bluuebunny/crossref_metadata_2025 huggingface dataset:
Severe Burn and Disuse Independently Contribute to Reduction in Bone Density in Rats
Authors: Baer Lisa , Wade Charles E , Silliman David T , Walters Thomas , Wolf Steven E , Wu Xiaowu
Published: June 2021

Patients with severe burns decrease bone mineral density (BMD). The decrease in BMD may be related to the whole‐body response to injury or bedrest (inactivity). In male rats, we evaluated these factors independently and in combination. Sprague‐Dawley rats (∼300g) were assigned to Sham‐burned/Ambulatory (SA; N=10), Sham‐burned/Hindlimb Suspended (SH; N=10), Burn/Ambulatory (BA; N=9), Burn/Hindlimb Suspended (BH; N=10). Burn groups received a 40% TBSA full‐thickness scald and disuse by hindlimb suspension was initiated immediately following burn. Food intake and urine was measured daily. Total mineral intake and urinary excretion (calcium (C), phosphate (P) and magnesium (M)) were measured for 14 days after injury. BMD was measured using DEXA on day 14. No differences were observed in total mineral (C, P and M) intake when adjusted for body weight. No differences in total urinary excretion in C or M, but significant increases were observed in P comparing BH to SA and SH (p<0.001) as well as BA and SA (p<0.02). Significant differences (p<0.002) in BMD were found between SH and BH when compared to SA and between BA and BH (Mean±SEM (g/cm): SA, 0.159±0.001; BA, 0.155±0.002; SH, 0.152±0.002; BH, 0.149±0.001). Burn and disuse were found to be independent contributors to BMD, however the primary cause is disuse. Support: US Army MRMC & Juvenile Diabetes Foundation
Eucapnic intermittent hypoxia increases Rho kinase‐independent myofilament Ca2+ sensitization in small pulmonary arteries
Authors: Snow Jessica , Kanagy Nancy , Walker Benjimen , Resta Thomas
Published: June 2021

Intermittent hypoxia (IH) associated with sleep apnea increases circulating concentrations of the vasoconstrictor peptide, endothelin‐1 (ET‐1), and elicits pulmonary hypertension. We hypothesized that, similar to chronic sustained hypoxia, eucapnic IH (E‐IH) enhances pulmonary vasoconstrictor reactivity to ET‐1 through Rho kinase (ROK)‐mediated vascular smooth muscle (VSM) Ca2+ sensitization. To test this hypothesis, we assessed vasoconstrictor responses to ET‐1 (10−11–10−7 M) in the presence or absence of the ROK inhibitor fasudil (10 μM) in endothelium‐disrupted, pressurized pulmonary arteries (158 ± 10 μ m inner diameter) from E‐IH rats (3 min cycles of 5% O2, 5% CO2/air flush, 7 hr/day, 4 wk) or air controls (air/air cycling for equal duration). VSM was loaded with fura‐2 AM to assess changes in VSM intracellular Ca2+ ([Ca2+]i) associated with ET‐1‐induced vasoconstrictor responses. Consistent with our hypothesis, E‐IH augmented ET‐1‐induced vasoconstrictor reactivity (10−9–10−7.5 M) without changing VSM [Ca2+]i responses. Surprisingly, ROK inhibition did not alter vasoconstriction or VSM [Ca2+]i responses in either group. We conclude that E‐IH enhances ET‐1‐induced Ca2+ sensitization in pulmonary VSM through a ROK‐independent mechanism.

(Supported by NIH grants HL‐07736, HL‐77876, HL‐58124 and HL‐63207)
Effect of chewing and taste on splanchnic blood flow
Authors: Someya Nami , Hayashi Naoyuki
Published: June 2021

To investigate the each role of chewing and taste in the control of splanchnic blood flow, which reportedly increased at initial phase of meal, we compared the blood flow responses in celiac artery (CA) to chewing of solid meal (MEAL, with chocolate taste) and paraffin wax (WAX, without taste). After five minutes of baseline measurement, twelve healthy subjects repeated chewing and expectorating the MEAL or WAX for 4 minutes followed by 10 minutes of recovery measurement. We measured blood velocity (BV) in CA and masseteric electromyogram (EMG). The BV in CA at the baseline and the masseteric EMG during chewing were not different between MEAL and WAX trials. The BV in CA significantly decreased at the 1st minute of chewing from the baseline in both trials. In the MEAL trial the BV in CA significantly increased at the 3rd minute of chewing from the baseline, whereas in the WAX trial the BV in CA returned to the baseline value. The BV in CA was significantly higher in the MEAL trial than the WAX trial at the 3rd minute of and after chewing. These results suggest that the taste of food plays a role in meal‐induced splanchnic hyperemia.
Prediabetes in the absence of the metabolic syndrome is associated with impaired brachial artery flow‐mediated dilation
Authors: Eskurza Iratxe , Fay Tara N. , Walker Ashley E. , Seals Douglas R. , Pierce Gary L.
Published: June 2021

Endothelium‐dependent dilation (EDD) is impaired with aging, but there is marked variability in EDD among older adults. The prevalence of prediabetes (Pre‐DB) increases with age, but it is unknown if Pre‐DB influences age‐associated reductions in EDD, particularly in the absence of other major cardiovascular disease risk factors. To determine this, brachial artery flow‐mediated dilation (FMD) was measured in 34 young (YN, age=23±1 yr, mean±SE) and 14 older (ON age=65±1 yr) healthy men with normal fasting blood glucose (FBG<99 mg/dl), and in 10 healthy older men with impaired FBG (FBG>100 mg/dl) (OPre‐DB, age=68±1 yr). FBG was similar in YN and ON (87.1±1.2 vs. 88.9±1.7 mg/dl, p>0.05), but higher in Opre‐DB (104.6±2.1 mg/dl, p<0.05). Brachial FMD was 30% lower in ON vs. YN (5.3±0.7 vs. 7.8±0.3 %, p<0.05). Most importantly, FMD was 43% lower in OPre‐DB (3.1±0.5 %) vs. ON and 60% lower in OPre‐DB vs. YN (p<0.05). Endothelium‐independent dilation did not differ among groups (p=0.86). The difference in FMD between OPre‐DB and ON was reduced by ∼half (3.9±0.7 vs. 5.0±0.7%) and was no longer statistically significant after correcting for waist‐to‐hip ratio (p>0.05). These results support the hypothesis that prediabetes, in the absence of the metabolic syndrome, modulates the age‐associated reduction in EDD, in part via increased abdominal fat. Supported by NIH AG013038, AG022241, AG006537, AG000279.
Role of TRPM2 Channels in Endothelial Ca2+ signaling and Transendothelial Migration of Neutrophil
Authors: Ahmmed Gias Uddin , Gao Xiao Pei , Hossain Md. Jamil , Hecquet Claudie , Vogel Stephen M. , Fukai Masuko U. , Malik Asrar B.
Published: June 2021

Adhesion of PMNs (polymorphonuclear leukocytes) to endothelial cells (ECs) may induce cross‐talk that contributes to inter‐cellular cooperation required for complex processes such as transendothelial PMN migration. Here we addressed the role of PMN activation of redox‐sensitive TRPM2 (transient receptor potential melastatin‐2) channels in activating Ca2+ signaling in ECs. ECs were loaded with Ca2+ indicator Fura‐2 and PMN‐specific agonist fMLP was used to induce adhesion and activation of PMNs applied to confluent EC monolayers. We observed an immediate PMN adhesion‐dependent spike in Ca2+ concentration [Ca2+]i in ECs lasting 2–3 min. TRPM2 siRNA‐suppression of TRPM2 in ECs reduced Ca2+ entry whereas TRPM2 overexpression augmented the response. Pretreatment of PMNs with superoxide dismutase or NADPH oxidase inhibitor DPI (dibenziodolium chloride) prevented the increase in EC [Ca2+]i. Inhibitors of adenosine diphosphate ribose (ADPR) generation (the endogenous TRPM2 activating agonist), DPQ (3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxyl]‐1(2H)‐isoquinolinone, 100 μM) and 3‐AB (3‐aminobenzamide, 1 mM), also prevented the rise in [Ca2+]i induced by PMNs. Measurement of inward current through EC TRPM2 channels showed that injecting ADPR into ECs or extracellular application of H2O2 both induced TRPM2‐sensitive cationic currents. siRNA‐suppression of TRPM2 in ECs reduced the transendothelial migration of neutrophil (TEM) comparing with control EC. Thus, PMN interaction with ECs activates Ca2+ entry through redox‐sensitive TRPM2 channels suggesting their critical role in transendothelial migration of neutrophils.
Structural damage to erythrocyte‐membrane proteins induced by Lonomia obliqua caterpillar bristle extract
Authors: Seibert Carla Simone , Tambourgi Denise V. , Santoro Marcelo L. , Sampaio‐Verssoni Sandra C , Takahashi Hilton K. , Peres Carmem M. , Sano‐Martins Ida S.
Published: June 2021

Lonomia obliqua caterpillar bristle extract shows in vitro hemolytic activity in human and rat erythrocytes (Er), and potent intravascular hemolytic activity in rats. Hemostatic disturbances are characteristic in human envenomation, and intravascular hemolysis have been observed in some cases.

Objective

To investigate the mechanism whereby L. obliqua caterpillar bristle extract cause lysis of human Er.

Methods

Human Er were treated with sublytic concentrations (250, 500 and 1000 μg/ml) of crude extract or saline (control). Afterwards, Er osmotic fragility was determined, and sialoglycoproteins of the Er‐membrane surface (GPA, GPC and band 3) were quantified using flow cytometry. Lipid bilayer composition was determined (fatty acids, triacylglycerols, cholesterol).

Results

Results are expressed as mean± standard deviation (n= 5). Er treated with 1000 μg/ml crude extract increased osmotic fragility (extract H50%= 90.5 ± 2.70, and control H50%= 75.0 ± 1.32 mM NaCl). Sialoglycoproteins were removed from the Er surface (26 % GPC and 23 % band 3). The ions Ca2+ and Mg2+ increased this activity, showing their greatest effect on band 3 (> 50 % removal). Nevertheless, the extract provoked no damage in the Er lipid bilayer organization.

Conclusion

Crude extract removes Er membrane proteins responsible for its flexibility and malleability, increasing osmotic fragility, and likely inducing hemolysis by rupturing Er when they are in blood stream.

FAPESP
Diarylheptanoids contribute to the estrogenic activity of Curcuma comosa.
Authors: Tantrakool Wipawee , Piyachaturawat Pawinee , Suksamrarn Apichart , Arao Yukitomo , Hewitt Sylvia C. , Korach Kenneth S.
Published: June 2021

Extracts of Curcuma comosa, a plant in the Zingiberaceae family, exert estrogenic activity in in vivo and in vitro assays. Diarylheptanoids, a major component of C. comosa, are estrogenic in rat uterine bioassays, in an estrogen responsive yeast two‐hybrid system, and in MCF‐7 (human breast cancer) cells. This study aims to characterize further the estrogenic activity of C. comosa‐derived diarylheptanoids. HepG2 cells expressing wild type or mutant forms of the mouse estrogen receptor‐α (ERα) and possessing an estrogen responsive reporter plasmid (3XERE‐TATA‐Luc) were exposed to increasing doses of diarylheptanoids. At 50 μM, the diarylheptanoids were equally efficient as 1 nM estradiol at stimulating ERE‐driven reporter activity, and this response was dose‐dependent. This activity was inhibited by the ER antagonist (ICI‐182780). A mutant ERα lacking the AF2 domain was unresponsive to the diarylheptanoids, while ERα lacking the AF1 domain exhibited wild type‐like activity. These data indicate that diarylheptanoids are active in an ER‐dependent system and this activity is dependent on ERα, more specifically its AF2 function. We therefore surmise that diarylheptanoids contribute to the estrogenic activity of C. comosa.

Supported by Thailand Research Fund through Royal Golden Jubilee PhD Program (PHD/0255/2546) to WT and PP; BIOTEC grant to AS; NIEHS Division of Intramural Research Funding to WT, YA, SCH and KSK.
Nitric oxide synthase inhibition into the rat locus coeruleus delays the LPS‐induced fever
Authors: Soriano Renato Nery , Ravanelli Maria Ida Bonini , Batalhão Marcelo Eduardo , Branco Luiz Guilherme Siqueira
Published: June 2021

This work was aimed at analyzing whether nitric oxide (NO) synthesis in the rat locus coeruleus (LC) is essential for mounting the LPS‐induced fever. Male Wistar rats (320–350g) were chronically, stereotaxically implanted with one guide cannula just above the LC and had a miniature temperature datalogger (SubCue) inserted in the peritoneal cavity. Unanesthetized, freely moving rats were unilaterally microinjected into the LC with either a nonselective nitric oxide synthase (NOS) inhibitor [L‐NMMA; 50 nmol in 100 nL of pyrogen‐free saline (PFS)] or just PFS as control, and were, 30 min later, intraperitoneally injected with either a polyphasic fever‐inducing dose of bacterial lipopolysaccharide [LPS; 100 μg/kg in PFS (1 mL/kg)] or PFS (vehicle), at an isothermal, subneutral ambient temperature (23°C). Under NOS inhibition, we observed an increased latency (1 h) for the onset of fever and also a mild, transient attenuation of its second phase (F2,18=5.334; p<0.001, vs control groups); a thermal index analysis showed a slighter fever (F2,18=5.482; p=0.0138, vs control groups). Moreover, measurements of LC nitrite/nitrate concentration revealed that the LC nitric oxide synthesis increased during the course of fever (p<0.001, vs saline group). These results indicate that the endogenous synthesis of NO into the rat LC mediates the onset of the LPS‐induced fever. Financial support: FAPESP, CNPq.
The effects of training on corticosterone levels in swimming and diving rats
Authors: McCulloch Paul , Connolly Tiffanny
Published: June 2021

Although there are many rodent experimental protocols that utilize water as a stressful stimulus, our objective was to determine if gradual training could reduce or eliminate the stressful component of water exposure in rats. Our hypothesis was that trained rats will show a reduction in corticosterone concentrations during swimming and diving. Male Sprague‐Dawley rats were randomly divided into four groups: Naïve rats (N=6) remained in their cage. Handled rats (N=4) were held daily. Trained rats (N=6) had 8 weeks of daily swim and dive training. Untrained rats (N=6) received no training. After completion of swim, voluntary dive and forced dive training, all Trained and Untrained rats were placed in the water for either 3 swim or dive trials. We found that corticosterone levels in swimming and voluntarily diving rats are lower in rats that have received daily training (160 ± 38 ng/ml and 115 ± 22 ng/ml for swim and dive, respectively), as opposed to rats that have received no training (232 ± 38 ng/ml and 224 ± 20 ng/ml for swim and dive, respectively). This habituation effect occurred for voluntary diving but not for forced diving (220 ± 13 ng/ml for Trained rats and 242 ± 22 ng/ml for Untrained rats). We conclude that the stressfulness associated with voluntary, but not forced, water immersion in rats can be reduced by gradual training, and that swimming and diving can be stressful to rats not trained to perform these behaviors. Supported by NIH R15 HL080007.
Long‐term Exercise Intervention Ameliorates Vasorelaxation Mediated by Insulin and Insulin‐like Growth Factor‐1 in Aortas of Diabetic Rats
Authors: Fu Keng‐Yeh , Su Chia‐Ting , Yang Ai‐Lun
Published: June 2021

In order to investigate whether exercise ameliorates insulin‐ and insulin‐like growth factor‐1 (IGF‐1)‐mediated vasorelaxation in the diabetes, male Wistar rats were randomly divided into non‐diabetic control (Con), diabetic control (DM), and diabetes with exercise (DM+Ex) groups. Diabetic groups were induced by the intravenous injection of streptozotocin. The DM+Ex group performed an 8‐week treadmill exercise program for 60 min/day, 5 day/week. At the end of experiments, the vasorelaxant responses to insulin, IGF‐1, acetylcholine (ACh), and sodium nitroprusside (SNP) were evaluated by the isometric tension of aortic rings in the organ baths. In addition, the roles of phosphatidylinositol 3‐kinase (PI3‐K) and nitric oxide synthase (NOS) in the vasorelaxation were examined by treating selective inhibitors. We found that 1) insulin‐, IGF‐1‐, and ACh‐induced vasorelaxant responses were significantly impaired in the DM group, compared with those in the Con group (P<0.05); 2) the exercise intervention significantly ameliorated the impaired vascular responses to insulin, IGF‐1, and ACh in the DM+Ex group, compared with those in the DM group (P<0.05); 3) these alternations of vascular responses affected by diabetes and exercise were mainly due to the release of PI3‐K and NOS; 4) no significant difference was found in SNP‐induced vasorelaxation among the three groups. Our results suggest that long‐term exercise intervention ameliorates vasorelaxation mediated by insulin and IGF‐1 in type 1 diabetic rats. The PI3K‐NOS‐dependent pathway is mainly involved in the exercise‐induced beneficial effects. This study was partly supported by the National Science Council, Taiwan (NSC95‐2314‐B‐006‐073‐MY2).
Spinal cord hemisection disrupts descending neuregulin input to phrenic motoneurons
Authors: Issa Amine Najib , Zhan When Zhi , Fang Yun‐Hua , Bailey Jeffery P , Sieck Gary C , Mantilla Carlos B
Published: June 2021

Spinal cord hemisection at C2 (SH) disrupts descending drive to ipsilateral phrenic motoneurons (PMNs) causing unilateral diaphragm muscle (DIAm) paralysis. Previously, we found a decrease in motoneuron size at 2 weeks post‐SH. Recently, we found neuregulin (NRG) in synaptic boutons surrounding PMNs. NRG is a trophic factor known to regulate development of the neuromuscular junction, but the effect of NRG on motoneurons is unknown. We hypothesized that 1) NRG input to PMNs is supraspinal in origin, and 2) SH disrupts NRG input to PMNs. Adult male Sprague‐Dawley rats underwent SH or sham‐surgery. PMNs were retrogradely labeled by intramuscular injection of Alexa488‐conjugated cholera toxin B. Using laser capture microdissection, we found that PMNs express mRNA for the NRG receptors ErbB2 and ErbB3, providing a functional substrate for a NRG effect. Two weeks post‐SH, labeled PMNs and NRG boutons were imaged using confocal microscopy. PMN morphology and the number of NRG boutons in the vicinity of PMNs were determined using a computerized, semi‐automated reconstruction algorithm. NRG boutons were fairly homogeneously distributed on motoneurons independent of size. Following SH, few NRG boutons were observed. We conclude that 1) NRG input to PMNs derives from a supraspinal source; and 2) SH removes NRG input to PMNs. It is possible that NRG input exerts a trophic influence on PMNs.

Supported by NIH grants HL37680, AR51173 and Mayo Clinic
Interleukin‐1 induced increases in blood pressure are mediated by cerebral‐vasculature‐associated perivascular cells
Authors: Schiltz Jennifer C.
Published: June 2021

Interleukin‐1 (IL‐1) induces a sympathetically mediated increase in mean arterial pressure (MAP) and heart rate (HR) in awake rats. The response can be attenuated with peripheral injections of non‐specific prostaglandin (PG) synthesis inhibitors, but a role for central PGs has not been established. ED‐2 positive perivascular cells (pvcs) are the sole source of Cox‐2 within the brain in response to low doses of IL‐1 and their loss has been shown to attenuate IL‐1 induced HPA activation suggesting central PG synthesis by pvcs mediates the response. This study tested the hypothesis that the increase in MAP and HR induced by IL‐1 was dependent on brain production of PG's by pvcs. Male Sprague Dawley rats were chronically instrumented with an iv jugular catheter, and an abdominal transmitter (Data Systems International) for recording MAP and HR. One week later, they received intraventricular injections of clodronate‐containing (clod‐lip) or saline and DiI‐containing liposomes (sal‐lip) to selectively deplete the brain of pvcs, or label them respectively. Clod‐lip injections produced a > 90% depletion of brain pvcs. Baseline MAP and HR values were not significantly different between groups (sal‐lip, 105 ± 4 mmHg, 413 ± 13 bpm vs. clod‐lip, 107 ± 4 mmHg, 408 ± 10 bpm). Recombinant rat IL‐1 (2 μg/kg, iv) increased MAP and HR by 10 ± 4 mmHg and 38 ± 24 bpm respectively at 10 minutes after injection and remained elevated for approximately 20 minutes. Liposome‐depletion of ED2‐positive pvcs significantly reduced the MAP response to −3 ± 6 mmHg and HR to 5 ± 11 bpm. These results suggest that the cardiovascular changes induced by IL‐1 are mediated by pvcs and implicate a novel signaling mechanism at the blood brain barrier used by cytokines to regulate the sympathetic nervous system.
Oxidative Stress and Exercise‐Induced Flow‐Mediated Dilation in COPD: Insight into Skeletal Muscle Dysfunction
Authors: Harris Ryan Alan , Nishiyama Steven K. , Wray D. Walter , Berkstressor Kimberly A. , Richardson Russell S.
Published: June 2021

The etiology of skeletal muscle dysfunction (SMD) in COPD is unknown. Oxidative stress (OS) and vascular dysfunction may be involved in the pathophysiology of COPD‐induced SMD. This study aimed to elucidate the association between exercise‐induced flow‐mediated dilation (FMD) and OS in COPD. Patients ingested either a placebo or an antioxidant cocktail (AOC) in a randomized, double blind fashion. The placebo and AOC consisted of two doses (AOC: totals = 1,000 mg of vitamin C, 800 IU vitamin E, and 600 mg of α‐lipoic acid). Using ultrasound, blood velocity and popliteal artery diameter were recorded at rest and following progressive plantar flexion (PF) exercise at 2, 4, and 6 Watts. The slopes of % change in FMD (5.0±0.9 vs. 2.1±0.0%) and absolute change in diameter (0.02±0.01 vs. 0.01±0.0 cm) induced by PF exercise were greater following an AOC compared to placebo. Although the slope for FMD/shear was similar between the placebo and AOC (0.049±0.004 and 0.055±0.009, respectively) there was an upward parallel shift in the slope observed following the AOC. Therefore, at any given skeletal muscle work rate, there appears to be an improvement in vascular function following the ingestion of an AOC in COPD. These results suggest a link between oxidative stress and vascular function in patients with COPD, which may play a role in the SMD associated with this disease. Supported by TRDRP 15RT‐0100 and Parker B Francis Fellowship.
Exercise improves endothelial nitric oxide synthase (eNOS) dimerization in diabetic rats
Authors: Edwards John G. , Hicks Steve , Grijalva James , Zhao Xiangmin
Published: June 2021

The dimeric form of eNOS catalyzes the rate‐limiting step in the synthesis of nitric oxide, while the monomeric form catalyzes the synthesis of peroxynitrite (ONOO), a highly reactive oxidant species (ROS). An increase in the monomer form in the hearts of diabetic animals has previously been reported and this may contribute to cardiac dysfunction. It is unclear if exercise may alter the functional coupling of eNOS. To investigate this question, Goto‐Kakizaki rats were exercise trained for 6‐weeks. Exercise training significantly increased both plantaris cytochrome oxidase activity as well as the left ventricle weight (sed: 0.72±0.02, train; 0.80±0.01 g). Exercise significantly improved glucose tolerance, decreased HbA1c, and fasting blood glucose levels (sed: 205±9.5 mg%; train: 174±9.5 mg%). Exercise training also increased both the total eNOS expression and the ratio of dimer to total eNOS in both the left ventricle (sed: 6.4±2.7%; train: 24.8±5.3%) and the kidney (sed: 12±2%; train: 20.7±3%). The increase in eNOS dimerization would be consistent with increased coupling of the enzyme to facilitate the production of NO at the expense of ROS generation, a shift that could serve to decrease diabetic‐related oxidative stress and that may serve to lessen diabetic‐related complications. Supported in part by the New York Medical College Research Endowment Fund.
Endothelin‐1 Inhibits Nox1 in Human Abdominal Aortic Endothelial cells: a Novel Function of ETB1 receptors
Authors: Dammanahalli Karigowda , Sun Zhongjie
Published: June 2021

Hypothesis.

Endothelin‐1 (ET‐1) inhibits Nox1 activity and decreases superoxide production and cell proliferation in human abdominal aortic endothelial cells (HAAECs).

Methods and Results.

HAAECs were exposed ET‐1 (10–30 nM) at different time intervals and superoxide production was measured by flow cytometry. Intracellular reactive oxygen species (ROS) was examined by confocal microscopy using 2′,7′‐dichlorodihydrofluorescein diacetate. NADPH oxidase activity was determined using lucigenin enhanced chemiluminescence in the presence and absence of siRNA for ETB1 receptors or Nox1. ET1 significantly attenuated Nox1 activity, ROS generation, and cell proliferation which could be abolished by silence of Nox1 gene. Furthermore, RNAi silence of ETB1 receptors significantly increased Nox1 activity and blocked the inhibitory effect of ET1 on Nox1 activity. ET1 enhanced bioavailability of intracellular NO measured using DAF‐2. ET1 did not affect Nox1 protein expression. Nox2 was not dectable in HAAECs. Activation of ETB1 receptors by ET1 suppressed protein phosphorylation of pyk2 (Y402) and Rac1, suggesting that ET1 inhibits Nox1 activity via ETB1‐Pyk2‐Rac1 pathway.

Conclusions.

This study demonstrated, for the first time, that that ET1 inhibited Nox1 activity, ROS production, and ell proliferation in HAAECs via ETB1‐Pyk2‐Rac1 pathway.
Mitochondrial movement is random and increased by hyposalinity in coelomocytes from an estuarine worm
Authors: Julian David , Rollins Allman , Predmore Benjamin L
Published: June 2021

Mitochondria typically move non‐randomly due to attachment to cytoskeletal components. We investigated whether variation in ambient salinity affects mitochondrial movement in coelomocytes (nucleated “blood cells”) from the estuarine annelid Glycera dibranchiata using high‐resolution fluorescence video microscopy of live coelomocytes labeled with MitoTracker Green FM. Cells were exposed to normal seawater (1000 mOsm/L) or seawater diluted to 750, 500 and 250 mOsm/L (all with 10 mM HEPES and 0.1% glucose). Cell area and mitochondrial motion were analyzed in 100 cells and 400 mitochondria in a split plot, fully nested design (5 mitochondria x 5 cells x 4 worms x 4 dilutions). Surprisingly, mitochondrial movement at 1000 mOsm/L was continuous and random, characteristic of Brownian motion. Decreasing osmolarity proportionally increased the cell volume (P<0.001), as expected, but also increased the length of each mitochondrial track (P<0.0001) and the average total displacement (P<0.001), with mitochondrial movement at 250 mOsm/L being 2.8‐fold greater than at 1000 mOsm/L. Furthermore, the change in the mitochondrial diffusion coefficient was 3‐fold greater than that predicted by the direct effect of salinity on water viscosity. We propose that changes in the composition of intracellular organic osmolytes associated with salinity adaptation substantially affect the motion of these organelles.
Increasing intracellular calcium in adipocytes increases fatty acid oxidation via the activation of AMP‐activated protein kinase
Authors: Gabler Nicholas Kurt , Davis Jeremy E , Walker‐Daniels Jennifer , Spurlock Michael E
Published: June 2021

Increased dietary calcium intake is associated with reduced adiposity in humans. We hypothesized that increased intracellular Ca++ would augment fatty oxidation in 3T3‐L1 adipocytes in an AMP‐activated protein kinase (AMPK)‐dependent manner. Adipocytes were grown under euglycemic (EG) or hyperglycemic (HG) conditions and treated with or without calcium ionophores (4x10‐6 M A23187 and 4x10‐6 M Ionomycin, [AI]). After 15–60 min, AI increased intracellular Ca++ concentrations by 250% (P<0.001) and 150% (P<0.05), in EG and HG cells respectively, vs. controls. P‐AMPK and P‐Acetyl CoA carboxylase protein abundance was up regulated by AI in a time‐dependent manner. This was associated with increased 14C‐palmitate oxidation under EG (267%, P<0.001) or HG (200%, P<0.02) vs. the controls. However, the AMPK inhibitor, adenosine 9‐beta‐d‐arabinofuranoside (araA), attenuated the AI‐induced increase in fatty acid oxidation (P<0.01) under both conditions. HG results in insulin resistance and impaired glucose uptake in adipocytes. Compared to the control, AI improved this glucose uptake by 210% (P<0.01), even in the presence of araA (180%, P<0.05). These data indicate that intracellular Ca++ is a significant determinate of fatty acid oxidation via the control of AMPK activity. In contrast, the ability of increased intracellular Ca++ to enhance glucose uptake in HG conditions is independent of the activation of AMPK.
Predictors of bone mineral density in rural and urban women in Mexico
Authors: Caamaño María del Carmen , García Olga Patricia , Anaya‐Loyola Miriam Aracely , Rosado Jorge Luis
Published: June 2021

The objective of the study was to identify the factors that are associated with bone mineral density in women. We evaluated 609 women between 35 and 60 y, 45% living in the city of Queretaro, Mexico and 55% living in rural communities. Bone mineral density (BMD) and body composition were measured with DEXA, and a fasting blood sample was taken to measure glucose and lipids profile. Anthropometry measurements included weight, height, waist and hip circumference. In a linear regression analysis age was associated negatively with BMD (95% CI: −0.0058, −0.0035 g/cm2). After controlling for age, a linear regression analysis showed a direct association of DMO with cholesterol (95% CI: 0.0000, 0.0003 g/cm2), fat free mass (95% CI: 0.0003, 0.0039 g/cm2) and BMI (95% CI: 0.0056, 0.0128 g/cm2). Fat percent (95% CI: −0.0083, −0.0040 g/cm2) and waist circumference (95% CI: −0.0034, −0.0007 g/cm2) were indirectly associated with DMO. After controlling for the previous variables, women living in rural communities had 0.028g/cm2 less BMD than women living in an urban area (95% CI: −0.0438, −0.0120 g/cm2). However, there were not significant differences in unadjusted mean DMO between rural an urban women nor between women with or without metabolic syndrome.
Bioactive compounds from Concord grapes prime γδ T cells and reduce DNA strand breaks
Authors: Nantz Meri P. , Rowe Cheryl A. , Nieves Carmelo , Percival Susan S.
Published: June 2021

Our overall objective is to determine if bioactive compounds can prime a γδ T cells and reduce inflammation. Previously, we have found that L‐theanine primes γδ T cells, thus, we asked if other small polyphenolic molecules will act similarly, perhaps because they resemble pathogenic secretions as hypothesized for L‐theanine. We tested the hypothesis that bioactive compounds from Concord grapes might also prime this particular cell, which is important in wound healing, pathogen eradication and cancer immunosurveillance. A randomized, double blind, placebo controlled human study was conducted for 9 weeks. We examined peripheral blood mononuclear cells (PBMC) and γδ T cell proliferation, and DNA strand breaks. A repeated measure 2‐way ANOVA with primary effect of Time and secondary effect of Treatment was performed. Despite no significant effect on PBMC proliferation, γδ T cell numbers were significantly higher after juice consumption (p=0.045) and their proliferation under ex vivo autologous serum was significantly greater than the placebo (p=0.044)). The number of intrinsic DNA strand breaks was reduced after consumption of either juice or placebo, however, only juice consumers had a reduced number of strand breaks when cells were challenged with H2O2 compared to placebo (p=0.017). Funded by Welch Foods Inc and the UF/IFAS Agriculture Experiment Station
Lipid Overload with Saturated and Monounsaturated Fatty Acids Has Differential Effects on MHC Class I Antigen Presentation
Authors: Shaikh Saame Raza , Li Matt , Schneck Jonathan , Edidin Michael
Published: June 2021

Lipid overload, associated with obesity, occurs when saturated fatty acids (SFAs) accumulate in non‐adipose tissues. Cells of these tissues use major histocompatibility (MHC) class I molecules to present antigen to cytolytic T lymphocytes (CTLs) in order to eliminate pathogens. Since obesity is associated with impaired immune responses, we tested if lipid overload with SFAs could affect MHC class I antigen presentation to CTLs. Antigen presenting cells (APCs) were fed either the saturated fatty acid palmitic acid (PA) or the monounsaturated oleic acid (OA), which are respectively major components of the high fat Western or Mediterranean diets. PA‐feeding lowered APC lysis by activated CTLs and inhibited the ability of APCs to stimulate naïve T cells. Inhibition of immune responses with PA was due to lowered MHC class I surface expression and inhibition in the rate of APC‐CTL conjugation. These changes were associated with a modification of plasma membrane acyl chain structure and F‐actin levels, which could modify the immunological synapse. OA‐treatment had no effect on antigen presentation and when fed together with PA, blocked the effects of PA. This block with OA correlated with its distinct cellular metabolic fate. OA‐treatment conferred protection by accumulating fatty acids into triglyceride‐rich lipid droplets of APCs. Our findings establish a link between lipid overload and antigen presentation and suggest that dietary SFAs in non‐adipose cells of obese individuals could impair immunity by affecting MHC I‐mediated antigen presentation; this could be prevented by accumulation of triglycerides rich in monounsaturated fatty acids.
Talin is a master regulator of platelet integrin activation in vivo
Authors: Petrich Brian Gary , Marchese Patrizia , Monkley Susan J. , Critchley David R. , Ruggeri Zaverio M. , Ginsberg Mark H.
Published: June 2021

Primary hemostasis following vascular injury is dependent on the integrin‐mediated adhesion of platelets to exposed subendothelium and subsequent aggregation. In vitro studies suggest that activation of β1 and β3 integrins depends on talin, a protein that links integrins to the actin cytoskeleton. To assess the role of talin in integrin function in vivo, we generated a platelet precursor‐specific talin1 KO mouse (fl/fl,Cre+). Platelets from fl/fl,Cre+ mice showed profound defects in agonist‐induced activation of both αIIbβ3 and β1 integrins and exhibited a profound bleeding diathesis. To test whether impaired talin‐dependent β1 integrin activation contributed to the hemorrhagic phenotype observed in flox/flox,Cre+ mice, the adhesion of talin‐deficient platelets to collagen was examined in flowing blood. Platelets from fl/fl,Cre‐mice, but not fl/fl,Cre+ mice, adhered to collagen and subsequently formed platelet‐rich thrombi, processes dependent on β1 and αIIbβ3 integrins, respectively. Consistent with ex vivo results, fl/fl,Cre+ mice showed markedly prolonged tail bleeding times and failed to form occlusive thrombi in response to ferric chloride injury of the common carotid artery. Together, our data show that talin is essential in platelets for normal hemostasis because it is required for the activation of β1 and β3 integrins and for platelet adhesion and thrombus formation in flowing blood.
Hypoxia‐ and VEGF‐Induced SDF‐1alpha/CXCR4 Expression in Glioblastomas: a potential Resolution of Scherer's Enigma
Authors: Zagzag David , Esencay Mine , Mendez Olga , Yee Herman , Smirnova Iva , Huang Yuanyuan , Lukyanov Eugene , Newcomb Elizabeth W.
Published: June 2021

In 1938, Scherer described the invasion of glioblastoma multiforme (GBMs) from the main tumor mass into the adjacent brain parenchyma, based on his morphological analysis of 100 GBMs. The patterns of glioma cell invasion are known as the “secondary structures of Scherer” and today are referred to as perineuronal and perivascular satellitosis, subpial spread, and invasion along the white matter tracts. We analyzed 31 GBMs for patterns of glioma cell invasion related to the expression of the chemokine stromal cell–derived factor‐1 (SDF‐1)α, its receptor CXCR4 and the angiogenic factor vascular endothelial growth factor (VEGF). SDF‐1α was highly expressed in neuronal cells, blood vessels, subpial regions and white matter tracts that form the basis of Scherer's secondary structures. In contrast, CXCR4, was highly expressed in invading glioma cells organized around neurons and blood vessels (perineuronal and perivascular satellitosis), in the subpial region (subpial spread) and along white matter (invasion along the white matter tracts). VEGF was expressed not only in tumor cells but also in neurons and blood vessels. In vitro assays demonstrated CXCR4 positive tumor cells migrated toward an SDF‐1α gradient and VEGF upregulated SDF‐1α expression in neuronal and endothelial cells. Taken together, these studies suggest a biological‐based mechanism that may help explain the formation of the secondary structures.
Pathogenic roles of Toll‐like receptor 2 and intracellular bacteria in intimal hyperplasia in apolipoprotein E‐deficient mice
Authors: Beasley Debbie , Celestin Flore , Moore Kathryn , Mendelsohn Michael
Published: June 2021

Atherosclerosis is an inflammatory disease, and recent evidence suggests that innate immunity may contribute to the inflammatory process. We recently found that vascular smooth muscle cells (VSMC) express Toll‐like receptor 2 (TLR2) and TLR4, and that activating TLR2 or TLR4 in VSMC induces marked phenotypic changes, including increased proliferation. Here we assessed the role of TLR2 in spontaneous and in injury‐ and bacteria‐induced intima formation in apolipoprotein E‐deficient mice. Mice having deficiencies of both ApoE and TLR2 (double knockout; DKO) were generated by cross‐breeding mice bearing single knockouts of each gene. Wire‐induced denudation injury elicited intima formation in ApoE KO mice, and this effect was inhibited by 37% in ApoE/TLR2 DKO mice (0.038±0.014 vs. 0.024±0.012 mm2, n=4). TLR2 deficiency also reduced spontaneous lesion formation in the lesser curvature of the aortic arch by 54% (12.1±2.2% vs. 5.6±0.9%; n=8). In addition, exposing the carotid arterial adventitia of ApoE KO mice in vivo to Chlamydia pneumoniae, an atherosclerosis‐associated intracellular bacterium that activates VSMC via TLR2 and TLR4 signaling, elicited similar intima formation in ApoE KO mice. The results reveal that TLR2 signaling may participate in injury‐induced intima formation, and establish a hypercholesterolemic mouse model of bacteria‐induced arterial pathogenesis. Supported by NIH HL47569.
Impact of Genetic Knockout of PEPT2 on the Pharmacokinetics, Renal Tubular Reabsorption, and Brain Penetration of Cefadroxil in Transgenic Mice
Authors: Shen Hong , Ocheltree Scott M. , Hu Yongjun , Keep Richard F. , Smith David E.
Published: June 2021

In this study, the disposition of the antibiotic cefadroxil was evaluated in the body, particularly the kidney and brain, of wild‐type and PEPT2 null mice after intravenous bolus administrations of cefadroxil (i.e., at 1, 12.5, 50, and 100 nmol/g body weight). Cefadroxil plasma pharmacokinetics were clearly nonlinear over the dose range studied (1–100 nmol/g), which reflected both saturable renal tubular secretion and reabsorption of the antibiotic. PEPT2 null mice exhibited a 3‐fold greater total clearance and 3‐fold lower systemic concentrations of drug compared with wild‐type animals after an intravenous bolus dose of 1 nmol/g cefadroxil. Renal clearance studies further demonstrated that the renal reabsorption of cefadroxil was almost completely abolished in PEPT2 null versus wild‐type mice (3% versus 70%, p < 0.001). Of the 70% of cefadroxil reabsorbed in wild‐type mice, PEPT2 accounted for 95% and PEPT1 accounted for 5% of reabsorbed substrate. Tissue distribution studies indicated that PEPT2 had a dramatic effect on cefadroxil tissue exposure, especially in brain where the cerebrospinal fluid (CSF)‐to‐blood concentration ratio of cefadroxil was 6‐fold greater in PEPT2 null mice compared with wild‐type animals. These findings demonstrate that renal PEPT2 is almost entirely responsible for the reabsorption of cefadroxil in kidney and that choroid plexus PEPT2 limits the exposure of cefadroxil and probably other aminocephalosporins in CSF.
Developmental Regulation of Glucocorticoid Receptor Processing in the Brain: Role of Specific E3 Ubiquitin Ligases
Authors: DeFranco Donald B , McCarran William , Monaghan‐Nichols Paula
Published: June 2021

Glucocorticoid hormones exert a variety of effects on the brain and impact memory, anxiety, and CNS responses to stress. The action of these hormones is mediated primarily by soluble receptors, the corticosteroid or glucocorticoid receptors (GRs), which primarily act directly in the nucleus to regulate select networks of target genes. Many in vitro and in vivo model studies have revealed a relationship between expression levels of glucocorticoid receptors and cellular responsiveness to glucocorticoid hormone, particularly in the brain. Various intrinsic and extrinsic factors influence the expression of the glucocorticoid receptors and thereby impact cellular output to glucocorticoid hormone exposure. We had previously shown that the downregulation of GR expression that is characteristic of adult neurons, particular in the hippocampus, does not operate in fetal hippocampal neurons as GR levels are not affected by chronic hormone exposure. This lack of downregulation can be reversed in vitro by the overexpression of a specific E3 ligase for the receptor, carboxyl terminus of hsp70‐interacting protein (CHIP). The impact of CHIP expression in neurons on GR regulated transcription will be examined as well as the pattern of CHIP expression in developing brain, with a particular emphasis on GR responsive regions.
Functional Tissue Engineering of Anatomically Shaped Cartilage Constructs
Authors: Ateshian Gerard A , Lima Eric G , Ng Kenneth W , Cook James L , Hung Clark T
Published: June 2021

Several important challenges remain to be met in the successful engineering of cartilage tissue constructs. In this study we address the need to engineer anatomically‐shaped constructs of entire articular layers for the treatment of joints with widespread degenerative disease. Reconstructing the anatomic contours can be achieved with a variety of techniques, including non‐invasive imaging such as MRI and CT. Calibrating the accuracy of such techniques can be performed on cadaver specimens using techniques such as stereophotogrammetry and laser scanning. A review of these methods will be provided in this presentation, along with recent advances in the engineering of mechanically functional cartilage tissue constructs.
How to make a limb: developmental paradigms
Authors: Niswander Lee Ann
Published: June 2021

The embryonic vertebrate limb is a classic paradigm for the study of pattern formation, cell growth and death, and skeletal formation. Our understanding of the molecular signals that regulate limb patterning is relatively complete. However, many questions remain as to how these signals are interpreted and translated into skeletal elements that are adapted to the needs of each animal on land, sea and air. Outstanding questions in patterning and skeletal formation include how and when are limb cells specified and acquire distinct cell fates in order to contribute to specific skeletal elements and what are the cellular and molecular mechanisms that lead to the establishment of the skeletal elements of the correct size and shape?

Our work is driven forward by two approaches. The first is the creation of mouse mutants with defects in limb development. We then clone the responsible gene that is mutated and discover its mechanism of action. The second approach uses a new imaging method to watch in real time as the limb mesenchyme undergoes the early steps of cartilage formation. We then combine this with the study of limb mesenchyme from mouse mutants to begin to understand the cell behaviors that are affected in mutants with too many digits, too few digits, or defects in cartilage formation. Furthermore, we have used our knowledge of limb development in models organisms, the mouse and chick, to begin to unravel the molecular mechanisms that underlie evolutionary change in the bat.
Multimodal Imaging of Embryogenesis
Authors: Lansford Rusty
Published: June 2021

Our research aims to dynamically visualize and characterize embryonic development at sub‐cellular resolution. We have used modified viruses to generate transgenic avians as an experimental system in order to study brain and heart formation. Our fluorescent toolbox relies on XFPs to label cells in order to dynamically follow their movement using multispectral imaging. The gene expression and cell migration data collected using multispectral imaging is subsequently integrated within MRI collected datasets in order to understand the complex informational interactions that are occurring during development within the spatial and temporal context of the maturing embryo.
Adding anatomy to the medical clerkships: experience with surgery and clinical skills
Authors: Jones Kenneth H , DePhilip Robert M
Published: June 2021

Reduced time for anatomy in the first year medical curriculum challenges anatomists to provide students with opportunities to learn the anatomy that is essential for clinical practice. One way to meet this challenge is to integrate specific topics of anatomy into the third year when they are clinically relevant. Previously we determined that third year students prefer a combination of demonstration by an instructor and “hands‐on” work with a cadaver to learn anatomy. More recently, we consulted with clinical colleagues to develop “need to know” clinical anatomy sessions that are now required for third year students in both the Surgery clerkships and in the Clinical Skills Immersion Experience (CSIE). Prosections are used to review specific anatomical relationships that are relevant to general surgery and to selected invasive procedures. Groups of 35 students rotate among the prosections and are assisted by anatomy staff or are self‐guided by exercises on computers. In the 2‐day CSIE, students first review relevant anatomy and then practice selected invasive procedures, e.g., chest tubes, paracentesis, thoracentesis, and kidney biopsy on unembalmed cadavers. Students report that they are “very satisfied” with these anatomy sessions. Invasive procedure training using unembalmed cadavers is greatly appreciated as it reduces anxiety and provides an authentic experience that cannot be matched using models.
Ezrin enhances survival of single metastatic osteosarcoma cells that reach the lung in an AKT‐independent manner
Authors: Hong Sung‐Hyeok , Ren Ling , Khanna Chand
Published: June 2021

To determine mechanisms of early metastatic advantage provided by ezrin, we examined adhesion, extravasation, and survival early after osteosarcoma (OS) cell arrival in the lung. We found no evidence ezrin‐dependent influences on tumor cell adherence. Confocal imaging of green tumor cells against a red fluorescent vascular map, demonstrated extravasation of both high and low ezrin expressing cells within 15 minutes of arrival in the lung. However, apoptotic rate in cells that expressed high ezrin was significantly lower than cells with low ezrin expression as early as 3 hours after the arrival of cells in the lung. These data suggest that the early metastatic survival associated with ezrin expression was the result of ezrin‐associated inhibition of apoptosis of tumor cells that had left the vasculature early after their arrival in the lung. To further examine mechanisms for this apoptosis we stably expressed an active Akt construct (actAkt) in the low ezrin cells. We found no rescue of the ezrin‐dependent single‐cell metastatic survival between actAkt clones (n = 4) and empty vector clones (n = 2). In addition, Akt inhibitor I/II did not induce apoptosis in the early time of survival novel ex vivo lung culture assay of metastasis. These data suggest that the Akt pathway is not responsible for the induction of apoptosis seen in low ezrin expressing OS cells early after their arrival in the lung.
Molecular Tipping Points in Neuroinflammation
Authors: Carson Monica J
Published: June 2021

Microglia, known as the brain macrophage are highly plastic in their phenotype, capable of performing a wide variety of cytoprotective (“wound healing”) and cytodestructive (pathogen‐defense) functions. We have begun to identify key molecular triggers that regulate microglial function and that may contribute toward tipping acute neuroinflammation toward chronic neurodegeneration. Here, we present the stage and pathology specific expression of two related receptor families: TREMs and TLTs. Due to the presence of ITIM domains, TLTs are postulated to trigger inihibitory intracellular signaling pathways, while TREMs are postulated to trigger activation via ITAM mediated intracellular signaling. Using overexpression and knock‐down strategies, we demonstrate the broad array of microglial functions regulated by these receptor families. We further find that despite being defined as “activating” receptors, TREMs may help to trigger neuroprotective and anti‐inflammatory responses within the CNS that have the potential to be exploited in therapeutic strategies.