bluuebunny/crossref_metadata_2025 huggingface dataset:
Carboxyspermidine Dehydrogenase of Spermidine Biosynthesis from Bacteroides fragilis
Authors: Todd Kaleb Jonathan , McFarlane Jeffrey
Published: April 2020

The human gut microbiome performs a host of metabolic processes which remain largely unstudied, many of which have implications for human health. For example, human cells uptake the microbial polyamine spermidine (
1), and increased spermidine levels are associated with the progression of pancreatic and colon cancers (
2,
3). Thus gut microbe spermidine biosynthesis is a potential therapeutic target. Spermidine is produced by the dominant gut microbes via a unique enzymatic pathway (
4). The first enzyme in this pathway is carboxyspermidine dehydrogenase (CASDH), an NAD(P)H‐dependent saccharopine dehydrogenase expected to catalyze the reductive condensation of either 1,3‐diaminopropane or putrescine with L‐aspartate semialdehyde to form carboxynorspermidine or carboxyspermidine, respectively (
5). CASDH enzymes involved in spermidine biosynthesis have not been structurally or kinetically characterized from any species. We propose the characterization of CASDH from a dominant gut microbial species using X‐ray crystallography and steady‐state kinetic analysis. The CASDH gene from Bacteroides fragilis was chosen as this species is known to adhere to colon cancer epithelial cells and has been associated with tumor progression. We have successfully expressed BfCASDH in an E. coli BL21 expression cell line and purified it via affinity chromatography. Crystallization trials and the development of kinetic assays are currently underway. X‐ray crystal structures of BfCASDH will reveal the active site structure and the amino acid residues involved in binding and catalysis. In addition, our kinetic assays will confirm the preferred substrate identities and enzymatic activities of the BfCASDH. These data will contribute basic science knowledge necessary for the future explorations of potential inhibitors.

Support or Funding Information

KT was supported by NIH BLaST Undergraduate Research Award RL5GM118990. BLaST is supported by the NIH Common Fund, through the Office of Strategic Coordination, Office of the NIH Director with the linked awards: TL4GM118992, RL5GM118990, & UL1GM118991.
SPARC: Topographical Organization and Morphology of Substance P‐IR Axons and Terminals in the Whole‐Mount Stomach of Rodents
Authors: Ma Jichao Mark , Chen Jin , Powley Terry L. , Cheng Zixi Jack
Published: April 2020

Afferent fibers innervate the stomach. However, very little is known about the topographical distribution and morphological terminal structures of gastric nociceptive axons and terminals in rodents. In this study, we used Substance P (SP) as a marker to label afferent fibers in the whole‐mount dorsal and ventral stomach including longitudinal and circular muscles and the myenteric nervous system in C57BL/6J mice and SD rats. The whole‐mount dorsal and ventral stomachs were processed with the substance P (SP) primary antibody followed by the secondary antibody, and then examined using confocal microscopy. We found that: 1) SP‐IR axons and terminals extensively covered the fundus, corpus and antrum of the dorsal and ventral stomach. 2) SP‐IR varicose axons ran in parallel with the longitudinal as well as circular muscles. 3) SP‐IR varicose axons formed dense rings of terminal varicosities around individual neurons in the myenteric plexus. 4) The density of SP‐IR axons and terminals varicosities was different in the fundus, corpus and antrum. Collectively, our data showed for the first time the comprehensive topographical distribution and morphology of SP‐IR axons and terminals in different regions of the stomach. This knowledge will provide an anatomical basis for differentiated nociceptive gastric afferent functions in rodents.

Support or Funding Information

NIH HEAL/SPARC Program NIH 1 U01 NS113867‐01 and NIH R15 HL137143‐01A1.
Stimulation of Resolution of Inflammation via Pro‐Resolving Lipid Mediators Prevents Ethanol‐Induced Tumor Growth
Authors: Verheul Sanne Margriet Liefke , Fishbein Anna , Hallisey Victoria , Yang Haixia , Deng Jianjun , Panigrahy Dipak , Serhan Charles
Published: April 2020

Alcohol consumption is a common cause of various cancers such as hepatocellular carcinoma (HCC) by inducing inflammation. Alcohol is oxidized in the body to aldehydes, mostly acetyldehyde which is a group 1 carcinogen. However, effective therapies to stimulate the resolution of inflammation to prevent alcohol‐induced cancers have not been elucidated. Ethanol and acetyldehyde generate apoptotic cell (“debris”) which may stimulate inflammation and tumor growth via an inflammatory response. Thus, controlling ethanol‐stimulated inflammation will be critical to prevent HCC and other tumor‐types caused by ethanol. This could be addressed with specialized pro‐resolving lipid mediators (SPMs), such as maresins and protectins, which are derived from essential fatty acids and stimulate the resolution of tumor‐promoting inflammation by activating macrophage clearance of cellular debris. In contrast to traditional anti‐inflammatories (e.g. NSAIDs and steroids), SPMs are not immunosuppressive and are active at nanomolar concentrations. SPMs are currently being evaluated in clinical trials for various inflammatory diseases. Here, we demonstrate that ethanol‐generated cell debris stimulates primary tumor growth in vivo by stimulating the release of pro‐inflammatory cytokines and bioactive lipid mediators. Aldehyde‐generated debris also stimulated tumor growth via oxidative stress and reactive oxygen species. Systemic administration of SPMs prevent ethanol‐induced cancers in mice via resolution of inflammation. Cytokine array analysis demonstrated that ethanol‐generated tumor and non‐tumor cell debris of multiple tumor types each triggered human or murine macrophages to release a storm of pro‐inflammatory and pro‐angiogenic cytokines. Maresins, maresin conjugates, and protectin conjugates stimulated macrophage phagocytosis of ethanol‐generated and aldehyde‐generated cell debris and suppressed the release of pro‐inflammatory, tumor‐promoting cytokines by debris‐activated macrophages. Thus, ethanol‐generated cell debris triggers a cytokine storm providing a potential mechanism whereby ethanol harbors the potential to stimulate or induce tumor initiation, growth, and/or metastasis. Uncovering the mechanism of ethanol‐induced cell death and debris‐induced tumor progression is paramount to preventing cancer induced by ethanol‐generated inflammation. Dead cells generated by cytotoxic ethanol or aldehyde exposure may inadvertently stimulate proliferation of dormant tumor cells in patients. While generation of cell debris may explain risk of alcohol causing cancer, enhancing endogenous clearance of cell debris via SPMs, such as maresin conjugates and protectin conjugates, represents a novel approach to prevent ethanol‐induced cancer.
Laryngeal Adductor Reflex Motor Bursts Rapidly Oscillate in the Cat
Authors: Shen Tabitha Y. , Rose Melanie J. , Olsen Wendy L. , Wang Wei , McLeod Justin C. , Segers Lauren S. , Nuding Sarah C. , Pitts Teresa G. , Poliacek Ivan , Gestreau Christian , Morris Kendall F. , Musselwhite M Nicholas , Kermit Kofi K. , Bolser Donald C.
Published: April 2020

The laryngeal adductor reflex (LAR) is a protective behavior induced by stimulation of upper airway afferents. Laryngeal afferent information reaches interneurons in the medulla via the superior laryngeal nerve (SLN). Expiratory laryngeal motoneurons (ELMs) are subsequently excited causing contraction of the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles. The LAR consists of a short latency (~10 ms), short duration (~10 ms) burst (R1) and a longer latency (~40ms) burst (R2) in laryngeal electromyogram (EMG) activity. We have shown that R1 LAR magnitude undergoes frequency‐dependent depression in response to electrical stimulation (eStim) of the SLN. Based on preliminary observations, we hypothesized that the R1 component of the LAR induced by SLN eStim oscillates. In anesthetized, spontaneously breathing cats (n=12: 7 males, 5 females), we recorded EMG activity from the TA bilaterally and the PCA unilaterally. The SLN was electrically stimulated at 2 or 20 Hz to evoke TA LAR ipsilateral to stimulation (iTA). No sex differences in TA or PCA LAR EMG magnitude were observed. Consistent with previous findings, TA LAR magnitude differed with breathing phase; normalized iTA LAR magnitude was 10% larger during expiration compared to inspiration. TA LAR contralateral to stimulation (cTA) and ipsilateral PCA LAR magnitudes were not breathing phase dependent. TA and PCA LAR EMG oscillations were highly variable across animals (range: 244 to 1140 Hz). Mean iTA EMG frequency was decreased at 20 Hz (2 to 20 Hz: 616 to 518 Hz, p=0.05). Mean cTA periodicity trended down at 20 Hz (2 to 20 Hz cTA: 647 to 531 Hz, p=0.07). In separate decerebrate, paralyzed cats (n=13), extracellular recordings of neurons in the ventral respiratory column (VRC) with multiple electrode arrays were conducted. Peristimulus time histograms provided evidence that ELMs oscillated at frequencies following SLN eStim similar to the lower range of periodicities in TA EMGs in unparalyzed animals. A subset of neurons recorded in the VRC were excited or inhibited by SLN eStim at short latencies. Spike train analysis suggested that ELM oscillations could be accounted for by synaptic inhibition and excitation from laryngeal‐responsive VRC inspiratory, expiratory, and non‐breathing modulated neurons. We conclude that the LAR is temporally regulated by a complex, anatomically distributed brainstem network that is capable of strong synchrony in response to stimulation of laryngeal afferents. We speculate that the function of this laryngeal‐responsive network is to exert tight regulatory control over LAR patterning.

Support or Funding Information

Supported by NIH HL131716 and 3OT2OD023854
Molecular Transducers of Physical Activity Consortium (MoTrPAC) Progress
Authors: Joseph Lyndon , Williams John P. ,   The MoTrPAC Consortium
Published: April 2020

Physical activity is beneficial to human health and well being across the lifespan. The numerous benefits of regular physical activity have long been recognized. Despite this, most exercise studies are associational and the molecular mechanisms that are the bases for the beneficial effects remain obscure as are the mechanisms of multi organ communications and benefits.

The NIH Common Fund initiated the Molecular Transducers of Physical Activity Consortium (MoTrPAC) in December 2016 by issuing 19 grants to 37 Principal Investigators from 23 institutions. Fundamentally, MoTrPAC is a large discovery project the goals of which are to:

Aim 1: Assemble a comprehensive map of the molecular changes that occur in response to exercise and provide insights into how they are altered by age, sex, body composition and fitness level.

Aim 2: Develop a user‐friendly database to facilitate investigator‐initiated studies and catalyze the field of physical activity research whereby researchers can develop hypotheses exploring novel mechanisms by which physical activity improves or preserves health.

This project will explore and document changes in molecules mobilized in blood, muscle and fat in humans as well as 15 additional tissues for rats in response to different exercise regimes. The human studies are a multi‐center clinical trial cohort of people of both sexes from 10–80 years of age and recruitment has begun. Preclinical Animal Study Sites (PASS) have conducted treadmill running experiments in male and female 6 and 18‐month old F344 rats and harvested tissues from control inactive rats and rats at seven time‐points following a single 30 minute acute bout of treadmill running (70% VO2max) in their active phase (dark). A separate group of rats were subjected to an intensive (also 70% VO2max) and progressive run training program of 5 day/week for 1,2, 4, or 8 weeks, also in their active phase, with 18 tissues collected per rat. Multiple state of art and omics platforms including genomic, transcriptomic, epigenomic, proteomic and metabolomics are being employed to define and discover the molecules mobilized in response to exercise that will serve as a valuable resource for the research community and support future investigator‐initiated studies to understand the molecular mechanisms of exercises effects. PASS tissues not analyzed are stored in the MoTrPAC BioRepository and will be available for ancillary studies. This presentation will summarize data from the initial public data release (November 15th, 2019; five most abundant tissues). Data will be available to the extramural community to collaborate with the MoTrPAC investigators to explore innovative mechanisms to expand the impact of the MoTrPAC studies.

Support or Funding Information

NIH Common Fund
Inhalation of Air Particulate Matter Accelerates Neuroinflammation and Amyloid‐beta Accumulation in APP/PS1 Mice
Authors: Mackos Amy , Sahu Bijayani , O’Piela Devin , Combs Colin , Wold Loren
Published: April 2020

Alzheimer disease (AD) which is a progressive, neurodegenerative disease characterized by neuroinflammation and accumulation of amyloid‐β (Aβ) fibers leading to neuronal death. It is theorized that a combination of host genetics and environmental factors contribute to AD development and progression, however the trigger for neuroinflammation and Aβ development has remained elusive. One such environmental exposure linked to AD development and progression is air pollution. Thus, we designed a study to test our hypothesis that air particulate matter exposure accelerates AD development and progression. Male, wild type (WT) C57Bl/6 and AD transgenic mice, APPswe/PSEN1dE9 (APP/PS1), were exposed to either filtered air (FA) or air particulate matter sized under 2.5 μm (PM2.5) for 6 hrs/day, 5 days/week for 3 months. Brain tissue was collected at the end of the 3 month exposure and was either flash frozen or formalin‐fixed. Results demonstrated a significant increase in hippocampal Aβ accumulation in PM2.5‐exposed APP/PS1 mice over that of FA‐exposed APP/PS1 animals. PM2.5‐exposed WT mice did not develop Aβ plaques. Immune markers CD68 (tissue macrophage) and IBA1 (activated microglia) and the glial marker GFAP (activated astrocytes), as assessed by immunohistochemistry, were also significantly increased in PM2.5‐exposed APP/PS1 mice over their FA‐exposed counterparts. PM2.5‐exposure did not lead to enhanced levels of CD68, IBA1, or GFAP in WT mice. Taken together, these data suggest that airborne particulate matter has the propensity to alter the brain environment in mice genetically predisposed to develop Aβ in a way that mimics early AD‐like pathology.

Support or Funding Information

Funding support from AG057046 to LEW and CKC.
Allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin‐5
Authors: Downs Micquel , Kim Catherine , Kim Elizabeth , Liu Liqiong , Parameswaran Sreeja , Kim Sunyoung , Wojcik Edward
Published: April 2020

Human Kinesin‐5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small‐molecule inhibitors of Eg5 are candidate anti‐cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S‐trityl‐L‐cysteine (STC) inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity.

Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5‐targeting inhibitors of Kinesin‐5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three‐dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.
Nutraceuticals Derived From Pomegranate Selectively Enhance Vitamin D Receptor Signaling to Amplify Key Vitamin D Target Genes
Authors: Livingston Sarah , Lucas Daniel , Sabir Marya S. , Mallick Sanchita , Purdin Hespera , Nidamanuri Sree , Whitfield G. Kerr , Haussler Carol A. , Haussler Mark R. , Jurutka Peter W.
Published: April 2020

The active vitamin D hormone, 1,25‐dihydroxyvitamin D (1,25D), mediates its biological effects by binding to the nuclear vitamin D receptor (VDR) and promoting heterodimerization with retinoid X receptors (RXRs). Various nutraceuticals, including resveratrol and curcumin, have been postulated to interact with the vitamin D signaling pathway. More recently, health benefits attributed to pomegranate have been associated with its high content of polyphenols, specifically ellagitannins, which are metabolized by the gut microbiota to produce Urolithin A (UA). In this study, we investigated the ability of UA to modulate 1,25D signaling via transcription and qPCR assays. We hypothesized that 1,25D in combination with urolithin will stimulate VDR activity more than 1,25D alone, increasing transcription of 1,25D target genes. The increased activation of anti‐oxidation genes by 1,25D and UA could help combat reactive oxygen species and attenuate cellular “aging”. Our results indicate that there is a significant increase in VDR transcriptional activity when HEK‐293 kidney cells are treated with UA in conjunction with 1,25D at several 1,25D andUA concentrations. A similar effect was also observed using three structurally‐distinct 1,25D response elements. The potentiation of VDR activity via UA retains receptor‐selectivity since UA did not promote further activation of other receptor‐ligand complexes including estrogen‐ER, rexinoid‐RXR and TO‐LXR. Finally, amplification of serotonin production in brain cells was greatest with a combined cocktail of 1,25D/UA/resveratrol via qPCR (TPH2 gene) and ELISA (5‐HT). In order to probe the molecular mechanism of UA amplification in VDR activity, we tested VDR‐RXR heterodimerization and observed no effect of UA. However, UA activates AMP‐kinase, and we found potential AMP kinase phosphorylation consensus sites in the VDR protein sequence. Collectively, our novel results position gut microbiota‐derived UA as a putative VDR modulator, suggesting that the influences of 1,25D and UA converging on VDR may potentially mediate anti‐aging and promote longevity.

Support or Funding Information

Financial support/grant sponsor: National Institutes of Health; grant numbers: DK033351 and CA140285
Characterizing the Interaction Between CowN and Nitrogenase
Authors: Lee Terrence , Chong Kiersten , Garcia Chloe , Strul Max , Wong Emily , Kharwa Ruchita , Bretzing Kevin , Owens Cedric
Published: April 2020

Nitrogenase catalyzes the conversion of atmospheric dinitrogen into ammonia. The enzyme produces ammonia under ambient conditions using the free energy of ATP to drive dinitrogen’s reduction. The most common form of nitrogenase is molybdenum nitrogenase (Mo‐nitrogenase). Mo‐nitrogenase is inhibited by the ubiquitous pollutant carbon monoxide (CO). To prevent inhibition of Mo‐nitrogenase by CO, nitrogen‐fixing bacteria produce the protein CowN and in its presence, Mo‐nitrogenase avoids inhibition by CO and remains active. However, the mechanism by which CowN protects Mo‐nitrogenase is unknown. Enzymatic assays suggest that CowN and Mo‐nitrogenase interact with a K‐d of approximately 5–10μM. Here, we present data from crosslinking and pulldown assays that were used to determine how CowN interacts with Mo‐nitrogenase. Crosslinking assays using the heterobifunctional crosslinker EDC showed no binding between CowN and Mo‐nitrogenase in the presence and absence of CO gas. Pulldown assays using Ni‐NTA resin and a His‐tagged CowN bait also did not show significant binding between CowN and Mo‐nitrogenase. These assays indicate that CowN may not have a strong binding affinity to Mo‐nitrogenase to be detected by crosslinking or pulldown assays. Future work will investigate potential nitrogenase‐CowN interaction using fluorescence anisotropy.

Support or Funding Information

This research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).
Deciphering the isoform code contained in Vo a‐subunit isoforms of the V‐ATPase
Authors: Kane Patricia M , Jaskolka Michael C. , Tuli Farzana , Mitra Connie , Banerjee Subhrajit , Tarsio Maureen
Published: April 2020

V‐ATPases are highly conserved proton pumps that acidify multiple organelles, including lysosomes, endosomes, the late Golgi apparatus, synaptic vesicles, and other regulated secretory granules. Precise tuning of the luminal pH of these organelles is critical for function, but the factors governing organelle pH control are not completely understood. V‐ATPases are multisubunit complexes consisting of a peripheral subcomplex (V1) that contains sites for ATP hydrolysis and an integral membrane subcomplex (Vo) that contains the proton pore. The Vo a‐subunit is the largest subunit. It is comprised of an N‐terminal cytosolic domain and a C‐terminal domain that forms part of the proton pore. Most organisms encode multiple isoforms of the Vo a‐subunit that exhibit organelle‐specific localization. We hypothesize that the N‐terminal (NT) domains of a‐subunit isoforms participate in distinct cellular interactions that are critical for isoform‐specific V‐ATPase localization, activity, and regulation. In yeast cells, there are two organelle‐specific isoforms of the Vo a‐subunit, Vph1 and Stv1. Vph1‐containing V‐ATPases transit through the secretory pathway en route to the lysosome‐like vacuole. Their activity is regulated by reversible disassembly in response to glucose levels, and they require interaction with the yeast RAVE (regulator of acidification of vacuoles and endosomes) for both their initial biosynthetic assembly and for glucose‐dependent reassembly. The Vph1NT domain binds directly to RAVE and is responsible for glucose‐sensitive interactions with the RAVE complex. In addition, the vacuolar lipid PI(3,5)P2 promotes assembly and activity of Vph1‐containing V‐ATPases. Mutations in Vph1NT compromise PI(3,5)P2‐induced activation, suggesting direct binding to lipid. In contrast, Stv1‐containing V‐ATPases assemble and function in the absence of the RAVE complex, and Stv1NT does not bind to RAVE. However, Stv1NT binds to the Golgi‐enriched lipid PI(4)P, and mutations that abolish PI(4)P binding compromise Golgi retention of Stv1‐containing V‐ATPases. The aNT domains of the four human Vo a‐subunit isoforms exhibit differential recognition of phosphoinositide lipids. Some human aNT domains interact with the human homologue of the RAVE complex. These data indicate that NT domains of Vo a‐subunit isoforms encode information for localization and regulation of V‐ATPase activity that help determine organelle pH and protect cells from stress.

Support or Funding Information

NIH R01 GM127364 and NIH R01 GM126020
Effect(s) of Self‐Assembled Structures of Phenylalanine on the Amyloid‐like Fibril Formation of the Human Acidic Fibroblast Growth Factor
Authors: Kupovics Gabriel , Al-raawi Zeina , Thallapuranam Suresh Kumar
Published: April 2020

Excessive concentrations of the natural aromatic amino acid, phenylalanine, is characteristic of the severe genetic abnormality known as phenylketonuria (PKU). However, the fundamental mechanism by which phenylalanine causes the disease is largely unknown. Similarly, the etiology of Alzheimer’s disease is widely debated. Previous research has found elevated levels of acidic fibroblast growth factor (FGF1), a prototypical member of the FGF superfamily of cell signaling molecules which assist in cell differentiation and proliferation, wound healing, and neurogenesis, in cerebrospinal fluid of Alzheimer’s patients. This suggests that FGF1 may exert a significant role in neuroprotection. Under certain conditions, however, FGF1 has been found to form amyloid‐like fibrils (Srisialam et al. 2003), rendering it biologically inactive. Thioflavin T fluorescence kinetics and transmission electron microscopy (TEM) data suggest the formation of higher ordered fibrillar structures by phenylalanine in vitro. Fluorescence kinetics of phenylalanine reveal a higher propensity to self‐assemble at pH 2. Seeding experiments suggest the potential of phenylalanine (at 6 mM) to inhibit fibril formation of FGF1 and the modulation of its neuroprotective activity. These data will be discussed in detail.
Thiamine Insufficiency Induced Hypoxia‐Inducible Factor 1‐α (HIF‐1α) Regulates Alzheimer’s Disease‐Like Neuropathology
Authors: Valle Maria Luisa , Zastre Jason
Published: April 2020

Vitamin B1 (thiamine) insufficiency (TI) promotes debilitating neurotoxicity that frequently accompanies the most prevalent and devastating age‐related neurodegenerative disorder, Alzheimer’s disease (AD). Age related decline in thiamine levels highly correlates with a deterioration in cognitive function and a potential biomarker for AD diagnosis. Congruent with established AD pathology, TI promotes cerebral energy hypometabolism, inflammation, oxidative stress, and plaque formation. Yet despite progress into the relationship between reduced thiamine levels and the neuropathology of AD, mechanistic insight into how chronic thiamine insufficiency promotes AD‐like pathology is lacking. Our previous work has shown that TI stabilizes Hypoxia Inducible Factor‐1α (HIF‐1α), the main transcription factor involved in hypoxic stress, through an oxygen independent mechanism (pseudo‐hypoxia). After cerebral ischemia, HIF‐1α has been shown to potentiate the amyloidogenic cascade via direct transcriptional activation of β‐secretase‐1 (BACE‐1). BACE‐1 cleaves the Amyloid Precursor Protein (APP) into 99‐residue membrane‐associated fragments (C99) involved in the generation of β‐amyloid peptides. Therefore, HIF‐1α may be a critical link between thiamine insufficiency and AD pathophysiology. Treatment with the thiamine antagonist Pyrithiamine (PT) activated HIF‐1α and enhanced the levels of BACE‐1 and C99 fragments in mouse hippocampal neuronal cells (HT22). In association with the increased expression, BACE‐1 activity was also enhanced. Supplementation of the Prolyl Hydroxylase (PHD) activator Octyl‐α‐ketoglutarate, attenuated HIF‐1α activation and reduced expression and activity levels of BACE‐1. This suggests that TIinduced HIF‐1α activation may occur through a pseudo‐hypoxic mechanism via PHDs inhibition. Additionally, pharmacological inhibition of HIF‐1α via YC‐1 or by a Dominant Negative HIF‐1α construct decreased BACE‐1 expression and activity, confirming that the BACE‐1 increase was HIF‐1α dependent. Overall, these findings suggest that HIF‐1α may be a key transcriptional regulator in TI mediated AD‐like neuropathology.

Support or Funding Information

Department funding
Effects of Oligosaccharide Supplementation on Intestinal Morphology and Enteric Glial Cell Marker Expression in a Neonatal Pig Model
Authors: Shapiro Lindsey D. , Ziegler Amanda , Odle Jack , Van Landeghem Laurianne , Blikslager Anthony
Published: April 2020

We have shown that oligosaccharide supplementation can modulate the gut flora in neonates, and the microbiome is known to drive the maturation of the enteric glial cell (EGC) network postnatally. Here, we test the effects of dietary oligosaccharides on postnatal changes in the EGC network and intestinal morphology in a neonatal pig model.After suckling colostrum for 24‐hours, one‐day‐old pigs were grouped onto one of three formula‐based diets: control, high oligosaccharide (1:1 mixture of galactooligosaccharide and polydextrose, 8g/L), or low oligosaccharide (4g/L). Small intestine and colon samples were collected at 1‐ 7‐ 14‐ and 21‐ days‐of‐age for western blot and histological analysis. Preliminary histological results indicate a trend toward a decreased small intestinal villus length in the high oligosaccharide group at 7‐days‐of‐age, indicative of accelerated intestinal maturity. Expression of the EGC marker glial fibrillary acidic protein is increased in the small intestinal mucosa at 7‐ and 14‐days‐of‐age in the high oligosaccharide group based on a preliminary western blot (n=1). Following ongoing work to assess crypt morphology and quantify additional EGC markers S100B, PLP‐1, Sox10 in the small intestine and colon, we expect to find increased mucosal expression of EGC markers earlier in postnatal development in the high oligosaccharide group, as well as histological changes consistent with enhanced rates of gut maturation in pigs fed a high oligosaccharide diet. Understanding how dietary inputs drive intestinal development postnatally may improve practices for managing optimal gut heath early in life.

Support or Funding Information

NIH K01 OD 028207, NIH P30 DK 034987, NIH‐NICHD R01 HD095876, USDA‐NIFA VMCG‐0065
Estimating Phase Angle Percentiles in Elite Athletes
Authors: Morin Thomas V. , Baller Daniel P. , Clark Nicholas J. , Watts Krista L.
Published: April 2020

Bio‐impedance analysis (BIA) is a common technique used to estimate body composition. BIA measures the electrical impedance in water contained in a subject’s body and uses this measurement to predict body composition. Phase angle is one of the components of body composition and is a major indicator of body fat percentage. Although BIA is less accurate when predicting body composition compared to dual‐energy X‐ray absorptiometry (DXA) or bod pods, it is cheaper, more portable, and does not require a licensed operator. For those reasons, athletes commonly use BIA to estimate body fat composition. Current BIA techniques are shown to be inaccurate for very athletic individuals
[1] since it is calibrated based on the general population. Previous studies have calculated phase angle distribution in elite athletes using DXA data, but none have examined BIA. Our research uses empirical Bayesian analysis with BIA data to create reference percentiles for phase angle by sport and gender among elite European athletes.
Cerebrovascular carbon dioxide reactivity is influenced by sex despite comparable hemodynamic and respiratory chemosensitivity in young fit individuals
Authors: Drapeau Audrey , Imhoff Sarah , Labrecque Lawrence , Paquette Myriam , Le Blanc Olivier , Malenfant Simon , Brassard Patrice
Published: April 2020

Cerebral blood flow (CBF) is generally higher in women compared to men at rest. Although accumulating evidence supports sex differences in CBF regulation, findings remain contradictory for dynamic cerebral autoregulation (dCA) and cerebrovascular reactivity to carbon dioxide (CVRCO2). Cardiorespiratory fitness represents an indicator of cardiovascular health. However, we have recently reported that dCA is reduced in young fit men compared to controls, and that dCA is attenuated in young fit women compared to fit men. However, the influence of sex on CVRCO2 in these young fit individuals remains misunderstood. The aim of this study was to examine the influence of sex on CVRCO2 in young fit individuals. Changes in middle cerebral artery mean blood velocity (MCAvmean; transcranial doppler), mean arterial pressure (MAP; photoplethysmography), heart rate (HR; ECG) and pulmonary ventilation (spirometer) were measured in 29 young fit individuals (women n=10, men n=19), in response to changes in end‐tidal partial pressure of carbon dioxide (PETCO2; gas analyzer) during the modified Duffin hyperoxic rebreathing test. Women were tested in the early follicular phase. CVRCO2, as well as hemodynamic and ventilatory chemosensitivity, were characterized using linear regressions on the rebreathing portion of the test after the determined ventilatory recruitment threshold. While age was not different between groups (26 ± 6 vs. 27 ± 5 years, P = 0.92), height: (1.64 ± 0.06 vs. 1.77 ± 0.08 m; P < 0.0001), body weight (60 ± 6 vs. 72 ± 10 kg; P = 0.0009) and maximal oxygen uptake (48.4 ± 3.8 vs. 57.9 ± 6.8 mL−1kg−1min−1; P < 0.0001) were lower in women. At rest, MCAvmean (72 ± 8 cm/s vs. 62 ± 11 cm/s; P = 0.02) and HR (73 ± 8 vs. 64 ± 11 bpm; P = 0.02) were higher in women, whereas MAP was not different between groups (100 ± 19 vs. 97 ± 10 mm Hg; P = 0.73). CVRCO2 was higher in women compared to men, both in absolute (1.59 ± 0.78 vs. 0.35 ± 1.51 cm/s/mm Hg, P = 0.01) and normalized terms (2.19 ± 0.97 vs. 0.54 ± 2.41 %/mm Hg, P = 0.02). Hemodynamic and respiratory chemosensitivity responses during CO2 rebreathing were not different between groups (all P > 0.05). These results indicate that the cerebrovasculature of young fit women is more sensitive to CO2 than men despite comparable hemodynamic and ventilatory chemosensitivity responses.

Support or Funding Information

S.I., L.L. and S.M. have been supported by a doctoral training scholarship from the Fonds de recherche du Québec ‐ Santé (FRQS)
The Autophagy Gene ATG9A as a HIF Target Essential for Intestinal Epithelial Barrier Function
Authors: Dowdell Alexander Shea , Cartwright Ian , Kostelecky Rachael , Ross Tyler , Welch Nichole , Glover Louise , Colgan Sean
Published: April 2020

A pathological hallmark of mucosal diseases such as inflammatory bowel disease (IBD) is disruption of the intestinal epithelium, a selectively permeable barrier that acts to absorb nutrients and water while simultaneously guarding against pathogens and environmental threats. The intestinal epithelium exists in a metabolic state of low oxygen tension termed “physiologic hypoxia” that is essential for its proper function as an absorptive and barrier organ. An important factor in maintaining hypoxic epithelial homeostasis is the transcription factor family HIF, which is stabilized in hypoxic conditions and mediates epithelial homeostatic response to low oxygen tensions. To identify HIF transcriptional targets in epithelia, chromatin immunoprecipitation (ChIP) was performed in Caco‐2 intestinal epithelial cells (IECs) using HIF‐1α and HIF‐2α specific polyclonal antibodies. ChIP‐enriched DNA was hybridized to a custom promoter microarray (termed ChIP‐chip) comprising a genome‐wide set of predicted transcription start site (TSS) flanking sequences at 50‐bp resolution. This unbiased approach identified autophagy as one of the major HIF‐1 targeted pathways. Binding of HIF‐1 to ATG9A, the only transmembrane autophagy protein, was significantly enriched by exposure of IEC to hypoxia. Validation of the ChIP‐chip revealed prominent induction of ATG9A mRNA and protein. Likewise, using luciferase promoter assays, we identified a functional hypoxia response element upstream of the TSS. Hypoxia‐mediated induction of ATG9A was lost in cells lacking HIF‐1. Strikingly, we found that lentiviral‐mediated knockdown of ATG9A in IECs prevents epithelial barrier formation and results in mislocalization of multiple junctional proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic differences in junctional protein expression, suggesting that ATG9A is essential for multiple steps of epithelial junction biogenesis. Our findings have novel applicability for inflammatory disorders that involve a compromised epithelial barrier, such as IBD, and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.
Mitochondrial fatty acid synthesis: not just another way to make fatty acids
Authors: Nowinski Sara M. , Solmonson Ashley D. , Rusin Scott F. , Bensard Claire , Bott Alex J. , Morgan Jeffrey T. , Fogarty Sarah , Lettlova Sandra , Berg Jordan A. , Gygi Steven P. , Winge Dennis R. , Deberardinis Ralph , Rutter Jared P.
Published: April 2020

Mitochondria are often dubbed “the powerhouse of the cell,” however this gross over‐simplification has arguably led to a paucity of knowledge concerning the many other functions of these incredibly diverse organelles. Fatty acid synthesis (FAS) is an essential cellular process, but it is a little‐known fact even among mitochondrial experts that eukaryotes actually harbor two distinct FAS systems – one in the cytoplasm, and one in the mitochondria. Although mitochondrial FAS (mtFAS) was discovered decades ago, its function(s) in mammals are still unclear. One well known product of the pathway is lipoic acid, an important lipid co‐factor for several mitochondrial dehydrogenases, however we now appreciate that mtFAS plays a far wider role in mitochondrial physiology. Here we show that while mtFAS does not directly contribute to the synthesis of structural lipids like PS, it instead is required for the production of lipids that facilitate assembly and stability of the mitochondrial electron transport chain (ETC). This novel regulatory role of mtFAS occurs via physical interaction of the acyl carrier protein and a family of late‐stage ETC assembly factors known as the LYR proteins. Notably, this effect is separate from lipoic acid production, as Lipt1 knockouts do not display the same strong ETC depletion phenotype. Proteomics and metabolomics data provide intriguing insights into the consequences of mtFAS depletion, and highlight promising future directions towards further understanding the role of this pathway in cellular metabolism.

Support or Funding Information

This work was supported partially by grants from the NIH (GM115174 and GM115129 to JR and GM97645 to SPG) and the Nora Eccles Treadwell Foundation (to JR), as well as HHMI (JR). Support for SMN was also provided by UMDF and ACS postdoctoral fellowships.
Identification of regulatory elements in the C‐terminus of the Protein Acyl Transferase, zDHHC9.
Authors: Ramadan Ahmed , Conniff Amanda Sales , Kirouac Lisa , Deschenes Robert
Published: April 2020

Mutations in zDHHC9, a Ras palmitoyltransferase, has been linked to X‐Linked Intellectual Disability (XLID). zDHHC9 catalyzes the palmitoylation of its substrates by a two‐step transferase reaction, the first being the autopalmitoylation of the enzyme with palmitoyl‐CoA serving a palmitoyl donor. The second step is the transfer for palmitate from zDHHC9 to its substrate. Several XLID mutations have been shown to be defective in autopalmitoylation or palmitoyl transfer. However, one mutation, R298*, causing a premature truncation of the C‐terminus of the protein. Interestingly, the zDHHC9(R298*) mutant protein is catalytically active, but exhibits a subcellular trafficking defect in neurons. This region of the protein is predicted to be intrinsically disordered. In this study, we test the hypothesis that there is a motif or motifs in the C‐terminal intrinsically disordered region that is required for subcellular location and substrate engagement by zDHHC9. This region includes a three cysteine cluster found in other members of the zDHHC family. Mutation of the three cysteine impairs location and is inactive in an orthotopic yeast zDHHC functional assay. We are testing whether one or more of these cysteines are palmitoylated. In addition, we are using BioID to identify proteins that interact with the zDHHC9 C‐terminus. We propose that identification of zDHHC9 interacting proteins will reveal how localization regulates palmitoyltransferase activity and contribute to our understanding of zDHHC9 dependent palmitoylation contributes to neuronal development and XLID.
An investigation of the pro‐inflammatory effects of wood smoke on human lung airway epithelial cells.
Authors: Enright Sarenna , Jones Shannon
Published: April 2020

Over two‐fifths of the world’s population uses solid biomass fuels such as animal dung, plastic, rubber or wood for the purpose of cooking and the heating of their homes. In some instances, especially in underdeveloped regions of the world, biomass smoke exposure can disproportionately impact women and children of color, as they are more than likely to be performing cooking duties and being exposed to this type indoor air pollution. Poor ventilation of homes can also contribute to poorer health outcomes associated with biomass smoke exposure. These individuals exposed to biomass smoke and particulate matter are also more likely to be suffer from negative health outcomes such as repeated, acute lower respiratory infections (ALRI) and asthma. The mechanisms by which biomass smoke exposure contributes to these pathologies of the lung remain largely unknown. The purpose of this research was to determine the general effects of woodsmoke on lung airway epithelial cells (A549). In this project, we are modeling the effects biomass smoke exposure using solubilized wood smoke. Biomass smoke is believed to have a significant impact on the innate immune system and the epithelial cells than line airways. To determine the pro‐inflammatory effects of woodsmoke, we used the polymerase chain reaction (PCR) to assess the expression of many pro‐inflammatory genes. Results thus far show that treated A549 cells displayed an increase in COX‐2 expression with increasing concentrations of woodsmoke. This induction of iNOS, TGF‐beta, and HO‐1 gene expression was also observed. The cytotoxicity of woodsmoke on A549 cells was also assessed using the lactate dehydrogenase assay (LDH) assay. Future studies are aimed at determining the impact of woodsmoke on cytokine secretion in A549 cells and whether smoke exposure is capable of inducing oxidative stress in these cells.

Support or Funding Information

University of Richmond’s Integrated and Inclusive Science Program
Identification of Candidate Genes Involved in the Survival of Drosophila in Extreme O2 Environments
Authors: Stobdan Tsering , Zhao Huiwen W. , Zhou Dan , Iranmehr Arya , Lu-Bo Ying , Bafna Vineet , Haddad Gabriel G.
Published: April 2020

The origin and evolution of life forms depended by and large on O2 availability over thousands of years. The mechanism of the adaptation process under strong selection pressure, however, is subject to some debate. For instance, it could be mediated by extant mutations or cryptic genetic variation that yield a fitness advantage in the new environment, or by mutations that arise de novo. For sexually reproducing organisms, multiple favored variants can also be acquired on a single haplotype via recombination to alleviate the effect of clonal interference and accelerate adaptation. It is however difficult to directly observe evolution in action. In the current study, we have identified the genomic intervals under selection in the high and low O2‐adapted Drosophila melanogaster. The adapted Drosophila melanogaster constitutes flies that could complete its life cycle in 90% and 4% O2 environments respectively, which otherwise is lethal for the naive flies. We used UAS‐RNAi x daGal4 system to functionally validate the role of specific genes of the top selected genomic interval. The top interval selected under high O2 environment consisted of 32 genes that includes four non‐protein‐coding genes. RNAi lines were available for 15 genes (15/28) that were evenly scattered across this interval. The high O2 tolerance assay indicate >98% eclosion rate in the adapted flies. Four genes i.e., CG15472, Klf15, CG2861 and CG42594, depicts a relatively higher number of pupation compared to the controls (12±0.6 to 14±2.5 versus <7 pupae counts per vial. The average pupae eclosed was higher for two of the genes, CG15472 and CG42594, although these numbers were significantly lower than the adapted flies. The percentage eclosion rate was significantly higher for CG15472 ‐(91.4 ±16.7%), which is close to the 98% eclosion seen in the adapted flies. Similarly, we have identified intervals under selection in the low O2 adapted flies. Remarkably, there was higher proportion of genes from the Notch signaling pathway in these selected intervals. Our results so far indicate that CG15472 from the top selected interval is one of the important gene playing critical role in the successful adaptation of flies to high O2 environment. Similarly, the Notch signaling pathway has an important role in the successful adaptation of flies to extremely low O2.

Support or Funding Information

NIH grant (R01 HL127403‐02, R21NS101652‐01) to GGH. NSF grant (NSF‐DBI‐1458557, NSF‐IIS‐1318386) and NIH (1R01GM114362) to VB and AA.
Infectious, Rhesus Monkey‐Derived Cellular DNA Sequences in Certain Stealth Adapted Viruses
Authors: Martin W. John
Published: April 2020

The risks of using monkey kidney cells to produce poliovirus vaccines have included the transmission of monkey viruses to humans. This occurred with SV‐40 virus from rhesus monkeys and with simian cytomegalovirus (SCMV) from African green monkeys. Vaccine contaminating viruses can undergo an immune evasion process referred to as stealth adaptation. This results from the deletion or mutation of the genes coding for the relatively few virus components normally targeted by the cellular immune system. Stealth adapted viruses can further incorporate cellular and bacterial genetic sequences, which become components of the replicating viruses. Cytopathic stealth adapted viruses have been cultured from many patients with the chronic fatigue syndrome (CFS). Some of the cultures were further tested using a low stringency polymerase chain reaction (PCR). When performed on stealth adapted virus cultures from three CFS patients, the PCR assay amplified various genetically unstable, rhesus monkey genome‐derived genetic sequences. Although slightly different due to genetic instability, the three cultures have some common rhesus monkey‐derived genetic sequences. This finding confirms the inclusion of these sequences in human to human transmission. In one of the patients, additional cellular‐derived sequences were of human origin, probably arising from homologous recombination. If this were to occur in germ cells, the substitution of a monkey for a human sequence in the human genome would have long‐lasting multi‐generational consequences. The cellular sequences so far identified in stealth adapted viruses are from non‐coding regions with no apparent ascribed cellular functions. Still, these viruses can potentially acquire oncogenic cellular sequences, which would then be transmitted between humans. In conclusion, stealth adapted viruses can act as interspecies carriers of genetically unstable cellular sequences. The available sequence data on the monkey and cellular DNA incorporated into stealth adapted viruses cultured from different CFS patients will be presented.