bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of senolytic treatment on expression of senescence‐associated miR‐126 expression in young and old female mice.
Authors: Lahde Collin , Nunes Allancer , Cavalcante Marcelo , Saccon Tatiana , Schneider Augusto , Yousefzadeh Matthew , Robbins Paul , Niedernhofer Laura , Kirkland James L. , Tchkonia Tamara , Masternak Michal
Published: April 2020

Cellular senescence represents one of the major risk factors for chronic liver disease and contributes to morbidity, mortality, and increased healthcare spending. A relevant strategy for understanding the role of senescent cells in liver pathogenesis is to use genetic or pharmacological strategies to eliminate senescent cells. The combination of the senolytic drugs, Dasatinib and Quercetin (D+Q), effectively reduces senescent cell burden in multiple tissues, extending both healthspan and lifespan in mice, and alliviating obesity and age‐associated hepatic steatosis. The aim of this study was to investigate effects of D+Q treatment on livers of young and old female mice. A total of 40 mice, 20 young females (3‐months) and 20 old females (18‐months), was divided into 4 groups: Young (Y) mice treated with either D+Q or placebo and old (O) mice treated with D+Q or placebo. The treatment was performed for 3 consecutive days every 2 weeks over 10 weeks. Comparative analysis of hepatic miRNAs indicated that D+Q treatment significantly decreased levels of senescence‐specific miR‐126 expression in livers of old mice treated with D+Q compared to vehicle‐treated old mice (p=0.025), As expected, there was no effect of D+Q treatment in young animals. Analysis of miR‐126 target genes indicated that PI3K gene expression was significantly increased in response to D+Q treatment (p=0.003). Additionally, Akt1 and mTOR mRNA levels were increased in D+Q‐treated mice (p=0.008 and p=0.02, respectively). In summary, our data suggest that the mechanism of PI3K signaling pathway activation upon elimination of senescent cells by treatment with D+Q is mediated through downregulation of senescence‐specific miR‐126.

Support or Funding Information

This work is supported by National Institutes of Health (NIH) (R56AG061414, R15AG059190 and R03AG059846)
Validation and Development of a Cell‐based Model of Acute Oxygen Sensing
Authors: LIU MIN , CHANG ANDY
Published: April 2020

Located at the bifurcation of the carotid artery, the carotid body senses a decrease in blood oxygen to increase breathing within seconds. This chemosensory organ is important for physiological adaptation to low oxygen (hypoxia), and its activity is increased in chronic diseases like sleep‐disordered breathing, chronic heart failure, and metabolic syndrome. In the carotid body, neuroendocrine cells called glomus cells respond to hypoxia to stimulate afferent nerves that signal to brainstem respiratory centers. Several important steps of the response of glomus cells to hypoxia have been identified, including the inhibition of oxygen‐sensitive potassium currents, membrane depolarization, intracellular calcium increase, and neurotransmitter release. However, the exact oxygen sensor(s) and how it integrates with these downstream steps remains controversial. A major limitation in studying glomus cell sensory signaling is poor recovery of primary glomus cells from tissue, and a significant fraction of cultured glomus cells are not responsive to hypoxia. We are exploring whether the PC12 cell line, derived from a rat pheochromocytoma, can serve as a model for carotid body oxygen sensing. Previously, other labs demonstrated that PC12 cells have similar acute responses to hypoxia as glomus cells, and more recent gene expression studies show that PC12 cells express some key signaling components implicated in carotid body oxygen sensing. Here, we confirm that hypoxia inhibits potassium currents and causes membrane depolarization in PC12 cells and describe the subtypes of potassium channels found in PC12. We hope to develop the PC12 cell line as a more tractable cell‐based model of acute oxygen sensing and to study the interactions of putative oxygen sensors with downstream signaling through ion channels and neurotransmitter release using this model.
Functional Regulation of Hsp70 Allostery by Post‐Translational Modifications (PTMs)
Authors: Meng Wenli , Clerico Eugenia , Gierasch Lila
Published: April 2020

The 70‐kDa heat shock proteins (Hsp70s) perform critical functions in the protein homeostasis network, such as assisting nascent chain folding, preventing protein misfolding and aggregation, facilitating assembly of complexes, and driving protein translocation. The function of Hsp70s relies on a conserved allosteric mechanism involving substrate binding and release modulated by ATP binding and hydrolysis, with the participation of co‐chaperones, including J‐proteins and nucleotide exchange factors. Many studies in recent years have demonstrated that post‐translational modifications (PTMs) play important roles in functional regulations of Hsp70s. As an example, a recent report showed that phosphorylation of Hsp70 T495 upon infection by Legionella pneumophila inhibits host protein translation. While the modulation of Hsp70 functions can be achieved by a variety of means, including tuning its allosteric landscape, altering its substrate binding properties, varying its co‐chaperone interaction, etc., the mechanism of functional regulation of Hsp70s through PTMs is still unknown. In the current study, by using nuclear magnetic resonance spectroscopy (NMR) and biophysical/biochemical assays based on phosphorylation‐mimicking mutant, we are able to elucidate the origin of functional regulation by T495 phosphorylation in Hsp70. Phosphorylation of T495 shifts Hsp70 to the conformation favored by ATP binding in which the nucleotide‐binding domain (NBD) and substrate‐binding domain (SBD) are intimately docked, thus reducing the substrate binding activity of the molecular chaperone. These results underline the regulation of Hsp70 function by modulating its allosteric landscape through PTMs.

Support or Funding Information

Supported by NIH grant GM118161
A pH Switch Controls Zinc Binding in Tomato Copper‐Zinc Superoxide Dismutase
Authors: Galaleldeen Ahmad , Taylor Alexander B. , Hart P. John
Published: April 2020

Copper‐zinc superoxide dismutase (SOD1) is a major antioxidant metalloenzyme that protects cells from oxidative damage by superoxide anions (O2−). Structural, biophysical and other characteristics have in the past been compiled for mammalian SOD1s and for the highly homologous fungal and bovine SOD1s. Here, we characterize the biophysical properties of a plant SOD1 from tomato chloroplasts and present several of its crystal structures. The most unusual of these structures is a structure at low pH in which tSOD1 harbors zinc in the copper‐binding site but contains no metal in the zinc‐binding site. The side chain of D83, normally a zinc ligand, adopts an alternate rotameric conformation to form an unusual bidentate hydrogen bond with the side chain of D124, precluding metal binding in the zinc‐binding site. This alternate conformation of D83 appears responsible for the previously observed pH‐dependent loss of zinc from the zinc‐binding site of SOD1. In addition, Analytical ultracentrifugation data suggest that it is a weaker dimer relative to human SOD1.

Support or Funding Information

St. Mary’s Faculty Grant and Biaggini Fellowship
The Pitching Problem: Proxy Measures for the Identification of Pitcher Fatigue
Authors: Aloma Zachary A. , Baller Daniel P. , Hughes David W. , Thomas Diana M.
Published: April 2020

Objective

This study assessed the utilization of accessible records and metrics in developing proxy measures for fatigue in professional starting pitchers, and to understand the associated effects within lapses in short‐term and long‐term performance.

Design and Methods

Leveraging MLB Statcast AI, we pulled relevant data relating to pitches thrown during the 2015 – 2018 regular and postseasons. The available data was then analyzed in three separate iterations to better understand and isolate the impact of critical metrics. This iterative approach isolated the data based on the following subcategories: anecdotal game, seasonal, and aggregate sets.

Results

Through the prior detailed approach, we established the significance of three unaccounted for workload factors that represented suitable proxies for the identification of fatigue in starting pitchers. These factors included: strike‐to‐ball ratio, rest period, and a hit‐to‐strike ratio. These metrics represent a means of analyzing fatigue in relation to a pitcher’s performance through the course of a single game, and subsequently, inform optimal strategies as to bullpen management.

Conclusions

As a starting pitcher approaches a pitch count of thirty, management staffs can begin to utilize the prior mentioned proxy metrics as indicators of the impact of fatigue on a pitcher’s performance. These measures represent an alternative to traditional methods and an accessible solution.
Bile Acid Composition Contributes to Metabolic Improvements after Sleeve Gastrectomy in Mice
Authors: Tu Jui , Liu Yanjun , Ding Lili , Zhang Eryun , Jin Lihua , Huang Wendong
Published: April 2020

Obesity and type 2 diabetes (T2D) can be managed by vertical sleeve gastrectomy (VSG), one of the most frequently performed bariatric surgeries. Although VSG can effectively reverse obesity and T2D, the underlying mechanism of how VSG does so is still under investigation. We found that VSG altered bile acid (BA) levels and composition in mice, resulting in gut microbiota change and concomitant metabolic improvements. CYP8B1 is a BA synthesis enzyme in the liver. We found that CYP8B1 was significantly downregulated in mice after VSG. To investigate whether or not CYP8B1 mediates the metabolic effects of VSG, we used Cyp8b1‐deficient mouse model. The results show that although VSG induced weight loss and improvement in glucose tolerance in wildtype mice, VSG barely improved the same parameters in Cyp8b1‐deficient mice. Our study indicates that alterations of BA and microbiota compositions contributes to metabolic improvements after sleeve gastrectomy in mice. CYP8B1 could be one of the mediators downstream of VSG. These results provide novel insight into the roles of bile acids and gut microbiota in VSG, and may help develop gut microbiota‐based therapeutic options to mimic VSG.

Support or Funding Information

Internal funding
EVALUATION OF MICRORNA‐155 AS A BIOMARKER OF MALIGNANT PROGRESSION IN PATIENTS WITH GLIOMA DIAGNOSIS
Authors: Cirino Mucio Assis , dos Anjos Caroline , Rodrigues Andressa , Peria Fernanda , Tirapelli Luis Fernando , Junior Carlos Gilberto Carlotti , Tirapelli Daniela
Published: April 2020

INTRODUCTION

Gliomas are neuroepithelial tumors and correspond to approximately 24.6% of all primary brain tumors. In the last decade, great effort has been made in academic circles to characterize gliomas in an attempt to describe clinical behavior, define prognosis and predict therapeutic response. Bioinformatics tools estimate that microRNAs can regulate about 60% of human genes, including a significant number of oncogenes, tumor suppressor genes, and chemo and radioresistance genes. A current problem in clinical practice is the absence of molecular tools to predict the evolution of gliomas classified as low grade, since during postoperative radiological follow‐up these patients may have tumor recurrence with histopathological confirmation of high glioma degree. Tumor transformation in high‐grade glioma has a worsening of prognosis and reduction of therapeutic possibilities

OBJECTIVES

To evaluate the expression of miR‐124a, miR‐138, miR‐155 and miR‐1275 in human primary and peripheral blood samples from patients diagnosed with low and high grade gliomas (astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, glioblastoma, oligodendroglioma grade II and oligodendroglioma grade III).

PATIENTS AND METHODS

MicroRNA expression analyzes were performed using the real‐time PCR technique. Sixty‐five adult patients (18–65 years of age) with confirmed histopathological diagnosis of glioma. Considering a level of significance of 5%, the association of categorical qualitative variables and microRNA expression were performed by the Mann‐Whitney test. Survival analysis was performed using the Kaplan‐Meier non‐parametric method.

RESULTS

The microRNAs under study did not present hyperexpression in tumor tissue of patients diagnosed with glioblastoma. Only miR‐1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and grade II oligodendroglioma. In addition, tumors of low and high grade oligodendroglial lineage demonstrated overexpression of miR‐1275. In peripheral blood, significant hypoexpression of miR‐1275, miR‐124 and miR‐138 were observed in a glioblastoma sample. There was also a marked hypoexpression of miR‐138 in samples of grade III oligodendroglioma.

CONCLUSION

The microRNAs miR‐124a, miR‐138, miR155 and miR‐1275 did not show hyperexpression in tumor tissue of patients diagnosed with GBM. MiR‐1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and miR‐155 was hyperexpressed only in samples of grade II oligodendroglioma. In addition, tumors of oligodendroglial lineage, of low and high grades, demonstrated hyperexpression of miR‐1275 and miR‐124a. In peripheral blood, significant hypoexpression of miR‐1275, miR‐124 and miR‐138 were observed in the GBM sample as well as marked hypoexpression of miR‐138 in samples of grade III oligodendroglioma.
mTOR Modulates Sodium and Potassium Conduction in Heterologous Expression System
Authors: Shabbir Waheed , Gleason Catherine E. , Saha Bidisha , Leite Dellova Deise C. A. , Pearce David
Published: April 2020

The role of mammalian target of rapamycin (mTOR) complexes mTORC1 and mTORC2 in ion transport in kidney tubule has been an area of increasing interest in epithelial research. We and others have shown both in cultured cells and in vivo that mTORC2 is the hydrophobic motif kinase for serum and glucocorticoid kinase 1 (SGK1) and that its activity is required for epithelial Na+ channel (ENaC)‐dependent sodium reabsorption in the aldosterone‐sensitive distal nephron (ASDN). However, recent evidence has shown that mTORC2 can also modulate the renal outer medullary K+ channel (ROMK) activity directly. Electrogenic sodium reabsorption through ENaC generates the main driving force in ASDN for potassium secretion through ROMK, and is a key locus of acute regulation of K+ secretion. We suspected that differences observed in various studies may be due to acute vs. chronic regulation. We therefore compared the effects on ENaC and ROMK activity of chronic (through gene‐knockout) and acute (through a pharmacological approach) mTOR inhibition. We used CRISPR‐Cas9 to generate Sin1 (an essential component of mTORC2)‐deficient HEK‐293T cells, which were compared with wild type cells. The cells were transiently transfected with ENaC (alpha, beta gamma subunits) and ROMK1 channels. After recovery, the cells were treated with either vehicle or the mTOR inhibitor AZD8055 for 30 min and subjected to whole cell patch clamp to assess ENaC and ROMK currents. We found that in Sin1 KO cells (chronic loss of mTORC2) ENaC current was unaffected, while ROMK was markedly reduced. In contrast, acute mTOR inhibition with AZD8055 inhibited ENaC but not ROMK whole cell currents. These data provide initial support for the idea that acute pharmacologic inhibition of mTORC2 and chronic selective inhibition of mTORC2 have opposite effects on ENaC and ROMK activities. However, the results are also consistent with the possibility that global inhibition of mTOR (blocking mTORC1 and mTORC2) has distinct effects from selective inhibition of mTORC2. These observations may provide further insight into such diverse processes as aldosterone escape and the distinct plasma K+ concentrations seen in various types of hypertension.

Support or Funding Information

This work was supported by Relypsa Nephrolgy and Cardiology fellowship 2019–2020 to WS and NIH RO1‐DK 56695 to DP
Using CRIPSR‐Cas9 to Study Repeat Variation in Azf1p
Authors: Parmenter Mackenzie , Stewart Taylor , Fuchs Stephen
Published: April 2020

Repetitive regions within the genome can code for proteins that have repetitive amino acid sequences. These genomic regions can expand or contract and, in the few cases studied, these changes can affect protein function. This project tests how variation in the repetitive regions of the prion‐forming protein Azf1p, cause changes in gene expression and prion formation. Azf1p has two repetitive regions, a polyQ and a polyN, and is a transcriptional activator. We used CRIPSR‐Cas9 gene editing technology to cut within repetitive regions and allow the inherent DNA repair mechanisms in budding yeast, S. cerevisiae, to fix the break and create expansions or contractions. Subsequently we tested the effect of the repeat variation in different assays, including spotting assays, growth assays, phenotypic assays and qPCR analysis. We generated several variants in the polyQ domain of the AZF1 gene which were confirmed via sequencing. The research presented here will assess how variation within these regions affects various functions of AZF1, including growth on different carbon sources, colony morphology and changes in target gene expression. This work hopes to both create ways of using CRISPR‐Cas9 on repetitive regions that can be useful in the study of the function of these repeats and identify new functional domains within an important transcription factor, AZF1.
Craniofacial Morphology of Juvenile Mice with Osteogenesis Imperfecta
Authors: Steele Ashley T. , Organ Jason M. , Menegaz Rachel A.
Published: April 2020

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder affecting the synthesis and structure of type I collagen (Col1) due to autosomal dominant mutations in proa1(I) or proa2(I) collagen genes (COL1A1 and COL1A2). Clinical manifestations of the severe OI type III include bone fragility, reduced physical stature, low bone mineral density, and midface hypoplasia resulting in a “triangle face” phenotype. To investigate the influence of Col1 on juvenile craniofacial morphology, we used the homozygous OI murine (OIM−/−) mouse model at the weaning stage (21 days).

The OIM−/− mouse has a nonlethal recessively inherited mutation of the COL1A2 gene and exhibits adult cranial and post‐cranial phenotypes similar to humans with severe OI type III. OIM−/− and wild type (WT) littermates were weaned at 21 days and scanned in‐vivo with a Skyscan 1176 micro‐CT system. Craniofacial landmarks were collected using 3D Slicer software. Interlandmark distances (ILDs) and centroid sizes were calculated using Past 2.17 software. ILDS were scaled against skull/mandible centroid size to account for the effect of overall body size on shape analyses. Mann‐Whitney U‐tests were used to compare both absolute and relative (scaled) ILDs between the genotypes.

Craniomandibular centroid sizes and absolute linear distances (skull, rostrum, palate, and mandible lengths) demonstrate that the OIM−/− mice are smaller overall than their WT littermates. When scaled to centroid size, juvenile OIM−/− mice have a decrease in nasal length, midface height, and mandibular diastema length and an increase in hemimandible length compared to WT mice. For a given skull length, OIM−/− mice have shorter faces in both the anteroposterior and dorsoventral dimensions.

The morphometric changes seen in the juvenile OIM−/− mice replicate the “triangle face” that is commonly seen in human pediatric populations with OI. This suggests that this mouse model can potentially be used to investigate the structural changes underlying the human OI phenotype and potential therapeutic interventions. The results of this study can be used to inform future studies of craniofacial developmental in older OIM−/− mice.

Support or Funding Information

This research was supported by an Indiana University Collaborative Research Grant and the Ralph W. and Grace Showalter Trust.
Differing Collagen Responses in the Right and Left Ventricle Following Postnatal Hyperoxia Exposure
Authors: Golding Regina , Braun Rudolf , Kumari Santosh , Goss Kara
Published: April 2020

Background

Preterm birth affects approximately 1 in 10 pregnancies, and extreme preterm birth is associated with a 17‐fold increased risk of heart failure during childhood and early adulthood. Whether this is related to increase cardiac fibrosis is currently unknown. The objective of this study was to determine if fibrosis is present in an animal model of cardiomyopathy of prematurity using postnatal hyperoxia exposure.

Methods

Postnatal hyperoxia exposure in an immature rat heart was used to mimic the relative hyperoxia exposure premature infants experience at birth. Timed‐pregnant Sprague Dawley rats (Rattus norvegicus) were allowed to deliver naturally, and pups randomized to either postnatal hyperoxia (85% FIO2) or normoxia (21%) exposure from 0–14 days of life. Dams were rotated daily to prevent maternal oxygen toxicity. On day 21 of life, cardiac tissue was harvested and prepped for Masson’s trichrome blue stain. Additional hearts were resected and the right ventricle (RV) and left ventricle (LV) were flash frozen. The trichrome stained tissue was analyzed using ImageJ to determine the percent of image that was collagen, and colorimetric collagen assays that measured the hydroxyproline were ran on the flash frozen tissue. Results were compared with t‐tests, with p<0.05 used to determine significance.

Results

Overall, presence of cardiac fibrosis was low. In the LV, trichrome analysis demonstrated no difference between the hyperoxic and normoxic groups (4.56x10−4% vs 3.01x10−4 %, p=0.58). Similarly, the collagen assay showed no difference between the hyperoxic and normoxic LV (1.24 ±0.20 μg/g vs 1.01 ±0.34 μg/g, p=0.231). However, in the RV there was decreased percent fibrosis in hyperoxia relative to normoxia (2.69x10‐3 % vs 4.88x10‐3 %, p=0.03). The collagen assay showed similar results of a trend towards decreased collagen in hyperoxic RV compared to the normoxic RV (2.01±0.45μg/g vs 2.57±0.27μg/g, p =0.09).

Conclusion

These results appear to suggest that cardiac fibrosis is not a prominent feature in the animal model of cardiomyopathy of prematurity. Whether this is true for humans born preterm merits further study in the function and structure of the human heart throughout its development. The increased risk of heart failure could be caused by factors other than collagen deposition, or collagen deposition that occurs later in life. Further testing is required to fully understand the relationship between hyperoxygenation and long‐term heart health.

Support or Funding Information

University of Wisconsin Clinical and Translational Science Award program, through the NIH National Center for Advancing Translational Sciences grant NIH UL1TR000427
Arf6 Heterozygosity Results in Impaired Glucose Tolerance Due to Abberant Hepatic Gluconeogenesis
Authors: Dutson Tavia , Islam Md Torikul , Cai Jinjin , Sun Zhonglou , Fang Daniel , Donato Anthony J. , Zhu Weiquan , Lesniewski Lisa A.
Published: April 2020

Adenosine diphosphate (ADP) ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in numerous cellular processes including proliferation and membrane trafficking. Arf6 has been found to be dysregulated in diseases such as diabetic retinopathy and tumor metastasis. Furthermore, genetic insufficiency or pharmacological inhibition of Arf6 is protective in the aforementioned diseases, suggesting that Arf6 may be a potential therapeutic target. Here, we assessed the metabolic consequences of a reduction in Arf6 expression by performing glucose (GTT: 2 g/kg, ip), insulin (ITT: 1 U/kg, ip) and pyruvate (PTT: 2 g/kg, ip) tolerance tests in 3–4 mo old whole body Arf6 heterozygote (Arf6+/−) and wildtype (Arf6+/+) littermate control mice. Both protein and gene expression of Arf6 in the liver were 40–50% lower (p<0.05) in Arf6+/− mice compared to Arf6+/+. Body mass was higher in normal chow fed Arf6+/− compared to Arf6+/+ mice (30.3±0.9 vs 36.4±2.0 g, p<0.05). Blood glucose at 15, 30, and 60 min after injection during the GTT were 20–30% higher in Arf6+/− mice compared to Arf6+/+ (each p<0.05). Likewise, the area under the GTT time response curve was ~15% higher in Arf6+/− compared to Arf6+/+ (p<0.05), indicating impaired glucose metabolism. To examine the underlying cause of this glucose intolerance, we performed an ITT and assessed insulin‐stimulated suppression of plasma free fatty acids 15, and 30 min after insulin injection. We found no groups differences between the ITT time response curves (p>0.05) and no difference in the percent decrease in plasma FFAs from fasted between Arf6+/− and Arf6+/+ mice (~50% vs ~50% p>0.05). Likewise, fasted and glucose‐stimulated (2g/kg, ip) serum insulin were not different between the groups (2.44±0.58 vs 1.73±0.28, fasted; 4.54±1.74 vs 4.03±1.68 insulin stimulated; both p>0.05), suggesting that reductions in Arf6 expression do not impact pancreatic beta cell insulin secretion. Collectively, these measures suggest that neither peripheral insulin resistance nor impaired pancreatic beta cell function underlie glucose intolerance in Arf6+/− mice. To determine the contribution of hepatic gluconeogenesis to the observed glucose intolerance in the Arf6+/− mice, we performed a PTT and found that Arf6+/− mice exhibited a 15–25% increase in blood glucose over time compared to Arf6+/+ mice (p<0.05), suggesting a role for elevated hepatic gluconeogenesis. Taken together, this study demonstrates that Arf6 heterozygosity impairs glucose metabolism that is associated with aberrant hepatic gluconeogenesis but is independent of peripheral insulin resistance or pancreatic beta cell dysfunction.

Support or Funding Information

NIA R01 AG048366, R01 AG050238 and K02 AG045339, R01 AG060395 and US Department of Veterans Affairs I01 BX002151, I01 BX004492.
Determining PARP14 ADP‐Ribosylation Dynamics and Site‐Specificity Using MALDITOF
Authors: Chihab Leila , Torres Yazmin , Wallace Sean , Yamasaki Miles , Yoshinaga Braden , Carter-O’Connell Ian O.
Published: April 2020

Poly (ADP‐ribose) polymerases, PARPs, are a family of 17 enzymes (in humans) that catalyze the transfer of ADP‐ribose onto target proteins from nicotinamide adenine dinucleotide (NAD+). ADP‐ribosylation occurs in a highly conserved PARP catalytic domain found in all seventeen PARPs. Significant effort using modern proteomic methods has led to the identification of thousands of potential PARP targets in the cell. Unfortunately, this analysis has not yet uncovered which sites are preferentially targeted in vivo nor the determinants for PARP family‐member specific targeting. Further, current analytical methods require proteolysis of the target protein, limiting the study of dynamic ADP‐ribosylation. Herein, we present a matrix‐assisted laser desorption/ionization time of flight (MALDI‐ TOF) method using thin‐layer chromatography (TLC) that facilitates population‐wide analysis of ADP‐ribosylation. Using this method, we observe divergent ADP‐ribosylation dynamics for PARPs 14 and 15, with PARP15 modifying more sites on itself (+4–5 ADP‐ribose) than the closely related PARP14 protein (+1–2 ADP‐ribose) ‐ despite similar numbers of potential modification sites. Using this method, we also identify, for the first time, a minimal peptide fragment (18 amino‐acids) that is preferentially modified by PARP14. We have identified the specific glutamate (E) residue that is targeted on this peptide, and we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARP14 prefers acidic residues. The development of this technique will not only be useful in identifying the basic biochemistry governing ADP‐ribosylation but should find utility in the identification of PARP family‐member specific peptide targets and inhibitors.
The Iron‐responsive, Uropathogenic E.coli Exometabolome Is Dominated by Multiple Enterobactin‐derived Catechols
Authors: Zou Zongsen , Robinson John , Steinberg Lindsey , Henderson Jeffrey P.
Published: April 2020

Uropathogenic E. coli possess numerous iron‐responsive genes that are transcriptionally upregulated during human and experimental cystitis. These gene products are involved in diverse functions, including transporters and biosynthesis of specialized metabolites with virulence‐associated functions. Here, we sought to identify iron‐responsive exometabolites in experimental cultures of the model uropathogenic E. coli strain UTI89. Using comparative metabolomics, we detected multiple iron‐regulated products related to the siderophore enterobactin. In addition to observing the canonical siderophores enterobactin and salmochelin, we also detected siderophore structurally related, lower molecular weight metabolites of unknown origins. We considered that these low molecular weight products may derive from premature release from the biosynthetic complex (anabolic products) or from degradation of the full siderophore (catabolic products). In siderophore import‐deficient UTI89ΔtonB, low molecular weight catechols were substantially attenuated, in particular for the glucosylated derivatives, which favors a catabolic source of low molecular weight catechols resulting from import and incomplete hydrolysis of ferric enterobactin and salmochelin. When siderophore biosynthetic mutants were cultured with purified enterobactin, enterobactin was consumed while low molecular weight catechol products were not generated. And isotope‐labelling experiments suggest that complete enterobactin hydrolysis products may be recycled by bacteria within the cells for subsequent rounds of enterobactin biosynthesis. The results then raise a new hypothesis of glucosylation resulting in different enterobactin and salmochelin catabolisms, which is currently being investigated. The secreted exometabolome of uropathogenic E. coli includes not only siderophores but siderophore‐related products that arise from transport, hydrolytic, and recycling pathways that may relate to these organisms’ pathogenic potential.

Support or Funding Information

Acknowledgements:

This work was supported by a CDC Prevention Epicenters Program Grant CU54CK000162 and National Institutes of Health R01DK099534 and R01DK111930 to JPH.
The Standardization of Water Filtering to Compare Concentrations of Colony Morphology and Diversity to Plastic Samples
Authors: Jensen Heather , Simmons Rachel
Published: April 2020

With an ever‐growing public interest in plastic pollution in the ocean, its widespread ecological effects are still poorly understood at all points in its life cycle; including coastal microbiomes and microfilms. However, making sensible comparisons to waterborne communities, which have a much lower bacterial density, requires some additional experimental design considerations. By utilizing the Most Probable Number (MPN) method, we will measure microorganism growth, colony diversity and colony morphology of the surrounding ocean water compared to plastic samples that have been incubated for at least 30 days. We will discuss the process of collecting samples, applying and testing the MPN method, growing microbial samples, and the identification and testing of bacteria colonies.
The Development of Novel Therapeutics Against Schistosomiasis
Authors: Alwan Sevan Najm , Jr Rolando Trevino , Romero Claudia Moreno , Taylor Alexander B. , Rhodes Jayce , Tidwell Michael , Becker Stanton F. , LoVerde Donald T.
Published: April 2020

Human schistosomiasis is a neglected tropical disease caused by parasitic worms. It affects over 250 million people globally. Most human infections are caused by S. mansoni, S. haematobium, and S. japonicum. Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Constant selection pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel.

Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated by a Sulfotransferase, an enzyme produced by S. mansoni (SmSULT). Although sulfotransferases are produced by S. haematobium and S. japonicum, OXA is not effective against these two species. By using information from the crystal structure of SmSULT bound to OXA, 250 OXA derivatives were designed and tested in vitro against the adult parasites. We were able to identify effective derivatives that kill Schistosoma mansoni (85%), S. haematobium (40%) and S. japonicum (83%). Recently, we identified CIDD‐149830 as an effective derivative that can kill all three schistosome species (100%) within 7 days in vitro. In vivo studies results show a significant reduction in the number of harvested worms from infected animals after treating them with CIDD‐149830.

In this study, we re‐engineered and tested 42 derivatives based on the structure of CIDD‐149830 and we were able to identify CIDD‐0150610 as a very powerful derivative that kills 100 % of Schistosoma mansoni overnight in vitro.

Support or Funding Information

R01 AI115691
Therapeutic Effects of Magnesium Biotinate on Propionic Acid‐Induced Autistic Features in Rats
Authors: Sylla Sarah , Komorowski James , Ojalvo Sara Perez , Orhan Cemal , Tuzcu Mehmet , Sahin Nurhan , Bozoglan Merve Yılmaz , Sahin Kazim
Published: April 2020

Objective

This study investigated the therapeutic effects of magnesium biotinate (MgB) on propionic acid (PPA)‐induced autistic features in rats.

Background

Biotin is involved in energy metabolism and various immune and nerve functions. MgB is a novel biotin complex that has enhanced absorption. Compared to D‐Biotin, MgB has superior tissue uptake and anti‐inflammatory effects. Biotin deficiency can result in neurological abnormalities. Low levels of biotin have been found in individuals with neurodevelopmental disorders such as autism spectrum disorder.

Design/Methods

Thirty‐five male Wistar rats (3 weeks old) were randomized into the following five groups; 1: Control (received saline), 2: PPA alone, 3: PPA + 10 mg human equivalent dose (HED) MgB, 4: PPA + 100 mg HED MgB, 5: PPA + 500 mg HED MgB. PPA treated rats were administered 500 mg/kg BW/day of PPA by subcutaneous injection for 5 days to induce autistic features. Treatment groups then received MgB for two weeks via oral gavage following PPA treatment. Neurobehavioral tests were performed on the seventh and eighth days. Serum and tissue samples were then collected for analysis.

Results

The neuronal loss and degeneration observed in the hippocampal CA1 region and cerebellum of PPA‐exposed rats were improved by treatment with MgB. Brain levels of oxidative stress and inflammatory markers were lower in all MgB groups compared to PPA alone (p<0.05). Brain levels of antioxidant markers were higher in the 100 and 500 mg HED MgB groups compared to PPA alone (p<0.05). These effects were most significant in the 500 mg HED MgB group. Sociability and social preference scores were greater in the 100 and 500 mg HED MgB treatment groups compared to PPA alone (p<0.05). The 500 mg HED MgB dose improved social preference score to the extent that it did not differ from normal controls.

Conclusions

Treatment with MgB significantly reduced brain toxicity and improved autistic features induced by PPA.

Support or Funding Information

This study was conducted at Firat University and funded by JDS Therapeutics, LLC.
Investigation of the pro‐inflammatory effects of biomass exposure on RAW264.7 macrophages
Authors: Williams A’Kayla , Jones Shannon
Published: April 2020

Biomass smoke is a common component of indoor air pollution that affects millions of people worldwide. Indoor air pollution has been demonstrated to induce inappropriate activation of the immune system and pro‐inflammatory responses. In some underdeveloped regions of the world, individuals are especially susceptible to the negative health effects associated with biomass smoke exposure and indoor air pollution. In many cases, which citizens’ homes do not have the proper air filtration, are they are therefore disproportionately affected by indoor air pollution exposure. Individuals chronically exposed to indoor air pollution often suffer from recurring upper respiratory infections and exacerbated asthma. The mechanisms that lead to these negative health outcomes are mostly unknown, but are thought to be mediated by activated cells of the innate immune system. The purpose of this research was to examine the impacts of woodsmoke on macrophages, a key mediator of innate immunity. RAW 264.7 macrophages were challenged with woodsmoke to and assessed for pro‐inflammatory responses. We hypothesized that an increasing concentration of woodsmoke would be damaging to the macrophages and would result in higher levels of cytokine production and release in nitric oxide. Multiple assays were performed to measure cytotoxicity, cytokine production and nitric oxide release. Some of the cytokines that were measured include MIP‐2 and TNF‐alpha, both of which are upregulated during inflammatory responses in macrophages. The LDH assay was used to assess the cytotoxicity of woodsmoke in treated macrophages. Our results thus far demonstrate, the following exposure to wood smoke, the release of MIP‐2 and TNF‐alpha was significantly increased following wood smoke exposure. Furthermore, the wood smoke exposure induced minimal amounts of cellular toxicity.

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University of Richmond’s Integrated and Inclusive Science Program
The role of miR‐146a‐5p on insulin signaling and inflammation in adipose tissue of longliving Ames dwarf mice.
Authors: De Carvalho Nunes Allancer Divino , Yu Lin , Lahde Collin , Noureddine Sarah , Saccon Tatiana , Schneider Augusto , Masternak Michal
Published: April 2020

MicroRNAs have been shown to be potent regulators of various biological processes. Multiple miRNAs are regulated during aging in several tissues from mice. Previous studies have indicated the MicroRNA 146a‐5p (miR‐146a) is increased during aging in the circulation, in a wide variety of tissues and different models of replicative aging. Importantly, this miRNA is also associated with cellular senescence, metabolic syndrome and inflammation. Thus, the aim of this study was to determine the mechanistic role of miR‐146a in insulin sensitivity and inflammation in visceral fat tissue in vivo. For this we used Long‐living Ames dwarf (df/df) and normal (N) female mice were subjected to five intraperitoneal injections of a miR‐146a mimetic (miR‐146a‐m) with two day intervals between each injection. Before the treatment, mice were randomly divide into five experimental groups: normal mice control, normal mice miR‐146a‐m 4 mg/kg, df/df control, df/df miR‐146a‐m 4mg/kg and df/df miR‐146a‐m 8 mg/kg. Overall, RT‐PCR analysis indicated that in vivo treatment with miR‐146‐m increased the expression level of miR‐146a in visceral fat tissue in both N and df/df mice. Importantly, in df/df the treatment with miR‐146a‐m 8 mg/kg significantly increased P16 gene expression, a known senescence marker, and reduced adiponectin, IRS2 and AKT1 gene expression. Moreover, the analysis of PI3K, IRS1, PPARƴ and PPARα gene expression in visceral fat showed a trend towards downregulation in response to treatment with miR‐146a‐m at 8 mg/kg. In summary, this study showed that miR‐146a regulated the expression genes related to insulin sensitivity in visceral fat tissue in long‐living df/df mice as compared to normal mice. Additionally, our data suggest that miR‐146a might suppress insulin sensitivity and stimulate inflammation through suppression of adiponectin levels.

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This research study is supported by National Institutes of Health (NIH) (R15AG059190, R03AG059846 and R56AG061414)
Renal hemodynamics and metabolism improve following a second Exertional Heat Stroke
Authors: Kolb Alexander Louis , Geddis Alyssa , Reynoso Mainaliz , Ward Jermaine , Dineen Shauna , Leon Lisa
Published: April 2020

Exertional Heat Stroke (EHS) occurs with rapid onset during strenuous exertion and results in hospitalization that is often associated with renal and hepatic injury. EHS may increase susceptibility to a subsequent EHS collapse if organ injury has not completely resolved prior to the second heat exposure. We investigated renal filtration and mitochondrial oxidative phosphorylation (OP) changes in radiotelemetered C57BL/6J mice that were forced to run in a heat chamber (37.5°C) until collapse (core temperature ~42.7°C). Mice were exposed to one (EHS) or two EHS events separated by 1 (EHS1), 3 (EHS3) or 7 (EHS7) days of recovery. Samples were collected 30 min, 3h, 1, or 7d after the first or second EHS exposure. Blood urea nitrogen (BUN; Abaxis VetScan analyzer) and a Mitochondrial Function Assay MitoPlate (Biolog) were used to assess renal filtration and OP changes, respectively. EHS1 mice experienced significantly greater thermal load (area under the heat curve) and dehydration compared to EHS, EHS3 and EHS7 mice (p<0.001), but hypothermia depth during recovery was significant less pronounced than the other groups (~35°C vs. 32°C, respectively; p<0.01). EHS1 mice showed 103% and 95% BUN decrease at 30 min and 3h of recovery compared with EHS, EHS3, and EHS7 groups, respectively (ANOVA, p<0.001). Analysis of OP kinetics revealed that the following Citric Acid Cycle enzymes increased: Pyruvate Dehydrogenase (78%), Citrate Synthase (77%), Isocitrate Dehydrogenase (66%), Aconotase (59%) Alpha‐Ketoglutarate Dehydrogenase (50%), Succinate Dehydrogenase (18%), Fumurase (37%), and Malate Dehydrogenase (50%) 3h following EHS1, EHS3 or EHS7 (p<0.05). All values were compared and normalized to naive control animals. The core temperature data indicate that EHS mice were partially acclimated prior to the second exposure on day 1 and this was sufficient to improve thermal tolerance (time in the heat before collapse) and reduce the severe sequelae during recovery. The minimal BUN changes EHS1 mice may be a consequence of increased glomerular filtration promoting normal renal hemodynamics. However, the kidney of EHS3 mice appeared to recover prior to the second exposure such that renal function was no longer adversely affected. The potential mechanism of these changes may be attributed to citric acid cycle kinetics of the renal mitochondria. These are the first data to indicate that partial heat acclimation in mice is sufficient to protect them against renal injury following a second heat exposure and this response has a 3d decay rate.

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The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.
Bone blood flow regulation in humans
Authors: Draghici Adina E. , Solinsky Ryan E. , Taylor J. Andrew
Published: April 2020

Bone is a highly vascularized organ with an extensive network of arteries, arterioles, and capillaries that is critical for bone development, maintenance, and repair. Despite the importance of bone circulation to bone health, the regulation of blood flow to bone is not well understood in humans. One of the potential factors controlling bone blood flow is the sympathetic nervous system, bone vasculature being innervated by a rich network of sympathetic nerves. While no human studies have been conducted, animal data suggests that sympathetic activation reduces bone blood flow. If this were not the case, bone vasculature would respond simply as a passive pressure system. Therefore, we characterized bone blood flow regulation in health young individuals (N=12 (8M; 4F), 23±0.7 years) in response to isometric handgrip to fatigue (IHE). Sustained IHE is a profound pressor stimulus, resulting in active vasoconstriction in the nonactive leg and progressive increases in blood pressure. During IHE at 30% of maximal voluntary contraction, we measured blood pressure, whole leg blood flow (via Doppler ultrasound), and tibial blood perfusion (via near infrared spectroscopy). Tibial blood perfusion was determined as changes from resting baseline in oxygenated and deoxygenated hemoglobin content. Data is presented as mean change from resting baseline ± standard error. To account for different IHE time courses, all variables were normalized to 100% duration and averaged across individuals. Blood pressure increased progressively to end of IHE (at 10%: 5.46 ± 1.30 mmHg; at 100%: 48.0 ± 4.32 mmHg). Whole leg blood flow decreased progressively throughout IHE, suggesting active sympathetic vasoconstriction in the leg (at 10%: −0.70 ± 0.28 cm/s; at 100%: −2.26 ± 0.50 cm/s). Similarly, tibial oxygenated hemoglobin decreased progressively throughout the test (at 10%: −0.21 ± 0.31 μM; at 100%: −1.19 ± 1.12 μM) whereas tibial deoxygenated hemoglobin did not change significantly throughout IHE (at 10%: 0.06 ± 0.07 μM; at 100%: −0.4 ± 0.32 μM). This indicates that arterial flow (oxygenated hemoglobin) declined while bone metabolism (deoxygenated hemoglobin) remained unchanged in response to IHE. It should be noted that the decline in oxygenated hemoglobin was slower and lesser than that in whole leg blood flow. This data suggests that sympathetic activity is involved in controlling bone circulation, but vasoconstrictor responses is not as fast or as robust in bone as it is in the whole leg.

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Paralyzed Veterans of America (AED)
Water Networks and Allosteric Scaffolds ‐ What Does Water Have to do with Protein Dynamics?
Authors: Taverner Keith , Prosser Robert Scott
Published: April 2020

The thermophilic enzyme Fluoroacetate Dehalogenase (FAcD) has been well characterized by crystallography and NMR. FAcD (RPA1163) is a homodimer alpha/beta hydrolase that breaks down haloacetates (including fluoroacetate, iodoacetate, and chloroacetate) into glycolate and a halogen ion. FAcD achieves this by half‐of‐sites activity, where the homodimer adopts a rare asymmetric state that is primed to capture substrate which must then diffuse along an 11 Å cavity before engaging the active site and adopting the Michaelis intermediate. This triggers release of ~30 bound water molecules, thereby entropically favouring the forward reaction. FAcD is thus an ideal model system to pursue paradigm shifting questions regarding allostery, interprotomer communication, and the role of hydrogen bonded water networks in catalysis ‐ all from an ensemble perspective. Here we examine how deuterium water (D2O) changes the dynamic and allosteric mechanisms of this system. Compared to water, D2O results in an increase in viscosity and has stronger hydrogen bonds. Through 19F NMR kinetic experiments we determine that despite the higher viscosity enzyme activity is increased in the D2O environment. Additionally, 19F NMR experiments reveal increased dynamics in D2O. This provides key insight into discrete hydrogen‐bonded water networks that are key to stabilization of allosteric processes associated with the reaction coordinate.

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Natural Sciences and Engineering Research Council (NSERC)