bluuebunny/crossref_metadata_2025 huggingface dataset:
Global Dysphagia Scale Detects Swallowing Disorder Following a Clinical Dose of Buprenorphine
Authors: Frazure Michael , Young Kellyanna , Musselwhite Nick , Greene Clint , Iceman Kimberly , Pitts Teresa
Published: May 2022

Opioid use is associated with increased risk of chest infection due to opioid suppression of immune function, ventilation, cough, and secretion mobilization. Buprenorphine is a highly lipophilic, semi‐synthetic partial agonist of the mu opioid receptor. Intramuscular buprenorphine is commonly used for post‐operative analgesia at a dose range of 0.01‐0.04 mg/kg in the cat. While buprenorphine is considered to have a low side effect profile, its effects on the pharyngeal phase of swallow are unknown. We hypothesized that administration of opioid would result in clinically meaningful decline of pharyngeal swallow function. Experiments were performed on healthy adult cats (12 months old). Animals were administered intramuscular buprenorphine (0.015 mg/kg) q12 hours for 48 hours. One hour after the last dose was given, videofluoroscopic swallow studies were performed to evaluate the effects of the drug on swallow function. Animals were presented with 40% by volume barium sulfate in tuna water mixed to a thin consistency, and all animals ate voluntarily. Images were recorded in the lateral plane at 30 frames/second and compared to control feeding assessments using the Global Dysphagia Scale (GDS), a novel tool developed to rate swallow function in the cat. The GDS consists of an Airway Invasion Scale (AIS) and relevant components of the Modified Barium Swallow Impairment Profile (MBSImP), a standardized protocol for swallow assessment. The AIS is a 10‐point scale where scores of 0‐1 are normal, scores of 2‐4 indicate laryngeal penetration, and scores of 5‐9 indicate tracheobronchial aspiration. Four components of the MBSImP were included: Initiation of the pharyngeal swallow, pharyngeal stripping wave, pharyngoesophageal segment opening, and pharyngeal residue. The GDS rating was derived by adding the AIS and MBSImP sub‐totals. During control assessments mean GDS was 5.5 ± 1.3, and aspiration was not detected in any animal. On buprenorphine, mean GDS was 11.25 ± 4.2, and 50% of animals exhibited large volume tracheobronchial aspiration. In conclusion, after 48 hours on buprenorphine, airway protection during swallow failed without appreciable response in 50% of animals (no cough, throat clear, or cessation of eating). Our success in producing dysphagia (significant but varying by animal) with relatively small doses of opioid has potential implications for predicting aspiration pneumonia in post‐surgical clinical cases.
Pharmacological Preconditioning by Corticotropin Releasing Factor (CRF) or ACTH Protects the Gastric Mucosa against Ulcerogenic Action of Indomethacin through Involvement of Glucocorticoids
Authors: Filaretova Ludmila , Morozova Olga , Komkova Olga , Yarushkina Natalia
Published: May 2022

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are widely used as analgesic and anti‐inflammatory drugs. However, adverse effects, including gastrointestinal, renal, cardiovascular side effects, seriously complicate NSAIDs use. Their gastrointestinal side effects are the most known serious complications in patients taking these drugs. Previously we have shown that preconditioning stress attenuates the ulcerogenic action of indomethacin (IM) on the gastric mucosa. According our findings glucocorticoids, released in response to stressor, are gastroprotective hormones and involved in stress preconditioning protective effects. Corticotropin releasing factor (CRF), a major mediator of stress response, stimulates ACTH release through CRF receptors of subtype 1 and ACTH, in turn, stimulates glucocorticoid production. Here we verify the hypothesis that pharmacological preconditioning by CRF or ACTH can protect the gastric mucosa against ulcerogenic action of IM in rats through mechanism associated with glucocorticoids. For this, preliminary fasted (24 h) rats were administered by CRF (2.5 or 5 µg/ kg, ip) or ACTH (1U/kg, ip) 30 min before IM injection (35 mg/kg, sc). Gastric lesions were examined 4 h after IM administration. Plasma corticosterone levels were measured before and 4 h after IM injection. Since the gastrointestinal injury is accompanied by changes in somatic pain sensitivity, we also measured tail flick latencies (tail flick test) before and 4 h after IM. Both CRF and ACTH by itself caused an elevation of plasma corticosterone levels, which was accompanied by an increase of tail flick latencies (analgesic effect). IM administration resulted in the gastric erosion 4 h later. Pretreatment with CRF or ACTH reduced an area of gastric lesions caused by IM (gastroprotective effect). IM‐induced gastric injury was accompanied by an increase of tail flick latencies, which was prevented by CRF (normalization of somatic pain sensitivity). To estimate the role of glucocorticoids in CRF‐induced gastroprotection an inhibitor of corticosterone synthesis metyrapone (30 mg/kg) or CRF receptor type 1 antagonist NBI 27914 (10 mg/kg) or glucocorticoid receptor antagonist RU‐38486 (20 mg/kg) was administered before CRF. Both metyrapone and NBI 27914 injected before CRF administration caused an inhibition of CRF‐induced corticosterone response and prevented protective effect of CRF on the gastric mucosa against the IM‐induced injury. The gastroprotective effect of CRF was also eliminated by the pretreatment with RU‐38486. Thus, CRF as well as ACTH can protect the gastric mucosa against ulcerogenic action of IM. Gastroprotective action of CRF accompanied by normalization of somatic pain sensitivity is provided by mechanism associated with glucocorticoids.
Actin‐Fence Enhancement Protects against Extra‐Pulmonary Infection of Pseudomonas Aeruginosa
Authors: Quadri Sadiqa K. , Guangchun Jin K. , Islam Mohammed N. , Bhattacharya Sunita K. , Bhattacharya Jahar K.
Published: May 2022

Lung‐endothelial membrane damage underlies acute lung injury (ALI), a major cause of mortality. ALI occurs after extra‐pulmonary infection by Gram‐negative bacteria. However, damage‐inducing bacteria‐endothelial interactions are unclear. Endothelial uptake of blood‐borne bacteria might induce cell‐membrane pores (CMPs), leading to membrane damage. Since the cytoskeletal involvement in this membrane damage remains unclear, we explored the role played by the membrane stabilizing, cell cortical f‐actin layer (PMID: 33749665). We induced extra‐pulmonary infection in mice, by intraperitoneal (i.p.) inoculation of fluorescent (GFP‐labelled) Pseudomonas aeruginosa (GFP‐PA) (3.5x106 cfu). In one group (n=5), after 40 hours we carried out real‐time confocal microscopy (RCM) of the lung to determine GFP‐PA interactions with calcein‐red (CR)‐loaded lung endothelium, per our reported approaches (PMID: 33749665). In another group, we carried out single‐blind assessments of survival in mice given no therapy (n=10), or the actin fence enhancer, TAT‐V12Rac1 (PMID: 33749665) by intravenous injection 7 hours after PA inoculation (n=10). RCM of live lung microvessels revealed GFP‐PA as green particles attached to the lung‐endothelial surface. Therefore, bacteria released from the peritoneum, reached the lung by the blood route. Confocal sections taken through the cell interior revealed internalized bacteria as yellow particles, reflecting the merged fluorescence of green (bacteria) and red (cytosol). Imaging indicated slow loss of CR fluorescence over the time course of an hour. As we reported, this slow loss of cytosolic dye indicates CMP formation (PMID: 22561462). In the survival assay, 80% of the mice died within 3 days of PA inoculation (p<0.01 versus PBS control). By contrast, in the group given TAT‐V12Rac1, mortality was markedly less at 30% (p<0.01 versus bacteria alone). We show for the first time, that within 2 days of i.p. inoculation, GFP‐PA internalized in lung endothelium, causing CMP formation. A day later there was high mortality that however, could be majorly abrogated by enhancing the endothelial actin fence by therapy with intravenous TAT‐V12Rac1. Thus, the CMPs may have promoted endothelial damage, an effect that was mitigated by fence enhancement. We suggest, the actin fence is a major protective factor against bacteria‐induced endothelial damage. Actin fence enhancement might be viable therapy against ALI due to septicemia.
Deficient Central Leptin Signaling “In Vivo” Alters Hypothalamic Levels of Circadian Clock Proteins Changing the Diurnal Eating Behavior in Rats
Authors: Mazuecos Lorena , Pintado Cristina , Villar Margarita , Rubio Blanca , Rodriguez Maria , Arribas Carmen , Andres Antonio , Gallardo Nilda
Published: May 2022

Background

Leptin, acting at CNS, decreases food intake (FI), body weight (BW) and adiposity, while circadian dysfunction induces hypothalamic leptin resistance associated to increased adiposity. Hence, we hypothesized that partial inhibition of hypothalamic leptin signaling “in vivo” would alter circadian rhythms resulting in changes in diurnal eating behaviors leading to increased adiposity and peripheral insulin resistance.

Methods

3‐months‐old Wistar rats were randomly assigned in two different groups: rats infused “in vivo” with saline (PBS) (n=12) or rats infused with a leptin antagonist (SLA) (0.2 micrograms/day) (n=12) via icv minipump insertion for 21 days. On day 0, rats were individually housed in metabolic cages and maintained for 21 days measuring daily BW, FI and locomotor activity. Hypothalamic proteomic analysis (n=6 per group) was carried out to determine if hypothalamic circadian proteins could be altered upon chronic SLA infusion. On day 21 and before sacrifice, one group of rats infused with PBS or SLA (n=6 per group) were injected icv with 100 ng of rat leptin in a volume of 2 microliters. 30 min later rats were euthanatized.

Results

SLA infusion decreased leptin‐induced phosphorylation of pY705‐STAT3 at the hypothalamus sustaining the induction of central leptin resistance upon chronic SLA administration. As a result, daily FI, final BW and visceral adiposity increased in SLA‐infused rats. Remarkedly, the diurnal eating behavior was altered in the SLA group of rats. In fact, daily FI was significantly increased by 48% only during the light phase in SLA infused vs PBS treated control rats, while in the dark phase FI increased less than 10% in the SLA group compared with control PBS‐infused rats. Moreover, SLA‐infused rats develop peripheral insulin resistance assessed by HOMA‐IR at the end of the treatment. Hypothalamic proteomic analysis revealed significant changes in 446 proteins, 266 were up‐regulated and 180 down‐regulated upon SLA infusion. Interestingly, proteins involved in the transcriptional activity of BMAL1/CLOCK were altered in SLA‐infused rats. Paraspeckle component 1, involved in the negative regulation of BMAL1/CLOCK, was over‐represented, while vasopressin‐neurophysin 2 copeptin, a positive regulator of circadian gene expression, was under‐represented.

Conclusion

Our results suggest that deficient hypothalamic leptin signaling is associated to circadian dysfunction which leads to changes in the diurnal eating behavior in rats resulting in increased adiposity and peripheral insulin resistance.
Early Life Stress Increases Lipid Storage in Female Mice Fed a High Fat Diet via MR Activation in Adipocytes
Authors: Leachman Jacqueline R. , Cincinelli Cole , Xu Mei , Morris Andrew , Hinds Terry D. , Loria Analia S.
Published: May 2022

We have previously shown that Maternal Separation and Early Weaning (MSEW), a mouse model of early life stress, exacerbates high fat diet (HF)‐induced obesity only in female offspring. Obese female MSEW mice display increased circulating and adrenal‐derived aldosterone and increased gWAT‐derived corticosterone compared with controls (C). Mineralocorticoid receptor (MR) activation has been shown to be critical in the regulation of the differentiation process of pre‐adipocytes into mature adipocytes and overall adipocyte homeostasis. The aim of this study was to determine the role of MR in the mechanism by which MSEW induces perigonadal hypertrophy in female mice fed a high fat diet (HF). MSEW and C mice were weaned onto a HF (60 % Kcal from fat). After 20 weeks, gonadal white adipose tissue (gWAT) was collected to conduct: 1) lipidomic analysis and markers of whitening, 2) collagenase digestion followed by magnetic activated cell sorting for isolation of preadipocytes for RNAseq, and 3) in vitro lipolysis assay. Another subset of mice was randomized to receive vehicle (50% Ora swift in water) or a MR blocker, spironolactone (Spiro, 100 mg/kg/day), for 2 weeks. Female MSEW mice showed increased adiposity along with reduced mitochondrial DNA density (0.81±0.09 and 1.08±0.07 mtDNA/gDNA, p<0.05 MSEW vs. C, respectively). Also, female MSEW mice displayed an increase of over 20 triacyclglycerols TAG, including a 6‐fold increase in 44:2/FA 18:2+NH4 (p<0.05 vs. C). Isolated mature adipocyte RNAseq revealed a 10‐fold downregulation of Aquaglycerolporin 3 (AQP3) in MSEW mice, a channel in adipocytes responsible for glycerol efflux (p<0.05 vs. C). Western blot analysis confirmed this reduction (0.40±0.08 and 1.0±0.14 AQP3/HSP90 p<0.05 MSEW vs. C, respectively). Spiro treatment induced a significant fat mass loss in obese MSEW females (‐3.5±0.9 and ‐1.0±0.5 delta fat mass (%BW) p<0.05 MSEW vs. C, respectively). Increased intracellular glycerol in vehicle‐treated MSEW mice was reduced to levels comparable to controls after Spiro treatment (1.08±0.2 and 1.64±0.12 mg glycerol/mg tissue, p<0.05 MSEW vs. C, respectively) while Spiro also decreased basal and stimulated released glycerol in MSEW mice (p<0.05). There were no differences in plasma glycerol between groups. It has been shown that aldosterone modulates AQP3 expression via MR signaling; however, Spiro treatment did not modify AQP3 protein expression. Overall, this data suggests that MR may play a role in adipose tissue hypertrophy as displayed by MSEW mice by preventing glycerol release in favor of triglyceride formation and storage, suggesting a role for impaired lipolysis that is independent of reduced AQP3 protein expression in adipocyte hypertrophy displayed by obese female MSEW mice.
Allosteric Probes for Modulating Essential Bacterial Chaperone‐Cofactor Interactions
Authors: Lupoli Tania
Published: May 2022

DnaK is the bacterial homolog of Hsp70, an ATP‐dependent chaperone that assists cofactor proteins to catalyze nascent protein folding and salvage misfolded proteins. In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactor proteins DnaJ1, DnaJ2 and GrpE are proposed drug targets. Despite the importance of chaperone function in human cancers and infectious disease, there are limited chemical probes or inhibitors that enable in vivo studies on the function of individual chaperones/cofactors. Here, we describe the discovery of small molecule inhibitors of mycobacterial DnaK from a re‐purposed drug library, most notably a peptidomimetic called telaprevir, which is able to inhibit chaperone function through interactions with the peptide‐binding cleft of DnaK. Binding of telaprevir leads to allosteric conformational changes that prevent ATP hydrolysis in a distal domain. We find that telaprevir also inhibits E. coli DnaK and human Hsp70 chaperones due to high conservation of Hsp70 sequences. Using in vitro and in vivo chaperone assays, we demonstrate that telaprevir modulates the function of mycobacterial DnaK and its cofactor protein DnaJ2 in cells, which disrupts cellular proteostasis. Co‐treatment of mycobacteria with telaprevir and aminoglycosides, which further stress the proteome, enhances the potency of these antibiotics. In addition, telaprevir combats mycobacterial resistance to the frontline TB drug rifampin, as DnaK‐DnaJ2 function is required for stabilization of protein mutants that confer drug tolerance. This work sets the stage for the design of peptide‐based inhibitors with higher selectivity for bacterial chaperones to probe chaperone‐protein interactions and explore resulting synergy with different classes of antibiotics.
Obesity‐related higher blood pressure is associated with augmented transduction of spontaneous muscle sympathetic nerve activity
Authors: Holwerda Seth W. , Gangwish Megan E. , Luehrs Rachel E. , Nuckols Virginia R. , Pierce Gary L.
Published: May 2022

Central obesity is associated with elevated adrenergic activity and arterial blood pressure (BP). Therefore, we tested the hypothesis that transduction of spontaneous muscle sympathetic nerve activity (MSNA) to BP, i.e., sympathetic transduction, would be augmented in central obesity (increased waist circumference) and positively related to resting BP. Young/middle‐age obese (n=14) or non‐obese (n=14) adults without hypertension (average ambulatory 24‐hr BP<130/80 mmHg) were included. MSNA (microneurography) and BP (finger cuff) were measured continuously for 10 min (supine) and bursts of MSNA were grouped into quartiles of burst clusters (1, 2, 3, 4+bursts) and burst amplitude. Beat‐to‐beat mean arterial pressure (MAP) was signal‐averaged over 10 cardiac cycles following each burst of MSNA. MSNA burst frequency was similar between obese and non‐obese (21±3vs.17±3 bursts/min, P=0.34). However, supine BP was significantly higher in obese compared with non‐obese (systolic: 127±3vs.114±3; diastolic: 76±2vs.64±1 mmHg, both P<0.01). Importantly, obese showed greater MAP responses to clusters of bursts compared with non‐obese (e.g.,4th quartile: 4.5±0.4vs.3.3±0.4 mmHg, P=0.05), but did not show greater MAP responses to higher burst amplitude (P=0.13). MAP responses to clusters of bursts were positively correlated with systolic BP when controlling for age, sex, and MSNA (R=0.43, P=0.03), but this correlation was abolished when also controlling for BMI (R=0.20, P=0.34). Diastolic BP was only weakly correlated with MAP responses to clusters of bursts (R=0.27, P=0.20). These findings suggest that the pressor response to clustered bursts of MSNA (i.e., sympathetic transduction) is augmented in central obesity and may contribute to the obesity‐related rise in supine systolic BP.
Elucidating The Nicotinic Acid Degradation Pathway In Bacillus niacini: Identification and Biochemical Characterization Of Proteins Of Unknown Function
Authors: Khal Sai K. , Helton Noah S. , Snider Mark J.
Published: May 2022

N‐Heterocyclic aromatic compounds (NHACs) are abundant in the manufacturing of solvents, pesticides, and dyes; however, accumulation of these compounds can lead to detrimental environmental impacts. To alleviate these impacts, it is critical to understand the mechanisms microbial species use to catabolize these types of molecules so that bioremediation techniques can be implemented. Nicotinic acid is commonly used as a model system for NHAC degradation because it is environmentally safe and can be degraded by a variety of bacterial species. Bacillus niacini is a soil‐dwelling bacterium that has been identified to degrade NA through an uncharacterized pathway. This makes B. niacini an organism of interest, as elucidation of the mechanism by which it catabolizes NA will reveal differences between these pathways that can be exploited to optimize bioremediation efforts. Bioinformatic analyses of genomic and transcriptomic data revealed a potential operon responsible for NA degradation in B. niacini. This operon contains 15 genes that encode for 7 enzymes that are necessary for NA degradation as well as three genes that have not yet been elucidated: a domain of unknown function (DUF), a flavin monooxygenase (FMO), and a hypothetical protein (HP). NA degradation in B. niacini is unique in that it involves two ring hydroxylating steps, forming the intermediate 2,6‐dihydroxynicotinic acid (DHNA). After decarboxylation of DHNA to 2,6‐dihydroxypridine (DHP), we show that the DUF hydroxylates DHP to 2,3,6‐trihydroxypyridine (THP). Separation of the DUF‐catalyzed reaction products by C‐18 chromatography (HPLC) and detection by UV‐Vis at pH 3 shows a new species with an Absmaxat 320 nm and 590 nm. Mass spectrometry (Q‐TOF) analysis of the reaction identifies the products as DHP‐ and THP‐ oxidized dimers (nicotine blue). Formation of these products are observed to be O2 dependent. When the DUF, FMO and HP genes are recombinantly expressed together in E. coli, the cell extract is able to degrade DHP without forming the DHP‐oxidized dimer. That observation suggests the possible roles of FMO and/or HP in catalyzing the conversion of the DHP‐dimer back to DHP. This study is the first to characterize the genes responsible for NA degradation by B. niacini and further establishes the NA degradation pathway.
No Effect of Permanent Tattoos on Local Sweat Concentration or Excretion Rate of Urea Nitrogen, Cortisol, Glucose, or Lactate During Exercise
Authors: King Michelle A. , Keyes David , Brown Shyretha , Engel Megan , Ciciora‐Gold Matthew , De Chavez Peter , Baker Lindsay
Published: May 2022

Permanent tattooing is common on areas of the skin typically sampled for sweat diagnostics, yet its potential to alter local sweat concentration and excretion rate (ER) of metabolites and other compounds is unknown. The purpose of this study was to investigate the influence of tattooed skin (TAT) on local sweat concentration and ER of urea nitrogen (UN), cortisol (CORT), glucose (GLU) and lactate (LAC) when compared to contralateral non‐tattooed skin (NT) during a group fitness exercise session. Moderately trained participants (males n=8, females n=8, 34±8 y) with at least one permanent tattoo (age of tattoo 7±5 y) on the upper body (arm, scapula, or thorax regions) underwent a ~60 minute outdoor (~27±3°C, 63±4% RH, 26±3°C WBGT) group fitness exercise session. Sweat was collected with absorbent patches (3MTMTegaderm+PAD) placed on the TAT and on NT. Patches were monitored during exercise and removed upon moderate saturation. Local sweat rate (LSR) was calculated from sweat mass over patch surface area (11.9 cm2) and exercise duration (56±2 min). Excretion rates (ER) were calculated as the product of analyte concentration and local SR. Sweat was analyzed by ELISA for UN (Invitrogen, ThermoFisher Scientific), CORT (Invitrogen, ThermoFisher Scientific), GLU (Caymen Chemical Co.) and LAC (LSBio). To evaluate differences between TAT and NT skin, paired t‐tests or Wilcoxon signed rank tests were used. Significance level was set at p<0.05. Data are presented as mean±SD or median±IQR for normally and non‐normally distributed data respectively. There were no differences in LSR for TAT vs. NT conditions (1.1±0.7 vs 1.2±0.6 mg/cm2/min; p=0.93). TAT and NT skin had similar concentrations for UN (19.2±6.0 vs 20.7±6.1 mg/dL; p=0.28), CORT (2.4±1.3 vs 2.5±1.4 ng/mL; p=0.41), GLU (0.22±0.3 vs 0.24±0.2 mg/dL; p=0.31), and LAC (5.8±6.3 vs 5.9±6.6 mM; p=0.83). ERs were also similar for all analytes: UN (252±150 vs 270±136 ng/cm2/min; p=0.34), CORT (1.8±3.4 vs 2.1±3.4 pg/cm2/min; p=0.46), GLU (2.3±1.6 vs 2.3±2.4 ng/cm2/min; p=0.40) and LAC (6.4±10.9 vs 7.1±10.1 ng/cm2/min; p>0.999). In conclusion, TAT does not appear to affect the LSR, concentration, or ER of select analytes during ~60 min of fitness exercise.
Uncovering the Conditionally Essential Roles of NAD Kinases in Human Cells
Authors: Flickinger Kyle M. , Cantor Jason R.
Published: May 2022

Forward genetic screens across hundreds of human cancer cell lines have been used to identify genetic dependencies of proliferating human cells and how these vary by genotype and cell lineage. However, while it is appreciated that environmental factors influence cell physiology, there has been little investigation into how nutrient availability impacts gene essentiality. Further, most such screens have been performed in vitro using culture media that poorly reflect the metabolic composition of human blood. Previously, we developed Human Plasma‐Like Medium (HPLM), a physiologic medium designed to more closely reflect the biochemical conditions in human blood. To determine how medium composition affects gene essentiality, we recently performed CRISPR‐based screens of K562 leukemia cells in HPLM versus RPMI, the medium historically used to culture human blood cells. Among the strongest scoring HPLM‐essential genes is Nicotinamide Adenine Dinucleotide Kinase (NADK), which encodes one of two human NAD kinases (NADK, cytosolic; NADK2, mitochondrial) that can catalyze the phosphorylation of NAD(H)to NADP(H). While most human cells co‐express both isozymes, only NADK was identified as HPLM‐essential, suggesting that endogenous NADK2 expression is not sufficient to complement NADK knockout in HPLM‐cultured cells. Interestingly, NADK has been annotated as an essential gene in less than 1% of over 1000 genome‐wide CRISPR‐based screens from the Broad Institute Cancer Dependency Map project. To begin to investigate the conditional CRISPR phenotype for NADK, we first engineered NADK‐knockout cells and then found that they showed a 40% growth defect versus control cells in HPLM relative to RPMI using a short‐term growth assay. Next, to test the hypothesis that cytosolic NAD kinase activity is required for the conditionally essential role of NADK, we performed similar short‐term growth assays with NADK‐knockout cells expressing cDNAs for wild‐type and cytosol‐restricted NADK2, as well as kinase‐dead NADK. These experiments revealed that neither kinase‐dead NADK nor NADK2 overexpression could rescue the growth defect of NADK‐knockout cells, but that cytosol‐restricted NADK2 could. Collectively, these results indicate that the conditionally essential role of NADK is linked to its cytosolic NAD kinase activity. Currently, we are applying systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene‐nutrient interaction, and to gain insights into the non‐redundant, context‐dependent role of NADK. Lastly, through further conditional essentiality profiling, we identified NADK2as DMEM‐essential versus HPLM. Consistent with two recently published studies, we traced this effect to the differential availability of proline between HPLM and DMEM. Collectively, our results demonstrate the human NADKs fulfill distinct cell‐essential roles and form distinct gene‐nutrient interactions that vary with their subcellular localization.
The Effects of Lavendustin‐A on Peripheral Nerve Degeneration in Hyperglycemic Danio rerio
Authors: MacDougall Madison
Published: May 2022

Diabetic peripheral neuropathy (DPN) results in degenerating peripheral nerves and affects over 50% of people with diabetes. Despite its high prevalence, DPN’s molecular pathways remain unknown, limiting treatment to pain management. The Clark lab has characterized two novel models of hyperglycemia in zebrafish in which DPN‐like phenotypes are recapitulated, including demyelination. In an attempt to elucidate the unknown molecular mechanisms of DPN, candidate small molecule drugs are currently being tested to accentuate or attenuate neurodegeneration. Lavendustin‐A, an EGFR tyrosine kinase inhibitor (TKI), was hypothesized to have a neuroprotective role, as overactivation of EGFR in hyperglycemic environments is associated with fibrosis and the inhibition of regeneration in various tissue types. Tg(mbp:gfpcaax) larval zebrafish were incubated in egg water (control 1) or 120mM glucose solution (control 2 and treatment) starting five days post‐fertilization (dpf) until 10dpf. From 10‐12dpf, control 1 remained in egg water + 1% DMSO, control 2 remained in glucose solution + 1% DMSO, and the treatment group remained in glucose solution + 1% DMSO + 65.5µM Lavendustin‐A. 12dpf, lateral images of myelinated (MBP+) peripheral motor nerves were taken on an EVOS imaging system. Fiji (NIH) software was used to measure the length and fluorescent intensity of the myelinated nerves. Both nerve length (*p=0.025) and MBP fluorescent intensity (**p=0.0013) were significantly greater in the treatment group compared to the hyperglycemic fish (control 2), and no significant difference was observed between the treatment group and control 1. To determine if function was impacted, a touch‐response assay was conducted before (10dpf) and after (12dpf) drug treatment. Escape response was significantly impaired in hyperglycemic fish (treatment and control 2) at 10dpf (***p=0.0007) and significantly improved in the treatment group by 12dpf (***p=0.0003), yet did not fully recover, as there was still a significant difference between the treatment group and control 1 (**p=0.0017). Our data suggests a possible role of EGFR in hyperglycemia‐induced neurodegeneration, getting us one step closer to identifying potential therapeutics for clinical application.
A Connection Between DNA Repair Protein APE1, Alpha‐Synucleinopathy, and Biological Sex in Rodents and Humans
Authors: Miner Kristin M. , Bhatia Tarun N. , Jamenis Anuj S. , Clark Rachel N. , Abraham Nevil , Rajasundaram Dhivyaa , Sauvaigo Sylvie , Stetler R. A. , Chen Jun , Sanders Laurie H. , Luk Kelvin C. , Leak Rehana K.
Published: May 2022

Lewy body disorders are characterized by proteostatic and redox disequilibrium, leading to deposition of α‐synuclein in hallmark inclusions and oxidative damage to DNA. One common pathway for repair of oxidative DNA damage is base excision repair (BER), which involves the coordinated activity of several enzymes, including apurinic/apyrimidinic endonuclease 1 (APE1). The main goal of this study was to assess APE1 in rodents and humans with α‐synucleinopathy, and to test the functional impact of APE1 in the preformed α‐synuclein fibril model. First, we report that knockdown of APE1 with two independent shRNA sequences or inhibition of APE1 DNA repair activity increased inclusions bearing pathologically‐phosphorylated α‐synuclein (pSer129) in preformed fibril‐treated primary hippocampal cultures. Second, we examined APE1 expression in a mouse model of limbic‐centered α‐synucleinopathy, in which α‐synuclein fibrils are infused into the olfactory bulb/anterior olfactory nucleus (OB/AON). Six months later, we observed a fibril‐induced decrease in APE1 expression in the brains of male mice and an increase in females.

Similar sex‐opposing patterns were noted for APE1 mRNA expression. Third, we demonstrated that the loss of APE1 in fibril‐infused male mice in vivo is mediated by oxidative stress, as APE1 loss was abolished by dietary administration of the antioxidant N‐acetylcysteine. Fourth, OB/AON tissues harvested from fibril‐infused male mice displayed higher cleavage of synthetic DNA lesions on a biochip than tissues collected in parallel from fibril‐infused female mice. However, OB/AON tissues harvested from fibril‐infused female mice had higher DNA repair activity compared to males. Assessments of open field activity measures and sucrose preference revealed a negative behavioral impact of α‐synuclein fibril infusions in male but not female mice. Fifth, men with Lewy body disorders displayed lower APE1 expression in the OB and amygdala compared to women with Lewy body disorders, similar to fibril‐infused mice. Finally, we infused α‐synuclein fibrils into the OB/AON of transgenic APE1 overexpressing rats and observed a reduction in pSer129 levels in male and female brains compared to fibril‐infused wildtype rats. Preliminary data suggest that APE1 overexpression in fibril‐infused rats improved olfaction and anxiety measures, especially in males. These findings reveal a sex‐biased impact of limbic α‐synucleinopathy on APE1 expression and function but also suggest that APE1 overexpression attenuates α‐synucleinopathy in both sexes.
A Tandem Activity‐Based Sensing and Labeling Strategy Enables Imaging of Transcellular Hydrogen Peroxide Signaling
Authors: Messina Marco , Chang Christopher
Published: May 2022

Hydrogen Peroxide (H2O2) is one of many reactive oxygen species (ROS) that plays vital roles in intracellular signaling and stress processes, but its transient nature has made deciphering its roles in transcellular signaling a significant challenge. We have designed boronate caged small‐molecule probes bearing a fluoromethylene pendant group that serves as a latent quinone methide source that can undergo a tandem activity‐based sensing and biomolecule labeling reaction cascade to produce a spatially localized fluorescence signal upon exposure to H2O2. This strategy enables us to capture and record spatial information on ROS fluxes and has enabled us to move beyond studying intracellular H2O2 signaling to identifying and mapping cell‐to‐cell H2O2 communication in a microglia‐neuron co‐culture cell model, where selective activation of microglia for ROS production increases H2O2 in nearby neurons.
Regulation of Vacuolar H+ ATPase Subunit Expression in the Mouse Brain by Developmental Stage and pH Changes
Authors: Rigkou Anastasia , Roussa Eleni
Published: May 2022

The vacuolar‐type H+ ATPase (V‐ATPase) is a ubiquitously expressed proton pump, essential for multiple cellular processes such as acidification of intracellular organelles. The V‐ATPase consists of a transmembrane domain (V0) responsible for proton translocation and a cytosolic domain (V1) which catalyzes ATP hydrolysis. The functional domains contain different subunits with tissue‐specific expression patterns. In some organs, such as the kidney and salivary glands, V‐ATPase is located in the plasma membrane and previous studies have shown that its subunits are regulated by acid‐base disturbances. It has also been proposed that V‐ATPase subunits may be regulated by growth factors. In the present study, QRT‐PCR was used to examine the expression of different V‐ATPase subunits in the mouse brain and kidney in different developmental stages. Additionally, we have examined the impact of Transforming Growth Factor β2 (TGF‐β2) on different subunit expression, as well as their regulation in mouse cortical astrocytes following extracellular acidosis. The results show that the subunits ATP6V1A1, ATP6V1C1 and ATP6V1B2 are more abundant in the mouse brain, compared to kidney. In contrast, expression of subunits ATP6V1C2 and ATP6V1G3 is higher in the kidney, compared to brain. Our data also reveal developmental changes of subunit transcriptional levels in the mouse brain. Subunit ATP6V1G1 expression is decreasing during brain development, while the expression of ATP6V1C1 is developmentally increasing in the mouse brain. Moreover, our data indicate differential regulation of distinct V‐ATPase subunits in mouse embryonic brain and kidney by TGF‐β2. Extracellular acidosis has no effect on ATP6V1A1, ATP6V1C1 and ATP6V1E1 transcript levels in cortical astrocytes. Our data provide first evidence on developmental regulation of V‐ATPase subunits in the brain.
Monocytes transition to monocyte‐macrophages within the inflamed vasculature via CCR2 on monocytes and endothelial TNFR2
Authors: Sathyanarayana Mysore Vijayashree , Tahir Suhail , Furuhashi Kazuhiro , Arora Jatin , Rosetti Florencia , Cullere Xavier , Yazbeck Pascal , Sekulic Miroslav , Lemieux Madeleine E. , Raychaudhuri Soumya , Horwitz Bruce , Mayadas Tanya
Published: May 2022

Objective

To understand the cellular pathways that drive the maturation of classical monocytes into macrophages in inflammation

Hypothesis

Monocytes play essential roles in homeostasis, infection and sterile inflammation and can acquire macrophage phenotypes by transitioning through intermediate states. The mechanisms leading to the accumulation of these monocyte intermediates within tissues is not well understood; in the intestine, it has been attributed to a disruption in the differentiation along the monocyte–macrophage continuum under steady state and following inflammation. We hypothesize that monocytes double positive for chemokine receptors, CX3CR1 and CCR2 are preferentially recruited to the glomerulus and transition to immature, inflammatory macrophages while still within the vasculature in a CCR2 dependent manner.

Methods

Monocyte recruitment and maturation was evaluated using mouse models of acute/ chronic glomerulonephritis, intravital microscopy of the kidney, cell sorting, multi‐parameter flow cytometry and adoptive transfer approaches. Mechanistic insights into this conversion were provided using in vivo intravascular staining, in vitro monocyte‐endothelial cell co‐cultures and single cell transcriptional profiling. Evidence of the conversion was also evaluated by flow cytometric analysis of blood and urine samples from SLE patients with active nephritis.

Results

We show that renal Ly6C+ monocytes acquire macrophage markers (MHCII, CD64, F4/80) upon renal inflammation and are distinguished by the expression of both CCR2 and CX3CR1. These cells, which we refer to as immature macrophages, may be derived from a CCR2+CX3CR1+ double‐positive monocytes in blood that are preferentially recruited and retained within glomerular capillaries, and acquire pro‐inflammatory characteristics. Mechanistically, the monocyte acquisition of macrophage markers occurs within the vasculature and relies on CCR2 on hematopoietic lineages and TNFR2 on parenchymal cells, findings that are recapitulated in vitro with monocytes co‐cultured with TNF‐TNFR2 activated endothelial cells, which generate CCR2 ligands. Single‐cell RNA‐sequencing defines a monocyte differentiation path associated with the acquisition of immune effector functions, and monocyte CCR2‐dependent generation of CCR2 ligands. Immature macrophages are detected in the urine of patients with lupus nephritis and their frequency correlates with clinical disease.

Conclusions

CCR2 dependent functional specialization of monocytes into immature macrophages occurs within the TNF‐TNFR2 activated vasculature and establishes a chemokine‐based autocrine, feed‐forward loop that may amplify inflammation and renal injury.
Lipidomics identifies novel circulating markers of CVD risk in African American and Caucasian women
Authors: Gonzalez Paula , Jain Raghav , Coe Chris , Ryff Carol , King Camille , Bersh Andy , Malecki Kristen , Simcox Judith
Published: May 2022

Cardiovascular disease (CVD) is the leading cause of mortality for women in the USA. Women with dyslipidemia have at increased risk of CVD. The parameters of dyslipidemia have primarily been defined using Caucasian male populations. Current lipid markers of dyslipidemia such as high density lipoprotein (HDL), low density lipoprotein (LDL), and triglycerides have been shown to be poor predictors of CVD risk in African American populations. We aim to identify marks of dyslipidemia in African American populations that better predict risk and development of CVD.

Untargeted lipidomics (total lipid #) was performed on sera from middle‐age African American and Caucasian women (n=872). Machine learning was used to identify predictive lipid markers of CVD risk. Findings were validated using an independent cohort of women (n=200).

We identified unique and shared signatures of CVD in women based on race. Arachidonic acid containing lipids are positively correlated with elevated blood pressure and cardiomyopathy. Because these lipids are important in inflammatory signaling, we also assessed interleukin‐6 and c‐reactive protein. Through machine learning we were able to determine that markers of inflammation and inflammatory lipids were predictive of CVD in both Caucasian and African American women. Next, we will perform targeted lipidomics on these lipids of interest.

Regardless of class, lipid species containing arachidonic acid were increased in circulation of women with hypertension. We propose the consideration of an inflammatory marker panel that includes arachidonic acid to assess CVD risk in women.
Neuromuscular Synapse Dynamics in Aged and Injured Skeletal Muscle
Authors: Aliya Berna , Mohiuddin Mahir , Choi Jeongmoon , Jeong Gunjae , Kang Innie , Castels Hannah , Jones Cade , Jang Young
Published: May 2022

It is evident that both aged and diseased skeletal muscle exhibit interesting neuromuscular dynamics, characterized by changes in morphology and functionality of the synapse. Although they display a high regenerative capacity, skeletal muscles are still susceptible to nervous system perturbations and impacts of aging. Aged neuromuscular junctions (NMJs) demonstrate a diseased phenotype following some form of muscle atrophy, indicating the presence of paracrine signaling between myofibers and neuronal cells. In this study, we investigated differences in neuromuscular dynamics in injured and aged skeletal muscle using whole‐mount immunostaining and imaging. It has previously been shown that disruption of one component of the muscle microenvironment, such as vasculature, disrupts the structure of NMJs, however it is unclear if aged skeletal muscle experiences similar changes. We hypothesized that both aged and injured NMJs would display similar morphological dynamics, characterized by denervation and fragmentation of postsynaptic acetylcholine receptors (AChRs). We demonstrated that aged and injured NMJs exhibit fragmentation of AChRs, Wallerian degeneration of the presynaptic motor neurons, decreased overlap of pre‐ and post‐synaptic sides, and decreased area of AChR clusters. Due to the prevalence of neuromuscular disorders in the aging population, it is pertinent to understand nerve‐muscle interactions in the healthy aging state in order to hypothesize how these dynamics may be altered in the age‐induced injury states. Ultimately, this will allow us to better comprehend the molecular and cellular mechanisms that are responsible for neuromuscular degeneration in order to develop strategies to improve mobility and quality of life.
Social Isolation Influences Endocrine and Metabolic Parameters in Female Mice Depending on Estrous State
Authors: Provost Terri , Dangler Brianna , Giblin Joshua , Hartness Kyle , Kuts Tanya , Khazratkulova Shakhrizoda , Marsh Alexandra , Phoeurng Katona , Keesom Sarah M.
Published: May 2022

Social distancing is a critical public health strategy employed to reduce the transmission of respiratory illnesses, such as COVID‐19. Studies conducted throughout the pandemic suggest that social distancing measures, including staying at home, simultaneously lead to an increased experience of isolation and loneliness. Social isolation is of potential concern because previous research in both humans and non‐human animals demonstrates that reduced social interaction has detrimental effects on mental and physical wellness, including an increase in mortality rate. Despite this background, less is known about how social isolation affects physiological parameters in a comprehensive manner, especially in females. We therefore conducted this study to investigate the extent to which social isolation simultaneously impacts multiple physiological and behavioral variables related to stress, metabolism, and reproduction. To do this, we housed three‐week‐old female Swiss‐Webster mice individually or in groups of three for five weeks. We measured body weight and food consumption each week of the experiment. During the last two weeks of treatment, we tracked the estrous cycle, using vaginal cytology to determine estrous states. After four weeks of housing treatment, we used closed system respirometry to measure oxygen consumption rate as an approximation of whole‐body metabolic rate. At the conclusion of the study, we collected blood in order to measure leukocyte counts, blood glucose concentration, and serum estrogen and progesterone concentrations. Individually housed females consistently consumed more food compared to group‐housed females. While there was no effect of social isolation on the length or proportion of time spent in the different estrous phases, the effects of isolation on other physiological variables depended on estrous state. Our preliminary results suggest that body weight, oxygen consumption rate, number of large lymphocytes, blood glucose concentration, and serum progesterone concentration were higher in individually housed females in diestrus, compared to group‐housed females in diestrus. In contrast, serum estrogen concentration was lower in individually housed females in diestrus, compared to group‐housed females in diestrus. Taken together, these findings suggest that social isolation has a substantial body‐wide effect on physiology and health in females, potentially mediated through a direct effect on ovarian hormone secretion. Furthermore, these findings highlight the importance of studying how stressors impact female physiology in naturally cycling animals.
Longevity Effects of DMSO‐Solubilized Rapamycin and Other Compounds in y w Male Drosophila melanogaster
Authors: Mockett Robin J. , Bearden Aaron A. , Nobles Amber C.
Published: May 2022

This study was performed to screen a number of potentially life‐extending supplements, including inhibitors of the TOR signal transduction pathway, while confirming life extension by other, previously tested compounds. This initial screen was conducted in male flies of a comparatively long‐lived y w strain of D. melanogaster. A hypothesis was that reports of life extension by some compounds would be confirmed and other longevity‐enhancing compounds would be identified, with optimum dosages that could be tested subsequently in males and females of multiple genotypes. The following supplements were delivered continuously throughout adult life, beginning 2 d posteclosion: rapamycin (0.1, 0.5, 1, 10, 100, 200 and 400 µM), spermidine (10 µM ‐ 10 mM), LY294002 (100 nM ‐ 10 µM), wortmannin, KU0063794 and PX‐866‐17OH (all 1 nM ‐ 10 µM), AZD8055, PI‐103 HCl and Torin 2 (all 100 pM ‐ 10 µM), WYE‐28 and WYE‐132 (both 10 pM ‐ 1 µM) and DMSO solvent (0.02‐0.2%). Unexpectedly, the main finding was a strong, dose‐dependent decrease in mean life span at high doses of rapamycin, ranging from a 29% decrease at 100 µM to 46% decrease at 400 µM (all P < 0.0005). Rapamycin had no significant effect at the lower doses. The highest dose of wortmannin (10 μM) also drastically decreased longevity (73%, P < 0.0005) and the highest dose of spermidine (10 mM) had a smaller adverse effect (11%, P < 0.0005), while the highest doses of PI‐103 HCl (10 μM) and AZD8055 (10 μM), an intermediate dose of AZD8055 (0.1 μM) and the lowest dose of WYE‐132 (10 pM) increased life span by 4‐6% (P = 0.001 ‐ 0.029). The other supplements had no significant effect on longevity at any dose. Possible explanations of the rapamycin result include use of a DMSO solvent in contrast to ethanol used for past studies, or storage of DMSO‐solubilized rapamycin in aliquots at ‐20ºC; however, the DMSO solvent at the low concentrations used to dissolve the supplements had no effect on life span. Experiments are in progress to test the effects of high dose rapamycin in male and female flies of several strains, comparing ethanol and DMSO solvents, different food recipes, lighting and storage conditions. A current conclusion is that rapamycin is not universally beneficial in Drosophila and that inhibitors of various kinases at the doses studied have limited or no effect on longevity.
Indole‐3‐glycerol phosphate synthase (IGPS) serving as a drug target in M. tuberculosis
Authors: Bhatti Maryum , Goodey Nina , Cho Sarah , Jefferson Natalie , Marin Patrycja
Published: May 2022

Tuberculosis (TB) is a disease that is caused by a bacteria called Mycobacterium tuberculosis and primarily attacks the lungs but can spread to other areas of the body as well. TB can develop resistance to drugs that may be used to cure the disease. Multi‐drug resistant TB, for example, does not respond to the two most powerful anti‐TB drugs, which are isoniazid and rifampicin. For this reason, finding new ways to target TB are highly needed. One possible way to do this is to target the enzymes in the tryptophan biosynthetic pathway of M. tuberculosis. Studies have suggested that the enzyme indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis(MtIGPS) coded for by the trpC gene could be a target (Shen et al. 2009). The interactions between ligands and MtIGPS were thus investigated by introducing mutations to residues that had been proposed to play a role in catalysis or binding. Specifically, E168 was mutated to Q and K119 was mutated to R. Predictions for these mutations were that K119 is a catalytic acid and that E168 plays a role in substrate binding. We investigated the impact of these mutations on steady state kinetics and rate‐pH profiles to obtain insights into the roles of these two residues in substrate binding and catalysis. These findings will facilitate the design of MtIGPS inhibitor candidates.