bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Mitochondrial Ribosomal Protein Expression impairs Oxidative Phosphorylation and Apoptosis in ER/PR(+) Breast Cancer Cell lines
Authors: Koc Emine C. , Kartal Funda , Tirona Maria , Koc Hasan
Published: April 2020

Remodeling of energy metabolism is described as one of the major hallmarks of cancer and contributes to its heterogeneity and survival in a dynamic environment with reduced nutrient and oxygen levels. Defects in oxidative phosphorylation (OXPHOS) can also cause a switch in energy metabolism from oxidative phosphorylation to glycolysis, referred to as the Warburg effect. Mitochondrial translation plays a crucial role in the biogenesis of OXPHOS complexes by synthesizing 13 mitochondrially‐encoded subunits of complexes I, III, IV, and V (ATP synthase). Interestingly, the changes in expression of mitochondrial translation components and single nucleotide polymorphisms (SNPs) of their corresponding genes have been associated with breast cancer. In this study, we studied the expression of OXPHOS components in 34 ER/PR (+) breast tumors. We found that the changes in the expression of mitochondrial ribosomal proteins (MRPs) contribute to the remodeling of oxidative energy metabolism and deregulation of apoptosis in breast cancer. We also investigated the expression of the pro‐apoptotic MRPs DAP3 and MRPS30 and their contribution to energy metabolism and apoptosis in ER/PR (+) breast tumors and cell lines. Our results showed reduced expression of both protein and impaired mitochondrial translation and therefore OXHOS. In the meantime, their ectopic expression resulted in increased apoptosis in ER/PR (+) MCF7 cell lines. We believe that a good understanding the role of MRPs in remodeling of energy metabolism and apoptosis will be essential in characterization of heterogeneity in breast tumors at molecular levels.
Characterization of phospholipid binding by the exocyst complex using lipid nanodiscs
Authors: Feyder Michael J. , Munson Mary
Published: April 2020

Proper regulation of exocytosis is crucial for cell function. Post‐Golgi vesicles require precise localization and temporal specification to fuse at correct sites on the plasma membrane to secrete their cargo
1. Secretion is essential for cell survival and is involved in intercellular communication and cell migration
2. Multisubunit tethering complexes (MTCs) are protein assemblies that coordinate interactions required for membrane fusion. Exocyst is a hetero‐octameric MTC responsible for targeting secretory vesicles to the plasma membrane by recognizing SNAREs, small GTPases, and phospholipid
2. Two exocyst subunits, Sec3 and Exo70, have been shown to bind phosphatidylinositol 4,5 biphosphate (PI(4,5)P2). Sec3 contains an N‐terminal Pleckstrin homology (PH) domain and there is a positively charged patch in the C‐terminus of Exo70
3,
4. These two domains mediate interactions with PI(4,5)P2. However, these interactions have only been shown using recombinantly purified individual subunits or domains. Combining exocyst purified from cryo‐milled yeast lysate with lipid nanodiscs, we are able to study binding of the wildtype and mutant octameric complex to various phospholipids. This has allowed us to characterize differential contributions of Sec3 and Exo70 to PI(4,5)P2 binding. Further, nanodiscs allow us to investigate interactions of the exocyst complex with a wide range of phospholipids, demonstrating the specificity of exocyst‐PI(4,5)P2 binding. This research is crucial to understanding the mechanism by which exocytic machinery is targeted to sites of exocytosis.
The nature of cell volume sensor in shrinkage‐induced apoptosis
Authors: Rana Priyanka , Kurokawa Manabu , Model Michael
Published: April 2020

Potassium loss and persistent shrinkage have been both implicated in apoptosis, but their relationship and respective roles remain obscure. We approached this problem by clamping intracellular sodium and potassium in HeLa, MDBK or MDCK cells by a combination of ionophores and ouabain. Although ionophores cause significant cell swelling, the initial volume can be restored and further reduced by applying external sucrose. We showed that swollen cells treated with ionophores remain viable for at least 4–8 hours without developing any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volume‐dependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation, cytochrome c release from mitochondria. Furthermore, overexpression of Bcl‐2 prevents mitochondrial loss of cytochrome c in shrunken cells. All these responses occurred to approximately the same degree regardless of whether the cells were kept in a high potassium/zero sodium or in a high sodium/zero potassium buffer, with only a slight enhancement under potassium‐free conditions. However, apoptosis induced by protein kinase inhibitor staurosporine in ionophore‐treated and swollen cells was strongly inhibited by as little as 5 mM potassium. Addition of 50 mM sucrose to restore the initial cell volume eliminated potassium dependence of staurosporine‐induced caspase activation. We have eliminated potassium as a stimulus in shrinkage induced apoptosis and are left with the choice between macromolecular crowding and cytoskeleton, which according to our unconfirmed preliminary data may be cytoskeleton. Thus, our results show that: (1) persistent shrinkage (presumably mediated through macromolecular crowding) acts as a direct proapoptotic signal independently of potassium; (2) strong potassium dependence of caspase activation is only observed in swollen cells with reduced cytoplasmic density.
Developing New Dopamine D2 Receptor Agonists and Partial Agonists for Parkinson’s Disease and Schizophrenia
Authors: Keck Thomas M. , Nguyen Catherine , Kurtyan Emily , Stephanie Bohn , Crowthers Raymond , Tharakan Jaslin , Briaguez Wendy , Ricchezza Joseph , Pino Michael , Brunt Dylan , Sullivan Holli-Joi , Wu Chun
Published: April 2020

Parkinson’s disease (PD) and schizophrenia are neuropsychiatric disorders that affect millions worldwide. Current medications target the dopamine D2 receptor (D2R). However, adverse side effects associated with these medications significantly reduce quality of life and drug compliance. Thus, new therapeutic strategies are clearly needed. D2R activation by agonists can activate cAMP and ß‐arrestin signaling pathways in neurons. Current evidence suggests that new biased D2R agonists—which preferentially activate one pathway over the other—and new D2R partial agonists could provide greater treatment specificity and avoid side effects. In order to develop new D2R‐selective agonists and partial agonists with potential biased signaling properties, new structural templates are needed. The objective of this project is to identify and investigate new D2R ligands with novel chemical scaffolds, with the long‐term goal of producing more effective medications for neuropsychiatric disorders. An initial virtual screen of a ZINC lead‐like library with >3.6 million molecular structures was performed using models of the D2R crystal structure refined into unbiased agonist and G protein‐biased agonist biding modes via molecular dynamics simulations. Following this in silico screen, 8 potentially novel D2R ligands were purchased and tested in radioligand competition binding screens using [3H]N‐methylspiperone and [3H]7‐OH‐DPAT. 6 of 8 purchased compounds (A01, A02, A03, A08, B03, B06) have detectable affinity for D2R (Ki = 0.75–51 μM), demonstrating the feasibility of our approach. New molecular dynamics simulations have been completed to expand in silico screens to include partial agonist and β‐arrestin‐biased agonist binding modes, and to evaluate a larger ZINC library (>17.9 million structures). Studies are ongoing to determine Ki values at D3R and D4R, and to characterize compound efficacy in cAMP and β‐arrestin signaling pathways for each hit compound. Collaborations are in development to build initial analogue libraries of each compound. Each hit compound is a new structural entity with no previously known dopaminergic activity. The identification of new chemical entities with D2R activity will build the foundation for future research to optimize pharmacological parameters for preclinical medications development.

Support or Funding Information

This research is supported by the Rowan University College of Science & Mathematics SEED Fund and the New Jersey Health Foundation. Travel support is provided by the Dean of the College of Science & Mathematics and the Rowan University Provost’s Office.
Rapid Diagnosis of Bacterial Pneumonia Using Volatile Organic Compounds (VOCs)
Authors: Dailey Allyson , Couch Robin
Published: April 2020

Timely diagnosis and treatment is essential for reducing morbidity, mortality, and inappropriate antibiotic usage often associated with bacterial pneumonia. Volatile organic compounds (VOCs) are a large and chemically diverse group of carbon‐based molecules, generally related by their volatility at ambient temperature. We have demonstrated the diagnostic potential of VOCs derived from biological samples
1–
4, and have established uniquely identifying microbial VOC fingerprints of several bacterial agents of pneumonia, including Francisella tularensis (Rabbit Fever) and Yersinia pestis (Plague). Additionally, we have created VOC fingerprints that distinguish wildtype from genetically engineered kanamycin‐resistant strains of select biothreat agents. We illustrate here the use of our rapid non‐invasive approach to the diagnosis of BALB/c mice infected with either Yersinia pestis CO92 or Francisella tularensis SCHU S4.

Support or Funding Information

Funding is provided by DTRA (Grant Number: HDTRA 1‐16‐1‐0040).
Activation of PACAP expression in mouse RTN neurons at birth protects postnatal breathing
Authors: Shi Yingtang , Stornetta Daniel , Reklow Robert , Sahu Alisha , Wabara Yvonne , Nguyen Ashley , Li Keyong , Zhang Yong , Perez-Reyes Edward , Ross Rachel , Lowell Bradford , Stornetta Ruth , Funk Gregory , Guyenet Patrice , Bayliss Douglas
Published: April 2020

The brainstem systems that regulate breathing are established prenatally but they remain immature and vulnerable to failure at the time of birth; this reflects, in part, underdevelopment of the respiratory chemosensory systems that typically monitor blood gases (O2, CO2) to regulate breathing. In the mouse retrotrapezoid nucleus (RTN), Phox2b‐expressing respiratory chemoreceptor neurons are a nexus for integration of multiple respiratory‐related inputs, homeostatically regulating blood gases by providing CO2‐modulated drive to brainstem respiratory rhythm generator and pre‐motor circuits. In this work, we demonstrate that postnatal expression of a neuropeptide in chemosensitive RTN neurons is precisely tuned to influence breathing at the time of birth. First, by using single cell qRT‐PCR and multiplex in situ hybridization, we define a striking postnatal burst of pituitary adenylate cyclase‐activating peptide (PACAP) expression in RTN neurons that is activated in neonates within minutes of exposure to the external environment. Further, by using whole body plethysmography, we show that mouse pups (P2‐P12) lacking PACAP in Phox2b neurons (Phox2b::Cre;PACAPfl/fl) have severely blunted CO2‐stimulated ventilation (reduced by >50%) and a striking ~3‐fold greater incidence of apneic episodes. PACAP expression in RTN neurons persists into adulthood, albeit at apparently lower levels. PACAP deletion/depletion in adult RTN neurons (in Phox2b::Cre;PACAPfl/fl mice or by shRNA‐mediated knockdown) also depresses CO2‐regulated breathing (by ~30–40%) and increases apneas (~3‐fold), while viral‐mediated re‐expression of PACAP in RTN neurons corrects these deficits. After PACAP knockdown in RTN, CO2‐dependent Fos expression was retained in RTN neurons but decreased in cells of the preBötzinger complex (preBötC), which contains respiratory rhythm‐generating neurons targeted by the RTN. Consistent with the preBötC as a site of action for RTN‐derived PACAP, we show that: 1) the PACAP receptor, PAC1, is expressed in preBötC; 2) PACAP injection directly into the preBötC stimulated central respiratory output, both in vitro and in vivo; and 3) shRNA‐mediated depletion of PAC1 in the preBötC phenocopied breathing deficits observed with RTN deletion of PACAP. Notably, PACAP variants are associated with sudden infant death syndrome (SIDS), and with a SIDS‐like phenotype in PACAP‐null mice. Collectively, these data demonstrate that well‐timed PACAP expression by RTN neurons protects the developmentally immature respiratory control system from hypoventilation and potentially dangerous apneas at birth, and identifies key molecular components of a circuit that supports breathing at this particularly vulnerable period in life.

Support or Funding Information

R01 HL108609; R01 HL074011
Integrating Pathophysiology to Enhance Learning and Information Literacy in Undergraduate Anatomy and Physiology
Authors: Brundage Cord M. , Khanbabaian Alina
Published: April 2020

Practical understanding of physiologic principals can be aided by exploring the process by which they become dysfunctional. This strategy is used in clinical education, but can also provide context to retain and reinforce abstract concepts for undergraduate anatomy and physiology students. Weekly assignments were added to a junior anatomy and physiology class that challenged students to identify a disease associated with the previous week’s physiologic principals. Students were asked to explain what physiologic process are disrupted and how normal physiologic function changed (with reference to a peer reviewed article) and include proper in‐text and bibliographic citations. Student performance on embedded course questions increased significantly in students completing pathophysiology assignments compared to previous semester (n = 48 for each; p < 0.05). Appropriate use of in‐text citations, reference formatting and the selection of primary literature were assessed using a 4‐point rubric for weekly assignments. Student performance improved in all categories over the course of the semester. Summative indirect assessment suggested that students felt more comfortable searching and referencing research. The majority of students also felt that the pathophysiology assignment helped to increase their overall understanding of course material. Collectively the integration of pathophysiology and primary literature may offer instructors an alternative means of enhancing student learning and information literacy in undergraduate anatomy and physiology courses.

Support or Funding Information

Support for this project provided by Cal Poly Pomona. Don B. Huntley College of Agriculture and a Cal Poly Pomona Special Projects for Intellectual Community Environment (SPICE) Grant for innovative approaches to instruction.
The Effects of Student Background and Peer Group Background on Anatomy Performance
Authors: Ngo Brandon , Kang Paul , Fisher Rebecca E.
Published: April 2020

As medical school curricula incorporate more active learning sessions, quality peer‐to‐peer interactions become increasingly important in the learning process. Students possess unique backgrounds and attributes that may help bolster not only their individual performance, but the performance of their peers as well. At the University of Arizona College of Medicine – Phoenix, the Clinical Anatomy (CA) block employs a hybrid approach: integrating lecture and lab activities to enhance active, peer‐driven learning. Five to six students are randomly assigned to a willed body donor for the entire course, with one instructor per table. The first half of each session consists of a slide presentation interspersed with stopping points to view structures on the right side of the donor, dissected the day before by allied health students. For the remainder of the session, the medical students dissect the left side of the donor and participate in quizzing activities with their table instructor. The objective of this study is to determine whether there is a correlation between a student’s pre‐medical school background, the background of their peer group, and CA exam performance. Retrospective, de‐identified data were collected from 285 first‐year medical students from four classes (2019–2022), including: age, gender, race, ethnicity, college, science and cumulative undergraduate GPA, undergraduate major, anatomy table group, and individual and cumulative CA exam scores. Univariate and multivariate linear regressions were utilized to assess the relationships between student characteristics and anatomy exam scores. Within the univariate analysis, students >30 years old scored lower than peers on exams 1 and 2 (p<0.01) and had a lower cumulative average (−5.7 mean difference, p<0.01), while students with MCAT scores ≥513 performed better than peers on exams 2 and 3 (p<0.05 and p<0.01, respectively) and had a higher cumulative average (+1.9 mean difference, p<0.05). Furthermore, students who had attended private undergraduate institutions scored higher than peers on exams 2 and 3 (p<0.01) and had a higher cumulative average (+2.6 mean difference, p<0.01), while students who had attended institutions outside Arizona scored higher than peers on exams 2 and 3 (p<0.05) and had a higher cumulative average (+1.3 mean difference, p<0.05). However, the multivariate analysis revealed significant relationships for only one parameter, with students >30 years old scoring lower than peers on exams 1 and 2 (p<0.01 and p<0.05, respectively) and demonstrating a lower cumulative average (−5.5 mean difference, p<0.05). Further analyses will elucidate the potential effect of peer group background on CA exam performance; however, these results indicate that most background characteristics do not correlate with performance in anatomy.
Protocol Development for the Production of Purified Matrix Metalloproteinase 1
Authors: DeMarco Angela Mary , Grove Laurie
Published: April 2020

Matrix metalloproteinase 1 (MMP1) is an enzyme involved in the degradation of the extracellular matrix of cells. In human MMPs, an active site containing three histidine amino acids uses zinc to catalyze peptide bond cleavage. Higher expression of this protein is associated with cancer and arthritis, so there is interest in using MMP1 to target drug delivery for these patients. Because there are twenty‐six MMPs, inhibiting the activity of one may influence the others causing unforeseen downstream affects. This is largely due to poor selectivity of the inhibitors, so more specificity is required to ensure only MMP1 is targeted.

The simple multicellular organism Volvox uses proteinases that are more active with copper than zinc. Zinc and copper have similar acid/base properties, so they should function in a similar way but don’t within these two systems. Volvox has two histidines and one glutamine in its active site. Exploring the functionality of the two proteinases with different metals may reveal the importance of the three histidines in its preference to zinc. Before experimentation can occur on MMP1, a method to produce large amounts of purified protein needs to be established.

Plasmids containing the gene for human full‐length proenzyme MMP1 with His tags was purchased and the identity was confirmed using restriction digest. These plasmids were then used to transform E. coli which was cultured to allow for protein expression. Sonication was used to lyse the cells. The lysate was then purified using Ni 2+ magnetic beads. SDS‐PAGE and kinetic assays were done on the MMP1 to ensure that it was an active enzyme sample. It was found that the E. coli was properly producing the protein, but further work is being done to ensure it is fully active. When this production method is perfected, the MMP1 collected here will be used in metal replacement assays and work with Crispr to generate multiple active site single point mutations.

Support or Funding Information

Wentworth Institute of Technology
Generation and characterization of a serum albumin‐humanized mouse model for biomarker discovery
Authors: Han Weiguo , Yin Lei , Sheng Jonathan , Fan Xiaoyu , Yang Weizhu , Zhang Qing-Yu , Turesky Robert , Ding Xinxin
Published: April 2020

The aim of this study is to develop a serum albumin‐humanized mouse model, for identifying human protein biomarkers of exposure to hazardous xenobiotics. The albumin‐humanized mouse was generated via cross‐breeding between a human albumin‐transgenic mouse and an albumin‐null mouse. The ability of human and mouse albumins to react with metabolites of the lung carcinogen 4‐(methylnitrosamino)‐l‐(3‐pyridyl)‐1‐butanone (NNK) was compared. Adult albumin‐humanized mice and wild‐type mice were treated with NNK (100 mg/kg body weight, IP). Blood, liver, and lung were analyzed 6 hours after NNK treatment. The NNK metabolite 4‐hydroxy‐l‐(3‐pyridyl)‐1‐butanone (HPB), free or as albumin‐HPB adduct, was detected in the plasma, whereas the O6‐methyl‐deoxyguanosine (O6‐mdG) DNA adduct, formed via NNK bioactivation, was detected in lung and liver. Results of quantitative analyses revealed lower levels of O6‐mdG in tissues of the humanized mice (10.4 and 85.5 pmol/μmol guanine, for lung and liver, respectively) than in wild‐type mice (18.4 and 240 pmol/μmol guanine, for lung and liver, respectively), a difference possibly explained by differing pharmacokinetic profiles of NNK. Interestingly, although a substantial genotype‐related difference was not observed in free HPB levels in the plasma (0.31 and 0.39 ng/ml, for wild‐type and humanized, respectively), human albumin‐HPB adduct levels in the humanized mouse were 6X higher than mouse albumin‐HPB adduct levels in the wild‐type mouse (per mg albumin). These data suggest that human and mouse albumins have different reactivities with reactive NNK metabolites and they illustrate the utility of the albumin‐humanized mouse model for biomarker discovery.

Support or Funding Information

Supported in part by NIH grant CA092596
Effects of Discriminator Length on Transcription Initiation by E. coli RNA Polymerase
Authors: Doughty Sarah , Plaskon Dylan , Record M. Thomas
Published: April 2020

Transcription initiation is a highly regulated step of gene expression. Here we investigate how different discriminator sequence lengths affect initiation. The discriminator is the region between the −10 element and the transcription start site (TSS) of the promoter and does not have a consensus sequence or length. Previous studies exchanged 6 and 7 base pair(bp) discriminators between promoters and found that the discriminator largely determines the lifetime of the binary RNA polymerase (RNAP)‐promoter open complex as well as the RNA‐DNA hybrid length at which RNAP escapes the promoter
1. Promoters with the 7 bp T7A1 discriminator have shorter lifetimes and escape points. Here we compare open complex lifetimes, initiation kinetics and RNAP escape points of the gcdp1 promoter (5 bp discriminator, a common length for E. coli promoters) with those of promoters with longer discriminators. Transcription start sites and discriminator lengths were determined by a primer extension assay. Fast‐mixing (RQF) and benchtop initiation experiments with α‐32P‐UTP or α‐32P‐GTP in the NTP mix were used to quantify transient amounts of short and long RNA products from productive complexes and stalling and abortive synthesis by nonproductive complexes. Results to date with the gcdp1 promoter/discriminator reveal a similar escape point to the λPR promoter. RQF studies with the T7A1 promoter show similar initiation kinetics to the λPR variant with the T7A1 discriminator, and an earlier escape point than for these promoters with the λPR discriminator. These results add to our understanding of the significance of the discriminator as a determinant of efficient initiation and promoter escape by RNAP.
Endothelial Ca2+ signals in Penetrating Arterioles Control Local Blood Flow in the Brain.
Authors: Mughal Amreen , Longden Thomas , Hennig Grant W. , Nelson Mark T.
Published: April 2020

Cerebral blood flow is exquisitely regulated to meet the fluctuating demands of active neurons. This activity‐dependent increase in blood flow (functional hyperemia) is managed by a number of molecular mechanisms collectively referred to as ‘neurovascular coupling’. Penetrating arterioles—the bridge between pial arteries and deep capillaries—serve as a high‐level control system to regulate local perfusion into the cortex. Here we show that dynamic Ca2+ signals in arteriolar endothelium, consisting of unitary and compound events with distinct spatiotemporal patterning, are dramatically increased by sensory stimulation and are followed by dilation of upstream arterioles and increased RBC flux in downstream capillaries. These arteriolar endothelial Ca2+ signals in the cortical vasculature are dependent on Gq‐protein coupled receptor signaling likely involving prostaglandin E2 (PGE2). PGE2‐dependent activation of EP1 receptors, results in IP3 receptor‐dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via TRPV4 channels. Our data highlight the pivotal role of endothelial Ca2+ signals in the regulation of cerebral blood flow.

Support or Funding Information

Supported by the National Institutes of Health, Totman Medical Research Trust, Fondation Leducq, EC Horizon 2020.
Reliability of a Novel Ultrasound Transducer Holder for Flow‐Mediated Dilation
Authors: Rodriguez Lisa A. , Morales-Acuna Francisco , Aguilera Juan , Gurovich Alvaro N.
Published: April 2020

Background

Flow‐mediated dilation (FMD) is the most common test to evaluate endothelial function. Although efforts have been made to incorporate FMD in clinical settings, guidelines still do not advocate for its use because the wide variability on results among testers. Strategies to overcome this problematic scenario include the use of transducer holders to increase the reproducibility on image acquisition. Therefore, the purpose of this study was to test the reliability of a novel ultrasound (US) transducer holder for brachial FMD.

Methods

Using 3D printing technology, an arm US transducer holder was created to assist the assessment of the vasculature via US imaging during FMD. A total of 15 young healthy subjects were evaluated for Intrarater and Interrater FMD reliability. Statistical analysis included Coefficient of Variation (CV) and Intraclass Correlation Coefficient (ICC). Based on the CV, reproducibility was categorized as excellent (0–10%), good (10–20%), moderate (20–30%), or poor (>30%). Regarding ICC, reproducibility was catalogued as poor (>0.5), moderate (0.5–0.75), good (0.75–0.9), and excellent (>0.90).

Results

The baseline and peak after deflation artery diameter images were analyzed using an automatic edge detection software. The following data was collected:

The results in table
1 show that the degree of reliability for an arm US transducer holder during FMD, between and within raters using artery diameter, %FMD, and delta‐FMD as markers, is excellent. The CV for all categories within the inter‐ and intrarater were under 7%, which supports achieving inter‐ and intrarater reliability. The ICC for all categories, with the exception of one, was 90% or better, this shows that the reproducibility of the holder was catalogued as excellent. The Interrater ICC for one category, delta‐FMD, was rated good at 0.89.

Conclusion

Excellent reliability was achieved with interrater and intrarater FMD using the arm US transducer holder device. This holder has the ability to improve the results of this type of noninvasive endothelial function testing.

Analyzed data collected during FMD

Reliability Test

Baseline Artery Diameter

Peak Artery Diameter

%FMD

Delta‐FMD

Intrarater CV (%)

0.26 (0.39)

0.40 (0.42)

6.44 (6.1)

6.27 (6.09)

Interrater CV (%)

0.23 (0.16)

0.52 (0.38)

5.75 (5.29)

5.82 (5.39)

Intrarater ICC

0.99

0.99

0.97

0.95

Interrater ICC

0.97

0.96

0.97

0.89
Dietary Fat and Sugar Differentially Affect Beta‐Adrenergic Stimulation of Cardiac ERK and AKT Pathways in Mice Subjected to High‐Calorie Feeding
Authors: Ashraf Sadia , Yilmaz Gizem , Chen Xu , Harmancey Romain
Published: April 2020

Introduction. High dietary fat and sugar promote cardiac hypertrophy independently from an increase in blood pressure. The respective contribution that each macronutrient exerts on cardiac growth signaling pathways remains unclear. The goal of this study was to investigate the mechanisms by which high dietary fat and sugar levels affect cardiac growth regulatory pathways in the mouse heart.

Methods. Nine‐week‐old male C57BL/6 mice (n = 20/group) were fed a standard rodent diet (STD; 3.0 kcal/g), a high‐fat diet (HFD; 5.24 kcal/g), a high‐fat and high‐sugar Western diet (WD; 4.73 kcal/g), a high‐sugar diet with mixed carbohydrates (HCD; 3.85 kcal/g), or a high‐sucrose diet (HSD; 3.85 kcal/g). Body composition was assessed weekly by EchoMRI. Whole‐body glucose utilization was assessed with an intraperitoneal glucose tolerance test. After six weeks on diets, mice were treated with saline or 20 mg/kg isoproterenol (ISO) and the activity of cardiac growth regulatory pathways was analyzed by immunoblotting. One‐ or two‐way ANOVA was used for statistical comparisons.

Results. Compared with STD, HFD increased body fat mass 4.5‐fold (P < 0.0001), induced glucose intolerance (P < 0.0001), and increased insulin levels 3.6‐fold (P < 0.0001), thereby enhancing basal and ISO‐stimulated AKT phosphorylation at both threonine 308 and serine 473 residues (+37–100%; P < 0.05). Both HCD and HSD potentiated ISO‐mediated stimulation of the glucose‐sensitive kinases PYK2 (>50%; P < 0.05) and ERK (>30%; P < 0.05), thereby leading to increased phosphorylation of protein synthesis regulator S6K1 at threonine 389 residue (≥70%; P < 0.05). All these anthropometric, metabolic and cardiac growth signaling alterations occurred simultaneously in WD‐fed mice.

Conclusions. The results indicate that dietary fat and sugar affect cardiac cell growth signaling pathways in C57BL/6 mice through distinct and additive mechanisms. The findings may provide new insights into the role of overnutrition in pathological remodeling of the heart.

Support or Funding Information

This work was supported by grants R01HL136438, P20GM104357 and P01HL051971 from the National Institutes of Health.
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 Information

St. 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

INTRODUCTION

Early 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.

METHODS

A 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.

RESULTS

The 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).

CONCLUSIONS

A 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 Information

MSU 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 Information

American 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 Information

National 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 Information

Supported 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 Information

National Institute for Drug Abuse (NIDA)