bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Vascular Calcification and Stiffness in Diabetic Mice with SMC‐specific STIM1 deficiency
Authors: Zhang Weiping , Sun Yong , Yang Youfeng , Chen Yabing 
Published: April 2020

Vasculopathy is a devastating complication in patients with diabetes mellitus, which increases the risk of morbidity and mortality. Vascular smooth muscle cells (VSMC) are the main vascular cells that regulate vascular tone and contribute predominantly to the development of vasculopathy. We have demonstrated an important role of VSMC in the development of vascular calcification and stiffness in diabetes. The present study investigated the function of stromal interaction molecule 1 (STIM1), an important regulator for intracellular calcium homeostasis, in regulating VSMC calcification in mouse model of diabetes, and uncovered the underlying molecular mechanisms.The effect of SMC‐expressed STIM1 in regulating vascular function in diabetes was determined in a SMC‐specific STIM1‐deficient mouse (STIM1Δ/Δ SMC), generated by breeding SM22α‐Cre mice with STIM1 floxed mice (STIM1f/f). SMC‐specific deletion in the STIM1Δ/Δ SMC mice was confirmed by immunohistochemical staining. Using the low‐dose streptozotocin (STZ)‐induced mouse model of diabetes, we found that SMC‐specific STIM1 deletion did not affect STZ‐induced hyperglycemia in mice. STZ‐induced vascular calcification and stiffness were enhanced in SMC‐specific STIM1 deficient mice comparing with their control littermates (STIM1f/f), which was associated with increased aortic expression of Runx2, the master osteogenic regulator. Furthermore, aortas from the STIM1Δ/Δ SMC mice exhibited increased protein O‐GlcNAcylation, an important post‐translational modulation that we have shown to promote vascular calcification and stiffness. Increased protein O‐GlcNAcylation was also determined in primary VSMC isolated from STIM1Δ/Δ SMC mice compared with those form the control littermates. STIM1 deficiency in VSMC promoted osteogenic differentiation and calcification of VSMC cultured in osteogenic media, which was attenuation by inhibition of protein O‐GlcNAcylation. Osteogenic media‐induced elevation of intracellular calcium flux was demonstrated in the STIM1 deficiency VSMC, which induced endoplasmic reticulum stress and activation of p38 MAPK pathway that contributed to increased O‐GlcNAcylation and VSMC calcification.In summary, our studies have determined a new causative effect of SMC‐expressed STIM1 on diabetic vascular calcification and stiffness, and identified a novel link connecting STIM1 and protein O‐GlcNAcylation in regulating endoplasmic reticulum stress and VSMC calcification.
Purification of Amyloid Fibers Formed by the Tetracycline Resistant Soil Bacterium, Microbacterium oryzae
Authors: Bovee Hannah June , Mechler Jack , Koloski Jordan , Berrus Hunter , Korn Abby , Olendzenski Lorraine , Marano Nadia 
Published: April 2020

Amyloid fibers are proteins formed by repeating protein monomers which stack to create a cross‐beta sheet quaternary structure. Though the unregulated production of amyloid fibers is often associated with diseases such as Alzheimer’s and Parkinson’s, amyloid fibers are also produced in a regulated manner by bacteria in biofilms. We isolated a number of tetracycline resistant soil bacteria from a pig farm in upstate New York and screened them for biofilm production with the crystal violet assay and amyloid formation using a Thioflavin T (ThT) assay. We focused on one species, Microbacterium oryzae isolate #29, which had the highest amyloid production. Five days of growth at ambient temperature in liquid R2A medium maximizes amyloid fiber recovery. An isolation procedure utilizing sonication and differential centrifugation to separate fibers from the cell (Jordal et al. 2009) was adapted for use with M. oryzae isolate #29. This procedure is being modified further to increase the percent recovery and improve purity. Two detergents, Triton X‐100 (TX‐100) and hexadecyltrimethylammonium bromide (CTAB), were assessed as a means of disrupting the cell membranes to separate fibers from soluble proteins during the procedure. The fluorescence of the detergents below and above their critical micelle concentrations in the presence of ThT was investigated to determine whether adding a detergent treatment step would interfere with the ThT assay, as is the case for the commonly used detergent SDS. Additionally, sonication time was assessed to see if more fibers could be released from the cell. Preliminary results suggest that while neither detergent interferes with the ThT assay only the TX‐100 appears to improve yield, and that a longer sonication treatment results in a greater percent recovery in the ultracentrifuge pellet. Fibers will be purified further using preparatory SDS‐PAGE and depolymerized using formic acid. The purified fibers can be imaged by atomic force microscopy and the amyloid protein monomers will be sequenced.Support or Funding InformationSt. Lawrence University Fellowship Program, Departments of Chemistry and Biology, St. Lawrence University, Canton, NY 13617
The Effect of Growth Restriction on the Gut Microbiome in Mice
Authors: Quinn Melissa Ann , Leszczynski Eric , Visker Joseph , Wellette-Hunsucker Austin , Quinn Robert , Ferguson David P. 
Published: April 2020

INTRODUCTIONEarly life growth restriction increases susceptibility to non‐communicable adult‐onset diseases. However, there is minimal mechanistic rationale for elevated disease risk. The gut microbiome assists in regulating host physiological processes, with emerging evidence suggesting microbiome dysregulation leads to increased risk of chronic disease. Thus, the purpose of this investigation was to determine differences in the gut microbiome of growth restricted mice as compared to non‐restricted mice.METHODSA cross‐fostering, protein‐restricted (8% protein) nutritive model in FVB mice was used to induce growth restriction during gestation (GUN; N=10 male,15 female) or lactation from days 1–21 of life (PUN; N=11 male, 12 female) along with a control group (CON, 20% protein, N=12 male, 11 female). At 21 postnatal days of age (PN21) all mice were weaned to a non‐restricted diet (20% protein), isolating undernutrition to early life windows. Fecal samples were collected weekly across the lifespan (until PN80) to determine longitudinal programming effects of growth restriction on the gut microbiome. Cecum samples were collected at PN21 and PN80. Microbiome analysis of fecal and cecum samples were conducted by first extracting the DNA using Qiagen Powersoil DNA extraction kits and conducting PCR amplification of the bacterial 16S rRNA gene amplicon profiles through the Qiita bioinformatics platform. Phenotypic growth markers of all three diet groups (CON, GUN, PUN) were analyzed using repeated measures ANOVA. Metabolomics data on the same samples is currently being analyzed and will be integrated with the microbiome dataset.RESULTSThe fecal microbiome was strongly separated by treatment group using the Bray‐Curtis distance measure (PERMANOVA p = 0.001). A random forest machine learning classification validated this separation showing an overall out‐of‐bag error rate of 18.6% across the three groups in the whole dataset. The PUN group was distinctly separated from GUN and CON, with a group error rate of only 15.0%. Differences in the microbiome were not evident through analysis at the Phylum level (Firmicutes/Bacteroidetes ratio) but was instead driven by differential abundances of specific rare taxa. Bifidobacterium was significantly elevated in the PUN group (Kruskal‐Wallis test p=0.00017), beginning as early as PN21 and remaining elevated in the PUN group through the lifespan (PN80).CONCLUSIONSA protein‐restricted nutritive model induces strong imprinting on the structure of the murine microbiome. Changes in the PUN group are primarily driven by differences in low abundance taxa. Understanding the mechanisms that lead to the microbiome dysbiosis shown here could lead to the development of therapeutic interventions to limit the development of a stunted or immature gut microbiota.Support or Funding InformationMSU Department of Biochemistry and Molecular Biology; MSU Department of Kinesiology
Bone Quality Analysis of the Effects of Maternal Prenatal Stress Exposure
Authors: Amugongo Sarah 
Published: April 2020

Unperturbed fetal development is essential for future health of an individual. Previous studies have linked diseases of aging to harmful alterations that happen during fetal development. Given the significant long‐term impact that intrauterine environment has on an individual's life, it was hypothesized that maternal stress during pregnancy will have negative effects on the offspring's bone density and cross‐sectional area. To test this, twenty‐eight female and seven male Wistar rats (Rattus norvegicus) were purchased and bred to produce 176 offspring. During pregnancy, dams were randomly divided into four groups (n=7, per group) and immobilization stress induced as follows; Group 1 (GW1): immobilization stress on days 1–7 of pregnancy, Group 2 (GW2): on days 8–14, Group 3 (GW3): on days 15–21, Group 4 (Controls): left undisturbed. Maternal cortisol hormone, food intake, and weight gain were monitored during pregnancy. Pups were raised under normal laboratory conditions and sacrificed at ages: 4, 8, 12, and 16 weeks to determine the effect of prenatal stress. At necropsy, the femur was removed and preserved for image analysis.Differences among groups were determined by T‐test or analysis of variance (ANOVA). P values ≤ 0.05 were considered significant. Cortisol hormone levels in controls were lower than those of stressed animals. Stressed dams consumed 12.5% less food per day compared to controls. Animals in GW1 and GW2 gained less weight during pregnancy but had larger litters than did GW3 or the control group. Offspring born to GW3 were heavier compared to all other groups. Preliminary MicroCT analysis of cortical bone shows the control males as having higher cortical thickness compared to male treatment groups at the age of 8 and 16 weeks. There was no significant difference in cortical thickness of females. Trabecular thickness in controls was higher at the age of 4 and 8 weeks compared to treatment groups. This difference was observed in both males and females. There was no significant difference observed among treatment groups. Analysis of the remaining samples is ongoing.Support or Funding InformationAmerican Association of Anatomists Visiting Scholarship.
Allosteric Modulation of SIRT1 Activity Affects Substrate Recognition and Protein Conformation
Authors: Wang Ningkun 
Published: April 2020

SIRT1 is a lysine deacetylase that plays an important role in regulating various cellular pathways such as neurodegeneration and insulin secretion. As such, it is of great interest to modulate SIRT1 activity as a therapeutic approach for diseases such as Alzheimer’s disease and diabetes. Several small‐molecule allosteric regulators of SIRT1, known as STACs (Sirtuin Activating Compounds) are studied, such as resveratrol. It is known that these regulators bind to the STAC Binding Domain (SBD) of SIRT1, distal from the enzyme active site, and regulates SIRT1 activity through allosteric effects. However, the molecular mechanism of these allosteric effects is not well understood, impeding more specific designs of SIRT1 modulator compounds. In this study we take a systematic approach to study the changes in Michaelis‐Menten kinetics and overall conformation of SIRT1 when its activity is allosterically decreased and increased. A previous end‐point screen has found that the effect of resveratrol on SIRT1 is substrate‐sequence dependent, where resveratrol can increase, decrease, or have no effect on the activity of SIRT1 against different subsets of peptide substrates. We have chosen several peptides from each of the above‐mentioned categories and determined the Michaelis‐Menten kinetics of SIRT1 against these substrates with and without resveratrol. Upon comparison, we find that the allosteric effect of resveratrol mainly changes the substrate recognition (KM) of SIRT1 for these peptides. In addition, we used Small Angle X‐Ray Scattering (SAXS) to monitor the overall conformation of SIRT1 in complex with the different peptide substrates with and without resveratrol and found that the protein conformation is altered upon addition of resveratrol. Combining these findings, we have a better understanding of the mechanism for allosteric regulation of SIRT1, which could shed light on other allosteric regulations in the cell and aide in guided design of small molecule SIRT1 modulators.Support or Funding InformationNational Institutes of Health NIGMS SC2GM132000‐01
Electrostatic and Chemical Properties in Natural and Designed Enzymes
Authors: Ondrechen Mary Jo , Coulther Timothy A. , Ngu Lisa , Watson Samantha , Beuning Penny J. 
Published: April 2020

Advances have been made in understanding how enzymes achieve their exquisite catalytic power. However there are still gaps in our understanding, as computationally driven enzyme design efforts are still in their infancy and must be followed by years of directed evolution in order to achieve reasonable turnover rates. The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic capabilities and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized in part by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, ornithine transcarbamoylase, and glycinamide ribonucleotide transformylase (GART). In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. These concepts can enhance current enzyme design protocols.Support or Funding InformationSupported by NSF MCB‐1517290.
A Medical Countermeasure Against Drugs of Abuse with Detoxifying Biomimetic “Nanosponge” Decoy Receptors Directed Against Opioid Drugs and Methamphetamines
Authors: Federico Isabella , Federico Francesca , Rojas Fernando , Zapf James , Esmaeli-Azad Babak 
Published: April 2020

Synthetic highly potent opioids (e.g., Oxycodone, fentanyl, carfentanil, & acrylfentanyl) are emerging as both national security & health threats. Every 15 minutes in the US, a person dies after overdosing on opioids, constituting a national crisis that affects public health as well as social and economic welfare. For opioid overdose, naloxone, an opioid receptor antagonist, is, to date, the only life‐saving treatment based on its acute reversal of respiratory depression/arrest.3 Critically, naloxone’s actions are short‐acting: when the opioid receptor again becomes accessible, residual opiates in the peripheral circulation or in the tissues can enter the CNS & “re‐narcotize” the patient causing an acute recurrence of life‐threatening respiratory depression/arrest – often before the patient in the field has been stabilized in a high‐acuity center. As such, naloxone therapy has potentially fatal shortcomings. In 2019, rates of natural opioid overdose deaths leveled out, yet overdose death rates involving synthetic opioids like fentanyl, which is 50 to 100 times more potent than morphine, have continued to increase. A major concern is that naloxone treatment could be insufficient against these synthetic opioids, and fatal.We designed and developed an acute opioid drug detoxifying treatment for overdose of any type of opioid, natural or synthetic. Our treatment can be administered with naloxone to compensate for nalaxone deficiencies and uses an opioid‐targeted biomimetic “nanosponges” that absorb overdosed opioids in the body. Called “NarcoBondTM” these 100 nm diameter nanospheres are made from a mixture of cholesterol & synthetic choline‐based phospholipids that mimic the lipid bilayer cell membranes.NarcoBond’s multifunctional surface displays an optimized milieu of human membrane proteins isolated from: a) erythrocytes to increase half‐life in peripheral blood & circulation, b) neurons to increase binding affinity for opioid receptor, & c) purified Mu opioid receptors to bind opioids in circulation. The NarcoBond’s repertoire of native opioid receptors (“Decoy Receptors”) broadly binds & captures opioids from microenvironmental niches of cells in tissues & blood resulting in an antagonistic pharmacological effect by rapidly reducing the concentration of the overdosed opioid.Our most recent in‐vitro and in‐vivo results, using this proteolipid nanosponge for detoxification of opioids, and our future plans for detoxification of methamphetamines using the same decoy receptor approach, will be presented. We will also present results of an informal survey of high school and college students (15–24 years of age), on intervention and adoption of countermeasures such as Naloxone and NarcoBond, in order to decrease the risk of licit and illicit drug overdose complications and fatalities.Support or Funding InformationNational Institute for Drug Abuse (NIDA)
Curcumin Improves Autoimmunity in Female Mice with SLE
Authors: Dent Elena L. , Taylor Erin B. , Ryan Michael J. 
Published: April 2020

Systemic lupus erythematosus (SLE) is a chronic multi‐system autoimmune disease characterized by the presence of circulating autoantibodies, inflammation, hypertension, renal injury, and cardiovascular disease. Current immunosuppressive therapies often impose unwanted side effects leaving patients searching for alternative therapeutic options. Curcumin has been used in Eastern medicine for its natural anti‐inflammatory and antioxidant properties. We hypothesize that the administration of curcumin will lessen renal inflammation and injury in female mice with SLE.Female NZBWF1 mice, an established experimental model of SLE, and female NZW (control) mice were administered curcumin (500 mg/kg/day) or corn oil (vehicle) by oral gavage for 14 days. This design is based on previously published experimental data using curcumin in mice. Plasma blood urea nitrogen (BUN), a clinical marker of renal function, was assessed at baseline (26 weeks of age) and again at the end of the study (28 weeks of age). To assess immune activity, we measured circulating anti‐dsDNA IgG autoantibodies, spleen weight, and used flow cytometric analyses of peripheral blood leukocytes to assess circulating B lymphocytes at the same time points.Compared with vehicle treated SLE mice, curcumin treated mice with SLE have significantly lower BUN at 28 weeks of age (13.7 ± 0.6 mg/dL SLE‐curcumin, n=14 vs. 36.2 ± 11.4 mg/dL SLE‐vehicle, n=5) (P = 0.0030). As expected, spleen weights are increased in female mice with SLE. However, curcumin treated SLE mice at 28 weeks of age have lower spleen weights compared to SLE vehicle treated mice (0.114 ± 0.009 g SLE‐curcumin, n=14 vs. 0.212 ± 0.03 g SLE‐vehicle, n=5) (P = 0.0008). In addition, the percentage of circulating CD45R+ B cells are decreased at 28 weeks of age in SLE curcumin treated mice compared to vehicle treated SLE mice (5.6 ± 1.6% SLE‐curcumin, n=4 vs. 36.4 ± 9.5% SLE‐vehicle, n=10) (P = 0.068). Despite fewer circulating CD45R+ B cells, plasma levels of anti‐dsDNA IgG autoantibodies are unchanged in curcumin treated mice with SLE compared to vehicle treated mice with SLE. These data suggest that curcumin modulates autoimmune activity and may lessen renal injury in female mice with SLE.Support or Funding InformationResearch reported in this publication was supported by the NIGMS of the NIH (5U54GM115428 and P20GM104357), NHLBI of the NIH (P01HL051971), VA Merit (BX002604‐01A2), and AHA pre‐doctoral fellowship (19PRE34380830).
Sex Difference In Kidney Electrolyte Transport III: Impact of Low K intake on Thiazide‐Sensitive Cation Excretion in Male and Female Mice
Authors: Xu Shuhua , Li Jing , Yang Lei , Wang Claire J. , Liu Tommy , Weinstein Alan M. , Palmer Lawrence , Wang Tong 
Published: April 2020

The thiazide sensitive Na‐Cl co‐transporter (NCC) in the distal tubule can be regulated by dietary potassium intake and is critical for electrolyte homeostasis. Previously we reported that there was higher NCC activity and expression in female over male mice and high‐K intake preserved the sex‐difference of NCC‐mediated sodium absorption due to comparable reductions of NCC activity and expression in both sexes. However, with high‐K intake hydrochlorothiazide (HCTZ)‐induced kaliuresis was greater in males than in female mice, suggesting a more powerful male adaptation to high‐K intake. In this study, we explored the role of NCC in adaptation to a low‐K (LK) diet. We measured HCTZ‐induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK) and fractional (FENa, FEK) cation excretion in male and female mice on control‐K (CK, 1% KCl) and LK (0.1%, KCl) diets for 7 days. We also examined LK‐induced changes in NCC, Na/H‐exchanger isoform 3 (NHE3), and ENaC protein expression by Western blotting. With CK, NCC‐dependent ENa and FENa were larger in females than males as we observed previously. However with LK HCTZ‐induced ENa and FENa increased in males but not in females. Thus K restriction abolished the sex differences in NCC function. Changes in total NCC protein followed a similar pattern. Under CK conditions females expressed more NCC protein, as previously reported. LK increased NCC abundance in both males (by 100%) and females (by 40%). The larger effect in males reversed the sex‐dependence of NCC expression, consistent with the measurements of function. LK intake did not change either NHE3 or NHE2 expression, but reduced the amount of cleaved (presumably active) forms of α and γENaC expression in males but not in females. Thus decreased Na reabsorption through ENaC may compensate for increased Na transport through NCC in males. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K‐secretory apparatus in the distal nephron is strongly suppressed under these conditions.Support or Funding InformationNIH NIDDK RO1 DK099284