bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationThis work is supported by NSF award IOS1558044This 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 InformationThe 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 InformationThis 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 InformationResearch 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 AwardThis 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 InformationNational Science Foundation IOS 15573244This 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

BackgroundNeural 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.MethodOne 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 & DiscussionBy 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 InformationThis 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 InformationNSF‐OIA‐1738707This 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 InformationThis 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.