bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism and regulation of DNA end processing
Authors: Symington Lorraine , Chen Huan , Deng Sarah
Published: June 2021

DNA ends exposed after introduction of double‐strand breaks (DSBs) undergo 5′‐3′ nucleolytic degradation to generate single‐stranded DNA (ssDNA), the substrate for binding by the Rad51 protein to initiate homologous recombination. We have shown that resection of DNA ends occurs by a two‐step process. In the first step, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. In a second step, Exo1 or Sgs1 with Dna2 rapidly process the minimally resected intermediate to generate long tracts of ssDNA that serve as substrate for Rad51. In the absence of Exo1 and Sgs1, the partially resected intermediates accumulate and are poor substrates for homology‐dependent repair (HDR). RPA plays an active role in resection by stimulating both Exo1 and Sgs1‐dependent end processing and controls the polarity of degradation. The initial processing step by the Mre11 complex and Sae2 is cell cycle regulated and appears to be important to promote HDR and to prevent repair by non‐homologous end joining. In the absence of end protection by Ku, we have shown that the requirement for the MRX complex is bypassed and Exo1 executes resection. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact but functionally compromised by elimination of the Mre11 nuclease activity. In addition, we show that replication associated DSBs need to be processed by Sae2 for repair by HR unless Ku is absent. Finally, we show that the presence of Ku or the MRX‐Tel1 damage checkpoint exacerbates DNA end‐processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.
Paternal folate status at mating influences fetal brain IGF‐2 expression in rats
Authors: Choi Yun Jung , Kim Hye Won , Kim Ki Nam , Mejos Karen Kay , Lim Eunmi , Chang Namsoo
Published: June 2021

We investigated the effect of paternal folate status on placental and fetal whole brain folate content and DNA methylation, and fetal whole brain insulin‐like growth factor‐2 (IGF‐2) expression in rats. Rats were divided into four groups: paternal folate‐supplemented (PS) or folate‐deficient (PD), and maternal folate‐supplemented (MS) or folate‐deficient (MD) and were fed with folic acid‐supplemented (8mg/kg) and folic acid‐deficient (0mg/kg) diets. After 4 weeks, rats were mated (PS×MS, PS×MD, PD×MS and PD×MD) and placenta and fetal whole brain were isolated at day 20 of gestation. Folate content, DNA methylation and the expression of IGF‐2 in the placenta and brain analyzed by microbiological assay, ELISA and western blot technique, respectively. Compared to the PS×MS group, placental folate content (P < 0.0001) and DNA methylation in placenta (P < 0.0001) and brain (P < 0.0001) was significantly decreased in the PD×MS group as much as the PS×MD group. Brain folate content was decreased in the PD×MS group as much as the level of PD×MD group (P < 0.05). As compared to PS×MS group, the expression of IGF‐2 in the PD×MS group was decreased as much as the level of the PD×MD group (P < 0.0001). These results suggest that the effect of paternal folate deficiency could be as important to the growth and development of placenta and brain of the fetus as maternal folate deficiency.
Effect of Dietary Genistein on Hormone‐dependent Rat Mammary Carcinoma Induced by Ethyl Methanesulphonate (EMS)
Authors: Ono Misaki , Koga Takaomi , Ueo Hiroaki , Nakano Shuji
Published: June 2021

Although genistein (GEN), a major soy isoflavone, has been shown to reduce the risk of breast cancer (BC) incidence, many experimental studies have yielded the inconsistent results. We investigated the effects of GEN on the development of BC using the EMS chemically induced rat model of hormone‐dependent BC. Female WKA rats were orally given EMS for 12 weeks and fed the soy‐isoflavone‐free NIH‐07PLD diets with or without GEN. All EMS‐treated rats fed either diet developed ER‐ and/or PgR‐positive hormone‐dependent mammary carcinoma by 24 weeks. Compared with GEN‐free diet, the addition of either low or high GEN to GEN‐free diet, which produced plasma concentrations of GEN comparable with those observed in humans consuming high or more soy diets did not show any preventive activity on the development of EMS‐induced hormone‐dependent BC. Soy‐containing food CE2, which resulted in the modest plasma concentration of isoflavones such as GEN, daidzein, equol, and glycitein, increased the latency periods, compared to either isoflavone‐free diets with (p=0.008) or without GEN (p=0.052). The body weight, total EMS uptakes, and urine estradiol concentrations were not significantly different between these 4 groups. These data indicate that GEN does not exert clear preventive effects on estrogen‐dependent mammary carcinogenesis and that isoflavones other than GEN may be responsible for the prevention of BC.
Chronic consumption of high‐fat diet stimulates tumor angiogenesis in the Lewis lung cancer allograft model
Authors: Song Hyerim , Cho Han Jin , Park Jung Han Yoon
Published: June 2021

Epidemiological studies indicate that overweight and obesity are associated with increased risk of several cancers. In the present study, we determined whether a chronic consumption of high‐fat diet (HFD) increases tumor angiogenesis and metastasis of Lewis lung cancer in the C57BL/6N mice. Four‐week old, male mice were fed a purified control diet (CD, 10 kcal% fat) or HFD (60 kcal% fat) for 16 weeks, and then Lewis lung carcinoma cells were subcutaneously injected into the animals’ left flank. The primary tumor was resected 3 weeks later, and all animals were killed 21 days after the surgery. HFD feeding increased solid tumor growth and lung metastasis. Immunohistochemical staining of tumor tissues revealed that the expression of hypoxia‐inducible factor 1, vascular endothelial growth factor, and CD31 (an endothelial cell marker) was significantly increased in the HFD group compared to the CD group. HFD feeding significantly increased the blood levels of angiogenesis‐related proteins including leptin, monocyte chemoattractant protein‐1, angiogenin, angiopoietin‐1, angiopoietin‐3, chemokine ligand 16, plasminogen activator inhibitor‐1, stromal cell‐derived factor‐1, and endoglin. These results indicate that the prolonged consumption of HFD enhances the expression and secretion of angiogenesis‐related proteins.
The citrus flavonone hesperetin inhibits growth of aromatase‐expressing MCF‐7 tumor and reduces the effective dose in letrozole‐treated mice
Authors: LI Fengjuan , Ye Lan , Chan Franky L. , Chen Shiuan , Leung Lai K.
Published: June 2021

Breast cancer is one of the most prevalent cancers affecting women. Aromatase is a key enzyme in estrogen synthesis, and aromatase inhibition is a contemporary adjuvant therapy for treating estrogen‐responsive breast cancer. In the present study, we examined the aromatase inhibitory effect of hesperetin and the combined effect of hesperetin and letrozole on an established postmenopausal breast cancer model. Athymic mice were ovariectomized and transplanted with aromatase‐overexpressing MCF‐7 cells. Our first experiment has shown that dietary administration of hesperetin at 1000 ppm and 5000ppm significantly deterred the xenograft growth. The combined treatment effect of hesperetin and letrozole was examined in a follow up experiment. Our result indicated that hesperetin could improve the potency of letrozole by about 10‐folds. Messenger RNA expression of the estrogen‐responsive gene pS2 was also decreased in the tumors of mice treated with hesperetin. Western analysis indicated that cyclin D1, CDK4 and Bcl‐x(L) were reduced in the tumors. This study suggested that hesperetin could be a potential co‐therapeutic agent to aromatase inhibitor.
The administration of Dioscorea batatas extract and fish oil inhibits the development of oxazolone‐induced atopic dermatitis‐like skin lesions in hairless mice
Authors: PARK JINAH , Ji Geun Eok , Sung Mi-Kyung
Published: June 2021

Atopic dermatitis (AD) is a chronically relapsing inflammatory disorder that is characterized by pruritic and eczematous skin lesions. In this study, we examined whether Dioscorea Batatas extract (DBE) and/or fish oil (FO) suppress the development of AD‐like skin lesions. SKH1 hairless mice were divided into eight experimental groups as follows: control, positive control, FO 0.09%, FO 0.18%, DBE 0.17%, DBE 0.33%, FO 0.18% + DBE 0.17% and FO 0.18% + DBE 0.33%. All animals except those in control groups were treated with OX for two weeks after the first experimental diet supply. Administration of FO and DBE inhibited the development of AD‐like skin lesions as exemplified by decreases in skin severity score, hypertrophy of the skin, infiltration of inflammatory cells in the epidermis and dermis. FO and DBE significantly reduced serum levels of both macrophage‐derived chemokine (MDC) and interleukin‐6 (IL‐6), and the mixture of FO and DBE markedly reduced IL‐6 concentration. In addition, FO and DBE mixture supplementation significantly decreased the mRNA expression of interleukin‐4 (IL‐4) in draining lymph nodes. These results suggest that FO and DBE effectively suppress the development of AD skin lesions, possibly by suppressing Th2 cell‐mediated immune responses.

* This work was supported by Gangwon Regional Innovation Agency, Ministry of Knowledge Economy and the Small and Medium Business Administration, Korea(S1072365).

Grant Funding Source : Gangwon Regional Innovation Agency, Ministry of Knowledge Economy and the Small and Medium Business Administration, Korea
Antiobesity and lipid‐lowering effects of Bifidobacterium spp. in high fat diet‐induced obese rats
Authors: An Hyang Mi , Kim Min Ji , Moon Jung Soon , Kang Joo Yeon , Lee Do Kyung , Lee Kang Ho , Shin Hea Soon , Ha Nam Joo
Published: June 2021

Background

Recent studies have reported the preventive effects of probiotics on obesity. Among commensal bacteria, bifidobacteria is one of the most numerous probiotics in the mammalian gut and are a type of lactic acid bacteria. The aim of this study was to assess the antiobesity and lipid‐lowering effects of Bifidobacterium spp. isolated from healthy Korean on high fat diet‐induced obese rats.

Methods

Thirty‐six male Sprague‐Dawley rats were divided into three groups as follows: (1) SD group, fed standard diet; (2) HFD group, fed high fat diet; and (3) HFD‐LAB group, fed high fat diet supplemented with LAB supplement (B. pseudocatenulatum SPM 1204, B. longum SPM 1205, and B. longum SPM 1207; 108 ~ 109 CFU). After 7 weeks, the body, organ, and fat weights, food intake, blood serum levels, fecal LAB counts, and harmful enzyme activities were measured.

Results

Administration of LAB reduced body and fat weights, blood serum levels (TC, HDL‐C, LDL‐C, triglyceride, glucose, leptin, AST, ALT, and lipase levels), and harmful enzyme activities (β‐glucosidase, β‐glucuronidase, and tryptophanase), and significantly increased fecal LAB counts.

Conclusion

These data suggest that Bifidobacterium spp. used in this study may have beneficial antiobesity effects.
Effect of Ginkgo biloba leaves on plasma and liver lipids, platelet aggregation and erythrocyte Na+ efflux channels in ovariectomized rats
Authors: Ryou Sung-Hee , Kang Young-Hee , Kang Jung-Sook
Published: June 2021

Forty 9 weeks old OVX rats ovariectomized at age of 5 weeks were divided into 4 groups and fed 0.2% cholesterol based control diet, 10% Ginkgo biloba (Gb) leaves powder, 3% Gb extract and 1% tanamin (EGb761) for four weeks. Total cholesterol and HDL cholesterol were significantly increased in rats fed diet with tanamin compared with the control (both p<0.01) with no difference in liver cholesterol. Liver triglyceride was decreased in Gb leave powder compared with no difference in plasma TG. Platelet aggregation in the maximum and the initial slope tended to increase in groups with Gb powder, Gb extract and tanamin compared with the control. Intracellular Na was increased in group with tamanin compared with other groups (p<0.05), and with no difference in intracellular K. Na‐K ATPase in group with Gb powder and Na cotransport in group with Gb extract were significantly increased compared with the control (both, p<0.05). Angiotesin converting enzyme (ACE) was significantly decreased in group with tanamin (p<0.05) and ACE activity was not correlated with any of Na efflux channels.

Bioactive compounds in gingko biloba in forms of powder, extract or tanamin (EGb761) seemed to play some roles on platelet aggregation and RBC efflux channels. Further studies including invitro experiment may be required for interpretation on their beneficial effects
Anthocyanins and cardiovascular disease prevention
Authors: Wallace Taylor C
Published: June 2021

In this review, recent studies on the CVD preventive activities of anthocyanins, including results from in vitro cell culture and in vivo animal model systems as well as data from clinical trials and human epidemiological studies are presented. Anthocyanins have become increasingly important to the food and dietary supplement industries as their use as natural alternatives to artificial colors, functional ingredients, and as botanical extracts has become widespread and knowledge of their health‐promoting properties is evident. Epidemiological studies suggest that consumption of anthocyanins lowers the risk of CVD, the most common cause of mortality. Anthocyanins frequently interact with other phytochemicals, exhibiting synergistic biological effects but making contributions from individual components difficult to decipher. Over the past 2 decades, many peer‐reviewed publications have demonstrated that in addition to their noted in vitro antioxidant activity, anthocyanins may regulate different signaling pathways involved in the development of CVD.
Dietary intake and appetite predict early treatment outcome among low‐BMI adults initiating antiretroviral therapy for HIV in Sub‐Saharan Africa
Authors: Heimburger Douglas C , Koethe John R , Bosire Claire , Blevins Meridith , Nyirenda Christopher , Kabagambe Edmond K , Zulu Isaac , Shepherd Bryan E
Published: June 2021

Low body mass index (BMI), common in patients with HIV in sub‐Saharan Africa, is a risk factor for early mortality on antiretroviral therapy (ART). Many persons in the region believe that ART produces hunger, and this compromises ART adherence. We measured dietary intake (24‐hour recall) & appetite (ordinal scale) in 142 adults starting ART in Lusaka, Zambia with very low BMI (<16 kg/m2) and/or advanced immunosuppression (CD4+ lymphocyte count <50 cells/μL). Relationships with 12‐week outcomes were analyzed with Cox models. Median age, BMI, & CD4+ count were 32 y, 16 kg/m2, & 34 cells/μL. Twenty‐five participants (18%) died before 12 weeks, and 33 (23%) were lost to care. A 100 kcal/day higher energy intake at any time after ART initiation was associated with 14% lower hazard of death (AHR=0.86, p=0.01); similar relationships were observed for higher intakes of protein (5 g/day, AHR=0.81, P=0.01) and fat (5 g/day, AHR=0.80, P<0.01), while that for carbohydrate fell short of significance (25 g/day, AHR=0.85, P=0.09). Higher energy intake was marginally associated with a reduction in the combined endpoint of mortality or loss to care (p=0.07). Among survivors, appetite tended to normalize gradually, and hunger was rarely reported. Intervention trials are needed to test the impact of dietary supplementation on early ART outcomes. Funding: NIH R21 AI 076430 & R24 TW 007988.
Iron status and predicted VO2max in Indian adolescents
Authors: Przybyszewski Eric , Udipi Shobha , Ghugre Padmini , Boy Erick , Haas Jere D.
Published: June 2021

The objective of this study was to determine whether iron status has a negative impact on predicted maximum oxygen consumption (VO2max, ml·kg−1·min−1) in Indian adolescents. School children (N=81 males, 74 females; age=12–16 y) were screened for indicators of iron status with a venous blood sample to assess ferritin (sFer), transferrin receptor (sTfR), hemoglobin (Hb), alpha‐1 acid glycoprotein (AGP), and C‐reactive protein (CRP) in rural Maharashtra, India. Anemia (N=10M, 15F) was defined as Hb<12.0g/dL; iron‐depletion (N=17M, 21F) was defined as sFer<16.0ng/mL. Each subject wore a Polar heart rate monitor and CosMed K4b2 indirect calorimeter while performing a YMCA sub‐maximal test on a Monark 874E cycle ergometer. VO2max was predicted by extrapolating the relationship between VO2 and heart rate to predicted maximum heart rate (195 bpm). Anemic boys had a significantly lower predicted VO2max (41.2ml·kg−1·min−1) than non‐anemic boys (45.9ml·kg−1·min−1) (p<0.05). Iron depleted (ID) boys had a significantly lower predicted VO2max (42.0ml·kg−1·min−1) than non‐ID boys (46.2ml·kg−1·min−1) (p<0.05). There were no significant differences in predicted VO2max between anemic and non‐anemic females (34.6ml·kg−1·min− 1,36.7ml·kg−1·min−1, respectively) or ID and non‐ID females (36.1ml·kg−1·min−1, 36.4ml·kg−1·min−1, respectively). Supported by HarvestPlus.

Grant Funding Source : HarvestPlus
NutriChip – A lab‐on‐a‐chip platform to investigate food absorption and underlying immune regulations
Authors: Schwander Flurina , Kopf-Bolanz Katrin Annika , Portmann Reto , Egger Charlotte , Silacci Paolo , Gijs Martin , Vergères Guy
Published: June 2021

The NutriChip project aims to develop a chip‐based technology that allows to monitor and compare the absorption of different food products in vitro regarding their immuno‐ modulatory properties. Dairy products were digested in vitro using a three step enzymatic digestion model, validated for its ability to degrade the macronutrients to physiological ranges. The nutrients released were applied apically to Caco‐2 cells cultured in a transwell system. The transported nutrients will be analyzed for their ability to modulate the inflammatory response of activated monocytes. This cellular co‐culture is integrated into a microfluidic chipsystem. Different products can be screened on their bioactivity, focusing on inflammation. The results obtained in the in vitro experiments will be compared with the results from human studies. In these studies, the effect of different test meals on postprandial inflammation is investigated by blood sampling before and after different time points of the test meals consumption. Blood samples are analyzed for glucose, insulin, triglyceride, CRP, different cytokines and metabolites. This project is funded by NanoTera.

Grant Funding Source : NanoTera
Dental Pulp Stem Cells, proliferation/differentiation switch reveals new insights in Oct4A dynamics
Authors: Spelat Renza , Ferro Federico , DˈAurizio Federica , Puppato Elisa , Pandolfi Maura , Beltrami Antonio Paolo , Cesselli Daniela , Falini Giuseppe , Beltrami Carlo Alberto , Ambesi-Impiombato Francesco Saverio , Curcio Francesco
Published: June 2021

Although the role played by transcription factors network, that includes c‐Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell pluripotency is known, its persistence and function in adult stem cells are still debated. To verify and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in proliferating and differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of Oct4A. Data demonstrate that: Oct4, Nanog, Klf4 and c‐Myc are expressed in adult stem cells and, with the exception of c‐Myc, they are significantly down‐regulated following differentiation. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. Last, cell differentiation was also associated with a reduction in both the Wnt/beta‐catenin/Lrh‐1 pathway and CD10, CD29 and CD117. Learning how to exploit the nuclear‐cytoplasm translocation of Oct4A could help in maintaining stem cells in an undifferentiated state, thus increasing opportunities to use adult stem cells in therapeutic applications.

Work supported by Agenzia Spaziale Italiana.
Impact of adenosine signaling on mutant LRRK2 induced neuronal injury
Authors: Steer Erin , Chu Charleen
Published: June 2021

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, affecting more than 1.5% of people over the age of 65. The most common genetic cause of PD is mutations in the gene for leucine‐rich repeat kinase 2 (LRRK2). Consumption of caffeine, a non‐selective adenosine receptor antagonist, has been linked to decreased incidence of PD. Additionally, activation of A1 adenosine receptors (A1AR) and blockade of the A2 adenosine receptors (A2AAR) have been shown to protect neurons against pathogenic stimuli. Therefore, we hypothesized that manipulation of the adenosine signaling pathway would attenuate mutant LRRK2 induced neurite retraction and cell death. Number of neuritic branch points and neurite length were measured in mouse cortical neurons expressing LRRK2 mutants following treatment with adenosine, a selective A1AR agonist [2‐chloro‐N6‐cyclopentyladenosine (CCPA)], or a selective A2AAR antagonist [7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2, 4‐triazolo[1,5‐c]pyrimidine (SCH 58261)].These data will indicate if adenosine signaling is able to protect against neurite retraction induced by mutant LRRK2.
Investigation of the Dravet syndrome using a mouse model
Authors: Tsai Ming-Shian , Lee Fang-Jen , Chen You-Tzung , Yu I-Shing , Liou Horng-Huei , Lin Shu-Wha
Published: June 2021

Objective

To identify genetic heterogeneity which influence the severity and mortality of the disease

Method

We first generated a point mutation allele of Scn1a in the R1 mouse ES cell using a recombineering approach. Germline chimeras were mated with 129/J females to generate mutant mice in a pure 129 stain. Some of the resulting heterozygous mutant males were outcrossed to C57BL/6 females to generate F1 hybrids that mutant progenies in the heterogeneous genetic backgrounds with various degrees of phenotypic presentations can be obtained by F1 intercrosses.

Result

In the pure 129 stain, heterozygous mutant mice were resistant to the disease. However, the homozygous mutant pups became malnourished and showed a significant reduction in their body weight and sizes within 2 weeks. They were also found to have an unstable gait and developed spontaneous seizures. All of them died within the third postnatal week. In contrast, when the mutant allele was brought to a heterogeneous 129 and C57BL/6 mixed stain, sporadic sudden death was observed in some heterozygous animals after the third postnatal week. The survival rate of the adult heterozygotes was reduced to 60% at 7 weeks. These results suggested that some modifier loci in the two different genetic backgrounds have different effects on the mutant phenotypes.

Conclusion

We successfully generated a mouse model for Dravet syndrome. The fact that Scn1a mutant seizure severity and gross lethality differ in 129 and C57BL/6 stains indicates an invaluable opportunity to identify modifier genes using these mutant strains.
Decreased basal ganglia activation in Chronic Fatigue Syndrome subjects is associated with increased fatigue
Authors: Unger Elizabeth R. , Miller Andrew H. , Jones James F. , Drake Daniel F. , Tian Hao , Pagnoni Giuseppe
Published: June 2021

Objectives

Conduct a functional magnetic resonance (fMRI) study using a monetary win‐lose gambling task that strongly activates basal ganglia to test hypothesis of decreased basal ganglia function in chronic fatigue syndrome (CFS).

Methods

Participants included 18 CFS subjects (1994 case definition) and 41 non‐fatigued controls matched on age, sex and race who were free of psychotropic medications and significant depression (Zung Depression score <60). The general activation pattern for win‐lose contrast across all subjects was determined. The resulting statistical parametric brain map thresholded at p<0.05, corrected for multiple comparisons, was intersected with basal ganglia regions of interest (ROIs). For each subject, the average value of win‐lose activation contrast in each ROI was extracted.

Results

CFS subjects showed decreased right caudate (p=0.01) and right globus pallidus (p=0.02) activation compared to controls. The decreased globus pallidus activation correlated with increased mental fatigue (r2=0.49, p=0.001), general fatigue (r2=0.34, p=0.01) and reduced activity (r2=0.29, p=0.02) Multidimensional Fatigue Inventory scores of CFS subjects but not controls.

Conclusion

Reduced basal ganglia activation may contribute to symptoms of fatigue in CFS subjects.

The findings in this report are those of the authors and do not necessarily reflect the views of the funding agency (CDC).
BETA3‐ADRENERGIC RECEPTOR REGULATION OF nNOS SIGNALING IN CARDIOMYOCYTES
Authors: Watts Vabren L , Sepulveda Fernando , Niu Xiolin , Miller Karen L , Barouch Lili A
Published: June 2021

β3‐AR plays a pivotal role in modulating cardiac function. We recently showed that β3‐mediated cardioprotection is lost in nNOS−/− animals under pressure overload as compared with WT. However, the manner in which β3‐AR regulates nNOS signaling at the cardiomyocyte level is unknown. Here, we used specific β3‐ AR agonist BRL37344 (BRL) to assess the role of β3‐AR regulation of nNOS in hypertrophied isolated neonatal rat ventricular cardiomyocytes. Hypertrophy was induced by 72‐hour norepinephrine pretreatment, which increased cell size and β3‐AR mRNA as compared with non‐hypertrophied cells. In hypertrophied myocytes, BRL increased NOS activity, nNOS phosphorylation at stimulatory site Ser1416, and dephosphorylation of deactivation site Ser847. NOS activity was attenuated by nNOS inhibitor LVNIO and phosphomemetic mutants Ser1416A and Ser847D. In addition, Gi/o and Akt inhibition suppressed BRL‐induced nNOS‐Ser1416 phosphorylation and NOS activity. Our data suggest that BRL regulates myocardial NOS activity via nNOS phosphorylation. This is first study to demonstrate a role for nNOS phosphorylation as a key factor in β3‐AR signaling. These results contribute significantly to our understanding of the negative inotropic properties of cardiac β3‐AR signaling during sympathetic overstimulation, and will ultimately aid in drug discoveries that target molecular mechanisms associated heart failure.
Flexible sensors to measure intravascular shear stress coupled with rapid and irregular atrial pacing
Authors: Jen Nelson , Yu Fei , Lee Juhyun , Hsiai Tzung
Published: June 2021

Atrial fibrillation (AF), the most common cardiac arrhythmia, is characterized by multiple rapid atrial depolarizations leading to rapid and irregular ventricular responses exceeding 100 beats per minute (bpm). The hemodynamic effects of AF on the vascular endothelium are poorly undefined. To simulate AF, we paced the left atrial appendage of New Zealand White (NZW) rabbits (n=4) at rapid irregular intervals. The pacing protocol was as follows: 1) baseline sinus rhythm at 121.8 ± 1.6 bpm, 2) regular pacing at 168.3±0.5 bpm, and 3) irregular pacing at 167.1 ± 57.4 bpm. Surface electrical cardiograms (ECG) were recorded for changes in atrial and ventricular rhythm while flexible MEMs sensors were simultaneously deployed to the aorta for real‐time changes in intravascular thermal profiles. AF pacing at 168±37 (from 120±5.4) bpm decreased cycle lengths by 28% (from 491.5±21.6 to 355.9±78.2 ms), shear stress by 28% (from 32.0±2.4 to 22.7±3.5 dyne/cm2), and slew rate by 17% (from 1275±80 to 1056±180 dyne/cm2•s). Systolic and diastolic pressures were also reduced by 7.22 ± 4.13 mmHg and 7.72 ± 6.05 mmHg, respectively (baseline: 99/75 mmHg; regular pacing: 92/75; irregular pacing: 92/67; n=4, P < 0.001). These findings provide new methodology to measure spatial and temporal variations in intravascular shear stress in response to rapid and irregular atrial pacing.
Protein expression of myostatin and follistatin in the myocardium of spontaneously hypertensive rats with heart failure
Authors: Damatto Ricardo Luiz , Martinez Paula , Guizoni Daniele , Lima Aline , Cezar Marcelo , Bonomo Camila , Nakatani Bruno , Campana Alvaro , Okoshi Katashi , Okoshi Marina
Published: June 2021

The aim of this study was to evaluate myocardial myostatin and follistatin protein expression in spontaneously hypertensive rats (SHR) with heart failure (HF).

Methods

18‐month‐old SHR's were evaluated to identify clinical features of HF: tachypnea/labored respiration and weight loss. After HF was detected, rats were subjected to transthoracic echocardiogram and euthanized. During euthanasia, we evaluated pathological evidence of HF such as pulmonary congestion and right ventricular hypertrophy. Wistar‐Kyoto (WKY) rats were used as controls. Myostatin and follistatin protein expression was assessed by Western blotting.

Results

All SHR's (n=8) presented tachypnea, lung congestion and right ventricular hypertrophy. No WKY rat (n=8) presented any feature of HF. Echocardiographic evaluation showed left chambers dilation (left atrial diameter: WKY 5.73±0.59; SHR 7.28±1.17 mm; p=0.004; left ventricular diastolic diameter/body weight ratio: WKY 19.6±3.1; SHR 27.7±4.7 mm/kg; p=0.001), and left ventricular systolic (fractional shortening: WKY 54.6±6.3; SHR 42.2±7.1 %; p=0.003) and diastolic dysfunction in SHR. Myostatin (WKY 1.00±0.16; SHR 0.77±0.23 arbitrary units; p=0.035) and follistatin (WKY 1.00±0.35; SHR 0.49±0.18 arbitrary units; p=0.002) protein expression was lower in SHR.

Conclusion

Myostatin and follistatin protein expression is reduced in myocardium of SHR with heart failure.
Estradiol replacement in ovariectomized female rats enhances mephedrone‐induced disruptions of nonspatial learning
Authors: Weed Peter Fletcher , Sankanarayanan Ananthakrishnan , Winsauer Peter J
Published: June 2021

Both ovarian hormones and mephedrone, a new substance of abuse, have been observed to affect learning and memory in rats. The interaction between mephedrone and estradiol on learning was examined by administering mephedrone to ovariectomized female rats with and without estradiol hormone replacement. To assess learning, rats were trained to respond under a multiple schedule with repeated‐acquisition and performance components. Ovariectomized rats received subcutaneous capsules containing 25% 17‐β estradiol at the start of the experiment to determine the dose‐effects of mephedrone in the presence of estradiol. These capsules were then removed to determine full dose‐response curves for mephedrone in the absence of estradiol. Acute intraperitoneal injections of mephedrone (1 – 10 mg/kg) were administered twice per week. In both the presence and absence of estradiol, mephedrone produced dose‐dependent rate‐decreasing and error‐increasing effects in the acquisition and performance components in all subjects; however, the higher doses of mephedrone produced greater error‐increasing effects when estradiol was present. Three out of the 5 rats were also more sensitive to the dose‐dependent rate‐decreasing effects of mephedrone in both components when estradiol was present. These data indicate that estradiol can enhance the disruptive effects of mephedrone on learning in female rats.
A comparison of methylphenidate enantiomers on delay discounting in rats
Authors: Slezak Jonathan , Katz Jonathan L.
Published: June 2021

Three forms of methylphenidate (MPH) are currently used in treating attention‐deficit hyperactivity disorder (ADHD): Ritalin (dl‐MPH), Focalin (d‐MPH), and Daytrana (dl‐MPH), a transdermal system producing higher levels of l‐MPH than dl‐ MPH as it avoids the first‐pass metabolism of oral administration. The current study compared MPH enantiomers using a delay‐discounting procedure in rats which is thought to assess “impulsivity” (a key characteristic of ADHD). Sprague‐Dawley rats (n=5) were trained to emit either of two responses, one producing an immediate 45‐g food pellet, the other producing four such pellets delivered at increasing delays (0, 8, 32 s). The percent selection of the larger food amount was graphed as a function of delay with the area under the curve (AUC) assessed. Increases in AUC are consistent with decreases in “impulsivity” (i.e., selection of the smaller, immediate over the larger, delayed reinforcer). Systemic (i.p.) administration of dl‐MPH and d‐MPH dose‐dependently increased AUC, while l‐MPH, morphine and pentobarbital did not change the AUC. The present results indicate that delay discounting can be used to identify treatments for ADHD, and that d‐MPH, but not l‐MPH, is active in this procedure, which is in agreement with the current understanding of the therapeutic actions of MPH.
TRPV1 Channels In The Heart: A Novel Redox Sensor?
Authors: Kmetz John G. , Ohanyan Vahagn , Enrick Molly , Kang Patrick T. , Chen Chewen-Lih , Chen Yeong-Renn , Bratz Ian N.
Published: June 2021

We have previously demonstrated TRPV1 coupling of myocardial blood flow (MBF) to metabolism is disrupted in diabetes. Accordingly, we hypothesized that enhanced oxidative stress in diabetic cardiomyopathy (DCM) modifies TRPV1 via an H2O2/20‐ HETE dependent pathway during DCM leading to modulation of MBF. Control, db/db and TRPV1 null mice (TRPV1−/−) were subjected to contrast echocardiography for quantification of MBF at baseline and during the infusion of the 20‐HETE (1–200 μg/kg). 20‐HETE increased MBF in controls but was inhibited by TRPV1 antagonism (SB366791) and significantly blunted in db/db mice. Robust dilation was observed in isolated coronary microvessels to H2O2 or 20‐HETE from controls. This response was blunted in microvessels from db/db, TRPV1−/− and in controls after SB366791, suggesting 20‐HETE and H2O2 induced vasodilation in part via TRPV1 activation. The redox status of the myocardium was measured by the redox activity of converting hydroxylamine (CM‐H) to stable nitroxide using EPR. We found myocardium from db/db mice are more oxidized, showing a 27% elevation in the redox status. Superoxide‐dependent and H2O2‐dependent redox status was increased by 18.4% and 124.5%, respectfully in the myocardium of db/db mice compared to controls. We suggest that enhanced redox activity in DCM alters TRPV1 signaling leading to disruption of the TRPV1‐mediated coupling of MBF to metabolism.
Increased tortuosity promotes platelet activation and thrombus formation in microvessels
Authors: Chesnutt Jennifer , Han Hai-Chao
Published: June 2021

Thrombosis is a major contributor to cardiovascular disease. Vessel tortuosity likely promotes thrombosis by high fluid shear stress. However, the underlying microscale physical mechanisms are poorly understood. The objective of this research was to use a new computational approach to elucidate physical mechanisms of thrombus formation in tortuous microvessels. A discrete element method was used to computationally simulate shear‐induced activation, receptor‐ligand adhesion, collision, and transport of individual platelets in thrombus formation within tortuous arterioles and venules. Computational results agreed with in vivo experimental studies, which showed that thrombus was initiated preferentially at inner walls of curved regions in venules. Increased venule tortuosity hastened thrombus initiation. Flow in an arteriole, compared with an identically‐shaped venule, generated higher amounts of mural thrombi and platelet activation. In arterioles with activated platelets, the location with the largest amount of thrombi switched from the outer wall to inner wall as tortuosity increased. In conclusion, tortuosity modified both trajectories and activations of platelets to regulate the process of thrombus formation in microvessels. This research was supported by NIH T32HL04776, HL095852, and NSF CAREER award 0644646.
Insulin ameliorates collagen remodeling after myocardial infarction
Authors: Tian Na , Zhang Yuan , Fu Feng , Yan Wenli , Gao Feng
Published: June 2021

Aim

To investigate whether insulin treatment ameliorates collagen remodeling after myocardial infarction (MI).

Methods

Adult male rats were subjected to the ligation of anterior descending coronary artery and randomized to receive one of the following treatments: Sham, MI+saline or MI+insulin. Myocardial collagen remodeling environment was simulated by TGF‐beta in cultured neonatal rat cardiac fibroblasts.

Results

Insulin significantly improved cardiac function by increased left ventricular ejection fraction after ischemia. Cardiac collagen contents were decreased post‐MI as evidenced by reduced hydroxyproline and collagen volume fraction (P<0.05). Meanwhile, immunohistochemistry staining and Western blot showed that the ratios of myocardial collagen I/III of 1 wk and 4 wk post‐MI were significantly higher than those in the sham hearts (P<0.05). Insulin treatment significantly reduced the ratios of collagen I/III compared with those in saline hearts 1 wk post‐MI (P<0.05), while there were no differences in the ratios of collagen I/III 4 wk post‐MI (P>0.05). Moreover, insulin reduced TGF‐beta induced a‐SMA expression and the phosphorylation of JNK in cardiac fibroblasts.

Conclusion

Insulin ameliorates cardiac collagen remodeling in post‐MI hearts via insulin‐suppressed JNK activation.

Supported by the State Key Program of National Natural Science Foundation of China (No. 81030005).
Identifying novel mechanisms of cardiac myofibroblast phenotype modulation
Authors: Dixon Ian Michael Charles , Cunnington Ryan H , Douville Josette M , Bathe Krista L , Rattan Sunil G. , Freed Darren H , Wigle Jeffrey T
Published: June 2021

Activation of TGF‐β1/Smad signaling plays a role in the phenoconversion of quiescent cardiac fibroblasts to secretory, contractile myofibroblasts. This event is associated with cardiac fibrosis, progressive cardiac dysfunction and heart failure. We have previously shown that c‐Ski, an endogenous inhibitor of TGF‐β1, is able to reduce myofibroblast contractility and collagen type I deposition. Further, c‐Ski overexpression was associated with a diminution of myofibroblastic phenotype. We have now determined that fibroblast ‐ myofibroblast differentiation is associated with a dramatic reduction in mRNA expression of homeobox proteins, Meox1 and Meox2. c‐Ski overexpression in first passage cardiac myofibroblasts was able to rescue Meox2 (but not Meox1) mRNA expression. The reduction of myofibroblast phenotype observed with c‐Ski was mirrored by Meox2 overexpression, while exogenous Meox1 had no significant effect on phenotype. To determine a putative mechanism of c‐Ski induction of Meox2, we examined expression of the Meox2 repressor protein, Zeb2 (eg, Smad‐interacting protein 1) in c‐Ski overexpressing cells. As c‐Ski caused a significant reduction of Zeb2 expression, we identified a putative mechanism for c‐Ski modulation of myofibroblast phenotype eg, that c‐Ski relieves Zeb2‐mediated inhibition of Meox2 thereby diminishing myofibroblast phenotype. Supported by the CIHR and HSFM.
AMP‐activated protein kinase (AMPK) enhances endothelial mediated vascular repair in endotoxin induced pulmonary edema
Authors: Jian Ming-Yuan , Lemley Zachary , Lewis Karen , Creighton Judy
Published: June 2021

Bacterial sepsis is a serious community acquired threat to human health. However, limited therapeutic options are available to treat pulmonary complications related to this condition. While development of vascular pathology associated with sepsis involves interactions of multiple vascular cell types at all anatomic levels of the vessel wall, endothelial cell dysfunction plays an integral role in increased permeability and fluid accumulation. AMPK is a molecular sensor for detection and mediation of cellular adaptations to vascular disruptive stimuli. Yet, little is known about AMPK function in lung endothelium. We hypothesized that AMPK enhances endothelial repair and resolves lung edema associated with vascular exposure to bacterial derived endotoxin lipopolysaccharide (LPS). Time‐lapse microscopy data indicate AMPK activation is necessary for wound resealing in capillary endothelial monolayers and restores repair in the presence of LPS. Indeed, the rate of gap resealing in LPS treated monolayers is similar to control levels following AMPK activation using the pharmacological tool 5‐aminoimidizole‐4‐carboxamide riboside (AICAR). Moreover, LPS‐induced increase in capillary endothelial permeability is decreased to control values by AICAR. In the isolated perfused rat lung, LPS increases the filtration co‐efficient (Kf) that is attenuated by AMPK activation. Supported by HL102296.
The βAR‐PI3K signaling pathway crosstalk: differential effects on phosphorylation of p70S6K and FoxO in newborn and adult hearts
Authors: Tseng Yi-Tang , Zhang Weizhi , Yano Naohiro , Mao Quanfu , Deng Minzi , Padbury James F
Published: June 2021

Cardiac β‐adrenergic receptors (βAR) and PI3K signaling pathway are highly regulated during development. They are both with low activity in adult heart but chronic βAR stimulation leads to hypertrophy, likely via activation of PI3K pathway. We have shown acute βAR stimulation results in cardiac‐specific activation of PI3K signaling pathway. They are both with high activity in fetal/newborn heart but the biological impact from the crosstalk is not clear. To further examine the signaling cascades induced by such crosstalk in developing heart, newborn mice were injected with propranolol (ip, 10 mg/kg, 30 min). As expected, acute βAR blockade induced significant decreases in PI3K activity and phosphorylation of Akt and GSK‐3α/β. However, there was paradoxical increases in phosphorylation of p70S6K (T421/S424), FoxO1 (S256) and FoxO3a (S318/321). While acute βAR stimulation in adults leads to an increase in Erk1/2 phosphorylation, βAR blockade in newborn did not have effect. Our preliminary results suggest a significant reduction in cardiomyocyte proliferation in newborns subject to acute βAR blockade. We speculate what we observed in the subtle differences in phosphorylation of downstream factors in PI3K signaling pathway may play critical in determining the biological outcome (hypertrophy or proliferation) following βAR stimulation or inhibition in the adult and newborn, respectively. (NIH 1 P20RR018728)
The NHE3 interacting PDZ protein NHERF2 determines the lipid raft association of the apical Na+/H+ exchanger NHE3 in murine small intestine
Authors: Sultan Ayesha , Riederer Brigitte , Xia Weiliang , Chen Mingmin , Lamprecht Georg , Lissner Simone , Yun Chris , de Jonge Hugo , Donowitz Mark , Gessner Johannes , Seidler Ursula
Published: June 2021

Background

Intestinal NHE3 is scaffolded and differentially regulated by Na+/H+Exchanger Regulatory Factor (NHERF) PDZ‐adaptors. NHE3 partially resides in lipid rafts of the Brush Border Membrane (BBM).

Aim

To investigate if there is a cross talk between NHERFs and membrane rafts, specifically if NHERFs associate differentially with the raft and non‐raft fraction of NHE3, and whether they influence the function and distribution of NHE3 in these microdomains.

Methods and Results

Murine BBM was detergent‐solubilised and lipid rafts isolated by density gradient floatation. NHE3 was partially, NHERF2 strongly and NHERF1 weakly raft‐associated, and NHERF3 exclusively in the non‐raft fraction. In absence of NHERF2, raft‐associated NHE3 was strongly decreased. Increasing the membrane sphingomyelin/ceramide ratio, increased NHE3‐dependent small intestinal fluid absorption in vivo, as well as overall NHE3 in the lipid rafts, suggesting a positive correlation between NHE3 function and raft association.

Conclusions

In murine small intestinal BBM, NHE3 differentially associates with the different NHERFs in the raft and non‐raft fraction. NHE3 raft‐association is NHERF2‐dependent. NHE3 and NHERF raft‐association and function are modulated by changes in the lipidic environment. This suggests that PDZ‐scaffolding proteins are involved in the retention of membrane proteins within lipid raft platforms.
Dietary Na+ restriction promotes release of an inhibitory tract from the γ ENaC subunit
Authors: Carattino Marcelo D , Mueller Gunhild M , Palmer Lawrence G , Frindt Gustavo , Hughey Rebecca P , Kleyman Thomas R
Published: June 2021

Maturation of ENaC involves furin‐dependent cleavage of the α and γ subunits. Cleavage of the γ subunit by furin at a single site (γRKRK143) and subsequent cleavage by a second protease at a distal site increases ENaC activity. We previously demonstrated that prostasin cleaves the γ subunit at a polybasic tract (γRKRK186). Deletion of the 43 residue tract between the furin and prostasin sites functionally mimics the effect of double cleavage. To determine whether this tract dissociates from processed γ subunits, we generated a polyclonal antibody (Ab) against this region (anti‐γ43). This Ab precipitates both full‐length (93 kDa) and furin‐processed (75 kDa) γ subunits with a C‐terminal V5 tag expressed in Xenopus oocytes. Co‐expression of prostasin resulted in a 70 kDa form detected with an anti‐V5 Ab. The 70 kDa band was not precipitated with anti‐γ43, suggesting that the intervening tract between the furin and prostasin sites had been released. ENaCs in kidney homogenates from rats fed a normal or low Na+ diet were precipitated with either anti‐γ43 or an anti‐γ C‐terminal (anti‐γ) Ab, and then immunoblotted with the anti‐γ Ab. Low Na+ diet increased γ subunit cleavage detected with the anti‐γ Ab. Anti‐γ43 Ab precipitated the full‐length but not the cleaved form of the γ subunit in both groups of rats. Our results suggest that the inhibitory tract dissociates from the γ subunit in kidneys from rats on a low Na+ diet.
Multidrug resistant proteins mediate extracellular ATP release in Caco2 cell monolayers
Authors: Rudenkyy Sergiv , Nakano Takanari , Akiba Yasutada , Kaunitz Jonathan D
Published: June 2021

Epithelial ATP release is an essential component of extracellular purinergic secretory regulation; ATP transporting channels, pannexin (Panx) hemichannels and multidrug resistant proteins (MDR/MRP) have been implicated. The robust intestinal ecto‐ATPase activity present in Caco‐2 cells impairs measurements of medium ATP. We thus transfected c‐Myc‐tagged GPI‐anchored plasma membrane luciferase (pmeLuc) to Caco2 cell monolayer to monitor ATP release at the cell surface. Confluent Caco‐2 cell monolayers were transfected with pmeLuc, confirmed by immunolocalization. Although transfection efficacy into Caco2 cell is ~ 10 %, much less than in HEK293 ~ 80 %, luminescence was measurable over the background (~ 3000 in Caco‐2, ~ 30,000 in HEK293). Luminescence was stable over 40 min, suggesting constitutive ATP release. Among the pharmacological reagents reported as inhibitors for ATP release, MK571, a specific MDR/MRP inhibitor dose‐dependently inhibited ATP release. Stilbene anion transport inhibitors or Panx inhibitors had no effect. Vesicles expressing P‐glycoprotein (Mdr1) and MRP4, but not MRP2 accumulated ATP inside. MDR/MRP mediates ATP release in intestinal epithelia, a novel mechanism for transmembrane ATP transport. Regulation of ATP release may have important implications for the physiological functioning of intestinal and other secretory epithelia. NIH‐NIDDK R01 DK54221 & R21 DK081199