bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamin and caveolae in cardiac ischemic preconditioning
Authors: Cipta Stephanie , Tsutsumi Yasuo M , Kidd Michael W , Niesman Ingrid R , Panneerselvam Mathivadhan , Roth David M , Patel Hemal H
Published: June 2021

Caveolae are specialized membranes enriched in lipids and proteins that regulate numerous signaling processes. Caveolae participate in clathrin‐independent endocytosis that is regulated by dynamin‐2, large GTPase proteins that induce membrane fission. We have previously shown that ischemic preconditioning (IPC) induces protection in cardiac myocytes via modulated formation of caveolae; however, the dynamics of caveolae formation and internalization by IPC is unknown. We hypothesize that IPC leads to the localization of dynamin to caveolae in cardiac myocytes. Adult male mice underwent 5 minutes of cardiac ischemia followed by 15 minutes of reperfusion or 20 minutes of sham surgery. For cell fractionation and SDS‐PAGE analysis, hearts were excised and homogenized in sodium carbonate buffer. The hearts that were used for immunofluorescence experiments were perfused and prepared for cryo‐sectioning. Western blot analysis of whole cell lysates confirmed equal dynamin expression in both samples. Following cell fractionation, there were higher levels of dynamin in caveolae rich fractions of IPC samples than in sham samples. Immunofluorescence images showed decreased cytosolic dynamin and increased amounts of dynamin along the plasma membrane, with caveolin‐3 co‐localization in IPC cardiac myocytes relative to sham cardiac myocytes. Our data suggests that IPC causes cytosolic dynamin to migrate to caveolae in cadiac myocytes, such migration may be key to modulation of caveolar mobilization from the plasma membrane that can affect cardiac protection.
Heme oxygenase‐1 induction in rat spleen following aniline exposure
Authors: Khan M. Firoze , Ma Huaxian , Wang Jianling , Ansari G. A. Shakeel , Boor Paul J.
Published: June 2021

Splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis and development of a variety of sarcomas in rats. However, mechanisms underlying splenic toxicity are not well understood. Previously we observed that aniline exposure causes oxidative damage to the spleen. To further explore the involvement of oxidative mechanism in aniline toxicity, we evaluated the role of heme oxygenase‐1 (HO‐1), which catalyzes heme degradation leading to the formation of biliverdin, carbon monoxide and iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1 or 7 days, while the controls received water only. HO‐1 quantitation by ELISA showed a 2.6 fold increase in spleens of rats treated with aniline for 7 days. These increases were supported by Western blot analysis (~2.5 fold increase in HO‐1 expression). No changes were observed in animals exposed for 1 day. Immunohistochemical analysis showed greater staining for HO‐1 in the red pulp areas of spleen from rats treated for 7 days. Increase in HO‐1 expression was associated with 1.9 fold increase in ferritin, 2.7 fold increase in total iron and, more importantly, a 3.5 fold increase in free iron in spleens of rats exposed for 7 days. It is evident from our data that increased HO‐1 expression and its association with iron accumulation could be an important pro‐oxidant mechanism leading to oxidative damage in the spleen. Supported by ES006476.
Development of LHRH‐Au@SPIONs for Biomedical Applications
Authors: Mohammad Faruq , Gopalan Balaji , Raghavamenon Achuthan C , Uppu Rao M , Kumar Challa S.S.R.
Published: June 2021

During recent years, Super Paramagnetic Iron Oxide nano particles (SPIONs) have been raised a great deal of interest because of their use in a number of bio‐medical applications such as targeted drug delivery, sensitive diagnostic tools, contrast agents for Magnetic Resonance Imaging (MRI), protein purification studies and lab‐on‐a‐chip devices. We have been interested in the development of ligand bound SPIONs as contrast agents for MRI of breast tumors and their metastases (Leuschner et al., Breast Cancer Res. Treat. 99, 163‐176, 2006). The Luteinizing‐Hormone Releasing Hormone (LHRH) has been found to be an excellent targeting agent for breast tumors and their metastases and hence LHRH‐SPIONs have been shown to enhance the contrast efficiency in MRI. In order to modulate magnetic properties of SPIONs through gold coating, we have synthesized gold coated SPIONs and established an approach to bind LHRH to gold surface through cysteamine linkage as shown below.

We have characterized the LHRH‐Au@SPIONs using TEM, X‐ray absorption spectroscopy and SQUID magnetometer. These particles are currently being evaluated for their cytotoxicity and inflammatory cytokine release using cell‐based assay systems. In addition to the toxicity results, investigations from their targeting ability in vitro using breast cancer cells followed by their MRI contrast efficiency will be presented.
Dietary n‐6 PUFA deprivation for 15 weeks alters expression and activity of enzymes of arachidonic acid and docosahexaenoic acid cascades in rat frontal cortex
Authors: Kim Hyung‐Wook , Igarashi Miki , Rao Jagadeesh S , Gao Fei , Rapoport Stanley I
Published: June 2021

n‐3 and n‐6 PUFAs are critical for brain function. Few studies have addressed the effects of n‐6 PUFAs deficient diet on brain, while many studies have examined n‐3 PUFA deficient diets. Previously, we reported that n‐3 PUFA deprivation for 15 weeks in rats increased the AA cascade enzyme ‐ cPLA2, sPLA2 and COX‐2‐expressions, but decreased the DHA cascade enzyme, iPLA2. We also reported that 15 weeks of n‐6 PUFA deprivation increased DHA and decreased AA in brain. Based on these, we hypothesized that n‐6 PUFA dietary deprivation would increase the DHA cascade enzymes while decreasing the AA cascade enzymes. We fed male rats an n‐6 PUFA adequate or an n‐6 PUFA deficient diet for 15 weeks after weaning, and then measured enzyme activity, expression levels of PLA2s and expression of their transcription factors. Fifteen weeks of n‐6 PUFA deprivation significantly increased the enzyme activity, expression levels of iPLA2 but decreased those of cPLA2 and COX‐2. These effects correlated with an increased protein level of an iPLA2 transcription factor SREBP‐1, and a decreased protein level of a cPLA2 transcription factor, NF‐κB. Expression levels of sPLA2 and COX‐1 were unchanged. The results suggest that dietary n‐6 PUFA deprivation leads to enzyme changes, at the transcriptional level, that would tend to reduce AA metabolic loss from brain, while increasing DHA metabolic loss. The increased iPLA2 expression and decreased cPLA2 and COX‐2 expression are in the opposite direction to changes caused by dietary n‐3 PUFA deprivation. Thus n‐3 and n‐6 PUFAs within brain have reciprocal effects, at the level of transcription, on expression of enzymes that regulate brain AA and DHA metabolism. This work was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.
Inter‐meal Snack Consumption of A Soybean Product Does Not Inhibit Post‐prandial Fat Oxidation
Authors: Iwashita Soh , Suyama Toru , Hamada Koichiro , Hujii Hisao
Published: June 2021

We presented that soybean consumption as an inter‐meal snack did not inhibit fat oxidation compared to wheat (EB2008). In this study, we tested whether inter‐meal snack of a soybean product which has low glycemic and insulinemic responses affect substrate oxidation. Thirteen healthy male volunteers (20.2 yrs, BMI= 22.3 kg/m2) attended three test sessions, either non‐snack (Non), rice cracker (R) or soybean product (S), in a randomized cross over design with one‐week of wash out period. Breakfast was served at 7:30 AM. Subjects started to be measured metabolic rates in the respiratory chamber at 11:15 AM after ~30 min or resting. Then subjects ate lunch at 12:00 PM and test snack (11.3 kJ/kg) at 3:00 PM in S and R sessions. Oxygen consumption and carbon dioxide production in the chamber had been monitored until 9:00 PM to calculate energy expenditure (EE) and substrate oxidation rates (OX). EE and substrate OX before test snack were similar between three treatments. Cumulative EE after snack timing was not different among three treatments. Fat OX in Non was maintained at pre‐snack levels. R inhibited fat OX at 120‐180 min after the consumption (P<0.05), while S did not inhibit during post‐snack phase. Thus, cumulative fat OX was lower in R (P<0.05), but not in S compared to Non. On the contrary, cumulative carbohydrate OX was higher in R than in Non and S (P<0.05). These findings suggested that the snack of a soybean product did not disturb the acceleration of postprandial fat OX.
Mice expressing ouabain‐sensitive α1 Na,K‐ATPase have increased susceptibility to pressure overload‐induced cardiac hypertrophy
Authors: Wansapura Arshani N , Lasko Valerie , Lingrel Jerry B , Lorenz John N
Published: June 2021

Endogenous cardiotonic steroids (CS) are elevated in a variety of cardiovascular disease states, but their (patho)physiological role is not well understood. In addition to their conventional role as Na,K‐ATPase inhibitors, studies suggest that CS binding to the Na+ pump may activate signaling pathways that induce cardiac growth, implicating a role in the development of cardiac hypertrophy. In the present study, we hypothesized that the hypertrophic response to pressure overload would be aggravated in mutant mice with a ouabain‐sensitive α1 Na,K‐ATPase subunit (αNKA). We performed transverse aortic constriction (TAC) surgery in α1‐resistant/α2‐resistant (α1R/R α2R/R), α1‐sensitive/α2‐resistant (α1S/Sα2R/R), and α1‐resistant/α2‐sensitive mice (α1R/Rα2S/S, wild type) to induce pressure‐overload left ventricular (LV) hypertrophy. At 4 weeks, echocardio‐graphy revealed a pronounced increase in LV mass and wall thickness in the α1S/Sα2R/R mice. Terminal analyses of histology and function demonstrated extensive perivascular and replacement fibrosis and significantly depressed LV systolic function in α1S/Sα2R/R mice compared to α1R/Rα2S/S, α1R/Rα2R/R and sham‐operated mice. These data reveal that mice expressing a ouabain‐sensitive α1NKA subunit have a dramatically enhanced susceptibility to pressure overload‐induced cardiac hypertrophy.

(NIH grants DK57552 & HL28573)
Relationship of resting energy expenditure to body composition and clinical health markers
Authors: Kerksick Chad , Beavers K , Chandran R , Jitomir J , Serra M , Shelmadine B , Wilborn C , Wismann J , Nassar E , Dove J , Galbreath M , Harvey T , La Bounty P , Campbell B , Cooke M , Iosia M , Rasmussen C , Wilson R , Kreider R
Published: June 2021

848 sedentary, obese women (47±11 yrs, 92±18 kg, 163±7 cm, 45±4% body fat) were medically screened prior to participation in an exercise and weight loss program. Medical data and fasting blood samples were collected and a correlation analysis was used to examine relationships between resting energy expenditure (REE), body composition and serum markers of health and safety. Baseline REE levels were significantly (all p<0.001) correlated to systolic blood pressure (r=0.093), diastolic blood pressure (r=0.199) and resting heart rate (r=0.166) in addition to higher levels of body mass (r=0.666), DEXA fat mass (r=0.569), DEXA fat‐free mass (r=0.694), DEXA % body fat (r=0.195),waist circumference (r=0.423) and hip circumference (r=0.549). In addition, REE values were positively correlated (p<0.001 to 0.010) to triglycerides (r=0.104), total cholesterol: HDL ratio (r=0.112), uric acid (r=0.130) and negatively correlated to HDL cholesterol (r= ‐0.161). REE levels were not correlated to total cholesterol (r= ‐0.038; p=0.29), LDL cholesterol (r= ‐0.012; p=0.75) and glucose levels (r=0.060; p=0.10). Data from this large sample provide continued support that management of REE levels is intimately related to several variables central to obesity and heart disease.
Gastric G cell‐based gene therapy for high‐fat induced type 2 diabetic mice
Authors: Lu Yu‐Chun
Published: June 2021

Transgenic G‐InsKi mice in which human insulin is released portally by gastrin‐producing antrum G cells in response to protein‐rich meals, but not glucose, exhibited prolonged lifespan when crossed to Akita type 1 diabetes mice. This study investigates the influence of gastric insulin in high‐fat (HF)‐induced type 2 diabetes mice. 6 G‐InsKi mice and 6 wild‐type (WT) mice were individually housed and fed either a lard enriched HF (60% en) or a low‐fat (LF, 20% en) diet ad libitum for 10 weeks. Our results showed that mice with a LF diet had a higher food consumption than HF, regardless of gastric insulin (HF‐WT: 8.4g/d; HF‐G‐InsKi: 7.8g/d; LF‐WT: 12.9g/d; LF‐G‐InsKi: 12.6g/d; p<0.05). However, no difference was found among groups when feed intake was expressed as kcal/day (p>0.05). All animals showed similar non‐fasting blood glucose levels and body weight change (p>0.05). Longer exposure to HF diet might be needed to induce diabetic abnormalities in this murine model.
Cardiac Output in Neonates, Noninvasively
Authors: Weaver Barbara Smith , Rodriquez Misael , Bozeman Andrew , Vogel Robert
Published: June 2021

Cardiac Output in Neonates, Noninvasively

Barbara Weaver, R.N.1, Misael Rodriguez, M.D.1, Robert L. Vogel, PhD.2, Andrew P. Bozeman, M.D.2, and Joseph M. Van De Water, M.D.2 Departments of 1Pediatrics and 2Surgery, Mercer University School of Medicine at the Medical Center of Central Georgia, Macon, GA, 31201.

Our objective was to assess a new noninvasive monitor for measuring cardiac output (CO) in neonates down to 590 grams. We used a new electrical impedance device, Aesculon® (Cardiotronic, Inc., 7855 Ivanhoe Avenue, Suite 226, La Jolla, CA 92037). The technology, Electrical Cardiometry, measures the differences in impedance presumably originating in red blood cell orientation between systole and diastole in the ascending and descending thoracic aorta in order to derive stroke volume (SV). Measurements on 75 neonates, ranging in weight from 590 to 4010 grams, were evaluated. Intra‐ and inter‐operator reproducibility was assessed from a sample of subjects. The correlation of both CO and SV to body mass (wt), body mass index (BMI) and body surface area (BSA) was computed. There were no problems using single electrode placement along the left side of the body at scalp, base of neck, xyphoid and thigh levels. Babys' tolerance was good and device operation was easy, facilitating parental consent. Reproducibility was good with a variance component for operator of less than 1% and for time of less than ½% of total variability for both CO and SV. Standards for CO in this sample of neonates (n = 75) correlated equally well with wt, BMI and BSA (r = 0.89, p = 0.0001). However, the best correlation was SV vs. wt and BSA (r = 0.93, p = 0.0001). This study suggests that a very useful noninvasive neonatal CO monitor now exists.
N‐acetylcysteine (NAC) Improves but does not Prevent the Appearance of Heart Failure in Syrian Cardiomyopathic Hamsters.
Authors: Cruz Nildris , Quidgley Jose , Escobales Nelson , Altieri Pablo , Crespo Maria J.
Published: June 2021

Oxidative stress has been postulated to contribute to the etiology of heart failure (HF). The efficacy of antioxidant therapy in HF, however, remains controversial. This study evaluates the effect of the antioxidant N‐acetylcysteine (NAC, 1g/kg/day) on cardiovascular function in 2‐ and 6‐month‐old Bio‐TO2 Syrian cardiomyopathic hamsters (SCH) after 1‐month and 5‐month treatment with this drug. Endothelial function, systolic blood pressure (SBP), and echocardiographic parameters were evaluated. One month of NAC administration normalized the increased SBP observed in 2‐month‐old SCH (n=5, P<0.05). Five‐month treatment with NAC, improved 24% (P<0.05) the EMax value from the Ach‐induced relaxation (from 45.8± 4 % to 56.1 ± 2 % n= 7) while did not modify EC50 or SBP. Ejection fraction increased 39±4 % (n=11, P<0.05) and cardiac output index improved but did not reach statistical significance. In addition, LVEDV and LVESV were significantly reduced by NAC (P<0.05), These results suggest that antioxidant therapy alone improves cardiovascular function in this animal model of dilated cardiomyopathy. The treatment, however, does not prevent the appearance of HF. Supported by NIH‐SCORE S06‐RR 08224.
Low‐dose aspirin and clopidogrel attenuate reflex cutaneous vasodilation in humans
Authors: Jennings John D. , Kenney W. Larry , Holowatz Lacy A.
Published: June 2021

Chronic low‐dose aspirin (ASA) significantly attenuates reflex vasodilation (VD) in humans potentially through vascular and platelet cyclooxygenase (COX) inhibition. We hypothesized that unlike aspirin, specific platelet ADP receptor inhibition with clopidogrel (Plavix) would not attenuate reflex VD. In a double‐blind crossover study, five subjects (50 ±2 years) were instrumented with two microdialysis (MD) fibers and heated to increase core temperature (Tc) 1°C (water‐perfused suit) during control, after 7 days of ASA (81 mg), and after 7 days of Plavix (75 mg) with a 2 week washout period. Skin blood flow was measured by laser‐Doppler over MD sites perfused with Ringers and 10mM L‐NAME, respectively. Data were normalized and presented as a percentage of maximal cutaneous vascular conductance (%CVCmax: 28mM SNP). During control, plateau CVC was 55 ±5 %CVCmax. ASA (ASA: 45 ±5%CVCmax; p<0.05) and Plavix attenuated reflex VD (Plavix: 33 ±4%CVCmax; p<0.01). L‐NAME attenuated VD in control (control: 31 ±5%CVCmax; p<0.01) but not in ASA or Plavix (ASA: 31 ±5; 30 ±7%CVCmax; both p>0.05). ASA and Plavix reduced the time required to increase Tc 1°C (control: 61±2 vs. ASA: 43±2; Plavix: 39±3 min; both p<0.001). ASA‐induced COX‐ and specific platelet ADP receptor‐inhibition attenuate reflex VD, suggesting platelet involvement in reflex VD through either the release of VD factors or through shear stress. R01‐AG‐07004‐19
Critical Role of Dendritic Cells in Wound healing after Myocardial Infarction in CD11c‐DTR‐GFP Mice
Authors: Pruthi Dafina , De Muinck Ebo , Fu Qiangwei , McGarr Robert , Moodie Karen
Published: June 2021

Dendritic Cells (DC) are known primarily for their function as antigen presenting cells. Recently it has been shown that DC acquire an endothelial phenotype and contribute significantly to tumor angiogenesis and were named vascular leukocytes (VLC). Our laboratory has shown in mice that VLC appear as early as one day after MI in the healing heart and that they reach their peak at day 7 in infarcted area which coincides with the peak of angiogenesis in this model. We asked if early depletion of DC which are the precursors of VLC, would result in changes in wound healing.

Methods

We investigated cardiac wound healing in CD11c‐DTR‐GFP transgenic mice at day 7 after inducing MI with and without depletion of CD11c DC (transgenic mouse has a transgene encoding simian Diphtheria toxin receptor under control of murine CD11c promoter, allows to specifically deplete CD11c DC by injecting Diphtheria toxin (DT)). Mice were randomized to injection with 8ng/g i.p. of DT just before MI surgery or PBS. We measured functional cardiac recovery (%ejection fraction‐ EF%) with echocardiography; cardiac diastolic and systolic function with pressure volume loops system (PV‐loops); morphometry to assess myocardial necrosis on the TTC stained slices.

Results

Echocardiography: average EF% at day 7 for mice with intact DC and depleted DC is 27% and 49% respectively, n=16, p=0.001. Morphometry: MI% at day 7, 57% and 19.4% respectively, n=16, p=0.001. PV loops: Cardiac Output (uL/min) at day 7, 7826 and 3161, End‐diastolic Pressure (mmHg) at day 7, 9.4 and 17.1

Conclusion

CD11c+ DCs play a significant and previously unrecognized role in post‐MI wound healing with DC depletion after MI resulting in a significant reduction of myocardial necrosis and a significantly better functional recovery of the heart.
Characterizing the role of RGS5 in regulating the myogenic vasoconstrictor response
Authors: Tirgari Sam , Chan Karen , Heximer Scott P
Published: June 2021

The myogenic response is an established, but poorly understood, mechanism mediated by arteriolar vascular smooth muscle cells (VSMCs). This mechanism is important for both the maintenance of resting vascular tone and the protection of microvascular beds from pressure‐induced injury. Recent evidence supports a role for G‐proteins in mediating this process. Accordingly we hypothesize that regulator of G‐protein signaling proteins that are highly expressed in VSMCs contribute to the regulation of the myogenic response. One such protein, RGS5, is highly expressed in mural cells of arterial vessels and microvasculature. To examine the potential role of RGS5 in the modulation of myogenic constriction, perfusion experiments were conducted on pressurized mesenteric arteries from wild type and RGS5 knockout mice. In support of a role for RGS5 regulating this process, RGS5‐deficient vessels show a trend towards increased sensitivity to elevated pressures and a greater extent of pressure‐induced vascular tone compared to wild type controls. The RGS‐dependent effect on vascular tone is specific to the myogenic response since wild type and knockout vessels responded similarly to phenylepherine and acetylcholine. These data suggest the need to further evaluate the molecular mechanisms whereby RGS5 regulates myogenic constriction in VSMCs.
Microvascular response to cell free Hb infusion in transgenic mouse model of sickle cell anemia
Authors: Tsai Amy G. , Cabrales Pedro , Young Mark A. , Winslow Robert M. , Intaglietta Marcos
Published: June 2021

Targeting O2 delivery to anoxic regions may reduce the incidence of complete deoxygenation of the RBC and thus be an effective therapy in sickle cell disease. Knockout transgenic mice (n = 6) were treated with an infusion of MP4, a blood substitute that targets oxygen delivery to hypoxic areas (malemide polyethylene glycol‐conjugated Hb, Hemospan®, Sangart Inc., San Diego, CA) and then subjected to a hypoxic challenge (FiO2 = 0.08). Results were compared to a control group which received saline (n = 5). A 10% blood volume (BV) topload was infused (BV estimated as 6% body weight). Changes of microvessel diameter and functional capillary density (number of capillaries perfused per area of tissue) were studied in vivo using the dorsal skin fold window chamber model. Mean arterial pressure was higher after treatment during normoxia and hypoxia challenge as compared to the control group. Arterioles dilated and venules remained unchanged from baseline during hypoxia in both study groups. FCD was statistically reduced from baseline during hypoxia; however MP4 infused animals maintained FCD at a level that was statistically higher than observed with saline infusion; 60 ± 15% and 27 ± 24%, respectively. Thus MP4 treatment reduces the severity of hypoxia‐mediated decline in FCD. Mechanistically the higher FCD could be due to the higher perfusion pressure in the absence of vasoconstriction. Reduced sickling and inflammatory responses (i.e., lesser leukocyte‐adhesion) are likely concomitant mechanisms which support and enhance capillary perfusion.
ASIC2 Subunits Target Acid‐Sensing Ion Channels to the Synapse via an Association with PSD‐95
Authors: Zha Xiangming , Price Margaret P , Costa Vivian , Harding Anne M , Reznikov Leah , Benson Chirstopher J , Welsh Michael J
Published: June 2021

Acid‐sensing ion channel‐1a (ASIC1a) mediates H+‐gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomomes. This localization depended on ASIC2a binding to PSD‐95 and matched that of ASIC1a, which does not bind PSD‐95. We found that ASIC2 and ASIC1a associated in brain, and through its interaction with PSD‐95, ASIC2 increased ASIC1a localization in dendritic spines. Loss of ASIC2 reduced the acid‐evoked elevation of [Ca2+]i in dendritic spines and impaired fear conditiong, both of which require ASIC1a. These results indicate that ASIC2 facilitates ASIC1a localization and function in dendritic spines and thereby fear‐related behavior.
Developing metacognitive skills through the use of blogs in an upper level undergraduate anatomy course
Authors: O'Loughlin Valerie Dean , Griffith Lauren Miller
Published: June 2021

Incorporating metacognitive approaches into classroom instruction helps students to monitor and adjust their learning strategies throughout the semester. Metacognitive tasks like journaling serve the dual functions of being reflection pieces for students and assessment instruments for instructors.

This research examined how the use of “blogs” (online journals) in an upper level human anatomy course (a) facilitated the development of metacognitive skills in the students and (b) provided the researchers insight about the ways students think about their own progression in understanding human anatomy. Specifically, did students become more self‐aware and cognizant of their strengths and weaknesses in mastering anatomy as the semester progressed? Twenty students reviewed 10 radiology online cases each throughout the semester and then reflected about their understanding of the anatomy, pathology and medical imaging interpretation in an online blog. Researchers utilized a grounded theory approach to develop a codebook that scored each line of each blog. Codes were mutually exclusive and Cohen's Kappa was used to test the inter‐rater reliability of the codebook.

Students became more confident and assessed their skill in identifying anatomy and pathology more positively as the semester progressed. Students started the semester with more anxiety and frustration comments in their blogs and primarily used guessing to evaluate cases. By the end of the semester, blogs had evidence of self‐confidence and positive, accurate reflections about their understanding of the anatomy. Furthermore, the students described and used a more methodical deductive approach to evaluate each case. The use of blogs made the development of students' metacognitive skills visible and allowed the researchers to methodically assess this development using a grounded theory approach.
DEP‐Induced Changes Observed in Early‐stage Volume Overload Heart Failure Cardiomyocytes
Authors: McAllister Mary Catherine , Wold Loren E , Lucchesi Pamela A
Published: June 2021

Increases in fatal cardiopulmonary dysfunction in cities with high levels of air pollution may be caused by the exacerbation of underlying heart conditions. The effect of Diesel Exhaust Particulates (DEP) in vitro on cardiomyocyte function was observed to determine if increased susceptibility occurs in heart failure animals. Aortocaval fistula (ACF) surgery was performed on rats to induce volume overload heart failure after 21 weeks. Myocytes were isolated 4 weeks post‐surgery, treated one hour with 1 μg/ml DEP, stimulated at 1 Hz, and analyzed based on sarcomere length and cytosolic calcium levels. No significant changes were observed during the cell contractile phase. DEP exposed ACF myocytes released significantly more calcium from the sarcoplasmic reticulum (SR) during contraction than Sham cells. Thus, Ryanodine receptors in strained ACF cells may be more sensitive to hyper‐phosphorylation. Calcium reuptake by the SR was also significantly faster in myoctyes from Sham versus ACF animals when treated with DEP. This indicates a possible decrease in SERCA2a function or expression. Unexpectedly, alterations in calcium transients did not correspond with changes in overall contractility. Future studies will be performed to investigate a compensatory mechanism involving the affinity of Troponin C for cytosolic calcium during contraction.

Funding was provided by the American Physiological Society.
Wnt3a/beta‐catenin increases proliferation in valve interstitial cells
Authors: Xu Songyi , Gotlieb Avrum
Published: June 2021

Background

Valve interstitial cells (VICs) mediate valve function in health and disease. It is suggested that valve repair processes are regulated by developmental pathways. We hypothesize that Wnt3a increases the amount of stabilized β‐catenin present in VICs, which then results in its nuclear translocation and subsequent increase in proliferation.

Methods

Porcine VIC cultures were maintained in standard media in 10% fetal bovine serum. Cultures were plated, grown to 4–6% confluence, then incubated with 100 ng/ml of exogenous Wnt3a for 3 days, and counted to measure proliferation. VICs were stained with antibody against β‐catenin. Whole cell, cytoplasmic and nuclear levels of β‐catenin were determined using Western blot.

Results

VICs treated with Wnt3a show increased cell number compared to control and vehicle‐treated cultures. Immunofluorescent images of Wnt3a‐treated VICs show slightly increased β‐catenin staining compare to control and vehicle‐treated cultures. Whole cell, cytoplasmic, and nuclear lysates of Wnt3a‐treated VICs show increased levels of β‐catenin compared to control and vehicle‐treated cultures.

Conclusion

Wnt3a may increase VIC proliferation through an increase in β‐catenin.

Supported by the Heart and Stroke Foundation of Ontario (grant NA6204) and Canadian Institute of Health Research (grant 84228).
Intense inflammatory response and high mortality rate are associated with dystrophin reduction in Trypanosoma cruzi‐infected mice
Authors: Prado Cibele Maria , Celes Mara Rubia , Malvestio Lygia MM , Pulici Erica C , Blefari Valdecir , Silva Joao S da , Jelicks Linda A , Tanowitz Herbert B , Rossi Marcos A
Published: June 2021

Heart failure is an important cause of morbidity and mortality in both acute and chronic phases of Chagas' disease. Taking into account that the contractile machinery inside the myocyte must remain intimately connected with the membrane and extracellular matrix, association provided by the dystrophin glycoprotein complex (DGC), this study was carried out to test the hypothesis that acutely infected murine hearts present changes of dystrophin expression. C57Bl/6 mice were infected with Y strain of T. cruzi. Histological analyses, immunohistochemistry (IH) for T. cruzi, immunofluorescence staining and Western blot for dystrophin were performed. Parasitemia peak at day 9 and the mortality rate peaked on day 20. The inflammatory infiltrate was present at day 14 and peaked at day 20. The IH for T. cruzi showed parasite persistence up to 20 days pi and then it became rare. Dystrophin was focally reduced at day 14 and completely lost 20 days pi. A correlation between the amount of dystrophin and mortality rate was observed. The peak of mortality was correlated with the focal loss of dystrophin. Our results lend support to the hypothesis that changes in cytoskeletal proteins and, in particular, dystrophin could represent the partial loss of the mechanical binding of the entire array of myofibrils to the extracellular matrix, thus providing a final common pathway for contractile dysfunction in these mice.
Early ventricular dysfunction in acute doxorubicin‐induced cardiotoxicity in rats
Authors: Polegato Bertha Furlan , Roscani Meliza Goi , Minicucci Marcos Ferreira , Azevedo Paula , Campana Álvaro , Pereira Elenize , Ferreira Ana , Matsubara Beatriz , Matsubara Luiz
Published: June 2021

Cardiotoxicity is the major side effect of Doxorubicin (Doxo). The acute damage to the myocardium is poorly understood. The aim of this study is to analyze in vivo and in vitro cardiac function early after Doxo injection in rats. Male Wistar rats received intraperitoneal injection of Doxo (20mg/Kg, n=13) or saline (n=13). In vivo cardiac function was assessed by echocardiography before and 48 hours after injection. In vitro study was performed using isolated heart according to Langendorff preparation. Collagen volume fraction was measured in histological sections of picro‐Sirius‐red‐stained myocardium. Metalloproteinase‐2 (MMP‐2) activity was assessed by zymography. Student t‐test was used for between groups comparisons (significance level: p<0.05). Acutely, Doxo induced a body weight decreasing (338±21g vs 318±18g). In vivo systolic (fractional shortening: 0.59±0.07 vs. 0.51±0.05) and diastolic function (isovolumetric relaxation time: 20.3±4.3ms vs 24.7±4.2ms) was impaired. In vitro study showed preserved myocardial contractility and increased diastolic stiffness (strain: 14.7±1.5% vs 12±2.2%). Collagen volume fraction was unchanged and MMP‐2 activity was increased in Doxo group. We concluded that ventricular dysfunction is present early after doxorubicin injection, although myocardial contractility is preserved. These effects are associated with MMP‐2 activation.
Cardiac myocyte‐specific caveolin‐3 overexpression sustains β‐adrenergic receptor response in basal and stressed conditions
Authors: Busija Anna Rebecca , Chun Beyong Jo , Roth David , Insel Paul A. , Patel Hemal
Published: June 2021

Caveolins are scaffolding proteins essential to the formation of caveolae, plasma membrane invaginations that compartmentalize signaling receptors and effector molecules. Cardiac myocyte‐specific overexpression of caveolin‐3 (Cav‐3 OE) increases the number of caveolae in cardiac myocytes. β‐adrenergic receptors (β‐ARs) are localized to caveolae and when stimulated by agonists undergo rapid desensitization to alter cardiac ionotropy and chronotropy. Whether increased expression of Cav‐3 alters β‐AR agonist response in the myocardium is unknown. To test this question, we exposed Langendorff‐perfused hearts from wild‐type (WT) and Cav‐3 OE mice to sublethal ischemic stress (10 min ischemia, 30 min reperfusion) or control conditions and to the β‐AR agonist isoproterenol (iso). In the basal setting, Cav‐3 OE mice had augmented iso‐stimulated left ventricular developed pressure (LVDP) and +dP/dt and also had reduced heart rate relative to WT mice. Following ischemic stress, which desensitizes β‐AR, the difference between Cav‐3 OE and WT mice was even more exaggerated; Cav‐3 OE mice maintained pre‐ischemic LVDP whereas the WT mice had a diminished response. Therefore, modulating expression of Cav‐3 in cardiac myocytes may provide a means to regulate β‐AR responsiveness in the setting of cardiac dysfunction secondary to ischemia.
Cardiac Muscle Ring Finger‐1 (MuRF1) alters myocardial ROS production in vivo
Authors: Willis Monte S , Rodríguez Jessica E. , Anderson Ethan
Published: June 2021

Muscle ring finger protein‐1 (MuRF1) is a muscle‐specific ubiquitin ligase that regulates cardiac metabolism at multiple points in vivo. We have identified these mechanisms using transgenic (MuRF1 Tg+) and knockout mice (MuRF1 −/−). Microarray analysis of the MuRF1 Tg+ hearts revealed significant alterations in genes involved in mitochondrial oxidative phosphorylation. We therefore investigated the effects of cardiac MuRF1 expression on mitochondrial function. Isolated permeabilized muscle fibers from MuRF1 Tg+ and MuRF1 −/− hearts were assayed for their oxygen utilization in the presence of a number of Kreb's cycle intermediates. Mitochondria O2 consumption from MuRF1 Tg+ and MuRF1 −/− mice did not significantly differ from wild type mice in the presence of pyruvate, malate, glutamate, and succinate (+/−ADP), although MuRF1 Tg+ trended to consume less oxygen. Mitochondria uncoupling was not detected in the presence of oligomycin in either mouse model. Quite unexpectedly, however, mitochondrial hydrogen peroxide production determined by Amplex Red was significantly reduced by >60% in MuRF1 Tg+ hearts, indicating increased MuRF1 inhibited the formation of reactive oxygen species. These findings are consistent with MuRF1's cardioprotective role in ischemia reperfusion injury, whereby the formation of ROS play a critical role in the pathogenesis of disease. Supported by NIH R01HL104129.
Regulation of the ubiquitin proteasome and calpain systems in a dog model of duchenne muscular dystrophy
Authors: Wadosky Kristine M. , Rodriguez Jessica E. , Li Luge , Bogan Dan , Kornegay Joe N , Willis Monte S
Published: June 2021

Duchenne muscular dystrophy (DMD) is an incurable and lethal genetic disorder characterized by progressive muscle wasting caused by the loss of functional dystrophin. Activation of both calpain and the proteasome degradation systems has been reported in the pathogenesis of DMD in skeletal muscle. In fact, calpain and proteasome inhibitors have been used as therapies in mouse models. Here we investigated specific calpain and proteasome activities of 5 skeletal muscles and the left ventricle (LV) in a more severe canine model of DMD (golden retriever muscular dystrophy [GRMD]). The expression of ubiquitin proteasome system components and calpain 1 and 2 were increased in GRMD skeletal muscle generally, but decreased in the LV. We identified significant increases in all three proteasome activities (trypsin‐, chymotrypsin‐, and caspase‐like) in only 1 GRMD muscle and no activation of any proteasome activities in the LV. Calpain 1&2 activities were increased in 2 GRMD skeletal muscles and the LV compared to controls. Furthermore, protease activity of most GRMD skeletal muscles studied correlate with the degree of atrophy and hypertrophy found in the individual muscles. As proteasome and calpain inhibitors continue to be considered as potential therapies for DMD, this work provides evidence for the variable activation of these systems in different skeletal muscles and the heart. Supported by NIH R01HL104129.
HIV Encephalitis: Atypical Biopsy for a Typical Presentation
Authors: Chapman Sherita Nicole , Kurukumbi Mohankumar , Adams Alice , Jayam‐Trouth Annapurni
Published: June 2021

Background

Human Immunodeficiency Virus is neurotropic; approximately two‐thirds of patients with HIV infection will develop central nervous involvement during the course of their disease. Biopsy is still considered the gold standard in diagnosing cranial lesions in HIV patients.

Case Presentation

36 year old male, with a history of HIV infection and recent CD4 count of 65, presented with complaints of weakness and numbness over left face and difficulty speaking for the four days. Clinical examination was normal except for mild flattening of the left nasolabial fold and decreased perception of pain and touch on left side of the face. Imaging revealed a right lateral frontal homogeneous enhancing lesion with surrounding edema. A craniotomy with biopsy was performed. Histopathology revealed acute and chronic vasculitis with focal tissue vacuolization, reactive gliosis and astrocytosis, consistent with HIV encephalitis. Special stains for fungi and Toxoplasma gondii were negative and there was no evidence of neoplasm.

Conclusion

This case report emphasizes the persisting challenge in evaluating cranial lesions in patients with HIV infection, including the gold standard brain biopsy.
Tigecycline attenuates apoptosis and the release of pro‐inflammatory cytokines in Lipopolysaccharide‐treated PC12 cells
Authors: Yagnik Radhi M , Benzeroual Kenza E.
Published: June 2021

Several inflammatory cytokines such as TNF‐alpha, and IL‐1beta have been involved in the neuroinflammation associated with Alzheimer's disease (AD). Tigecycline, a glycylcycline antibiotic and an analog of Minocycline, is shown to exert anti‐inflammatory effects that are distinct from its anti‐microbial activity. In this study, the inhibitory effects of tigecycline on TNF‐alpha and IL‐1beta release in lipopolysaccharide (LPS) treated PC12 (a neuronal cell line) cells were examined using Western blot and ELISA, respectively. Further, the effect of tigecycline on nitric oxide (NO) release was also evaluated. To determine the involvement of apoptotic machinery, cellular caspase‐3 levels as well as the pro‐apoptotic Bad levels were measured. The results showed that tigecycline significantly attenuated the levels of TNF‐alpha and IL‐1beta, as well as NO levels in LPS‐treated PC12 cells. The caspase assay revealed a significant decrease in the caspase‐3 levels in the drug treated cells. The results of pro‐apoptotic Bad levels showed a protective mechanism of the drug. The findings of our study suggest that tigecycline may be considered as a potential therapeutic agent in the neuroinflammation associated with AD or other neurodegenerative disorders.
Histopathological changes in septic mouse brain: effect of the free‐radical scavenger edaravone
Authors: Yokoo Hiroki , Hattori Yuichi
Published: June 2021

Despite the fact that neuronal dysfunction can occur in the course of sepsis, sepsis‐associated histopathological changes in the brain are poorly understood. We found that mRNA expression levels of proinflammatory cytokines, including tumor necrosis factor‐alpha, interleukin‐1beta, and interleukin‐6, were up‐regulated in brain tissues from mice with cecal ligation and puncture‐induced sepsis, but to a much lesser extent when compared with those in peripheral tissues such as lungs. Importantly, blood‐brain barrier permeability was greatly increased in septic mice, as determined by the measurement of sodium fluorescence or Evans Blue content. This was associated with increased immunostaining for nitrotyrosine, a stable biomarker of reactive nitrogen species, in cerebral vessels. Light and electron microscopic examination of septic mouse brain showed the presence of many neuronal cells that appeared deformed, shrunken, and densely‐stained, which were mitigated by treatment with edaravone, a free‐radical scavenger. We thus suggest that the histopathological derangement of neuronal cells in septic mouse brain is associated with enhanced generation of free‐radical species.
BAY 41‐2272 increases VASP phosphorylation via increases in both cAMP and cGMP in rat primary VSMCs
Authors: Adderley Shaquria , Joshi Chintamani , Martin Danielle , Mooney Shayna , Tulis David
Published: June 2021

We recently reported that the guanylate cyclase agonist, BAY 41‐2272 (BAY), has anti‐proliferative properties in rat A7R5 vascular smooth muscle cells (VSMCs); however, its mechanism of action is not clear. Here we sought to elucidate the mechanisms of BAY‐mediated growth inhibition and we hypothesize that BAY inhibits VSMC proliferation through selective phosphorylation of vasodilator‐activated serum phosphoprotein (VASP). In rat primary VSMCs, BAY increased cAMP (~9500%,1nM, 30″) and cGMP (233%,100nM, 15Prime;), and at 10μM significantly increased VASPSer239 and VASPSer157 phosphorylation (pSer239 and pSer157), respective markers for active PKG and PKA. The PKA inhibitor PKI augmented BAY‐induced pSer239 and pSer157, whereas the PKG inhibitor DT2 increased pSer239 but had no effect at pSer157. These findings suggest that BAY operates through both PKA and PKG to phosphorylate VASPSer239 and through PKA to phosphorylate VASPSer157. Interestingly, the PKC inhibitor Calphostin C increased BAY‐induced pSer239 but had no effect on pSer157, again suggesting involvement of multiple kinases in BAY‐mediated pSer239. In comparison, in the presence of a cGMP analog, 8Br‐cGMP, only DT2 increased pSer239, suggesting that PKC and PKA phosphorylation induced by BAY are via increases in cAMP. These data suggest that BAY induces differential phosphorylation of VASP by cross‐talk between the cAMP and cGMP pathways.
Kappa opioid receptor (KOR) signaling on peripheral sensory neurons in vitro and in vivo
Authors: Rowan Matthew , Sanchez Teresa , Silva Yamille , McGuire Blaine , Clarke William , Berg Kelly
Published: June 2021

Considerable interest has developed in understanding the regulation of peripheral opioid receptors to avoid central nervous system‐mediated side effects of opioid pharmacotherapy. However, little is known about peripherally restricted KOR‐mediated analgesic responses. Here we studied KOR agonist‐signaling responses in primary cultures of sensory neurons and in a rat model of thermal allodynia. The KOR agonist, U50488, did not inhibit PGE2‐stimulated adenylyl cyclase (AC) activity in vitro nor PGE2‐induced thermal allodynia in vivo unless cells/tissue were pretreated with the inflammatory mediator bradykinin (BK). In contrast, U50488 stimulated ERK activity without BK pretreatment, which suggests that not all KOR‐mediated responses require pre‐exposure to an inflammatory mediator. Interestingly, the duration of U50488‐induced anti‐allodynia was inversely related to dose, suggesting that the KOR system desensitizes or that KOR activation may initiate both pro‐ and anti‐nociceptive pathways. These results indicate that KOR signaling to AC and ERK is differentially regulated in peripheral sensory neurons. A better understanding of how KOR are regulated on peripheral sensory neurons may provide for improved approaches for the treatment of pain that are devoid of CNS adverse effects. Supported by DA026619.
The mechanism of activation of G‐protein coupled estrogen receptor induced coronary artery smooth muscle relaxation
Authors: Yu Xuan , Li Fen , Sellers Minga , Stallone John , White Richard , Prossnitz Eric , Han Guichun
Published: June 2021

The G protein‐coupled estrogen receptor1 (GPER) may contribute to the non‐genomic, beneficial vascular effects of estrogen; however, the mechanism of GPER action in coronary arteries is unclear. Our objective was to determine how GPER influences coronary artery reactivity. In vitro isometric force recordings were performed on endothelium‐denuded porcine coronary arteries, and were augmented with single‐cell patch‐clamp experiments. RT‐PCR and immunoblot confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G1 (selective GPER agonist) relaxed endothelium‐denuded porcine coronary arteries, suggesting a direct effect coronary artery smooth muscle (CASM). This response was inhibited by G15 (GPER selective antagonist), or by inhibiting large‐conductance calcium‐activated potassium (BKCa) channels with iberiotoxin, but inhibition of NO signaling had no effect. Interestingly, ICI182,780 (ERα/ERβ antagonist, but a GPER agonist) also relaxed coronary arteries. Lastly, whole‐cell and cell‐attached patch‐clamp studies demonstrated that G1 stimulates BKCa channel activity in CASM cells. In summary, GPER activation relaxes CASM by increasing potassium efflux via BKCa channels. This novel action of estrogen‐like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.
Enhanced Expression of G(alpha)q and PLCβ1 in Aortic Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats: Role of Endogenous Angiotensine II and Endothelin‐1
Authors: Emehdi Atef Mohammed , Anand‐Srivastava Madhu
Published: June 2021

Gαq and Gαq associated signaling play an important role in the regulation of vascular tone and cardiac hypertrophy. We have earlier shown that hyperglycemia, induced enhanced expression of Gαq and PLCβ proteins in vscular smooth muscle cells (VSMC) which was attributed to the endogenous levels of angiotensin II (Ang II) and endothelin‐1 (ET‐1). The present study was undertaken to investigate if the expression of Gαq and PLCβ1 proteins is upregulated in aortic VSMC from 16 week‐old spontaneously hypertensive rats (SHR) that has been shown to exhibit cardiac hypertrophy and to further explore the implication of endogenous Ang II and ET‐1 in this upregulation. The expression of Gαq and PLCβ proteins in VSMC was determined by Western Blotting analysis, whereas vascular hypertrophy and prolifeartion was determined by 3H‐leucine and 3H‐thymidine incorporation respectively. The expression of Gαq and PLCβ1 proteins was significantly increased in VSMC from 16‐week old SHR and not from 12‐week old SHR as compared to their age mached WKY rats. The incresead expression of Gαq and PLCβ1 was attenuated by AT1 receptor antagonist, losartan, and ETA receptor antagonist, BQ123, but not by ETB receptor antagonist, BQ788. Protein and DNA synthesis was significantly augmented in VSMC from 16 week‐old SHR as compared to WKY rats. However, the antisense of Gαq attenuated the hypertrophy but not the prolifeartion. These results suggest that VSMC from 16‐week old SHR exhibit enhanced expression of Gαq and PLCβ1 which may be attributed to enhanced levels of Ang II and ET‐1 and may be responsible for the vascular hypertrophy observed in 16 week‐ald SHR.