grant GM72623 awarded to VB.
Structural Comparison of Firefly Luciferase with the Latent Luciferase CG6178
Authors: Adams Spencer , Bohn Markus , Schiffer Celia , Miller Stephen
Published: June 2021

Firefly luciferase shares high sequence identity to insect fatty acyl‐CoA synthetases (ACSLs). These homologous enzymes operate through a similar two‐step mechanism. In the first step, the substrate is adenylated to form an AMP ester. Both firefly luciferase and ACSLs can accept fatty acid substrates. In the second step, CoA displaces AMP to form a fatty acyl‐CoA. Uniquely, firefly luciferase can adenylate D‐luciferin, and in the second step the adenylated D‐luciferin substrate is oxidized to an excited‐state oxyluciferin which emits a photon of light. ACSLs are unable to catalyze light emission from D‐luciferin. Recently, work in our lab demonstrated that an ACSL from the fruit fly Drosophila melanogaster (CG6178) can function as a luciferase when supplied with the synthetic luciferin CycLuc2. We sought to understand the structural basis for CG6178′s discrimination between luciferin substrates. Using x‐ray crystallography, we have solved a structure of CG6178. This structure shows conservation of the overall fold relative to luciferase, but the active site environment differs between the two enzymes. The structural differences between the two enzymes provide clues about how CG6178 adenylates CycLuc2 but not D‐luciferin. Engineering of the CG6178 enzyme based on these structural studies provides a novel avenue for future dissection of the fundamental requirements for bioluminescence and understanding how luciferases evolved from ACSLs.
Quantifying dynamic host‐microbiota signatures of antibiotic‐associated GI infection: What can the host proteome tell us?
Authors: Elias Joshua , Lichtman Joshua , Sonnenburg Justin
Published: June 2021

OBJECTIVE

Quantify and compare the interactions between resident gastrointestinal (GI) microbiota and host proteins shed into the gut lumen under conditions of antibiotic‐induced infection.

METHODS

Using mouse models of antibiotic‐associated Salmonella typhimurium and Clostridium difficile infection, we conducted mass spectrometry‐based host‐centric surveys of stool proteins, and microbial community profiling in parallel. Time course profiles allowed us to compare the rates and extents of perturbation and recovery of both microbiota and host proteomes following the administration of two antibiotics, with or without concurrent infection with the enteric pathogens. The influence of fecal transplant on recovery profiles was determined. The resulting pathological proteome and microbiome signatures were further compared with a chemically‐induced colitis model.

RESULTS

By assessing the protein component of the intestinal mileau contributed by the host in diverse perturbed states, we identified differentiating proteomic signatures that allowed us to stratify recovery states that were not evident through microbe enumeration alone.

CONCLUSIONS

We conclude that the host‐derived intestinal proteome provides an important route towards discovering mechanisms by which host epithelia shape, and are shaped by changes in the commensal microbiota.

This work was supported in part by the Stanford Digestive Disease Center (JEE), the NSF GRFP Fellowship (JSL), and NIH grant (R01‐DK085025)
The Anti‐Proliferative Effect of Doxorubicin is Catalyzed through the Proteolytic Activation of CREB3L1
Authors: Denard Bray , Chen Qiuyue , Lee Ching , Pavia‐Jimenez Andrea , Brugarolas James , Ye Jin
Published: June 2021

Doxorubicin is an extensively used chemotherapy agent, but the drug response rate is low owing to an unclear mechanism of action. Here we report that the anti‐proliferative effect of doxorubicin is dependent upon the proteolytic activation of CREB3L1. CREB3L1 is synthesized as a membrane‐bound precursor. The N‐terminal domain of CREB3L1 projects into the cytosol and can function as a transcription factor. Doxorubicin stimulates the proteolytic cleavage of CREB3L1 to release the N‐terminal domain from the membrane so that it can enter the nucleus to transcribe anti‐proliferative genes. TM4SF20, a polytopic transmembrane protein, plays an important role in the regulation of CREB3L1 proteolytic activation. Under normal conditions, TM4SF20 prevents the proteolytic activation of CREB3L1 precursor. Doxorubicin activates CREB3L1 cleavage by inducing the de novo synthesis of ceramide. The accumulation of ceramide reverses the membrane topology of TM4SF20 by preventing the N‐terminal peptide of TM4SF20 from functioning as a signal sequence. The topology reversal of TM4SF20 leads to the proteolytic activation of CREB3L1. These findings are clinically significant as only cancer cells expressing CREB3L1 are sensitive to doxorubicin both in vitro and in vivo. Our data suggests that CREB3L1 may be used as a biomarker to predict the effectiveness of doxorubicin‐based therapy.

Sources of Research Support: National Institutes of Health (AI‐090119, HL‐20948, and AI 70116‐6)
Luminescence Lifetime Imaging Microscopy For Analysis Of Oxygen In The Cancer Cell
Authors: Dmitriev Ruslan
Published: June 2021

Molecular oxygen (O2), the key component of aerobic metabolism, plays important roles in normal and cancer cells and tissues. A real‐time and quantitative O2 imaging in 3D cell models would allow to discriminate complex effects of hypoxia on cell function in complex (heterogeneous) cell populations at subcellular resolution. Here, we used luminescence lifetime imaging (FLIM, PLIM) to develop and evaluate phosphorescent O2 probes with 3D spheroid and multi‐cellular aggregate models. We tested rat embryonic neurosphere culture with cationic nanoparticle O2 probes (MM2, NanO2) and developed O2 imaging protocol, multiplexed with analysis of distribution of specific protein markers such as HIF‐2α. To address the problem of efficient in‐depth staining, we designed two novel intracellular O2 probes: small molecule Pt‐Glc and nanoparticle PA2. Both probes displayed high analytical performance (brightness, photostability, O2 calibration) and were evaluated with rat pheochromocytoma PC12 and human colon cancer HCT116 cell aggregates. Resting cells cultured in 3D aggregates of >50 μm size displayed significant deoxygenation even under ambient atmospheric O2 levels. Still, they showed high viability and experienced different levels of oxygenation, upon treatment with mitochondrial uncouplers, inhibitors or pharmacological drugs. This data confirms the importance of probing of cellular O2 for experiments with 3D cell models. When combined with other live cell imaging dyes, such as markers of necrosis and apoptosis, the method allows for multi‐parameter quantitative assessment of cell physiology within relevant 3D micro‐environment.

Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.
Hospital Acute Malnutrition Before and After a Three‐Year Multidisciplinary Hospital Nutrition Intervention in a Major Urban General Hospital, Vietnam
Authors: Huong PTT , LAM NT , Thu NN , Quyen TC , Lien DTK , Anh NQ , Henry EG , Oliver L , Collier S , Gura KM , Young LS , Manders A J , Apovian CM , Ziegler TR , Lenders CM
Published: June 2021

Objectives

Change in % ideal body weigh (IBW) and risk of acute malnutrition after a 3y hospital nutrition intervention.

Methods

Cross‐sectional data from patients aged 31d‐90y hospitalized in seven key wards were obtained using a one‐day survey method in 2010 and 2013, Bach Mai Hospital. Data included age, sex, admission type, ward, length of stay (LOS), actual/recall weight and height. Exclusions criteria were discharge <24h, unavailability, or lack of nutrition status data (n=85/1474). Acute malnutrition was defined as pediatric wasting or thinness and adult underweight (WHO). Regression analyses were used.

Results

624 (57% male) patients in 2010 and 765 (58% male) in 2013. At baseline, IBW was 90.8+0.6% with 17%(18/104) pediatric wasting or thinness and 34% (175/520) adults underweight. After adjusting for potential confounding variables including sex, admission type, ward, LOS, and missing actual weight or height, patients admitted in 2013 showed a 3% increase in IBW (3.3+0.8%, p<0.0001) and were 29% less likely to have acute malnutrition (OR: 0.71 [0.55‐0.92]).

Conclusion

A reduction in hospital acute malnutrition was observed after a nutrition intervention. (Support: the Abbott Fund)
Trends in Nutrition Screening for Hospitalized Patients in Hanoi, Vietnam During a Four‐year Clinical Nutrition Demonstration Project
Authors: Henry Elizabeth , Lien Dinh Thi Kim , Anh Nguyen Quoc , Ly Lai , Miller Jessica , Thu Vu Thi , Lenders Carine
Published: June 2021

Objective

To assess trends in documentation of medical nutrition practices over the course of a four‐year international partnership between hospital, academia and government, launched to address malnutrition at the main tertiary care hospital in Hanoi, Vietnam.

Methods

This study used a cross‐sectional time‐series design. Data were collected in 7 wards of Bach Mai Hospital in Hanoi by systematically extracting information from medical records of all patients in the ward on 1 day each month. Descriptive analyses were conducted and data was analyzed with run charts to identify trends and shifts, using rules that are based on probability theory.

Results

Since the start of the project, 5 of 7 wards showed significant positive shifts in recording weights of patients at admission, and 4 of 7 showed positive shifts for recording height, indicating positive changes in nutrition screening behaviors. Figure 1 illustrates a positive shift in the Respiratory Ward in percentage of patients with weight taken and documented at admission. Additional significant shifts were found in the ICU and Surgery wards for use of nutrition screening tools and nutrition consults.

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Conclusion

Data is critical to understand behavior change in a hospital intervention and allows practitioners to advocate for more support from the leadership

Funded by the Abbott Fund.
NUTRITION STATUS OF CHILDREN WITH CONGENITAL HEART DISEASE: IMPACT OF CARDIAC SURGERY
Authors: Tin HT , Nhan LNT , Phuc VM , Linh NPM , Chau TTT , Dung LTK , Cam DT , Hien VTT , Nga TT , Gura KM , Collier S , Ziegler TR , Lenders CM
Published: June 2021

Objective

To determine the prevalence of acute malnutrition and low biological markers in children with congenital heart disease (CHD) before and after corrective surgery, Children's Hospital 1, Ho Chi Minh City, Vietnam.

Methods

Prospective observational study of 30 consecutive children <24 month old with CHD admitted for corrective surgery, 5‐9/2013. Data included age, cardiac diagnosis, length of hospital stay, among others. Weight, height, serum markers of nutrition, inflammation and immunity were measured before and 1m after surgery. Anthro‐software (WHO) and STATA 12 were used.

Results

The sample consisted of 30(40% male) patients aged 8.5±4.0m. The interval between diagnosis and surgery was 6.7±3.8m and hospital length of stay 27±14d. A total of 24(80%) children were wasted before and 18(60%) 1m after surgery (P<0.001). At baseline, 14(47%) had anemia, 8(27%) low retinol, 7(23%) low thiamine, and 20(67%) low IGG, among others. There was a significant improvement in the prevalence of low IGG (p=0.018) but not of other biological markers (p>0.05) after surgery.

Conclusion

Earlier surgery and comprehensive nutrition intervention might further improve these results. (Support: Abbott Fund)
Weight, Body Composition and Infertility in Men and Women
Authors: Reyes Martin Karen , Mendoza López María del Mar , Gutiérrez‐Gómez Yareni
Published: June 2021

There are many causes for infertility, among which body composition is very relevant; it is estimated that the worldwide prevalence for this problem is around 15%. The aim of this review is to analyze the existing evidence which relates infertility with body weight and composition. A literature search was conducted in electronic biomedical databases (MEADLINE, EMBASE). It has previously been observed that low weight impacts negatively in fertility, nevertheless overweight and obesity do too, this finding being even more striking given the increasing obesity prevalence around the world. Several mechanisms are believed to relate body composition and infertility, among which we can find sperm disturbances, insulin resistance and hormonal change, amongst others. Exercise is also an important factor to consider because of its capacity to change body composition; however, it is important to note that excesses in both weight and physical activity not only are risky in regards to fertility but also on general health. Hence is logical to conclude that, the first line treatment for infertility should be an adjustment in body composition being this the healthiest, cheapest and most effective approach.
Evaluating Growth Regulating Hormones in Rural Gambian Children
Authors: Nabwera Helen , Bernstein Robin , Jallow Amadou , Jallow Momodou , Liberatore Jennifer , Prentice Andrew
Published: June 2021

Objectives

To evaluate the hormonal status in malnourished children during nutritional rehabilitation in order to identify markers of nutritional recovery.

Methods

Children 6‐24 months of age were recruited from the Medical Research Council‐ Keneba clinic. Anthropometric measurements done at baseline and on alternate days. Children were assigned to a group based on their WHO weight/height z scores (WHZ): WHZ<‐2 “malnourished” and WHZ>‐2 “well‐nourished”(WN). Children received WHO F‐75 test meal at baseline. Malnourished children received milk‐based formulas and/or supplementation with ready‐to‐use therapeutic food. We used Quantakine ELISA kits to measure the plasma hormonal levels.

Results

Sixty one children were recruited. Median age was 15.4 months. There were significant differences in the levels of leptin, sOBR, IGF‐1, IGFBP3 between malnourished and WN children, (P<0.005). Nutritional supplementation increased WHZ in the malnourished children and induced increases in the levels of leptin, IGF‐1, IGFBP3 and decreases in the levels of (sOBR) and the sOBR: Leptin molar ratio. The levels of leptin at Day 28 in the malnourished children remained below those of the WN, (p<0.005). However, the sOBR, sOBR: Leptin molar ratio, IGF‐1, IGFPB3 and IGF‐1:IGFPB3 molar ratios in the malnourished children on Day 28 were similar to the levels in the WN.

Conclusion

Our findings suggest that in rural Gambian children leptin alone is not be a good biomarker for nutritional recovery. The sOBR, sOBR: leptin molar ratio, IGF‐1, IGFPB3 and IGF‐1:IGFPB3 molar ratios appear to be better biomarkers.
A Longitudinal Quantification of Total Protein in Human Milk From Three Global Cohorts: a GEHM Study
Authors: Labhart Beau , Gray Michael , Maria Sarah , Phillips Shay , McMahon Robert , Davidson Barbara , Morrow Ardythe
Published: June 2021

The quantity of protein in human milk is believed to be important for growth and development of breastfed infants. This study, in support of the Global Exploration of Human Milk (GEHM) collaboration, reports total protein concentration as a weight to weight percentage based on Dumas combustion in longitudinal samples collected from three global cohorts (Mexico, China, and USA) at 4, 13, 26, and 52 weeks lactation. Average total protein decreased rapidly from 4 to 13 weeks of lactation (1.40 ± 0.14% to 1.16 ± 0.16%) and then remained relatively stable to 52 weeks (1.17 ± 0.13%). Similar trends have been reported and are further strengthened by data generated from these samples collected using standardized techniques. Since some components of human milk, including protein, are known to vary diurnally, samples for the GEHM study were collected in the morning by emptying an entire breast via electric pump. Additionally, commercially available human milk standards (HMS) and NIST Standard Reference Material (SRM) 1849a were incorporated into analyses to further verify the accuracy of Dumas combustion for determination of total protein in human milk. The HMS analyzed by combustion all fell within protein certified ranges determined by Kjeldahl methodology. SRM was analyzed before, during, and after each analytical run as a check sample. These added assessments of accuracy, as well as the integration of samples from different geographical populations across multiple points of lactation, strengthen the foundation of knowledge into total protein concentration in human milk. This research was supported by Mead Johnson Pediatric Nutrition Institute.
Process Evaluation Findings of B'More Healthy Communities for Kids: A Multi‐Level, Multi‐Component Obesity Prevention Program
Authors: Yang Tracy , Anderson Steeves E , Rastatter S , Kharmats A , Shipley C , Perepezko K , Welsh C , Gittelsohn J
Published: June 2021

B'More Healthy Communities for Kids (BHCK) is a multi‐level, multi‐component obesity prevention trial directed toward low‐income African American youth in Baltimore. BHCK works with small food stores, recreation centers, carryout restaurants, wholesalers, policymakers and families. Process evaluation is conducted by monitoring 88 implementation standards based on reach, dose delivered and fidelity. Guidelines rank each standard in terms of low, medium or high delivery. Here, we report on findings to date based on two feedback periods. Food store interactive sessions had low reach to the target audience (10‐14 year old youth) with 7.4 + 6.2 interactions/session compared to higher reach for adults (17.1 + 6.6). Dose delivered of food samples (28.5 + 14.4) and giveaways (16.2 + 6.2) distributed was medium to high. Carryout restaurant menu redesign obtained medium to high fidelity with 20% healthy side options and 10% healthy entrée options provided on posted menu boards. Recreation centers achieved high reach with 11.6 + 6.1 youth attending educational sessions. The agent‐based policy model (ABM) had low to medium reach with one local policymaker including the ABM on a formal agenda in the past year. Wholesalers obtained overall high fidelity by stocking food items that met BHCK nutrition guidelines. Social media and text messaging achieved high reach with 70% of BHCK‐enrolled families joining the program. Dose was high with an average of 3.2 text messages sent per week. These findings are used to monitor and modify components during the trial and to assess implementation quality post‐intervention.