bluuebunny/crossref_metadata_2025 huggingface dataset:
The extracellular matrix signals through protein kinase MEKK‐3 and transcription factor ATF‐7 to activate cytoprotective genes
Authors: Wimberly Keon , Choe Keith
Published: June 2021

Animal cells secrete proteins and carbohydrates to create an extracellular matrix (ECM) that provides mechanical support for tissues. Rather than being an inert scaffold, the ECM is a dynamic, physiologically active component in living tissues. Barrier ECMs stand at the surface of animals and are the first of line defense against many environmental stressors such as osmotic pressure changes, toxins, and biological pathogens. Activation of cytoprotective genes is the second line of defense against environmental stress. Cytoprotective genes are conserved and influence adaptation to climate change, disease progression and pathogenesis, and aging. Therefore, a molecular understanding of fundamental signaling mechanisms that control cytoprotective genes has broad relevance. The molecular signaling pathways that sense and regulate cytoprotective responses within cells have been studied in detail, but far less is known about how barrier ECMs interact with environmental stress to influence cytoprotective gene regulation. The nematode model organism Caenorhabditis elegans is covered by a complex barrier ECM termed the cuticle. Given that the cuticle directly interacts with the environment, we hypothesize that it may include a sensor for stress. We recently used RNAi screening, collagen mutations, and RNAseq in C. elegans to demonstrate that disruption of specific bands of collagen, called annular furrows, significantly induce organic osmolyte, detoxification, and immunity cytoprotective genes (p<0.05) (Dodd et al., 2018 Genetics 208:1467). To identify regulators of cytoprotective gene transcription downstream of annular furrows, we conduced an RNAi screen of all C. elegans' protein kinases and transcription factors in annular furrow mutants expressing fluorescent cytoprotective gene reporters. This screen and subsequent RT‐qPCR analysis identified protein kinase MEKK‐3 and transcription factor ATF‐7 as significantly required for expression of cytoprotective genes* in annular furrow mutants (p>0.001; *atf‐7 knockdown did not significantly repress immunity gene induction). Previous C. elegans' literature indicates that MEKK‐3 regulates longevity with dietary restriction and that ATF‐7 regulates innate immunity. Importantly, both of these proteins are conserved in humans where they regulate diverse stress responses and are implicated in tumorigenesis. We are now leveraging the experimental tractability of C. elegans to determine how these two conserved signaling genes function to confer protection to environmental stress in this novel ECM‐initiated context.

Support or Funding Information

NSF, UF Graduate School Fellowship, McKnight Doctoral Fellowship, William Townsend Porter Predoctoral Fellowship from the American Physiological Society

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrated Meta‐omics reveals a fungal‐associated bacteriome and distinct functional pathways in C. difficile infection
Authors: Amaniera Isabella , Stewart David B. , Wright Justin R. , McLimans Christopher J. , Fowler Maria , Baker Owen , Wong Hoi‐Tong , Tokarev Vasily , Brabec Jeff , Druker Rebecca , Lamendella Regina
Published: June 2021

Clostridium difficile is a bacterial pathogen of the gut that causes nearly 500,000 infections per year in the United States, with 20 to 30 percent of cases re‐occurring. Little is known about how C. difficile modulates the gut's fungal community and how this dysbiosis may perpetuate its reocurrence. This study aimed to contribute to the understanding of the disease's mechanism by identifying bacterial and fungal community structures and the bacterial‐fungal interactions in C. difficile infected (CDI) patients. Forty‐nine diarrheal stool samples, 18 CDI and 31 non‐CDI, were collected from hospitalized patients. The taxonomic marker, or “thumbprint,” regions of DNA, 16S rRNA for bacteria and ITS for fungi, were isolated and sequenced to determine the microbial communities. Metagenomic and metatranscriptomic analyses were also preformed to further characterize gut microbial structure and function. Bioinformatic analysis of the 16S rRNA and ITS data revealed a greater number of fungal taxa enriched in CDI samples than non‐CDI samples. Further, co‐occurrence network analysis using the program CoNet displayed negative correlations between the fungal taxon Candida and several bacterial taxa in CDI samples. These findings indicate that CDI infections may create an environment that allows fungal communities to bloom and possibly suppress commensal bacteria. Additionally, bioinformatic analysis of the metatrancriptomic and metagenomic data revealed that pathways involving biofilm formation, inflammation, flagellar assembly, and two‐component systems involving osmotic regulation were enriched in CDI samples. These functionalities may allow C. difficile to persist in the gut, and therefore may lead to failed treatments and reoccurrences. Our data suggest that the differential bacterial and fungal communities in CDI and non‐CDI patients may lead to high recurrence rates. The enriched fungal community in CDI patients, may be perpetuating the dysbiosis, thus leading to the development of new therapeutic approaches.

Support or Funding Information

This research was supported by a grant from the American Society of Colon & Rectal Surgeons in 2017 (ASCRS Research Foundation Benign Colorectal Disease Grant RFP‐002). This research was also supported by a grant to Juniata College from the Howard Hughes Medical Institute (http://www.hhmi.org) through the Precollege and Undergraduate Science Education Program, as well as by the National Science Foundation (http://www.nsf.gov) through NSF award DBI‐1248096.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Seasonal Shifts in Gut Microbiota‐Mediated Nitrogen Recycling in 13‐Lined Ground Squirrels
Authors: Regan Matthew D. , Verdoorn Kristen M. , Chiang Edna , Gugel Sadie R. , Assadi‐Porter Fariba M. , Carey Hannah V.
Published: June 2021

Hibernation is a complex phenotype that enables animals to fast for several months during periods of food scarcity. During this time, less of the hibernator's protein is degraded than would be predicted and, in the 13‐lined ground squirrel (Ictidomys tridecemlineatus), muscle volume and protein synthesis rise near the end of the hibernation season despite continued fasting and loss of body mass. With no exogenous nitrogen intake, one nitrogen source to support protein synthesis in late winter could be from urea nitrogen salvage (UNS), a nitrogen recycling process mediated by the ureolytic activity of the hibernator's gut microbiota. We hypothesized that the contribution of UNS to skeletal muscle growth in 13‐lined ground squirrels increases over the hibernation season, and tested this by comparing UNS processes in summer, early hibernation (1 month), and late hibernation (>3 months) squirrels. Data collected thus far suggest that expression of the urea transporter (UT‐B) in cecal tissue is 3‐fold greater in late winter than in summer squirrels (P<0.001; n=12 per group), indicating a greater capacity for transport of urea into the cecum during winter fasting. Real‐time measurement of microbial ureolytic activity using cavity ring‐down spectrometry on squirrels I.P.‐injected with 13C,15N‐labeled urea confirmed the bacterial origin of UNS, because δ13C in breath is substantially reduced in antibiotic‐depleted microbiotas. Isotope ratio mass spectrometry showed that tissues of squirrels treated with 13C,15N‐urea accumulated up to 200x more 15N as saline‐treated control squirrels, confirming that urea‐derived nitrogen is recycled and incorporated into host tissues. Together, these results corroborate UNS in active and hibernating 13‐lined ground squirrels and suggest that this mechanism for nitrogen recycling is a key aspect of the host‐microbe symbiosis in hibernating mammals that helps prepare the squirrels for spring emergence.

Support or Funding Information

This work is supported by NSF award IOS1558044

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Intermittent Hypoxia and Respiratory Motor Plasticity after Cervical Spinal Cord Contusion
Authors: El‐Chami Mohamad , Holland Ashley E. , Mitchell Gordon S. , Gonzalez‐Rothi Elisa J.
Published: June 2021

Chronic intermittent hypoxia (CIH), characterized by > 100 hypoxic episodes per day, is a hallmark of moderate sleep apnea, a condition highly prevalent in individuals with spinal cord injury. Both 1 day (Huxtable et al., 2013) and 7 days (Gonzalez‐Rothi, in preparation) of CIH (15 episodes/hour; 8 hours/day) abolish phrenic motor plasticity in intact rats due to neural inflammation. Although a milder CIH protocol (40 episodes/day) enhances phrenic activity in acutely injured rats (Fuller et al., 2003), the effects of CIH protocols that more closely approximate moderate sleep apnea have not been explored in animal models of chronic spinal injury. Thus, we are evaluating the impact of moderate CIH (2 min hypoxic episodes, 2 min intervals; 8 hrs; single day) on phrenic nerve activity and the capacity to elicit phrenic motor plasticity in rats with chronic C3/4 midline contusion injury (12 wks). Neurophysiology experiments conducted one day post‐CIH are ongoing and include assessments of: 1) phrenic nerve activity during baseline and maximal chemoreflex activation; and 2) moderate AIH‐induced phrenic long‐term facilitation (pLTF). Preliminary analysis suggests reduced pLTF in contused rats exposed to CIH. These experiments are the first to explore the effects of CIH on respiratory outcomes following chronic spinal contusion. Further experiments are required to explore the impact of: 1) prolonged CIH exposures on breathing ability after chronic cervical contusion; and 2) whether reducing neural inflammation will enhance phrenic plasticity and translate into improved functional recovery following chronic spinal cord injury.

Support or Funding Information

The Department of Defense: SCI160123 (EGR); and the National Institutes of Health: K12 HDO55929 (EGR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Mechanisms by which the Methyl‐CpG Binding Protein ZBTB38 Identifies Interacting Partners and Mediates Transcription
Authors: Buck‐Koehntop Bethany A. , Hudson Nicholas O.
Published: June 2021

Cytosine methylation in CpG dinucleotides is an essential epigenetic modification utilized for regulating transcriptional processes within the eukaryotic genome. Consequently, misappropriation of DNA methylation patterns is associated with many diseases, including cancer. At many gene promoters, DNA methylation mediates expression through recruitment of methyl‐CpG binding proteins (MBPs). These specialized transcription factors read the methylated DNA signal and subsequently recruit chromatin remodeling complexes; leading to alterations in chromatin architecture and transcriptional responses. Thus, MBPs represent important functional intermediaries between these two key epigenetic events. ZBTB38 is the largest member of the ZBTB family of MBPs and contains two sets of five Cys2His2 zinc finger (ZF) domains distributed into N‐ and C‐terminal clusters, as well as an N‐terminal BTB/POZ protein interaction domain. There is increasing evidence that ZBTB38 regulates transcription for several core cellular processes in both normal cells as well as disease states. However, full mechanistic insight for how ZBTB38 identifies binding partners to elicit cellular function remains to be determined. Using combined structural and biochemical approaches, specific DNA binding partners have been identified and characterized for both the N‐ and C‐terminal ZF domains of ZBTB38. Importantly, we recently discovered that in addition to the N‐terminal ZFs, the C‐terminal ZFs also exhibit methyl‐selective DNA binding through the use of an alternative mode for methylated DNA recognition. Cell based studies have been utilized to begin delineating how these two ZF domains may function together or independently to modulate transcription. In addition to DNA interactions, we have begun to evaluate protein interacting partners for ZBTB38 using a combination of biophysical and cell based methodologies. Together, these findings expand our understanding for ZF recognition of methylated DNA, as well as illuminate the molecular basis by which ZBTB38 preferentially recognizes its biomolecular binding partners to translate transcriptional outcomes into specific cellular responses.

Support or Funding Information

This work was supported in part by MCB‐1715370 from the National Science Foundation and by RSG‐14‐185‐01‐DMC from the American Cancer Society.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Analysis and Characterization of the Metal Bound Sco Protein from Thermus thermophilus
Authors: Hofman Cristina , Hunsicker‐Wang Laura
Published: June 2021

Cytochrome c oxidase (CcO) is the final complex of the respiratory electron transport chain, which is a set of complexes that participate in the production of adenosine triphosphate (ATP), the main form of energy used by cells. CuA is the initial electron acceptor of CcO and consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine and the carbonyl backbone of a nearby glutamine. Sco proteins have been implicated in the assembly of the CuA, site but the exact role that it plays is the subject of much debate and study. Previous work has found that Sco proteins share a fold similar to thioredoxins, pointing toward the possibility of thiol‐disulfide oxidoreductase activity as a function of the protein. The ability of Thermus thermophilus Sco (TtSco) to act as a thiol‐disulfide oxidoreductase has been probed using an experiment to trap the protein‐protein intermediate between TtSco and its target, Thermus thermophilus CuA (TtCuA). The effect of nearby amino acids and metal binding prior to reaction have been determined for the formation of the protein‐protein intermediate, the mixed disulfide intermediate (MDI). The formation of MDI was monitored with either UV‐Visible spectroscopy by observing an absorbance increase at 412 nm as a chromophore tag, TNB, was released upon reaction between TtSco and TtCuA or by non‐reducing SDS‐PAGE gels. Altering Arg 48, which is adjacent to the attacking cysteine to an Asp has a very large effect on MDI production. TtSco also has metal binding ability, found to bind Cu(I), Cu(II), Ni(II), Cd(II) and Co(II) with varying relative affinities, in addition to disulfide reductase activity. To further characterize MDI formation with TtSco, the impact of conserved residues involved in metal binding on MDI formation were probed and showed that changing the conserved histidine did not affect MDI formation. However, binding of metal to Sco prior to incubating with TtCuA abrogated MDI formation. In characterizing the metal bound protein, preliminary studies to determine the binding affinity of Cu(II) to TtSco have been completed. The impact of conserved cysteines on metal binding has also been studied and the results indicate that one cysteine binds much more tightly to the Cu(II) than the other. While this work allows for a better understanding of TtSco function, much work remains to be done to understand the impacts of metal binding and to further characterize the Sco family of proteins.

Support or Funding Information

Research Corporation (Cottrell College Science Award 7963), the Welch Foundation (W‐0031 [Trinity Chemistry Department]), FASTER grant SURF‐ National Science Foundation DUE S‐STEM Award 1153796 and the Arnold and Mabel Beckman Foundation Beckman Scholars Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Novel Homologs to the C‐terminal Domain of the Orange Carotenoid Protein
Authors: Wei Derek T , Bao Han , Dominguez‐Martin Maria A , Kerfeld Cheryl A
Published: June 2021

The biochemical pathways involved with the photosynthesis of industrially and agriculturally relevant organisms are of paramount interest to researchers. Feeding and fueling the rising generations poses a challenge that will require novel understanding of these pathways. Photoprotection is one such mechanism where insights have led to improved production of resources. The cyanobacterial photoprotective mechanism is primarily officiated by the orange carotenoid protein (OCP). The OCP is photoactivated under blue‐green light allowing it to attach to cyanobacterial light harvesting antennae ‐ phycobilisomes. This diverts excess photosynthetically absorbed energy to be given off as heat. The OCP is a modular protein consisting of two domains: the N‐terminal domain (NTD) and C‐terminal domain (CTD). In addition, a carotenoid involved in the photoprotective mechanism spans the interior of both domains. Recently, a new family for homologs to the NTD were described as the Helical Carotenoid Proteins (HCPs). Similarly, Homologs to the CTD were classified as such (CTDHs). Both families have the ability to bind carotenoids. While the HCP is unique in cyanobacteria, the CTDH contains the nuclear transport factor 2 (NTF2) fold, which is ubiquitous. Bioinformatics revealed new NTF2‐fold containing proteins in the model cyanobacteria Fremyella diplosiphon and in the model algae Chlamydomonas reinhardtii. Structural analysis suggested the conservation of amino acids important for binding carotenoid in these proteins. The current study is investigating the carotenoid binding ability of two proteins from both F. diplosiphon and C. reinhardtii. These four proteins have been cloned with an N‐terminal histidine tag and overexpressed heterologously with the carotenoid Canthaxanthin in Escherichia coli. These proteins have been purified through affinity chromatography and their overexpression confirmed by SDS‐PAGE gel analysis. They have been characterized via various biochemical and spectroscopic methods showing oligomerization states and the correct folding of the proteins. Unfortunately, they did not show binding of Canthaxanthin. Future work includes in vitro reconstitution of purified proteins with a variety of carotenoids as well as overexpression of other identified putative homologs to investigate carotenoid binding ability.

Support or Funding Information

National Science Foundation IOS 15573244

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of neural networks of Caenorhabditis elegans by functional cellomics
Authors: Yamauchi Yuji , Matsukura Hidenori , Aoki Wataru , Ueda Mitsuyoshi
Published: June 2021

Background

Neural networks generate complex behaviors, but relationships between neural networks and complex behaviors have not been understood well. Although optogenetics has been a powerful tool which can provide knowledges on mechanisms of neural computation, conventional optogenetics has mainly three difficulties: 1. A hypothesis is necessary to conduct experiments in advance; 2. Optogenetics is low‐throughput because different transformants are required to test different hypothesis; 3. Single cell analysis is difficult due to lack of single‐cell‐specific promoters.

Therefore, we have developed a method called ‘functional cellomics’ in which we can annotate functions of neural networks of Caenorhabditis elegans in hypothesis‐free, highthroughput, and single‐cell‐resolution manner.

Method

One of the core technologies of ‘functional cellomics’ is a stochastic Cre/lox system. By applying Cre/lox system, we have designed a methodology to enable stochastic labeling of any effectors to C. elegans neurons. To develop ‘functional cellomics’, we constructed four plasmids. In the initial state, pan‐neuronal F25B3.3 promoter drives production of mCherry in all neurons. After induction of Cre recombinase by heat shock, Cre catalyzes recombination of DNA exclusively either between loxP sequences or between lox2272 sequences. If a recombination occurs between lox2272 sequences, a transcription factor QF2w is produced. Then, QF2w drives production of ChR2::GFP under QUAS promoter. In this way, we can obtain a C. elegans library in which ChR2 is labeled in a stochastic manner.

Results & Discussion

By applying heat shock against the C. elegans library, we confirmed that ChR2::GFP was labeled in a stochastic manner. To demonstrate feasibility of ‘functional cellomics’, we chose the egg‐laying behavior of C. elegans as a model. The C. elegans library was illuminated with the blue light. We identified that ChR2::GFP was produced in HSNs (hermaphrodite‐specific neurons) in all of egg‐laying individuals, but it was not in the non‐egg‐laying individuals.

Our results that HSNs can be identified by ‘functional cellomics’ in a high‐throughput manner proves the feasibility of proposed ‘functional cellomics’ [1].

Support or Funding Information

This work was supported by PRESTO, JST (grant No. JPMJPR16F1), JSPS KAKENHI (grant No. JP17K19452), and Kyoto University Live Imaging Center.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of lipoprotein lipase glycosylation from adipocytes in lean and obese Zucker rats
Authors: Cook Allison B. , Frost Leslie M. , Rakus John F.
Published: June 2021

C‐mannosylation is a type of protein glycosylation involving a covalently attached mannose sugar to the C2 carbon of an indole ring on tryptophan. Lipoprotein lipase (LPL), is an enzyme involved in lipid metabolism, hydrolysis of triglycerides in chylomicrons and very low‐density lipoproteins. LPL is mainly expressed in adipose tissue, muscle tissue, and macrophages, and it has been shown to contain a C‐mannosylated tryptophan in LPL‐overexpressing cell lines. Metabolic disorders, such as diabetes and obesity, leads to a deficiency of LPL function. LPL can be regulated at post‐transcriptional levels in a tissue specific manner that also depends on the nutritional state and hormonal level of the tissue. The goal of this project is to determine whether LPL is C‐mannosylated in adipose tissue from obese and lean Zucker rats. The Zucker fatty rat is a well‐established model of obesity and insulin resistance. Obese Zucker rats have high levels of lipids and cholesterol in their bloodstream, and their adipocytes are increased in size and number. LPL was isolated by immunoprecipitation (IP) using a monoclonal antibody specific for the LPL protein and by using heparin‐sepharose affinity chromatography. The isolated protein was analyzed by mass spectrometry utilizing the peptide mass fingerprint technique and low‐energy CID tandem MS/MS to identify C‐mannosylated tryptophan residues. The results of this experiment further the knowledge of the role that obesity plays in the post‐translational modification of LPL in adipose tissue.

Support or Funding Information

NSF‐OIA‐1738707

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AGE/RAGE Signaling in Diabetes‐Mediated Vascular Calcification in Vascular Smooth Muscle Cells
Authors: Kay Amber M. , Stewart James A.
Published: June 2021

Advanced Glycation End‐Products (AGEs)/Receptor for AGEs (RAGE) signaling cascade comprises many parts such as the complex nature of the receptor and ligand specificity. The intersecting pathways of AGE/RAGE signaling is still not well understood. AGE/RAGE signaling influences both cellular and systemic responses to increase bone matrix proteins through p38 MAPK and ERK1/2 signaling pathways in hyperglycemic and calcification conditions. AGE/RAGE signaling has also been shown to increase oxidative stress by promoting diabetes‐mediated vascular calcification through NOX‐1 activation and decreased SOD‐2 expression to promote a phenotypic switch of vascular smooth muscle cells (VSMCs) to osteoblast‐like cells. The purpose of this research is to understand AGE/RAGE mediated vascular calcification as a complication of diabetes. Calcification was induced in primary mouse VSMCs of non‐diabetic (db/wt), diabetic (db/db), non‐diabetic RAGE knockout (db/wtRKO), and diabetic RAGE knockout (db/dbRKO), and then subsequently treated with AGEs to activate RAGE. Intracellular calcium levels were quantified and showed a pronounced calcification in db/wt VSMCs and loss of RAGE resulted in a decrease in calcification in db/wtRKOVSMCs. Western blotting analysis revealed VSMC marker protein (a‐smooth muscle actin) was decreased in db/wt calcified cells. In addition, the presence of the bone marker, osteopontin (OPN), was found in both db/wt and db/wtRKO, but OPN expression was decreased with the loss of RAGE. This result indicates an osteoblast‐like phenotypic switch and this switch was negated in db/wtRKOVSMCs. These data demonstrated that RAGE has a role in diabetes‐mediated vascular calcification. By understanding the role, the AGE/RAGE signaling cascade plays diabetes‐mediated vascular calcification will allow for possible targets for pharmacological intervention to be identified that may decrease the severity of this diabetic complication.

Support or Funding Information

This research was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 2015202674. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systematic Characterization and Conservation of Adjacent Gene Co‐Regulation in Fungi
Authors: Eldabagh Reem , Foley Jay , Arnone James
Published: June 2021

It is essential that cells orchestrate gene expression, which oftentimes requires regulated expression of large gene sets. Previously it has been observed that spatial positioning influences transcription, including in rRNA and ribosome biogenesis (RRB) and ribosomal protein (RP) regulons. Both include a disproportionate number of immediately adjacent gene pairs, which results in tighter transcriptional coordination among adjacent paired genes compared to unpaired genes. This non‐random genomic distribution has been seen in a handful of functionally related gene families and many of these pairings are conserved across divergent fungal lineages. To date, the significance of these observations has not been extended in a systematic way to characterize how prevalent and significant the role of adjacent gene co‐regulation is in transcriptional regulation. Therefore, we statistically analyzed genome‐wide expression profile data under five stressors for the RP, nitrogen metabolism, carbohydrate metabolism, toxin response and heat shock regulons, using Python to compute Pearson's correlation coefficient values. Surprisingly, it seems there is not an overall transcriptional advantage to functional pairing of genes in S. cerevisiae – although there are specific conditions that this pairing appears advantageous for, which could be the driving force behind this arrangement. Divergent fungal lineages were similarly analyzed to allow for comparison of newer evolved pairings to ancestral pairings. Our results demonstrate that the longer two genes have co‐existed as neighbors, the tighter their transcriptional response is to broad stress and nutritional conditions. Moreover, we systematically characterized the genomic distribution of the 140 gene families defined by the G.O. Slim designation, where we found that ~25% of these functional groupings can be found as functionally related, immediately adjacent gene pairs. This suggests that adjacent gene co‐regulation is much more wide‐spread than previously realized. Moreover, hypergeometric distribution analyses of gene expression were conducted, enabling the identification of potentially influential histone remodelers on the adjacent gene pairs in S. cerevisiae. Such analyses have been completed on the RP, nitrogen metabolism, heat shock, and toxin response regulons. Future work will focus on extending this analysis to genes of other regulons to better understand gene expression, and verifying these results experimentally using knockout strains of yeast.

Support or Funding Information

This research was supported by funding from the Garden State Louis Stokes Alliance for Minority Progress. The GS‐LSAMP is supported by a grant from the National Science Foundation. It was also supported by the Center for Research and ART at William Paterson University of New Jersey.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic Studies of 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase substrate analogs
Authors: Kedir Dean Reshad , Watkins Linette M.
Published: June 2021

2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) is an enzyme that catalyzes the cleavage of the carbon‐sulfur bond as the final step of the desulfurization of 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) producing sulfite and 2‐hydroxybiphenyl (HBP). This reaction is used to study the biodesulfurization of dibenzothiophene, the major organosulfur compound found in fossil fuels/petroleum. Previous studies in the Watkins lab suggested that halogenated analogs of HPBS are competitive inhibitors of DszB, so a coupled assay was used to test their effect on kinetic activity. The relative activity of these analogs was tested measure both products of the reaction. The rate of HBP formation was monitored using a fluorometric assay with an excitation at 288 nm and measuring emission at 414 nm. The rate of sulfite product formation was measured in the coupled assay with Sulfite Oxidase (SOX) by analyzing a change in absorbance at 540 nm. When testing the analogs HPBS‐Cl and HPBS‐Br for activity, no product was detected. When measuring enzyme activity in the presence of the halogenated analogs and the substrate HPBS, an increase in DszB activity was observed, with HPBS‐Cl and HPBS‐Br showing an increase product formation of 475% and 213%, respectively. Individually these HPBS analogs did not act as catalytic substrates or competitive inhibitors, however when coupled with HPBS the analogs acted as activators. Based on these results, we are exploring the structure of DszB to further understand how HPBS analogs can affect the desulfinase kinetics.

Support or Funding Information

NSF CHE‐1757874

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reduced amino acids: the placenta after maternal vertical sleeve gastrectomy in the rat
Authors: Welch Bradley , Himel Alexandra , Duncan Brittany , Spann Redin , Grayson Bernadette E
Published: June 2021

Vertical sleeve gastrectomy (VSG) is a surgical weight loss procedure used to reduce the comorbidities of Metabolic Syndrome improving obesity, diabetes and heart disease. Both in humans and rodents, maternal bariatric surgery prior to pregnancy results in challenges during pregnancy including increased small‐for‐gestational age offspring that we have shown in the rodent model of VSG develop greater metabolic disease later in life compared to obese controls. A common gestational cause of poor growth in utero is maternal protein deficiency. Previously, we published that VSG dams had normal levels of plasma amino acids in the fasting condition and following a protein shake gavage. We did not find any gross abnormalities in plasma amino acids under these conditions. In the current work, we hypothesized that the maternal levels were sufficient and that the transporters within the placenta may be differentially transporting amino acids to the fetus. Using gestational day 19 placenta from pregnant Lean, Obese and post‐VSG dams, we performed analysis of the amino acid content of the placenta and prepared placental mRNA for quantitative PCR analysis of genes associated with amino acid transport. Aliphatic Amino acids: isoleucine, leucine, proline and valine were statistically reduced in comparison to Lean dam levels (p< 0.05). Phenylalanine, aspartic acid and glutamic acid, arginine, histidine and methionine were also significantly reduced (p< 0.05). Furthermore, amino acid transporter SLC38A1 (SNAT1) and SLC38A4 (SNAT4) showed trends towards reductions in both VSG and Obese dams. On the contrary, SLC38A2 (SNAT2) and SLC7a7 (LAT1) was significantly elevated (p< 0.05) in VSG in comparison to Lean and Obese. Collectively, these data suggest that despite healthy ingestion of proteins by the mother, alterations in amino acid availability to the fetus may be obstructed by the placenta after bariatric surgery. More work is needed in order to determine the relevance of these findings to the human condition.

Support or Funding Information

The research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1P20GM121334‐01, and BEG is also supported DOD contract W81XWH‐16‐1‐0387.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sequence specificity of amyloid propagation in the yeast model
Authors: Chernoff Yury O , Chen Buxin , Gyoneva Stefka , Romanyuk Andrey V
Published: June 2021

Amyloids are highly ordered homogenous fibrous aggregates produced by many proteins in vitro and associated with some diseases in vivo. Self‐perpetuating amyloids (prions) cause infectious neurodegenerative diseases in mammals and transmit heritable traits in yeast. The ability of a yeast protein to generate and propagate an amyloid state is controlled by a specific region of the protein termed prion domain (PrD). Amyloid propagation is a sequence‐specific process. Divergence of PrD sequences impairs transmission of amyloid state to the orthologous proteins from other species in some combinations, leading to so‐called “species barrier” in prion transmission. Such a barrier is detected even between prion proteins of the closely related species of Saccharomyces yeast. These proteins are capable of coaggregating, thus demonstrating that conversion to a prion state does not represent a mechanical consequence of coaggregation and requires a higher level of sequence identity. Moreover, slight sequence differences may result in “poisoning” of amyloid growth by a divergent protein. Amyloids of different species exhibit differential patterns both in vivo and in vitro. Detailed analysis of the yeast prion protein Sup35 revealed that specific changes within different amino acid stretches of Sup35 PrD are responsible for the species barrier in different cross‐species combinations.

(Supported by NSF.)
Targeting Mutant V600EB‐Raf in Melanoma Interrupts Immunoediting of Leukocyte Functions and Melanoma Extravasation
Authors: Liang Shile , Peng Hsin‐Hsin , Robertson Gavin P , Dong Cheng
Published: June 2021

Previous studies have shown that neutrophils (PMNs) could facilitate melanoma transendothelial migration mediated by endogenously produced chemokine interleukin 8 (IL‐8) from the tumor microenvironment. The objective of this study was to investigate the role of B‐Raf, the most mutated gene in malignant melanomas, in this process. Using short interfering RNA (siRNA) to reduce expression/activity of mutant V600EB‐Raf in melanoma, we showed knockdown of mutant V600EB‐Raf inhibited melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inhibition of V600EB‐Raf significantly reduced the constitutive secretion of IL‐8 from melanoma cells, as well as the capacity of endogenous IL‐8 production from the melanoma‐PMN microenvironment. Furthermore, a reduction in ICAM‐1 expression on melanoma cells was detected following mutant V600EB‐Raf knockdown. Together, these results suggest that targeting mutant V600EB‐Raf reduces melanoma cell extravasation by decreasing IL‐8 production and interruption of ICAM‐1‐β2 integrin binding of melanoma cell to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant V600EB‐Raf to inhibit melanoma extravasation and subsequent metastasis development.

This study was supported by NIH CA‐97306, NSF BES‐0138474 and American Cancer Society RSG‐04‐053‐01‐GMC.
Maternal Body Composition of Postpartum Women in Rural Bangladesh by Deuterium Oxide Dilution and Bioelectrical Impedance Analysis
Authors: Schulze Kerry Jean , Shaikh Saijuddin , Labrique Alain , Kurpad Anura , Ali Md Hasmot , Rashid Mahbubur R , Shamim Abu Ahmed , Wu Lee S‐F , Christian Parul , West Keith P
Published: June 2021

Rural South Asia contributes greatly to the global burden of malnutrition and poor pregnancy outcomes. However, little is known of the relationship of maternal body composition (BC) to reproductive health for mothers or infants—in part because assessing BC in rural settings is challenging. Bioelectrical impedance analysis (BIA) is a field‐applicable approach for BC assessment, but population‐specific prediction equations derived from direct measures of BC, such as deuterium oxide (D2O) dilution, are required to interpret resistance (R50) data from BIA. We assessed BC at 19.3 ± 9.3 wk postpartum in 152 women who had participated in a placebo‐controlled randomized trial of weekly vitamin A or beta‐carotene supplementation. Saliva was collected at baseline and 3‐h after dosing women with a 0.15 g/kg body weight dose of ~95% D2O; its enrichment was measured by isotope ratio mass spectrometry to determine total body water (TBW). Women were 20.4 ± 5.3 y old, 41.2 ± 6.4 kg, and 148.7 ± 5.1 cm tall. Assuming hydration of fat free mass (FFM) was 73.2%, mean BC parameter estimates were: TBW, 22.6 kg, 55.3% of body wt; FFM, 30.9 kg, 75.5%; fat, 10.3 kg, 24.5%. A prediction equation for TBW from R50 (TBW (kg) = 4.588 + 0.174*weight (kg) + 0.360*height (cm)2/R50, R2=0.79, n=84) was derived, providing the means to readily estimate maternal BC by BIA methods of postpartum mothers in rural Bangladesh. Support: Gates Foundation, USAID, IAEA
A high protein diet maintains glucose production and enhances gluconeogenesis during exercise induced energy deficit
Authors: Smith Tracey J , Pikosky Matthew A , Schwartz Jean‐Marc , Rood Jennifer , Castaneda‐Sceppa Carmen , Glickman Ellen
Published: June 2021

Glucose production (GP) decreases when energy expenditure (EE) exceeds energy intake (EI), whether dietary macronutrient content modulates this response is unclear. We determined if increasing dietary protein (PRO) mitigates down‐regulation of glucose production (GP), glycogen breakdown (GB) and gluconeogenesis (GNG) during exercise‐induced energy deficit. Thirteen men (22 ± 1 yrs, VO2peak 60 ± 2 ml·kg−1·min‐1) increased EE 1000 kcal·d‐1 for 7‐d (D1‐7), following 4‐d baseline (BL1‐4) of normal EE (i.e. pre‐study). Volunteers consumed BL EI all 11‐d, producing 1,000 kcal d−1energy deficit on D1‐7. Subjects ate a 55% carbohydrate diet with either 0.9 (DEF, n=7) or 1.8 (DEF‐HP, n=6) g pro kg−1 d−1. GP and GNG were measured in the fasted, rested state on BL4, D3 and D8 using 4‐h primed, continuous infusions of [6,6‐2H2]glucose (dilution method) and [2‐13C]glycerol (MIDA technique), respectively. DEF experienced 17% decline in GP (2.3 ± 0.3 mg·kg−1·min−1), due to 24% decline in GB (1.4 ± 0.1 mg·kg−1·min−1) with unchanged GNG compared to BL4. Conversely, DEF‐HP maintained GP, attributed to 7% increase in GNG (1.0 ± 0.1 mg·kg−1·min−1), but no change in GB compared to BL4. Rate of GP for DEF‐HP was higher than DEF on D8 (1.1 ± 0.1 mg·kg−1·min−1 and 0.9 ± 0.0 mg·kg−1·min−1, respectively). Additional dietary protein mitigated effects of exercise‐induced energy deficit on GP. Research funded by MRMC.
Transplacental induction of fatty acid oxidation in newborn pigs by clofibrate
Authors: Xi Lin , Jacobi Sheila , Odle Jack
Published: June 2021

This study examined the effect of maternal feeding of clofibrate on fatty acid oxidation in newborn pigs. Pregnant sows were fed a commercial diet with or without 0.8% clofibrate for 7 days. Blood samples were collected from the utero‐ovarian artery of the sows and the umbilical vein of the intact term fetuses as they were removed from the sows by c‐section on day 113 of gestation. HPLC analysis clearly identified clofibric acid was present in the plasma of the clofibrate fed sow and the fetuses. The maternal‐fed clofibric acid had no impact on the liver weight of the newborn pigs, but hepatic fatty acid oxidation examined in fresh homogenates showed that clofibrate significantly increased 14C‐accumulation in CO2 and ASP by 2.9‐fold from [1‐14C]‐oleic acid and 1.6‐fold from [1‐14C]‐lignoceric acid. Correspondingly, hepatic carnitine palmitoyltransferase (CPT) and fatty acid oxidase (FAO) activities were increased also by 36% and 42% compared to the newborn pigs from the control sow. These data demonstrate that clofibrate crosses the placental membrane of the sow and enters fetal circulation to induce hepatic fatty acid oxidation by increasing CPT and FAO activities of the newborn. Supported by CSREES, USDA NRI program award 2007‐35206‐17897.
Superior Nutraceutical Properties of High Proanthocyanidin, High Flavonoid Sorghum Brans
Authors: Hartle Diane K. , Greenspan Phillip , Hargrove James L.
Published: June 2021

We are studying bioactivities of high proanthocyanidin‐containing (HPC) sorghum brans in a variety of experimental systems. HPC sorghum brans and their extracts are rich sources of proanthocyanidins, flavonoids and phytosterols for use in functional food/beverage, food supplement, and cosmeceutical industries. The HPC sorghum brans have a total phenolic content (50–70 mg phenolics/g) that is similar to spices. As a result, the phenolic content, antioxidant potential, anti‐inflammatory activity, and anti‐glycation activity are superior to rice, wheat and oat brans. HPC sorghum bran extracts inhibit a variety of target enzymes such as amylase, hyaluronidase and aromatase. Anti‐inflammatory activities have been tested in vitro (mixed monocyte functions) and in vivo (mouse TPA model of edema and inflammation). Anti‐glycation activities are relevant to anti‐diabesity and anti‐aging formulations. Most activities tested to date correlate positively with total phenolic content. Some bioactivities are in the proanthocyanidin fraction of polyphenolics, others are in the non‐proanthocyanidin fraction and some are in both. Bioactivities found in HPC sorghum bran were markedly higher than non‐HPC varieties of sorghum brans and other commercial brans with low total phenolic content. Supported by USDA #5854304363
Role of reverse‐mode sodium‐calcium exchange to serotonergic contractility in pulmonary arteries of hypoxic sheep
Authors: Goyal Ravi , Nguyen Dan , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Serotonin (5HT) is a prominent vasoactive substance in the lung with increases in intracellular Ca2+ being important to 5HT elicited pulmonary arterial (PA) contractility. L‐type Ca2+ channel (CaL) activation concomitant with membrane depolarization contributes significantly to 5HT dependent PA contractility and Ca2+ signaling. Chronic hypoxia (CH) is a risk factor in the development of pulmonary hypertension (PH) and we find CH reduced the role for CaL during 5HT‐mediated contractility in PA from fetal sheep, even though the contractility was unchanged when normalized to 125 mM KCl. Evidence indicates membrane depolarization sufficient to activate CaL will also shift the sodium‐calcium exchanger (NCX) into “reverse‐mode”, which would augment extracellular Ca2+ entry. Using wire‐myography on endothelium‐denuded PA rings from CH fetal and adult sheep, we tested the hypothesis that CH reduces “reverse‐mode” NCX in PA from fetus. PA rings were stimulated with 10 ?M 5HT and reverse mode NCX selectively inhibited with 10 ?M KB‐R 7943. In adult, 10 ?M KB‐R 7943 reduced 5HT‐elecited PA contractility to 76 ± 7 % of control while in fetal there was no affect on 5HT‐mediated PA contractility, being 97 ± 7 % of control. This work provides evidence that CH reduces “reverse‐mode” NCX activity in PA of fetal sheep, suggesting this process may contribute to CH‐related vascular dysfunction in newborn. (Support from NIH, NSF and UM)
STAGING FOR SIX DAYS AT 2200 M INDUCES PHYSIOLOGIC ADJUSTMENTS THAT MAY BE RESPONSIBLE FOR DECREASING ACUTE MOUNTAIN SICKNESS (AMS) AT 4300 M
Authors: Beidleman Beth A , Fulco Charles S , Muza Stephen R , Rock Paul B , Staab Janet E , Money Alison , Forte Vincent , Brothers Michael , Cymerman Allen
Published: June 2021

Staging at moderate altitude minimizes AMS following ascent to high altitude. However, few studies have reported physiological adjustments following staging that may be responsible for decreasing AMS. This study determined the effectiveness of 6 d of staging at 2200 m on physiological adjustments and AMS during rapid ascent to 4300 m. Eleven sea‐level resident men (mean±SE, 21±1 yr) completed resting measures of end‐tidal CO2 (PETCO2), arterial oxygen saturation (SaO2), and heart rate (HR) at sea level (SL) and within 1 h of exposure to 4300 m in both a hypobaric chamber (preSTG) and the summit of Pikes Peak following 6 d of staging at 2200 m (postSTG). AMS‐C (Environmental Symptoms Questionnaire) was measured following 8 h exposure at preSTG and postSTG. Resting PETCO2 (mmHg) was unchanged from SL (39.8±0.8) to preSTG (39.3±1.0) but decreased (P<0.05) from preSTG to postSTG (32.8±0.8). Resting SaO2 (%) decreased (P<0.05) from SL (97±1) to preSTG (80±1) and increased (P<0.05) from preSTG to postSTG (83±1). Resting HR (bpm) did not change from SL (69±2) to preSTG (70±2) but decreased (P<0.05) from preSTG to postSTG (65±2). AMS‐C decreased (P<0.05) from preSTG (0.94±0.14) to postSTG (0.59±0.13). These results suggest that staging for 6 d at 2200 m induces physiologic adjustments characteristic of altitude acclimatization that may be responsible for the reduction in AMS during rapid ascent to 4300 m. Funding provided by USAMRMC. Authors’ views; not official U.S. Army or DoD policy.