bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic acid induces a metabolic switch in SH‐SY5Y cells from glycolysis to oxidative phosphorylation
Authors: Xun Zhiyin , Lee Do-Yup , Lim James , Canaria Christie , Barnebey Adam , Yanonne Steve , Bowen Ben , Northen Trent , McMurray Cynthia
Published: June 2021

Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The basis for its therapeutic efficacy is unknown, however, mitochondria (MT) have been implicated as key effectors in RA‐mediated differentiation process. Here we use the SH‐SY5Y human neuroblastoma cell line as a model to examine the contribution of MT to the physiological effects of RA treatment. We find that RA induces a dramatic increase in the energy metabolism of SH‐SY5Y cells, and shifts the dependence on energy production from glycolysis to oxidative phosphorylation. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment elevates the production of intracellular pyruvate as fuel for mitochondrial consumption, and significantly enhances the mitochondrial respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state.
S. pyogenes is reliant on salvage of host pyridine precursors for NAD synthesis: implications for pathogenesis and antibacterial intervention
Authors: SORCI LEONARDO , Blaby Ian , de Crécy-Lagard Valérie , Osterman Andrei
Published: June 2021

The essential coenzyme nicotinamide adenine dinucleotide (NAD) plays important roles in metabolic reactions and cell regulation in all organisms. In bacteria, NAD metabolic subsystem has been recently investigated as a potential source of novel antibacterial targets. Our comparative genome analysis across nearly a thousand of bacterial species revealed that all Streptococcus pyogenes and pneumoniae strains – but not other Streptococci – have a disrupted de novo NAD biosynthesis making these bacterial pathogens completely reliant on the salvage of host pyridine precursors. Rather puzzlingly, while the two genes responsible for the conversion of L‐aspartate to quinolinate (nadB and nadA) are missing, the final gene of de novo pathway (nadC), encoding for quinolinate phosphoribosyltransferase, is present. Growth experiments confirmed that S. pyogenes is not able to grow on a chemically defined medium, while significant growth was observed when adding nicotinic acid, nicotinamide, and quinolinate. Biochemical characterization of NadC demostrated its activity in vitro, while the inability of our nadC knockout mutant to grow on quinolinate as opposed to wild type strain directly implicated NadC in quinolinate auxotrophy (in vivo). We propose that quinolinate auxotrophy might be linked with the pathogenesis of streptococcal diseases.
Autoinhibition and Salt Sensing are Linked in the WNK1 Kinase
Authors: Moon Thomas Matthew , Correa Fernando , Kinch Lisa , Cobb Melanie , Gardner Kevin , Goldsmith Elizabeth
Published: June 2021

This research focuses on a 230kDa serine/threonine protein kinase known as WNK1 (With No lysine {K}), isoforms of which have been associated with a monogenic form of hypertension. Structural analysis of the autoinhibitory (AI) domain from WNK1 by NMR indicates that the RFXV binding site from the PASK/FRAY homology 2 (PF2) domain is conserved in the WNK1 AI domain. Titration data shows that incubation of RFXV peptides display extensive chemical shift perturbation as assessed by 1H, 15N‐HSQC. Size exclusion chromatography of the tethered construct of the AI and kinase domains show substantial conformational changes when dialyzed from high to low [NaCl]. Differential scanning fluorimetry conducted on the kinase domain shows that the domain undergoes a substantial increase in stabilization as the concentration of salt is increased. Further analysis of this phenomena points toward evidence of anion sensing. To corroborate this, the crystal structure of the activated WNK1 kinase domain was solved in low salt conditions. The kinase displays disorder of many key structural elements such as αC which is unsurprising given the DSF data. The coupling of the information gathered points to a mechanism of salt sensing and self‐contained signaling control in the WNK1 kinase.
Temporal Control of Nuclear Envelope Assembly by Phosphorylation of Lamin B Receptor
Authors: Chen Rey-Huei , Tseng Li-Chuan
Published: June 2021

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Herein, we use time‐lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin‐binding ability of LBR together with Aurora B activity contribute to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.
Proposal for international nutrition criteria for school lunches for children aged 5–18
Authors: van Elk Kathelijn Neeltje , Eilander Ans , Roodenburg Annet JC
Published: June 2021

The nutritional status of children aged 5–18 is often suboptimal and across the globe, school lunches have an important contribution to children's daily diet. Therefore, providing a nutritious school lunch can likely have a significant impact on children's health. However, for the development of nutritious meals, international science‐based nutrition criteria are currently lacking for both primary and secondary schools.

Aim

To develop international criteria for the nutritional composition of school lunches.

Methods

FAO/WHO recommendations for total daily diets were translated into nutrient criteria for school lunches. Specific criteria were developed for total energy, proteins, total fat, PUFA, SAFA, trans fatty acids, carbohydrates, added sugars, and sodium, as well as for the critical micronutrients folate, zinc, iron, iodine, vitamin A, and vitamin D, which are often suboptimal in children's diets.

Results

Criteria were defined so that a lunch meal delivers 30% of the daily energy intake and 50% of the micronutrient recommendations for children aged 5–18. Tolerable upper intake levels were taken into account.

Conclusion

While criteria were developed globally, implementation will differ between countries based on differences in dietary habits, affordability and local food composition. Also, feasibility of developing recipes based on the criteria needs to be further explored.
Higher Bifidobacteria counts in male offspring exposed to supplemental levels of vitamin D in utero and during suckling in IBD‐prone mice
Authors: Glenn Andrea J. , Li Sophia , Fielding Kristina A , Chen Jianmin C , Comelli Elena M , Ward Wendy E
Published: June 2021

Vitamin D deficiency has been linked to an increased risk of inflammatory bowel disease (IBD), and microbial dysbiosis has been implicated in IBD. Our objective was to determine if exposure to supplemental levels of vitamin D can favourably modulate microbiota composition pre‐inflammation in the male interleukin‐10 knockout (IL‐10 KO) mouse that spontaneously develops intestinal inflammation at 6–8 weeks of age. The mice were randomized to a diet containing 25 IU (low group) or 5000 IU (high group) of vitamin D/kg of diet in utero and during suckling, and fecal samples were collected at 5 weeks of age. Fecal microbiota composition was determined by qPCR. Mice in the supplemental group had higher (p = 0.01) counts of Bifidobacteria than mice in the low group. Total bacteria, Bacteroides, Clostridium leptum, Clostridium coccoides and Escherichia coli counts were unaffected. Bifidobacteria have been found to sustain intestinal homeostasis and inhibit Th1‐driven inflammation and may favourably alter gut microbial composition to a more health‐promoting phenotype. Further investigation is needed to determine if this results in protection against developing intestinal inflammation.
Biomarker in human breast cancer cells
Authors: Schmidt Jane , Schmidt Jane
Published: June 2021

Multiplex measurement of biomarkers in human breast cancer cell culture supernates

Jane Schmidt, Ph.D, Michael Hall, Richard Fuerstenberg. R&D Systems, Inc. Minneapolis, Minn.

The mixture of biomarkers that is produced in the tumor microenvironment has an important role in cancer pathogenesis. For example, cytokines that are released in response to infection, inflammation and immunity can function to either promote or inhibit tumor development and progression. The multiplex immunoassay has become an efficient tool both for rapid screening and more in‐depth analysis of cancer cell supernates, while requiring only a single, small sample volume. In this study, the VersaMAP™ Development System was used to rapidly and simultaneously screen for human biomarkers in supernates from six human breast cancer cell lines. Fifteen positive biomarkers were identified and biomarker profiles were established for each of the cell lines. The VersaMAP Development System, developed by R&D Systems,Inc., is a customized assay system designed for the simultaneous measurement of multiple biomarkers from a single 50 μL sample. It offers the ability to measure up to 10 biomarkers chosen by the user from a continuously expanding library of 39 available analytes. The assay is available in a custom premixed form, giving the user total flexibility to choose their multiplex panel. The assay system is a bead‐based multiplex immunoassay designed for use with a Luminex® or Bio‐Rad® Bio‐Plex® analyzer.
Influence of physical exercise on cardiac structure and function of spontaneously hypertensive rats
Authors: Pagan Luana Urbano , Damatto Ricardo Luis , Cezar Marcelo Diarcadia Mariano , Gimenes Rodrigo , de Campos Dijon Henrique Salomé , Lima Aline Regina Ruiz , Bonomo Camila , Guizoni Daniele Mendes , Okoshi Marina Politi , Okoshi Katashi
Published: June 2021

The aim of this study was to evaluate the influence of physical exercise (PE) on cardiac structure and function of spontaneously hypertensive rats (SHR) before clinical evidence of heart failure (HF).

Methods

16 month old SHR were divided into two groups: sedentary (SED, n=14) and exercised (EX, n=14). Physical training was performed 5 times/week for 16 weeks in a treadmill. Arterial pressure (AP) was measured by tail‐cuff method. Echocardiogram (Echo) was performed to evaluate in vivo cardiac structures and left ventricular (LV) function. In vitro myocardial function was analyzed in LV papillary muscle preparations. Statistics: Studentˈs t test (p<0.05).

Results

AP did not differ between groups (SED: 212±22; EX: 225±20 mmHg). Echo did not show differences in cardiac structures or LV systolic function. Late diastolic mitral annular velocity (Tissue Doppler) was lower in EX group (3.3±0.7 vs 4.2±1.1 cm/s). The in vitro study showed increased contractile function in EX group (developed tension: 7.6±1.8 vs 6.2±1.1 g/mm2; +dT/dt: 67±18 vs 53±10 g/mm2/s).

Conclusion

PE started before clinical evidence of HF is beneficial on LV diastolic and myocardial contractile functions in SHR. Support: CAPES, CNPq.
The prostate in DMBA administration – Morphometric evaluation
Authors: Barandas Gustavo Teixeira , Pereira Filipe , Costa Eduardo , Farinha Rodrigo , Cabrita Silverio
Published: June 2021

7,12‐Dimethylbenz(a)antracene (DMBA) is a xenobiotic found in cigarette smoke and exhaust gases of combustion engines. It is an aromatic polycyclic hydrocarbon, metabolized by P450 enzymes producing reactive metabolites which form covalent adducts with DNA and the formation within DNA of depurinated abasic sites. The aim of this study is the evaluation of DMBA action on prostate by morphometric and histopathological analysis.

Ten Wistar male rats, 8 weeks old, were submitted to DMBA administration, 20 mg/Kg/day, three times a week. The control group was kept without manipulation. All the animals were killed after two weeks, and a complete necropsy was performed. The prostate fragments were stained by H&E and the pieces lined by epithelia were measured using the software Image J. The frequencies distribution of the control group were achieved and considered as a reference value, for test group comparison. No statistic significance was found. The histology evaluation of the prostate of test group show no morphologic lesions.

These results allow us to suggest that the acute intoxication with DMBA, as in this model, has no morphologic translation.
Chronic corticosterone treatment does not elicit depression‐like behavior in adolescent rats
Authors: Meyer Teresa Jo , Smith Stephanie R. , Middlemas David S.
Published: June 2021

Major Depressive Disorder (MDD) is a debilitating emotional disorder that affects people of all ages. The etiology of MDD is unknown and the mechanisms of action of antidepressant drugs are not well understood. MDD affects adolescents differently than adults. Furthermore, adolescents respond differently to antidepressant drugs. Recently, an animal model of MDD involving repeated corticosterone (CORT) injections in adult rats has been reported. This model elicits not only a behavioral change congruent with MDD, but also shows a depressed rate of hippocampal neurogenesis, a potential marker of MDD. However, to our knowledge, no such model of MDD exists for adolescents. The purpose of our study was to establish a model of MDD in adolescent rats. Male Sprague‐Dawley rat pups were injected with 30 mg/kg CORT or vehicle for 29 days, followed by behavioral testing. The elevated plus maze, used to detect anxiety‐like behavior, and the open field test, used to detect changes in locomotion, found no differences between rat pups treated with CORT or vehicle. The forced swim test, used to detect depression‐like behavior, also found no differences in behaviors between CORT treated and vehicle treated rats. Our results indicate that exogenous CORT treatment does not induce depression‐like behavior in adolescent rats and would not serve as a sufficient adolescent model of MDD. Research support provided by the ATSU Graduate Program.
Highly palatable food normalizes the HPA axis activity and modulates anxiety‐like behaviors in rats that experienced neonatal maternal separation
Authors: Jahng Jeong Won , Kim Jin Young , Park Eunyoung , Lee Joo Young , Lee Jong-Ho
Published: June 2021

In this study, we have examined the effect of highly palatable food (HPF) on the psycho‐emotional behaviors of rats that experienced neonatal maternal separation. Male SD pups were separated from dam for 3 h daily during PND 2–14 (MS) or left undisturbed (NH). Half of NH and MS pups had free choices of cookie (HPF) and chow from PND 28, the rest half chow only, and were subjected to behavioral tests during young adulthood. Plasma corticosterone levels were analyzed by radioimmunoassay, and tissue contents of ΔFosB and pCREB in the nucleus accumbens by western blot. Caudal grooming was reduced in MS‐control and NH‐HPF compared with NH‐control. HPF suppressed defecation activity in MS, but not in NH. HPF increased anxiety‐like behaviors during elevated plus maze test and depression‐like behaviors during swim test in NH, and these behaviors were not further affected by HPF in MS. Stress‐induced corticosterone increase was blunted by MS experience, and HPF appeared to normalize it. pCREB in the nucleus accumbens was increased, and ΔFosB decreased, by MS experience, and HPF normalized them. Results demonstrate that long‐term HPF may normalize the HPA axis activity and modulate anxiety‐like behaviors in MS rats, likely in relation with neural plasticity in the nucleus accumbens.

Supported by the 21st Century Frontier Research Program (2009K001269) and the NRF (2010‐0003642) funded by MOEST
Synthesis of Some New Iminoethyl‐benzopyran‐2‐ones, Benzylideneimines and Iminoethyl‐phenoles and their antimicirobial aktivity
Authors: Hoti Ramiz , Krasniqi Vlora , Mulliqi-Osmani Gjyle , Ismaili Hamit , Thaçi Veprim , Kolshi Valmira
Published: June 2021

Novel benzylideneimines, iminomethyl‐phenoles and iminoethyl‐benzopyran‐ 2‐ones were synthesized by condensation reactions of benzaldehyde analogues and acetyl‐benzopyran‐2‐one and heterocyclic amines. Condensation of 3‐acetylbenzopyran‐2‐one 1a and 2‐aminopyridines and 2‐aminopyrimidine 2(a‐c) resulted by synthesis of corresponding derivates of [2‐ylimino)‐ethyl]‐benzopyran‐2‐one 3(a‐c). By catalytic condensation of benzaldehyde and their analogues 1(b‐d) and 2‐amino‐5‐bromothiazole, 2‐aminobenzothiazoles and 2‐aminopyrimidines 2(d‐h), novel benzylideneimine derivatives 3(d‐h) were syntesized. The antibacterial activity of products 3(a‐h) against S. aureus, E. coli dhe Clebsiella were investigated and reported. Results of inhibition zones were indicated for considerable activity of these compounds against these microorganisms. Compounds 3f and 3c were more active against Staphylococcus aureus. Compounds 3c and 3g exhibited the strongest activity against Clebsiella, whereas 3d was more active against Escherichia coli. In general there was a driect positive correlation between increasing concentration of the compound and anitbacterial activity.
Isolation, purification, structural characterization and functional elucidation of novel antihyperglycemic compound from the bark of Pterocarpus santalinus
Authors: kondeti vinay kumar , Badri Kameswara Rao , Maddirela Dilip Rajasekhar , chippada Apparao
Published: June 2021

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. In accordance with the recommendations of the WHO expert committee (2002) on diabetes mellitus, an investigation of hypoglycemic agents of plant origin used in traditional medicine seems important. Additionally, in recent years, there is an increasing demand by patients to use the natural products with antidiabetic activity. Earlier studies from our laboratory have shown that the ethanolic extract of the bark of P. santalinus (PS) produced a significant antihyperglycemic activity. In the current study we have isolated, purified and characterized the active principle with antidiabetic activity from the bark of PS by bioassay guided fractionation. The mass of the compound was found to be 250 by mass spectroscopy. On the basis of the data obtained from 1H NMR, 13C NMR and IR spectra, the chemical structure of the active principle has been derived, and it is identified as derivative of butanoic acid. Further this novel active principle showed maximum antihyperglycemic activity (71% fall in blood glucose) when administered at a concentration of 100 mg/kg body weight in streptozotocin induced diabetic rats. Our results conclude that butanoic acid derivative present in PS is a novel and effective antihyperglycemic agent.
Transgenic humanized mice carrying the human CYP1A1‐luc promoter show augmented transcriptional activation and increased susceptibility to lung damage by hyperoxia
Authors: Moorthy Bhagavatula , Wang Lihua , Couroucli Xanthi I , Jiang Weiwu
Published: June 2021

Supplemental oxygen administration is administered to infants having pulmonary insufficiency. In this study, we tested the hypothesis that hyperoxia induces human cytochrome P450 (CYP)1A1 enzyme by transcriptional activation of the CYP1A1 promoter in vivo, and humanized mice expressing the human CYP1A1 promoter would be more susceptible to hyperoxic lung injury than those on a wild type (WT) background. Adult CD‐1 (8 week‐old) mice, transgenic mice carrying the human CYP1A1 promoter and the luciferase (luc) reporter gene on a WT or Cyp1a1‐null background (humanized) were maintained in room air or exposed to hyperoxia ( 24–72 h). Hyperoxia exposure of mice expressing hCYP1A1 promoter for 24 and 48 h resulted in 3‐ and 1.9‐fold increases, respectively in luc signal intensities, compared to room air controls, and humanized mice showed a greater extent of induction, suggesting that CYP1A1 metabolizes endogenous Ah receptor ligands. The hCYP1A1‐luc mice showed the greater susceptibility to lung injury than WT mice, and this was potentiated in humanized mice, suggesting that endogenous Cyp1a1 enzyme is protective against injury. (Supported by grants RO1HL070921 and RO1HL087174.)
Role of nNOS in the progression of systemic inflammatory response induced by lipopolysaccharide
Authors: BAIG MIRZA SAQIB , Bakhshi Farnaz , Ye Richard D , Gao Xiaopei , Malik Asrar B , Minshall Richard D , Ganter Ben , Bonini Marcelo
Published: June 2021

Nitric oxide synthases (nNOS, eNOS & iNOS) are a family of enzymes that catalyze the production of nitric oxide (NO) from L‐arginine. NO is an important cellular signaling molecule, having a vital role in many biological processes. The roles of eNOS (endothelial NOS) and iNOS (inducible NOS) in mediating inflammatory processes have been appreciated but little is available about the role of nNOS (neuronal NOS) especially in what pertains to the initiation of the inflammatory process. Our recent findings suggest that nNOS‐KO mice are resistant to LPS induced inflammation. To gauge the molecular mechanism we have identified that inhibition of nNOS leads to over expression of SOCS1 and elevated SOCS1 binding to P65 subunit of NFkB within the nucleus. SOCS1 operating as a ubiquitin ligase, targets nuclear p65 to degradation. In addition, serine phosphorylation at various sites of the p65 subunit has been shown to be important in initiating transcription. We have also shown that decreased phosphorylation of p65(536) in nNOS‐ko cells after LPS stimulation results in transcriptional inactivation of p65 subunit of NfkB. Thus these molecular events provide terminated expression of NfkB inducible genes and LPS tolerance in nNOS‐ko mice. Taking together our recent studies pointing towards the important role played by nNOS in the initiation of inflammatory response and ultimately in lung injury, sepsis and mortality.
Inhibitory effects of caspase inhibitors on the activity of matrix metalloproteinase (MMP)‐2
Authors: de Castro Michele Mazzaron , Fuah Jason , Fan Xiaohu , Holt Andrew , Schulz Richard
Published: June 2021

Matrix metalloproteinase (MMP)‐2 plays a harmful role during the onset of ischemia and reperfusion (I/R) in the heart. Caspases are a group of cysteine‐dependent aspartate‐directed proteases which regulate cellular apoptosis. The role of caspase activity in I/R is controversial, but there are protective effects of caspase inhibitors which are independent of apoptosis. The protective actions of caspase or MMP inhibitors in ameliorating I/R injury had led us to hypothesize that caspase inhibitors also reduce MMP‐2 activity. In vitro degradation assay using human recombinant MMP‐2 and troponin I (TnI) as its substrate showed that caspase inhibitors (Z‐IE(OMe)TD(OMe)‐fmk, Ac‐DEVD‐CHO, Ac‐LEHD‐cmk, Z‐VAD‐ fmk, Ac‐YVAD‐cmk), in comparison to a bond fide MMP inhibitor, significantly inhibited MMP‐2‐mediated TnI degradation in a concentration‐dependent manner (10 μM: p<0.0003 and 30 μM: p<0.0001 vs. DMSO control). A quantitative kinetic assay using a fluorogenic MMP substrate revealed that these caspase inhibitors blocked MMP‐2 activity in a concentration‐dependent manner with similar IC50 (all values in μM range). Inhibition of MMP‐2 activity may be an additional pharmacological action which could contribute to the protective effects of caspase inhibitors seen in heart injury models. Canadian Institutes for Health Research, Heart & Stroke Foundation of Canada and Alberta Innovates – Health Solutions.
Genetic Variation of Phenylethanolamine‐N‐Methyltransferase at Position −182 Does not Influence the Catecholamine Response to Albuterol in Healthy Humans
Authors: Martinez Marina , Baker Sarah , Wheatley Courtney , Snyder Eric
Published: June 2021

We have previously demonstrated that an inhaled β2‐adrenergic agonist (albuterol) increases norepinephrine (NE) and decreases epinephrine (EPI) in humans. Phenylethanolamine N‐methyltransferase (PNMT) converts NE to EPI. We have shown that genetic variation of PNMT influences resting cardiovascular (CV) function and the catecholamine and CV response to exercise. An adenine (A) to guanine (G) substitution at position −182 in the promoter region of PNMT has been associated with differences in blood pressure and cardiac function. We sought to determine the influence of genetic variation of PNMT at position −182 on the catecholamine response to albuterol. Thirty‐one subjects were recruited and stratified according to PNMT genotype (n=6, 11, 14; age=29±8, 30±11, 24±6years; height=174±13,174±12,171±8cm; and weight=70±7, 76±16, 68±12kg, for AA, AG, and GG). We found that both genotype groups had an increase in NE, a drop in EPI, and a drop in EPI/NE at 60min following the administration of albuterol, but no differences were found according to genotype (baseline: NE=289±69, 310±77, 257±81pg/ml; EPI=53±18, 62±46, 59±39pg/ml; and EPI/NE=18±7, 20±14, 25±17: 60min: NE=368±98, 366±87, 295±97pg/ml; EPI=40±16, 30±11, 35±23pg/ml; and EPI/NE=10±7, 9±4, 14±12, for AA, AG, and GG). These data suggest that genetic variation of PNMT at position −182 does not affect the catecholamine response to albuterol.
Protease‐activated receptor 1 expressed in the human corpus cavernosum induces phosphorylation of endothelial nitric oxide synthase at threonine 495
Authors: Mantey Jane Ama , Motley-Johnson Evangeline
Published: June 2021

Protease‐activated receptor 1 (PAR1) can induce phosphorylation of endothelial nitric oxide synthase (eNOS) at site threonine 495 to inhibit production of nitric oxide. The objective of this project is to elucidate whether PAR1 is a potential therapeutic target for improving availability of nitric oxide in patients with erectile dysfunction and related vascular diseases. PAR1 has not been shown to be expressed in the penile endothelium, however. I hypothesize that PAR1 is expressed in the endothelium of the corpora cavernosa of the penis and, once activated, leads to significant phosphorylation of eNOS‐Thr495. Human corpus cavernosum endothelial cells (HCCECs) were lysed and probed for PAR1 expression using western blot and immunocytochemistry. The cells were then stimulated with thrombin or TFLLR, a PAR1 agonist, and phosphorylation of eNOS was examined by western blot and enzyme‐linked immunosorbent assay. Expression of PAR1 was detected in the HCCECs. Treatment with thrombin and TFLLR induced concentration and time dependent phosphorylation of eNOS‐Thr495. This study shows that PAR1 is expressed in the penile endothelium. Future studies include analyzing any differences in expression or functionality of PAR1 in HCCECs from males with erectile dysfunction.

This project was supported, in part, by National Institute of General Medical Sciences (NIGMS) Grant 5SC3GM086336‐02.
Conditional knockout of Deaf1 in neuronal precursors produces anxiety behavior in adult mice
Authors: Rajamanickam Shivakumar , Bulinski Joseph , Jarrett Kelsey , Bowman Kaitlin , Huggenvik Jodi I , Collard Michael W
Published: June 2021

DEAF1 is a DNA binding protein and the first transcription factor to be linked to suicide and depression. In humans with major depressive disorder (MDD), DEAF1 is reported to have altered expression in the prefrontal cortex and the dorsal raphe nucleus of females but not males, and may therefore function in sex‐specific depression. To better understand the role of DEAF1 in the regulation of behavior, we have produced a mouse line with conditional gene targeting (Deaf1‐flox). Deaf1‐flox mice were bred to congenic status onto a C57BL/6 background, and were then bred to mice transgenic for the nestin‐Cre gene to produce embryonic knockout of Deaf1 in neuronal precursor cells. Adult mice were tested for anxiety behavior using the Elevated Plus Maze and Open Field Exploration tests. Relative to control mice, both male and female mice with homozygous deletion of Deaf1 in brain displayed decreased exploration of open spaces while staying preferentially near enclosed spaces, as measured by the amount of time spent and distance traveled in those spaces. Our results indicate that conditional knockout of Deaf1 in neuronal precursors produces anxiety behavior in adult mice, and that this new mouse model may be useful in understanding the molecular mechanisms resulting in MDD. This work was supported by National Institutes of Health Grants CA89438, CA137556, and HD060122.
Novel mechanisms to ATP‐dependent glucose uptake in skeletal muscle cells
Authors: Osorio-Fuentealba Cesar , Contreras-Ferrat Ariel , Klip Amira , Jaimovich Enrique
Published: June 2021

Contraction and insulin stimulate glucose influx into muscle, and contraction further sensitizes muscle to insulin. Insulin signals via α,β class I phosphatidylinositol 3‐kinase (PI3Kα,β) to Akt, but contraction signals remain elusive. Interestingly, ATP is released by contracting muscle, and it was recently reported that extracellular ATP can act auto/paracrinely on muscle cells. Our aim was to investigate if ATP induces glucose uptake in muscle cells and promotes insulin action.

Rat primary myotubes transiently transfected with GLUT4myc and L6 myoblasts stably expressing GLUT4myc were exposed to 9s 6V electric stimulation (ES); insulin (100 nM, 5 min) or ATP (100 uM, 5min). Uptake of the fluorescent hexose 2NBDG was assayed in real time. Surface GLUT4 was measured by anti‐myc antibodies in fixed, unpermeabilizad cells. Phosphorylation of Akt and its target AS160 was detected with site‐specific antibodies.

ES, insulin and ATP each stimulated glucose uptake, GLUT4 translocation, Akt and AS160 phosphorylation. ATP added prior to insulin increased glucose uptake beyond insulin's effect. Inhibitors of Akt (AktVIII), PI3Kα–γ (LY294002), PI3Kγ (AS605240) or dominant‐negative PI3Kγ and Akt inhibited the ATP‐effect. In sum, ES or ATP promote glucose uptake via GLUT4, and ATP adds to insulin action. These effects may be mediated by PI3‐Kγ and Akt and contribute to glucose uptake during muscle activity.

Support: FONDAP 15010006, AT‐24100067, CIHR‐MT12601.
The activity of some enzymes in the frog's liver (Rana ridibunda Pall.) in the different natural conditions of thermal water regime
Authors: Bislimi Kemajl Rexhep , Gashi Agim , Bojniku Hazbije , Grapci Linda , Halili Jeton , Mazreku Ilir , Bajraktari Ismet
Published: June 2021

During the period of March–July 2011, the activity of aspartate aminotranspherase (AST), alanine aminotranspherase (ALT), lactate dehidrogenase (LDH) and alkaline phosphatase (ALP) enzymes was measured in the liver of the Rana ridibunda Pall. natural frog population.

Altogether 52 frogs were collected for the research purposes in the two rivers of a different water temperature regime respectively, the thermal water spring of Banja of Malishevë (24.7°C – 29.0°C) and river Tërdec (7.9°C – 19.7°C).

There were not recorded great oscillations of AST and ALT activity in the liver of frogs from the thermal water, and ALT activity from the river Tërdec as well during the period of investigation. At the same time in the liver of frogs from the river Tërdec, the lower activity of AST has been recorded in March (12.7±2.6 ), whereas a higher activity in May (41.4±8.4 ), and in July (46.6± 7.6). The distinctive characteristic of this research is that only in March, the activity of AST in the frogs liver from the river Tërdec is significantly at lower level compared with values of frogs from the thermal water.

Despite AST, in the liver of the frogs of two localities there were not recorded large oscillations of the ALT, LDH and ALP activity values during the entire period of research.

According to the research results we could conclude that the water temperature (the cold stress), the seasonal changes of the environmental temperature, the nutrition, and the metabolic activity of the frogs, affects the activity of AST.
Discovery of Novel Functional Link between Mediator Complex and Histone Modification Enzyme
Authors: Tsutsui Taiki , Shinmyozu Kaori , Ohkuma Yoshiaki
Published: June 2021

The Mediator complex has been thought as a key regulatory complex of RNA polymerase II (Pol II) and it has been known as bivalent, positive and negative, transcriptional regulator. Mediator complexes were reported to form at least two subtypes, large‐Mediator and small‐Mediator, depending on those subunit compositions. In previous studies, large‐Mediator has been thought to act negatively and small‐Mediator to act positively in transcription. Compared to its roles in transcriptional activation, little has been known in its roles in transcriptional repression. Here I show novel mechanisms of the Mediator complex in transcriptional repression in C/EBPβ target genes. To study a repressive mechanism by large‐Mediator, I purified CDK8 which is the large‐Mediator specific subunit forming complex and discovered PRMT5 as an interaction factor. PRMT5 is known to catalyze repressive histone modification (e.g., H4R3me2s) and recruits DNMT3A to target regions. Therefore, Large‐Mediator specific subunit CDK8 recruits PRMT5 and DNMT3A to certain C/EBPβ target gene promoter regions and represses those genes transcription via epigenetic machinery. This finding highlights a link of the Mediator complex to epigenetic regulation machinery and shows that the Mediator complex acts not only as Pol II recruiter but also as epigenetic regulator in transcription.

This study supported by Grant‐in‐Aid for JSPS Fellows.
Dietary format alters faecal bacterial phyla in the domestic cat (Felis catus)
Authors: Bermingham Emma Natasha , Young Wayne , Kerr Katherine R , Kittelmann Sandra , Swanson Kelly , Roy Nicole , Thomas David
Published: June 2021

The domestic cat evolved to metabolise high protein (HP):carbohydrate (CHO) ratio diets. In adult cats, low protein (LP):CHO diets alter intestinal microbiota composition and increase weight gains, possibly due to the microbiota extracting more energy from the diet. We hypothesise that pre‐ and post‐natal exposure to different LP:CHO ratio diets alters the intestinal microbiota composition of kittens. Pregnant queens were maintained either on commercial LP:CHO (32.5% CHO; 37.5% Protein, 21.8% Fat) or HP:CHO (2.5% CHO; 48.5% Protein; 40.6% fat) diets throughout pregnancy and lactation. Kittens were weaned from 4 to 8 weeks of age onto either HP:CHO or LP:CHO diets in a nested design. Faecal samples were taken at weeks 8 and 18 of age and the microbiota composition was profiled by next generation sequencing of 16S rRNA gene amplicons. Energy expenditure was determined at 18 weeks of age using a doubly labelled water bolus. Expression levels of key genes in glucose and insulin pathways were determined by RT‐qPCR in adipose and reproductive tissue taken at neutering and will be discussed. Kittens on HP:CHO diets had higher growth rates and levels of energy expenditure. Pre‐natal exposure to diet had no effect (P>0.05) on intestinal microbiota. Kittens fed LP:CHO showed higher levels of Actinobacteria (P<0.05) and Firmicutes (P<0.001) whereas kittens fed HP:CHO had higher levels of Fusobacteria (P<0.001).
Transcriptional Activation by Pax3‐FOXO1 is influenced by the presence of FOXO1 promoter elements
Authors: Johanson Kelly E. , Blunt Endia L. , Collins Chantelle L , Skripnikova Elena
Published: June 2021

The translocation of FOXO1 with either Pax3 or Pax 7 has been implicated in the development of Alveolar Rhabdomyosarcoma (ARMS). These translocations yield chimeric proteins containing amino‐terminal portions of either Pax transcription factor fused to carboxy terminal regions FOXO1. Cellular transformation by Pax3‐FOXO1 is dependent on the presence of the FOXO transactivation domain; by contrast, little attention has been paid to the DNA‐binding ability of FOXO1 fusions. The FOXO1 fusions retain a significant portion of the conserved DNA‐binding domain necessary for DNA contact, and Pax3‐FOXO1 can interact with FOXO1 recognition sequences in vitro. The objective of this study to determine if FOXO1 recognition sequences influence Pax3‐FOXO1 gene targeting and transcriptional activation. We examined the proximal promoter region of putative Pax3‐FOXO1 targets and determined that Pax3 and FOXO1 recognition sequences could be found in close proximity in 75% of these genes. We further examined four known targets of Pax3‐FOXO1 and again identified FOXO1 elements adjacent to the identified Pax3‐FOXO1 binding site. Of these, CNR1, is also a transcriptional target of FOXO1 and was therefore chosen for further study. These data indicate that FOXO1 fusions may target genes normally regulated by FOXO1 as well as those targeted by their fusion partner.
Sonic hedgehog gene expression is increased in adult skeletal muscle in ovarian hormone deficient mice that wheel exercise
Authors: Spence Madoka , Childs Thomas E , Ferreira J Andries , Brown Marybeth
Published: June 2021

Estrogen (E2) affects skeletal muscle pathway activation; however, the mechanisms by which E2 regulates skeletal muscle are not yet fully known. There is increasing evidence that (1) Sonic hedgehog (Shh) pathway is involved in myogenesis and hypertrophy, and (2) estrogenic compounds affect Shh pathway in vivo. The purpose of this study was to examine the relationship between E2, physical activity, and Shh pathway in adult skeletal muscle. In this study, we tested the hypothesis that both E2 and physical activity would increase Shh pathway gene expression. Female C57/BL6 mice (4 mos) were divided into 4 groups: Intact control, Intact run, Ovariectomy (Ovx) control, and Ovx run. Mice were on running wheels for 6 weeks. Ovx was done prior to wheel running. Shh pathway activation was measured in gastrocnemius muscle by mRNA expression of Shh signaling proteins, Gli1 and Ptch1. Gli1and Ptch1 mRNA expression was significantly increased in the Ovx run group compared to Ovx control, however exercise did not affect the intact mice. Ovx mice ran 38% the distance of Intact run. Muscle size was unaffected by running in both groups. However, muscle force/area (Po/mass) in Ovx run was significantly less than Intact run (p<0.05). Our results indicate that Shh pathway is upregulated in adult skeletal muscle when E2‐deficient animals undergo voluntary exercise. Support: MU Research Board Grant
Atractylodes macrocephala regulates water channel protein expression by hypertonic stress in renal collecting duct cells
Authors: Lee Yong Pyo , Lee Yun Jung , Lee So Min , Yoon Jung Joo , Kim Dae Hwan , Li Bin , Kang Dae Gill , Lee Ho‐Sub
Published: June 2021

Edema is a symptom that results from the abnormal accumulation of fluid in the body. The cause of edema is related to the level of aquaporin (AQP)2 protein expression, which regulates the reabsorption of water in the kidney. Edema is caused by overexpression of the AQP2 protein when the concentration of Na+ in the blood increases. The rhizome of Atractylodes macrocephala has been used in traditional Oriental medicine as a diuretic drug; however, the mechanism responsible for the diuretic effect of the aqueous extract from A. macrocephala rhizomes (AAM) has not yet been identified. We examined the effect of the AAM on the regulation of water channels in the mouse inner medullary collecting duct (mIMCD)‐3 cells under hypertonic stress. In the mIMCD3 cells, the AAM attenuates a hypertonicity‐induced increase in AQP2 expression as well as the trafficking of AQP2 to the apical plasma membrane. Tonicity‐responsive enhancer binding protein (TonEBP) is a transcription factor known to play a central role in cellular homeostasis by regulating the expression of some proteins, including AQP2. Western immunoblot analysis demonstrated that the protein and mRNA expression levels of TonEBP also decrease after AAM treatment. These results suggest that the AAM has a diuretic effect by suppressing water reabsorption via the downregulation of the TonEBP‐AQP2 signaling pathway
Vegetable consumption linked to decreased hepatic fat deposition in overweight Latino youth
Authors: Cook Lauren T , O'Reilly Gillian A , Goran Michael I , Weigensberg Marc J , Spruijt‐Metz Donna , Davis Jaimie N
Published: June 2021

We have previously shown that 38% of overweight Latino youth have Non‐alcoholic fatty liver disease (NAFLD). This study examines the relationship between diet, specifically vegetable consumption, and NAFLD in this high‐risk population.

This cross‐sectional study of 172 overweight ([ge]85th percentile BMI) Latino youth (8–18 y) had measures of dietary intake (multiple 24‐h recalls), total body fat (dual‐energy x‐ray absorptiometry), and adipose tissue distribution (magnetic resonance imaging). Analysis of covariance was used with the following a priori covariates: age, gender, energy intake and total body fat.

Quartiles of non‐starchy vegetable (NSV) intake were associated with lower hepatic fat fraction (HFF, p=0.01); those who consumed the most NSV (mean intake = 1.7±1.0 serv/d) compared to the least (mean intake = 0.1[plusmn]0.1 serv/d) had 44% less liver fat (10.0[plusmn]8.5 vs. 5.6[plusmn]8.7 % fat, p=0.01). No relationship was found with fruit. These findings suggest that approximately 2 servings of NSV per day by overweight Latino youth may help decrease the risk of NAFLD. Research is supported by U54 CA 116848, RO1 HD/HL 33064, P60 MD002254, R01 HL07953, 1K01DK078858–01.

Grant Funding Source: U54 CA 116848, RO1 HD/HL 33064, P60 MD002254, R01 HL07953, 1K01DK078858–01
High Dietary Sodium Reduces Flow Mediated Dilation Similarly in Humans with Salt Sensitive & Salt Resistant Blood Pressure
Authors: Lee Matthews Evan , DuPont Jennifer J , Greaney Jody L , Lennon‐Edwards Shannon L , Edwards David G , Farquhar William B
Published: June 2021

Recent studies demonstrate that high dietary sodium (HS) impairs endothelial function in those with salt‐resistant (SR) blood pressure (BP), suggesting that the effect of dietary sodium on endothelial function may be independent of BP. We hypothesized that HS would impair flow mediated dilation (FMD) more in those with salt sensitive (SS) BP compared to those with SR BP. Five SS (2M 3W, 43±5yrs) & 7 SR (3M 4W, 45±2.4yrs) subjects were enrolled in a controlled feeding study consisting of a run‐in diet followed by a two phase randomized crossover 7‐day diet perturbation: low dietary sodium (LS) (20 mmol/d) & HS (300 or 350 mmol/d). Brachial artery FMD and 24‐hr BP were assessed on the last day of each diet. SS BP was defined as a change in 24‐hr mean arterial pressure of >; 5 mmHg between LS & HS diets. HS attenuated FMD in both the SS (LS: 12.8±2.4%, HS: 8.2±1.4%, p<0.05) & SR (LS: 10.5±1.8%, HS: 6.9±1.1%, p<0.05) groups. The decline in FMD was not different between groups for the change in FMD (SS: Δ‐4.6±1.2%, SR: Δ‐3.6±1.2%, p>;0.05). These data indicate a HS diet impairs FMD to a similar extent in SS & SR individuals, providing additional evidence suggesting a BP independent effect of dietary sodium on endothelial function.

Research funded by NIH grants 2 P20 RR016472–11 & R01 HL104106.
MiR‐133a ameliorates cardiac stem cells survival and differentiation in Insulin2 mutant diabetic mice
Authors: Chavali Vishalakshi , Diaz Noel Leiberh , Tyagi Suresh C , Mishra Paras Kumar
Published: June 2021

The Ins2 +/− (orthologous to human Insulin) mutation predisposes to diabetic complications and mitigates cardiac stem cell differentiation into cardiomyocytes that foster cardiomyopathy. MicroRNA (miR)‐133a, a muscle specific miRNA that regulates cardiac hypertrophy and fibrosis, is attenuated in diabetic hearts. However, the role of miR‐133a in cardiac stem cell survival and differentiation is unclear. We hypothesized that miR‐133a improves cardiac stem cell survival and differentiation and mitigates diabetic cardiomyopathy. To test the hypothesis, miR‐133a was overexpressed in WT (C57BL/6J) and diabetic Akita (Ins2+/‐) through lentivirus. The levels of miR‐133a, c‐kit and differentiation markers (Actn2, Oct4 and Tnnt2) were determined by individual miRNA assay, RT‐QPCR, Western blot and confocal microscopy in four groups: 1) WT, 2) Akita, 3) WT +miR‐133a, and 4) Akita + miR‐133a. The cardiac dysfunction was assessed by M‐Mode echocardiography. The results revealed that miR‐133a, c‐kit, Actn2, Oct4 and Tnnt2 were up regulated in Akita + miR‐133a as compared to Akita. The % FS is increased in Akita after miR‐133a treatment (Akita‐ 38.6±1.2; Akita +miR‐133a ‐ 50.8 ±5.1). These findings elicit a novel mechanism of miR‐133a mediated cardiac stem cell survival and differentiation and cardiac protection in diabetic mice.
Cellular mechanims for the protective effects of prior exercise on statin‐associated muscle force loss in mice
Authors: Godar MIchelle , Chamney Carissa , Huey Kimberly
Published: June 2021

Exercise accustomization may protect against statin‐associated losses in muscle force. Exercise‐induced increases in Hsp25 and αB‐crystallin may contribute to this protection by helping reduce muscle breakdown and oxidative stress. We tested the hypothesis that 1 or 3 days of prior exercise protects against statin‐induced muscle force loss and this will be associated with reduced contractile protein breakdown and oxidative stress. Mice received daily atorvastatin or saline for 14d, with/without wheel running (RW) (Novel & Sedentary (Sed)) and prior RW groups completed 1 or 3d of RW before saline or statin + RW. Plantarflexor force, fatigue, contractile proteins, protein carbonyls, and Hsp25 and αB‐crystallin protein were quantified. Statins reduced force in Sed, novel, and 1d RW groups vs saline (21, 35, & 21%, respectively, p<0.05), while 3d RW prevented statin‐associated force loss. Hsp25 and αB‐crystallin increased with RW vs. Sed, independent of statin treatment (p<0.05). Statins were associated with lower contractile protein levels in Sed and novel groups vs. saline (18 & 26%, respectively, p<0.05). Total protein carbonyls were lower than Sed‐saline in novel, 1d, & 3d RW groups with either treatment (p<0.05). Results suggest 3d prior exercise does not reduce oxidative stress vs. novel exercise, but may preserve contractile proteins and help prevent statin‐associated force loss with exercise.
Vitamin D modulation of diaphragm muscle strength in mice
Authors: Ray Andrew D , Pesonius Kirkwood J , Hershberger Pamela A
Published: June 2021

Recent data provides evidence for an important role of vitamin (Vit) D in respiratory muscle health. We tested the hypothesis that Vit D deficiency will reduce diaphragm muscle strength. Fifteen A/J mice were exposed to diets containing either 100 IU/kg Vit D3 (deficient diet), 1,000 IU/kg (reference diet), or 10,000 IU/kg (supplemented diet) (n=5/group) for eight weeks. At the end of the 8 weeks, plasma Vit D and diaphragm and EDL muscle in‐vitro contractile properties were measured. Mice fed the deficient, reference and supplemented diets had plasma 25(OH)D3 levels equal to 7, 21, and 59 ng/mL respectively. Calcium levels remained stable. Diaphragm force was significantly weaker (25%) in Vit D deficient mice compared to mice from either of the Vit D replete groups (p < 0.05). Diaphragm fiber diameter was also reduced in Vit D deficient mice compared to the reference and supplemented groups 684±293μ2, 769±297μ2 and 753±304μ2, respectively (p<0.001). There were no changes in EDL muscle force. These data suggest a critical threshold for Vit D levels with respect to diaphragm force production, because supplementation (59 ng/ml) does not offer an apparent performance enhancing effect beyond 20 ng/mL. These data also establish that diaphragm muscle responds differently to Vit D deficiency compared to peripheral muscle (EDL).