bluuebunny/crossref_metadata_2025 huggingface dataset:
ACTIVATION OF ALC1 (CHD1L) NUCLEOSOME REMODELING BY PARP‐1‐DEPENDENT POLY(ADP‐RIBOSYL)ATION
Authors: Ooi Soon-Keat , Sato Shigeo , Tomomori-Sato Chieri , Conaway Ronald C. , Conaway Joan W.
Published: April 2020

ALC1, also known as CHD1L, is a macrodomain‐containing SNF2‐family ATPase that has been implicated in various cancers and has apparent roles in DNA repair. ALC1 displays robust ATP‐dependent nucleosome remodeling activity only in the presence of PARP‐1 and NAD+. However, the mechanism by which PARP‐1 stimulates ALC1 nucleosome remodeling activity through NAD+‐dependent poly(ADP‐ribosyl)ation (‘PARylation’) remains less well understood. PARP‐1 is a multi‐domain enzyme that catalyzes most protein PARylation in cells. PARP‐1 consists of an N‐terminal region that includes three zinc finger domains (Zn1, Zn2 and Zn3), a BRCA1 C‐terminal domain (BRCT), a tryptophan‐glycine‐arginine‐rich domain (WGR) and a C‐terminal catalytic domain (CAT) that is responsible for protein PARylation. Previous studies have shown that DNA or nucleosome binding by domains within the PARP‐1 N‐terminal region allosterically activates CAT for poly(ADP‐ribose) (‘PAR’) synthesis.

To explore the functions of PARP‐1 domains in stimulating ALC1 nucleosome remodeling activity, we performed a structure‐function analysis of PARP‐1. As expected, the PARP‐1 CAT domain, which is essential for PAR synthesis, is also needed for ALC1‐dependent nucleosome remodeling. To determine whether CAT is sufficient to stimulate nucleosome remodeling by ALC1, we took advantage of a gain‐of‐function mutation in the PARP‐1 CAT domain that renders PARP‐1 PARylation activity independent of DNA binding. CAT with this gain‐of‐function mutation exhibits robust PARylation activity but is unable to activate ALC1 nucleosome remodeling activity, arguing that one or more PARP‐1 N‐terminal domains are needed in addition to CAT for maximal ALC1‐dependent nucleosome remodeling. In further experiments, we tested the abilities of PARP‐1 mutants lacking individual domains within the PARP‐1 N‐terminus to activate ALC1. Consistent with previous studies indicating that the Zn2 and BRCT domains of PARP‐1 are dispensable for protein PARylation, we found that these domains are also dispensable for ALC1 nucleosome remodeling activity. In addition, results of experiments performed with constitutively active PARP‐1 mutants lacking Zn1, Zn3 or WGR domains suggest that each of these domains contribute to ALC1‐dependent nucleosome remodeling. Studies exploring the mechanism(s) by which Zn1, Zn3, and WGR domains contribute to nucleosome remodeling are underway.
SPARC: Different MicroRNA Regulatory Networks Are Active In The Nucleus Of The Solitary Tract During The Development Of Hypertension In Male And Female Spontaneously Hypertensive Rats
Authors: Moss Alison , Kuttippurathu Lakshmi , Achanta Sirisha , Schwaber James S. , Vadigepalli Rajanikanth
Published: April 2020

Hypertension is a chronic disease affecting millions of people, often leading to heart disease or stroke and is known to affect males and females differently. Previous work in our lab examined the microRNA networks in male Spontaneously Hypertensive Rats (SHRs) in comparison to the wild type Wistar Kyoto (WKY) rats (DeCicco et al., Physiological Genomics 2015). Our results revealed a panel of microRNAs in the nucleus of the solitary tract (NTS) that showed differential expression between the SHR and WKY. Specifically, a subset of microRNAs regulating inflammation and angiotensin pathways were found to have higher expression in the SHR than in the WKY rats at the onset of hypertension. In our present work, we have conducted a similar time series microRNA profiling analysis in female rats allowing us to compare and contrast the results with previously obtained data in males. Rats were sacrificed over the development of hypertension at 8, 12, and 16 weeks of age and brainstems were removed and stored in OCT. Brainstems were sectioned on a cryostat and the NTS was extracted using a micropunch. As previously reported in DeCicco et al. (2015), total RNA was extracted and samples were analyzed for expression of over 400 miRNAs using the Nanostring nCounter system. Our analysis revealed extensive differences in the differential expression of miRNAs in males vs. females. Consistent across both sexes is the finding that miRNA levels in SHR and WKY differ primarily at the onset of hypertension. However, differential regulation in SHR vs. WKY was opposite in direction between males and females. Male SHR showed upregulation of a subset of miRNAs at the onset of hypertension, which were not statistically significant in the female SHR vs. WKY at the same stage. By contrast, female SHR showed downregulation of a distinct set of miRNAs that were not differentially regulated in the male SHR vs. WKY, at the onset of hypertension. These results suggest that miRNA regulatory networks operate in a sex‐specific manner during the development of hypertension. Examination of these regulatory networks lends important insight into the different autonomic control mechanisms driving hypertension in males and females.

Support or Funding Information

Supported by the NIH Common Fund SPARC Program award OT2 OD023848 and NIH NHLBI Multiscale Modeling award U01 HL133360.
Aerobic Exercise Training Prevents Obesity and Insulin Resistance Independent of Changes in the Skeletal Muscle ACE2/Ang 1‐7/Mas Axis.
Authors: Evangelista Fabiana S. , Vecchiatto Bruno , Américo Anna Laura V. , Martucci Luiz Felipe , Ferreira Marilia M. , Muller Cynthia R.
Published: April 2020

Aerobic exercise training (AET) has been widely used for the prevention and treatment of obesity and insulin resistance (IR). Improvements in the oxidative capacity of skeletal muscle can mediate the protective effect of AET. Considering the role of angiotensin converting enzyme 2 (ACE2)/angiotensin 1‐7 (Ang 1‐7)/receptor Mas axis activation in reducing cardiometabolic diseases, the present study aimed to evaluate if the prevention of obesity and IR through AET is associated with changes in the ACE2/Ang (1‐7)/Mas axis in the skeletal muscle. Adult male C57BL6/J mice were assigned into groups (n=10/group): chow‐fed controls (C), chow‐fed trained (T), cafeteria diet (CAF), and cafeteria diet and trained (CAFT). AET was performed simultaneously with diet and consisted of 8‐wk running session of 60 min at 60% of maximal speed, 5 days/wk. Experimental procedures were approved by Ethics Committee from School of Arts, Science and Humanities, University of Sao Paulo, Brazil (#001/2016). The CAF group presented higher body weight gain and adiposity, glucose intolerance, IR and increased lipid deposition in the liver while AET prevented such damages in the CAFT group. Food consumption did not change among the groups. During the maximal physical exertion test, the T group increased VO2máx and both T and CAFT groups showed higher maximal speed, higher speed on the VO2máx and lower relative running cost compared to C and CAF groups. The enzymatic activity of citrate synthase and β‐hydroxyacyl‐CoA dehydrogenase (β‐HAD) in the soleus muscle increased in the T (352 ± 21.5 mmol/min/mg and 80.1 ± 6.6 mmol/min/mg) and CAFT (350 ± 22.4 mmol/min/mg and 87.9 ± 7.3 mmol/min/mg) groups compared to C (272.7 ± 9.5 mmol/min/mg and 62.7 ± 3.9 mmol/min/mg) and CAF (254.6 ± 15.7 mmol/min/mg and 46.7 ± 1.6 mmol/min/mg) groups. Protein expression of mitofusin 1, mitofusin 2 and dynamin related protein 1 (Drp1) did not change among groups. Also, no differences were observed in the ACE2 and Ang (1‐7) levels nor in the receptor Mas protein expression. In conclusion, the AET prevented the increase in body weight, adiposity and IR induced by cafeteria diet, improved the aerobic capacity and promoted the increase in oxidative enzymes activity in the soleus muscle. However, the adaptations induced by AET were not associated with changes in the ACE2/Ang (1‐7)/Mas axis in the skeletal muscle.

Support or Funding Information

São Paulo Research Foundation (FAPESP #2015/04948‐4; #2016/23783‐9; #2016/20659‐5).
Calcium Chloride Enhance Crystal Growth of an Uncharacterized GNAT Enzyme
Authors: Robles Alyssa , Arada Angelika , Baumgartner Jackson , Houseman Charles , Variot Cillian , Kuhn Misty L.
Published: April 2020

Gcn5‐related N‐acetyltransferases (GNAT) are widespread across all domains of life and are capable of acetylating an assortment of substrates. However, many of their structures and functions remain unknown in bacteria. Our laboratory currently studies uncharacterized GNATs from Pseudomonas aeruginosa. We previously found the enzyme PA3944 acetylated polymyxin antibiotics and observed there was a duplicate GNAT gene (PA3945) with approximately 75% identity on the same operon. This protein has not been structurally or functionally characterized. Therefore, we screened this enzyme for activity using a broad‐substrate screening assay and set up crystallization screens to obtain protein crystals. Unfortunately, the PA3945 enzyme did not acetylate any substrates in the kinetic screening assay, which indicates it has a separate role compared to the PA3944 enzyme. We identified crystallization conditions that yielded microcrystals in the presence of calcium chloride. We further optimized these conditions using calcium chloride as an additive, which yielded larger crystals. Currently, we are investigating whether calcium chloride plays a role in enzyme activity and whether we can further optimize the crystallization conditions to yield suitable crystals for X‐ray diffraction and structure elucidation.

Support or Funding Information

Research reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).
Connexin43 as a Therapeutic To Restore Cardiac Structure in Arrhythmogenic Heart Disease
Authors: Zhang Jing , Zanella Fabian , Lyon Robert , Mezzano Valeria , Roberts Jason , Ursell Philip , Carromeu Cassiano , Gu Yusu , Martin Jody , Muotri Alysson R. , Scheinman Melvin , Peterson Kirk L. , Sheikh Farah
Published: April 2020

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is termed a fatal “disease of the desmosome” as 40–50% of patients harbor mutations in genes associated with the desmosome, a mechanical based cell‐cell junction structure found between cardiac muscle cells. Connexin43, the most dominant gap junction protein in ventricles, has been classically associated with electrical functions between adjacent cardiac cells, is consistently downregulated and considered an early disease hallmark of ARVD/C. However, limited efforts have focused on dissecting the role of connexin43 in ARVD/C, especially during end‐stage cardiac disease, where structural alterations are integral in facilitating ventricular failure, arrhythmias and premature death. Here we show that genetic restoration of connexin43 in human and mouse models of ARVD/C is sufficient to bypass desmosomal structural deficits as well as circumvent disease and premature death. In vitro studies demonstrated the ability of human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes to recapitulate classic variations in desmosomal structural defects and reveal connexin43 loss as a molecular predictor of physiological severity within ARVD/C hearts in a donor‐specific manner. Adenoviral restoration of connexin43 could rescue baseline and catecholamine induced electrical and contractile physiological deficits in ARVC hiPSC derived cardiomyocytes in absence or presence of desmosomal structural alterations. Furthermore, in vivo cardiac‐specific connexin43 gene therapy could prolong lifespan and restore cardiac structure (desmosomal protein expression) in a mouse model with end‐stage ARVC, in a dose‐dependent manner. Our results provide evidence for non‐canonical and independent functions for connexin43 in mechanical modulation of junctions. We anticipate that our findings will have broad implications in exploiting connexin43 as a viable therapeutic target in advanced diseases associated with underlying structural defects.

Support or Funding Information

Department of Defense (PR170125); National Institutes of Health (NIH R01 HL142251‐A1); American Heart Association Postdoctoral Fellowship (18POST34060228)
Evaluation of the Integrity of the Respiratory Chain in Rooster Sperm after Freezing
Authors: Nikitkina Elena , Krutikova Anna , Musidray Artem , Shapiev Ismail
Published: April 2020

One of the causes of cell death is a violation of electron transfer through the respiratory chain of mitochondria. The aim of study was to assess the integrity of the respiratory chain sperm after freezing in roosters. The effect of 2.4 dinitrophenol (2.4 DNP) on NADH fluorescence in fresh and cryopreserved semen of roosters was investigated. If the transfer of electrons and protons along the respiratory chain functions normally 2.4 DNP enhances the nonphosphorylating oxidation of NADH and its fluorescence decreases. The sperm of 30 cocks was frozen. The NADH was measured on a LUMAM fluorescence microscope. The luminescence excitation wavelength of NADH is 365 nm. Registration of NADH was carried out at a wavelength 465 nm. The amount of NADH was expressed in conventional units and the change in fluorescence after addition of 2.4 DNP was calculated as a percentage of the initial value taken as 100 %. The fluorescence of NADH after adding 2.4 DNP to frozen semen was 9% less than in fresh semen. A relationship between the magnitude of the change in NADH fluorescence after the addition of DNP and egg fertilization was established (r =0.65; P<0.05). Respiratory stimulation by 2.4 DNP is an useful and informative indicator in assessing sperm quality.

Support or Funding Information

The study was supported by the Russian Science Foundation (project No. 18‐16‐00071)
No Sex Differences in Cerebral Blood Velocity Responses During Resistance Exercise
Authors: Sherman Sara R. , Rosenberg Alexander J. , Schroeder Elizabeth C. , Lefferts Wesley K. , Lima Natalia S. , Grigoriadis Georgios , Baynard Tracy
Published: April 2020

Introduction

The brain requires intricate regulation of blood flow to maintain adequate perfusion to the tissue. Resistance exercise (RE) causes large increases in mean arterial pressure that challenges the regulation of cerebral blood flow. It is unknown, however, if the sex differences in blood pressure regulation may alter how cerebral blood flow responds to the large increases in blood pressure induced during RE.

Purpose

To determine if the cerebral blood velocity response during an acute bout of RE differs by sex.

Methods

Twenty‐nine young, healthy adults (M/F: 17/12; 25 ± 5 yrs; 23.9 ± 3.3 kg/m2) performed 3 sets of 10 repetitions maximal isokinetic knee flexion/extension exercise. Systolic (SBP), diastolic (DBP), and mean (MAP) arterial pressure were measured using finger photoplethysmography and used to calculate pulse pressure (PP). Transcranial Doppler ultrasound (2 MHz) was used to measure right middle cerebral artery mean blood velocity (MCAmv) and calculate pulsatility index ([MCAsystolic ‐ MCAdiastolic velocity]/MCAmv). Conductance was calculated as MCAmv/MAP. The last 30‐sec of a 10‐min baseline and last 10‐sec of the 3rd RE set were averaged for analyses.

Results

Overall, females had higher MCAmv and conductance compared to males (p<0.05). SBP, DBP, MAP, PP, MCAmv increased and conductance decreased similarly for males and females during RE (p<0.05). Pulsatility index did not change during RE in either sex (p>0.05).

Conclusions

The blood pressure and cerebral hemodynamic response during RE was not different between sexes. These data suggest sex may not effect cerebrovascular responses to a large increase in blood pressure incurred during maximal RE in young healthy adults.

Blood pressure and cerebral blood velocity responses at baseline and during an acute bout of resistance exercise in young adults.

Male (n= 17)

Female (n= 12)

p for Interaction

Baseline

Resistance Exercise

Baseline

Resistance Exercise

SBP (mmHg)
*

124 ± 10

172 ± 18

123 ± 7

170 ± 27

0.92

DBP (mmHg)
*

73 ± 7

99 ± 10

74 ± 7

103 ± 17

0.58

MAP (mmHg)
*

93 ± 8

128 ± 13

96 ± 7

132 ± 10

0.80

PP (mmHg)
*

52 ± 7

72 ± 11

50 ± 6

67 ± 14

0.42

MCA Mean Velocity (cm/sec)
*,
‡

55 ± 9

65 ± 12

70 ± 15

81 ± 30

0.89

MCA Pulsatility Index

0.90 ± 0.16

0.82 ± 0.14

0.81 ± 0.15

0.82 ± 0.27

0.27

Conductance (cm/sec/mmHg)
*,
‡

0.60 ± 0.09

0.51 ±0.10

0.74 ± 0.17

0.62 ± 0.24

0.39

All data are presented mean ± standard deviation

*Main effect for time (p<0.05)

‡Main effect for sex (p<0.05)
Effects of “Junk‐Food” on Excitatory Transmission and NMDA Receptor Subunit Expression in the NAc of Females
Authors: France Amanda M. , Alonso-Caraballo Yanaira , Ferrario Carrie R.
Published: April 2020

Eating diets high in fats and sugars (i.e., “junk‐food”) produces alterations in the function of brain regions like the nucleus accumbens (NAc) that promote food‐seeking and over‐eating. For example, consumption of “junk‐food” increases the expression and function of high conductance calcium permeable AMPA receptors (CP‐AMPARS) in the NAc of obesity‐prone, but not obesity‐resistant male rats. These alterations persist for up to 30 days after initial “junk‐food” exposure. In addition, blockade of CP‐AMPARs within the NAc prevents enhanced food‐seeking in obesity‐prone rats. Thus, obesity‐prone rats are more sensitive to diet‐induced enhancements in NAc glutamatergic transmission that promote food‐seeking. However, whether similar experience‐induced alterations occur in females is unknown. Therefore, the goal of the studies presented here is to determine the effects of “junk‐food” on NAc glutamate receptor expression and function in females. Males were included for comparison and to replicate initial findings described above. Rats were given the “junk‐food” diet (10 days) followed by a return to ad lib standard chow for 14 days (i.e., “junk‐food” deprivation) in order to examine long‐lasting effects. Controls remained on standard chow throughout. Biotinylation or BS3 crosslinking followed by western blotting was then used to measure changes in the expression of AMPAR and NMDA receptor (NMDAR) subunits. In addition, whole‐cell patch clamp recordings from ex vivo brain slices were used to measure effects on AMPAR and NMDAR‐mediated transmission. Furthermore, the estrus cycle was monitored throughout and recordings were conducted in the metestrus/diestrus phase of the cycle. For biochemical studies, post hoc analyses are used to assess potential effects of the cycle on protein expression. In males, we found increases in GluA1, but not GluA2 surface expression and enhanced CP‐AMPAR‐mediated transmission following “junk‐food” consumption, replicating previous results. However, in females no effects on GluA1 or GluA2 surface expression or on AMPAR‐mediated transmission (CP‐AMPARs or non‐CP‐AMPARs) were found. However, there was a “junkfood” induced increase in the AMPA/NMDA ratio in females that was due to a reduction in NMDAR‐mediated transmission in the absence of any change in AMPAR‐mediated transmission. Thus, initial results show that there are sex differences in “junk‐food” induced alterations in CP‐AMPARs, and suggest a reduction in NMDA‐mediated transmission in females. Ongoing studies are examining effects of “junk‐food” on NMDAR subunit expression (GluN1, GluN2, GluN3). This will provide insights into which NMDAR populations may be affected. Together, these data shed light on potential sex specific mechanisms by which “junkfood” diet alters NAc function.

Support or Funding Information

NIH R01 DK106188 (CRF)

NIH F99 NS108549 (YAC)
HL‐60 Cells Alternative Response to an Ionomycin PMA Cocktail
Authors: Sternberg Lauren , O’Donnell Robert W.
Published: April 2020

The combination of Ionomycin and phorbol 12‐myristate 13‐acetate (PMA) cocktail has been used to increase the proliferation of normal mouse and human T cells and PMA alone has been used to cause the differentiation of the myeloid leukaemia HL‐60 cells in culture to a macrophage‐like adherent population. Contrariwise, the cocktail was linked to apoptosis in acute HL‐60 cells. This distinction has also been noted in the literature by Wang, et al. (2014), who concluded that Jurkat T cells were inhibited at S‐phase and G2/M‐phase by the cocktail. When we cultured the HL‐60 cells for 48 hours in the presence of the cocktail, we also saw no growth and significant death. Originally, we suspected this distinction was due to the biological differences between normal cells and tumor cells; however, further investigation revealed a discrepancy within the literature. While HL‐60, Jurkat, and Glioma cells (Han 2013) are inhibited by the cocktail, breast cancer cell lines were MDA‐MB‐435 and MDA‐MB‐231 had enhanced invasion (Yiu 2006). Future experiments including a cell cycle analysis and apoptosis assay will be used to test other cancerous and non‐cancerous cell lines in hopes of elucidating a pattern for the effects of the ionomycin PMA cocktail.

Support or Funding Information

Carl Savino and Olga Gerych‐Savino Endowment, The Geneseo Foundation, and David Wolf, D.O.
Alleviation of Metalloid‐Containing Methionine Analog‐Induced Toxicity Using Chemically Defined Media Supplementation
Authors: Hill Austin Reece , Boles Jeffrey Oakley
Published: April 2020

X‐ray crystallography continues to be the most widely used method for protein structure determination. Most naturally occurring atoms lack the mass to sufficiently diffract electrons and thus, multiple isomorphous replacement (MIR) via the incorporation of metalloid‐containing analogs, such as selenomethionine, remains a valid solution to solve the phase problem associated with x‐ray crystallography. However, selenomethionine adversely affects cells in vivo and is considered cytotoxic. Through a proteomic study performed in 2013, differentially expressed proteins were identified from both telluromethionyl and selenomethionyl‐incorporated E. coli DL41 (DE3) (pcock), a methionine auxotroph. The cytotoxic effects of the heavy metal analogs caused the complete downregulation of various vital proteins, such as: fructose‐bisphosphate aldolase, UPase, phosphoserine aminotransferase, and others. It is hypothesized that through chemically defined media (CDM) supplementation of compounds integral in these downregulated proteins’ pathways, the cytotoxic effects incurred from these heavy metal analogs can be partially alleviated. Compound addition in optimal concentrations should increase overall cell culture growth and positively impact current methodologies pertaining to x‐ray crystallographic analysis.
Pharmacology of Metabotropic Glutamate Receptor 2 Homo‐ and Heterodimers
Authors: McCullock Tyler William , Kammermeier Paul
Published: April 2020

Metabotropic Glutamate Receptors (mGluRs) are a family of class C g‐protein coupled receptors (GPCRs) that respond to glutamate as their native agonist. The eight members of the mGluR family are widely expressed across the nervous and are primarily charged with shaping and fine‐tuning neuronal excitability and synaptic efficacy. Like most class C GPCRs, mGluRs are obligate dimer proteins. All mGluRs maintain the capacity to homodimerize, but recent work has demonstrated that heterodimeric mGluRs can form and exist natively. Further, studies of the mGluR2‐4 heterodimer have demonstrated that this heterodimeric receptor exhibits a distinct and unpredicted pharmacological profile compared to either the mGluR2 homodimer or the mGluR4 homodimer. These findings underscore the importance of better understanding mGluR heterodimer pharmacology, particularly with preclinical studies showing promising results for leveraging mGluRs as therapeutic targets for a wide variety of conditions. To develop a better understanding of the behavior of mGluR2, we used a multifaceted approach to investigate mGluR2s dimerization preferences, whole cell signaling, and individual receptor signaling when expressed by itself, or with other mGluRs. To test dimerization preference, we utilize a split luciferase assay. To test whole cell signaling, we utilize a bioluminescent resonance energy transfer (BRET) based G‐protein Beta‐Gamma sensor. Finally, to detect and determine the pharmacological profile of specific mGluR2 heterodimers, we combine the split luciferase technique with BRET, allowing us to isolate signals from a specific dimer within a population of receptors. These studies will help define the pharmacological profile of various mGluR2 containing heterodimers, as well as develop new tools to help further interrogate the pharmacology of other dimer complexes. These new tools are also high‐throughput compatible, allowing for the development of more advanced drug screens to identify new molecules that specifically target mGluR heterodimers.

Support or Funding Information

University of Rochester Harold C Hodge Memorial Fund and the University of Rochester Drug Targets and Mechanism Pilot Award
Cell Death Induced by Airborne Particulate Matter in Murine Microglial Cells
Authors: Street Mikayla , Feiner Kailey , Mazzer Paula
Published: April 2020

Airborne particulate matter has been linked to neurodegeneration in recent epidemiological studies. Previous work in our lab has shown that different types of particulate matter elicit different reactions. We set out to investigate two different particulate matter types in microglial cells to see whether they would have the same effect in different types of neurological cells. We combined caspase‐3 assay with lipid profiling to determine the mode of cell death induced by several different types of airborne particulate matter. Some types of particulate matter induced cell death via apoptosis and others induced cell death by neuroinflammatory methods.

Support or Funding Information

NIH P20GM103443
RIP‐3 and HMGB‐1 are upregulated in adult hypertensive male spontaneously hypertensive rats (SHR).
Authors: Abdelbary Mahmoud A. , Gillis Ellen E. , Musall Jacqueline , Sullivan Jennifer C.
Published: April 2020

Background

Necrotic cell death is a hallmark of hypertension. We previously published that male SHR have greater renal necrosis compared to age matched female SHR. Consistent with this, inhibition of necrosis attenuated maturation induced increases in blood pressure (BP) in male, but not female SHR. However, the mechanisms underlying sex differences in renal necrotic cell death in SHR are unknown. The goal of the current study is to test the hypothesis that maturation in male SHR drives increases in necrosis and upregulation of key proteins mediating necrotic cell death, receptor‐interacting protein kinase 3 (RIP‐3) and high mobility group box 1 (HMGB‐1).

Methods

Male and female SHR were euthanized at 6 (pre‐hypertensive, sexually immature; n=6) or 15 weeks of age (hypertensive, sexually mature; n=6). The right kidney was isolated and processed for flow cytometric analysis of cell death using 7AAD. The left kidney was isolated for Western blot analysis of total RIP‐3, phosphorylated RIP‐3 and HMGB‐1. Protein expression was normalized to β‐actin. All data are expressed as mean ± standard error.

Results

Renal necrosis was comparable between 6 week old male and female SHR (expressed as % total kidney cells: 2.4±0.2 vs. 1.8±0.1, respectively) and increased with age only in males (5.4±0.2 vs. 1.7±0.3, respectively; Psex<0.0001; Page<0.0001; Pinteraction<0.0001). To gain insight into what mediates sex differences in necrosis in 15 week old SHR, protein expression of RIP‐3 and HMGB‐1 were determined in 6 vs. 15 week old male SHR and in 15 week old male and female SHR. Protein expression of RIP‐3 (1±0.1 vs. 0.7±0.1; P=0.008; n=5–6), p‐RIP‐3 (1±0.1 vs. 0.7±0.03; P=0.008; n=5–6) and HMGB‐1 (1±0.06 vs. 0.7±0.1; P=0.005; n=5–6) were all greater in 15 week old vs 6 week old male SHR. 15 week old male SHR also had greater protein expression of RIP‐3 (1±0.1 vs. 0.4±0.1; P=0.003; n=6), p‐RIP‐3 (1±0.2 vs. 0.4±0.1; P=0.02; n=6) and HMGB‐1 (1±0.2 vs. 0.6±0.1; P=0.02; n=6) compared to age matched females.

Conclusion

In contrast to adult, hypertensive SHR, sex differences in renal necrosis were absent in young, sexually immature pre‐hypertensive SHR. Consistent with greater renal necrosis, key proteins of necrotic cell death machinery were also upregulated in hypertensive male SHR compared to either age matched hypertensive females or young, pre‐hypertensive male SHR. Future studies are needed to test the hypothesis that RIP‐3 activation and the subsequent HMGB1 release potentially underlie greater renal necrosis in hypertensive male SHR compared to age matched females.
Spatiotemporal Patterning of ABC Multidrug‐Resistance Transporters During Germ Layer Specification
Authors: Schrankel Catherine S. , Vyas Himanshu , Espinoza Jose A. , Hamdoun Amro
Published: April 2020

Xenobiotic transporters are important for the protection of differentiated somatic cells, yet less is known about the role of these proteins in rapidly changing embryonic cells. A surprisingly large number of ABC transporters (80%) are expressed from fertilized egg to gastrulation, but their spatial and temporal patterns of expression during early embryogenesis remain incompletely understood. Here I report a pattern of ABC transporter expression that emerges during the early differentiation of mesoderm, endoderm, and ectoderm of the sea urchin. These included orthologs of six major xenobiotic transporters, including ABCB1, ABCB4, ABCC1, ABCC4, ABCC5, and ABCG2. In‐situ hybridization (ISH) revealed that transporters followed one of three patterns of early developmental expression: The first, seen with protective and homeostatic genes such as ABCB1 and ABCC1, was the early and ubiquitous expression across all three germ layers, and later enrichment in ectoderm‐derived sensory and feeding structures of the larva. The second, seen with the transporters ABCC4 and ‐C5 that are known to transport signal molecules, is a spatially restricted expression domain within a ring of mesodermal cells of the early blastula; ABCC4 then becomes restricted to primordial germ cells (PGCs) and their embryonic niche during gastrulation. Third, ABCB4 and ABCG2 show expression only in endoderm fated cells, particularly in the hindgut (presumptive intestines) at the onset of gut development; both genes eventually expand to the midgut region (presumptive stomach). To further refine the ISH map, I used fluorescent substrates to assay the functional outputs of transporter mRNA localization. ABCC1 and ‐C4 specific substrates are strongly effluxed out of the ring of mesodermal cells. This overlaps with robust ABCC5 transport activity in blastulae, suggesting a potential ABCC‐mediated signaling center in early mesoderm, where the PGCs are born. In the endoderm, ABCB4 and ABCG2‐specific efflux activity begins several days before the larva feeds. Collectively, this work is the first systems‐level description of the emergence of xenobiotic transporter expression patterns across the germ layers during embryogenesis. The resulting map of transport reveals unanticipated developmental functions for these genes and provides a framework for understanding the genetic regulatory controls of transporter expression. This will have additional impact on predicting and mitigating the effects of xenobiotic exposures during vulnerable windows of early development.

Support or Funding Information

Supported by NIH R01ES027921 and R01ES030318 to AH, and NIH F32ES029843 to CSS.
Press Pulse Therapy in MMTV‐PyMT Murine Breast Cancer
Authors: Poff Angela , Moss Sara , Soliven Maricel , Noboa Karina , D’Agostino Dominic
Published: April 2020

Background

Tumors can be viewed as populations of cells which may succumb to eradication principles observed in nature. For example, population reduction or eradication occurs when populations are exposed to simultaneous press and pulse pressures. Press disturbances are pressures which produce chronic stress in populations, while pulse disturbances produce acute stress. We recently proposed a novel Press‐Pulse therapeutic strategy for the metabolic management of cancer that is based on these principles (
https://nutritionandmetabolism.biomedcentral.com/articles/10.1186/s12986‐017‐0178‐2). We hypothesize that specific press‐pulse protocols will be effective in managing even aggressive cancers, while allowing for the use of low dose and low‐ or non‐toxic metabolic‐targeted therapies.

Methods

In this project, we are testing the press disturbances of the ketogenic diet and ketone supplementation, and the pulse disturbances of 2‐deoxyglucose (glycolytic inhibitor), sodium phenylbutyrate (glutamine targeting agent), hyperbaric oxygen therapy, and pharmacological ascorbic acid, in the MMTV‐PyMT mouse model of breast cancer.

Preliminary Results

To date, we have tested the proposed metabolic treatments as monotherapies and combination therapies. Tumor growth rate and survival time were largely unaltered by the treatments tested. Ketogenic diet and sodium phenylbutyrate were the only treatments to affect survival time as a monotherapy. Ketogenic diet treated mice lived 8.7% longer than standard diet fed mice (p<0.05). Sodium phenylbutyrate treated mice had a decrease in survival of 8.1% from the standard diet fed mice (p<.05). Data analyses pending for the effects of combination therapies.

Conclusions

We are not yet able to determine if Press‐Pulse targeting is effective in this model system as we have only tested and analyzed monotherapies. Ketogenic diet therapy appears to elicit a minor therapeutic effect as a stand‐alone, but the other metabolic‐targeted treatments utilized were not effective as monotherapies. Studies to test combinations of these therapies have been completed, and the analyses of the data is ongoing.

Support or Funding Information

Donner Foundation, Florida High Tech Corridor
SPARC: Time Series Transcriptomic Analysis of the Nucleus of the Solitary Tract During the Development of Hypertension
Authors: Moss Alison , Kuttippurathu Lakshmi , Achanta Sirisha , Schwaber James S. , Vadigepalli Rajanikanth
Published: April 2020

Essential hypertension affects millions of people and is known have a neurogenic component where regions in the brain and brainstem involved in the autonomic nervous system govern the regulation of blood pressure set point. While some work has been done to profile gene expression levels in the nucleus of the solitary tract (NTS) in males, to date no work has specifically looked at how gene expression changes in the NTS drive hypertension development in females. We have conducted a time series mRNA profiling analysis in female spontaneously hypertensive rats (SHR) in comparison to wild type Wistar Kyoto (WKY) allowing us to compare and contrast the results with previously obtained data in males. Rats were sacrificed over the development of and during chronic hypertension at 8, 12, 16, and 24 weeks of age and brainstems were removed and stored in OCT. Brainstems were sectioned on a cryostat and the NTS was extracted using a micropunch. Total RNA was extracted and samples were submitted for RNAseq. We used a data analysis approach focusing on comparative pattern counts (COMPACT) to exhaustively identify the dominant and subtle differential expression patterns (Kuttippurathu et al., BMC Genomics, 2016). Over 2000 genes that show both strain and age dependent differential expression were examined further to interrogate differences in expression patterns between SHR and WKY. Broad analysis reveals that genes involved in the adaptive immune response are differentially regulated in the SHR compared to WKY. Using COMPACT to examine specific patterns, we found that expression levels of many genes involved in transport and secretion of catecholamines do not change over time in WKY but are specifically downregulated in SHR before the onset of hypertension at 8 weeks of age. Interestingly, pathways such as arachidonic acid metabolism that have been implicated in hypertension development in males did not show significant differences between SHR and WKY in females. These results provide valuable insight into the regulatory networks governing hypertension development in females and allows us to compare and contrast this to what is known in males.

Support or Funding Information

Supported by the NIH Common Fund SPARC Program award OT2 OD023848 and NIH NHLBI Multiscale Modeling award U01 HL133360.
Modest reductions in face skin temperature do not improve tolerance to a simulated hemorrhagic challenge in exercise heat stressed individuals.
Authors: Tourula Erica , Lenzini Miramani , Rhodes Addison , Hetz Sarah E. , Pearson James
Published: April 2020

Face cooling is associated with autonomic and cardiovascular alterations that accompany an increased blood pressure. Following an exercise heat stress, even minor increases in heart rate and vascular resistance may permit an improved blood pressure control during a simulated hemorrhagic challenge (Lower Body Negative Pressure, LBNP) and improve tolerance. Our aim was to examine whether modest reductions in face skin temperature would improve LBNP tolerance in exercise heat stressed individuals. Ten healthy subjects (Age: 29 ± 13 yrs; Ht: 177 ± 9 cm; Wt: 72.4 ± 8.0 kg) completed two counterbalanced trials (Trial A and Trial B) cycling at 63 ± 6% of their predetermined VO2Max whilst wearing a warm water perfused suit and face mask until core temperature had increased 1.5°C. Participants then underwent LBNP to pre syncope. LBNP tolerance was quantified as cumulative stress index (CSI; mmHg*min). Face skin temperature represents the average of forehead, cheek and chin skin temperatures. Face skin temperature was maintained at exercise heat stress values throughout LBNP in Trial A but was reduced via facial fanning in Trial B after 1 minute of LBNP. Arterial blood pressure (MAP, Penaz method), heart rate (HR, ECG), stroke volume (SV, Pulse Contour Analysis) were continuously measured and total peripheral resistance (TPR) was calculated. Following exercise heat stress, core (38.6 ± 0.2 °C), mean skin (38.2 ± 0.6 °C) and face skin (36.4 ± 0.5 °C) temperatures were elevated relative to baseline (all P < 0.05) and not different between trials (all P > 0.05). Face skin temperature remained elevated throughout LBNP in Trial A (36.1 ± 0.5 °C) but was reduced in Trial B (31.3 ± 1.6°C, P = 0.0001 between trials). Following exercise and prior to LBNP, HR was increased (63 ± 8 to 121 ±15 BPM) while MAP (86 ± 7 to 70 ± 6 mmHg) and TPR (12.5 ± 2.0 to 7.7 ± 1.6 mmHg/L/min) were similarly lowered relative to baseline in both trials (all P < 0.05). Mean arterial pressure was similarly reduced at pre syncope in both trials (54 ± 11 and 57 ± 5 mmHg; P < 0.0001 relative to prior to LBNP). TPR and HR increased during LBNP in both trials relative to baseline (both P < 0.05) but were not different between trials 2 minutes after the onset of LBNP (TPR: 7.8 ± 1.7 vs. 7.8 ± 1.8 mmHg/L/min and HR: 131 ± 17 vs. 137 ± 22 BPM, main effect of trial both P > 0.05). CSI was not different between trials (Trial A: 332 ± 176 vs. Trial B: 393 ± 261 mmHg*min; P = 0.482). LBNP tolerance was not improved by face skin cooling in exercise heat stressed individuals. These results suggest that modest reductions in face skin temperature are not accompanied by an improved blood pressure control during a simulated hemorrhagic challenge in exercise heat stressed individuals.
Using Diffusion Tensor Imaging to Find Optimal Stimulation Targets in Obsessive‐Compulsive Disorder Treatment
Authors: Finger Laura , Thompson John A. , Davis Rachel
Published: April 2020

Obsessive‐compulsive disorder (OCD) is a common psychiatric condition that can be severe and treatment‐resistant in some patients. Treatment‐resistant OCD (tr‐OCD) can now be managed with deep brain stimulation (DBS), an invasive neuromodulation therapy. However, due to the small population of tr‐OCD patients being treated with DBS, there is little consensus on the best target for stimulation. Past research targeted the anterior limb of the internal capsule (ALIC), the subthalamic nucleus, and the nucleus accumbens (NAc). The purpose of this study is to use diffusion tensor imaging (DTI) to refine the optimal target for DBS in tr‐OCD patients, when targeting the ALIC, specifically studying the NAc (COMIRB #14‐0554). We performed tractography analysis on fiber tracts going through the volume of tissue activation (VTA), using each patient’s DTI. We then related the change in OCD symptom severity on the Yale‐Brown Obsessive‐Compulsive scale (Y‐BOCS) from pre‐surgery, up to one‐year post‐surgery. Preliminary results (n=1) show there was a 6.45% increase in OCD symptom severity, and that was correlated with putatively activated tracts within the VTA, or area of stimulation. Comparing two time points we found that there was change in the diffusion coefficients of 1.16% for FA and 2.62% for MD on the left, and 28.96% for FA and 3.07% for MD on the right. Changes in these DTI coefficients between the two‐time points suggest that modification in stimulation parameters that led to clinical benefit was associated with activation of putatively distinct fiber populations and thus different neural pathways.
Congenital Heart Defects in the Context of Typical Development and Circulation
Authors: Cassidy Keely
Published: April 2020

Solid foundational knowledge of the normal embryological development of the heart and great vessels is essential for understanding the etiology of various congenital heart defects (CHDs). As an increasingly diagnosed category of pathologies and overall the leading cause of neonatal morbidity, CHDs are often well tolerated when the developing human is in utero. Clinical sequelae, the signs and symptoms of patients with CHDs, become apparent after birth when the fetus no longer has access to a placenta and must use solely its own anatomy to provide and circulate the appropriate blood gases. CHDs can be divided into two major categories: critical, those which are life‐threatening and require surgery in infancy, and non‐critical, those which are not immediately life‐threatening and do not necessarily require surgery immediately or at all. One fundamental piece of information for the diagnosis of any CHD is whether or not the individual is cyanotic, and furthermore which specific tissues are subjected to this oxygen deficiency. Taussig’s classification of congenital heart defects includes those with no shunting of blood, those with a left to right shunt and acyanosis, and those with a right to left shunt and subsequent cyanosis. In the typical fetus, blood enters the right atrium of the heart at fairly high pressure. Some of this blood is entering via the inferior vena cava, which in the fetus contains some highly oxygenated blood via the ductus venosus and some blood of a low oxygenation rate that flowed through the developing liver tissues. Due to higher pressures on the right side of the fetal heart than the left, most blood passes through the foramen ovale and into the left atrium. This is a normal right to left shunt only in the fetus. When the fetus is born and air is introduced to the respiratory system the pressures change. Absent placental circulation leads to a decrease in pressure on the right side of the heart and increased pulmonary blood flow leads to an increase in pressure on the left side of the heart, specifically between the atria. This ex utero pressure differential closes the foramen ovale to form a fossa ovalis and no patent communication between the atria. The normal fetal right to left shunt should no longer be present. Since pulmonary vascular resistance is now lower than systemic vascular resistance, the blood flow in the ductus arteriosus between the pulmonary trunk and arch of the aorta reverses, the shunt constricts, and then forms into the ligamentum arteriosum. When these processes or those of cardiac septation occur incorrectly then the result may be a CHD. Proper identification of the pathological anatomy and etiology is relevant for understanding both normal and abnormal developmental processes in cardiac and great vessel morphology.
The effects of eating a high fat diet on sensitivity of female rats to ethanol
Authors: Galindo Kayla I. , Castillon Alex Serna , Serafine Katherine M.
Published: April 2020

Eating a diet that is high in fat or sugar can impact dopaminergic systems, the same pathways targeted by drugs of abuse. Rats eating a high fat laboratory chow are more sensitive to the behavioral effects of drugs of abuse, including cocaine and methamphetamine. However, it is not known if eating a high fat diet also enhances sensitivity of rats to alcohol. To address this gap in knowledge two experiments were conducted. In Experiment 1, 24 female Sprague‐Dawley rats eating either standard (18% kcal from fat) or high fat (60% kcal from fat) chow for 5 weeks were trained to drink ethanol solution. Training consisted of a daily 4‐hour session for 28 days. Following training, during the test phase, two bottles were presented (one containing 8% w/v ethanol and one containing water) while animals had continuous access to food. In Experiment 2, the development of locomotor sensitization to once weekly cumulative doses of 15% ethanol (0.5–2.0 g/kg; i.p.) was studied in 16 female Sprague Dawley rats eating the same types of chow used in Experiment 1 (high fat or standard). In Experiment 1, rats eating high fat chow consumed more milliliters of ethanol on average daily than rats eating standard chow; however, when accounting for body weight, this difference was no longer apparent. Experiment 2 is ongoing; however, based on previous work with psychomotor stimulants, it is anticipated that rats eating high fat chow will be more sensitive to ethanol‐induced locomotor sensitization. These results provide additional information related to the growing obesity epidemic and its potential relationship substance use disorder. Future directions will investigate the impact of diet on sensitivity of male rats to ethanol, in order to examine potential sex differences.

Support or Funding Information

Disclosures: The authors have no conflicts to declare. The present study was supported by the National Institute on Drug Abuse (R25‐DA033613).
Neuropeptide Y Inputs to Neurons in the Dorsomedial Hypothalamus Tonically Inhibit Brown Adipose Tissue (BAT) Sympathetic Nerve Activity (SNA) via NPY Y1 Receptors
Authors: Madden Christopher J.
Published: April 2020

Hypothalamic Neuropeptide Y (NPY) has been implicated in metabolic homeostasis. For example, NPY acts in the paraventricular nucleus of the hypothalamus to decrease BAT SNA. NPY neurons within the dorsomedial hypothalamus (DMH) have also been implicated in energy balance, however the role of NPY inputs to BAT regulatory neurons in the DMH has not been assessed. The present study tested the hypothesis that neurons in the DMH receive a NPY‐containing input that contributes to the regulation of BAT SNA via activation of NPY Y1 receptors. First, administration of a retrograde tracer (cholera toxin B) into the raphe pallidus area (RPa) and immunohistochemistry for NPY demonstrated that CtB‐labeled (RPa‐projecting) DMH neurons receive close appositions from NPY‐positive fibers. In addition, bilateral nanoninjections of the NPY Y1 receptor antagonist BIBO3304 (2mM, 60nl) into the DMH increased BAT SNA +424±110% baseline, TBAT (+2.2±0.5 °C) and expired CO2 (+1.2±0.1%). In conclusion, NPY inputs to neurons in the DMH tonically inhibit BAT SNA and BAT thermogenesis via activation of NPY Y1 receptors.

Support or Funding Information

NIH DK112198