bluuebunny/crossref_metadata_2025 huggingface dataset:
The Coffin‐Lowry syndrome‐associated protein RSK2 regulates neurite outgrowth through phosphorylation of PLD1 and synthesis of phosphatidic acid, a membrane curving fusogenic lipid.
Authors: Vitale Nicolas , Ammar Mohamed Raafet , Bader Marie‐France
Published: June 2021

Most of the mutations associated with mental retardation (MR) induce limited visible morphological alterations in brain organization and the molecular bases underlying the neuronal clinical features remain elusive. We show here that neurons cultured from mice deficient for ribosomal S6 kinase 2 (Rsk2), a model for the Coffin‐Lowry syndrome (CLS), exhibit a significant delay in development in a pattern very similar to that shown by neurons cultured from phospholipase D1 (Pld1) knockout mice. We found that gene silencing of Pld1 or Rsk2 as well as acute pharmacological inhibition of PLD1 or RSK2 strongly impaired neuronal growth factor (NGF)‐induced neurite outgrowth from PC12 cells. NGF triggered RSK2‐dependent phosphorylation of PLD1 leading to the synthesis of phosphatidic acid at the site of neurite growth. NGF‐induced neurite outgrowth and VAMP‐7 vesicle exocytosis was severely inhibited in PC12 cells silenced for RSK2 expression. However, expression of a phosphomimetic PLD1 mutant or provision of lysophosphatidic acid, an inverted cone‐shaped lipid that favors membrane curvature were able to rescue the growth defect in RSK2 silenced cells, revealing that PLD1 is the major target of RSK2 in neurite formation. We propose that the loss of function mutations in RSK2 that lead to CLS and neuronal deficits are related to defects in neuronal growth due to impaired RSK2‐dependent PLD1 activity.
Acceptance of modified food by low income audiences
Authors: Roy Heli , Urala Nina , Cater Melissa , Thomas Bipin
Published: June 2021

Objective

We assessed the acceptance of modified foods by low income audiences before and after nutrition education sessions.

Methods

Participants were recruited trough the on‐going community nutrition education programs to assess the intake of modified foods (low salt, whole wheat products, low fat milk, yogurt with probiotic bacteria, and fruit and vegetable intake) by flyers at client recruitment sites in 15 parishes. The intake and liking of modified and non‐modified food products, eating‐related attitudes, and eating choices were assessed by a questionnaire. The subjects were randomized into intervention and control groups receiving either 4–6 lessons on nutrition or on family financial management.

Results

211 urban and rural participants were recruited in the control group and intervention group. The urban participants had a significant increase in vegetable intake after the intervention (P=0.0049). In the rural participants, there was a significant decrease in whole milk intake (P=0.0442), and a significant increase in probiotic yogurt intake.

Conclusion

Low income audiences adjust their food intake of modified foods after 4–6 nutrition educational sessions.
Leucine‐enriched essential amino acid mixture suppresses muscle soreness induced by eccentric exercise in rats
Authors: Kato Hiroyuki , Mimura Masako , Suzuki Hiromi , Inoue Yoshiko , Sugita Mayu , Suzuki Katsuya , Kobayashi Hisamine
Published: June 2021

Exercise‐induced muscle damage causes to muscle fatigue, muscle soreness, and so on. Recovery of the muscle damage may be accelerated by an enhancement of skeletal muscle protein synthesis (MPS). Leucine‐enriched essential amino acid mixture (LEAA) increases MPS. Therefore, we investigated the effect of LEAA on MPS and on the muscle soreness after eccentric exercise in rats.

LEAA (1 g/kg BW), in which 40% of total amount of amino acids was leucine, or water as control was orally administered after running exercise (−13.5°, 17 m/min for total of 130 min). MPS (%/h) in vastus lateralis muscle was measured by the incorprating L‐[ring‐2H5]phenylalanine into protein 20 minutes following LEAA administration. LEAA significantly increased MPS (Control : 0.084±0.006, LEAA : 0.119±0.007 %/h, p < 0.05).

Muscle soreness, which was induced by eccentric contraction (EC) to the tibialis anterior muscle (TA), was investigated by means of the behavioral pain test (Randal‐Selitto test applied to the TA) 1 to 3 days after EC. LEAA (1 g/kg BW) was orally administered 30 minutes before and 1 hour after EC. LEAA significantly suppressed muscle soreness (Control : 85.1±2.5, LEAA : 92.8±1.7 % of the value before exercise, p < 0.05).

These data suggest that LEAA improves muscle soreness following eccentric exercise by enchancement of MPS.
Genetic regulation of impaired fasting glucose in congenic mice
Authors: Kim Jung Han , Mao Xia , Stewart Taryn , Dillon Kristy
Published: June 2021

Previously, we identified a quantitative trait locus (QTL) linked to 4‐hour fasting plasma glucose levels on mouse chromosome 4, Tanidd4, using F2 progeny of normal C57BL/6 (B6) and type 2 diabetic TALLYHO/Jng (TH) mice. For this locus, the TH allele was associated with hyperglycemia. Subsequently, we have generated congenic mice that carry the tanidd4 QTL derived from TH mice on a B6 background. In this study, we characterized the glucose metabolism, body composition, and energy balance in the congenic mice. Overnight fasting plasma glucose levels were significantly increased in the congenic mice compared to B6 control mice [140±6 vs. 113±3, 13 weeks of age (wk); 165±9 vs. 114±11, 24 wk; means±SE; males]. However, there was no difference in glucose tolerance between the two strains (by IPGTT). Interestingly, the congenic mice were significantly heavier than B6 mice, and the body weight difference was accounted for by an increase in lean body mass rather than fat mass (by MRI). The congenic mice also showed increased energy expenditure (heat production; kcal/hour) and locomotor activity compared to B6 mice (by Comprehensive Laboratory Animal Monitoring System; 19–20 wk). There was no difference in fuel source preference, as respiratory exchange ratio. In summary, tanidd4 congenic mice offer a useful model for identifying genetic determinants of impaired fasting glucose, a characteristic of pre‐diabetes in humans.
Pathway of leukocyte transendothelial migration across an in vitro human blood brain barrier model
Authors: Winger Ryan C. , Koblinski Jennifer E. , Muller William A.
Published: June 2021

Leukocyte transendothelial migration (TEM) can occur at endothelial junctions (paracellular) or directly through the endothelial cell body (transcellular). Both pathways involve similar molecules but the factors governing the migratory pathway choice are unknown. Transcellular TEM may preferentially occur in vasculature where the junctions are complex and tight, such as the blood brain barrier (BBB). Therefore, to study the pathway of TEM at the BBB in vitro, we constructed a human BBB model by culturing human umbilical vein endothelial cells in astrocyte conditioned media supplemented with cAMP modulators. Our model exhibits >;5 fold greater barrier function as measured by transendothelial electrical resistance and permeability to FITC‐Dextran. We investigated the pathway of TEM using 3D confocal imaging of fixed immunostained cells. Despite enhanced barrier function, paracellular TEM predominated to the same extent as under standard conditions. Interestingly, directly activating the leukocytes with apical TNF or MCP‐1 (CCL2) increased the proportion of transcellular TEM equally under both standard and tight junction conditions. Thus, the route of TEM is not altered by the tightness of endothelial junctions under these conditions, but can be influenced by the activation state of the leukocytes. This research was supported by T32 Al07476 to RCW and RO1 HL046849 and R37 HL064774 to WAM.
Macrophage Dysfunction and Impaired Wound Healing in Corneas of Heme Oxygenase (HO)‐2 Deficient Mice
Authors: Bellner Lars , Marrazzo Giuseppina , Castellano Kirkland , Dunn Michael W , Schwartzman Michal L
Published: June 2021

Corneal epithelial injury in HO‐2 null (KO) mice leads to delayed wound healing, exaggerated inflammatory cell infiltration, unresolved inflammation, ulceration, perforation and neovascularization. We examined the effect of systemic macrophage (MØ) depletion, using clodronate liposomes, on corneal wound healing in WT and KO mice. The corneal epithelium was removed in control and MØ‐depleted WT and KO mice. MØ depletion of WT mice delayed corneal wound healing when compared with the control group by 15% at day 4 after injury (p<0.05). Surprisingly, MØ depletion of KO mice had no additional effect on an already impaired corneal wound healing. Furthermore, MØ depletion caused a 100% (p<0.05) increase in the number of PMNs in the corneas of WT mice, whereas no significant change in the number of accumulating PMNs was observed in the corneas of KO mice. HO‐2 silencing of RAW 264.7 MØ using HO‐2 shRNA reduced phagocytic capacity by 40% (p<0.05), whereas HO‐induction using SnCl2 increased phagocytic capacity by 85% (p<0.05). Together these data indicate that HO‐2 deletion impairs MØ function and that an intact HO‐2 is required for ordered inflammatory and repair response.

Supported in part by NIH grant EY06513 (MLS).
Chronic Absence of Baroreceptors Induces Left Ventricular Dysfunction and Reduced Coronary Blood Flow in Spontaneously Hypertensive Rats
Authors: Souza Pamella R , Moreira Edson D , Mostarda Cristiano , Jorge Luciana , Monteiro‐de‐Moraes Wilson Max , Guimarães Fabiana , Oliveira Edilamar Menezes , Flues Karin , Silva Maikon Barbosa , Irigoyen Maria Claudia
Published: June 2021

Hemodynamic and cardiac morphological variables were measured in normotensive Wistar (N), intacts hypertensive (H) and SHR submitted to sinoaortic denervation (DH) to evaluate the chronic baroreceptor dysfunction on left ventricular (LV) functions and coronary blood flow. Blood (BP) and end‐diastolic pressure (PD2) were obtained by direct quantification. The coronary blood flow was measured by the infusion of colored microspheres. The ratio of left ventricular weight to body weight, natriuretic atrial peptide (ANP), alpha‐skeletal actin (α‐skelectal), alpha and beta myosin heavy chain (α‐MHC, β‐MHC) genes mRNA expressions were evaluated by real time PCR to assess ventricular hypertrophy. The results were reported as means ± SEM. DH rats did not change BP values compared to H. PD2 of DH (9.91±1.78) was higher than H group (4.30±0.53) while an inverse result regarding coronary blood flow was observed (0.94±0.15 vs 1.80±0.25, respectively). The LV adaptations in DH were higher than in H (2.98 ±0.07 vs 2.15±0.05), as well as the expression of ANP (3.35±0.19 vs 2.39 ± 0.74), β‐MHC (3.36±0.18 vs 2.39 ± 0.37). The α‐MHC genes expression also were decreased (0.62±0.10 vs 0.79±0.10). Therefore, the chronic absence of baroreflex control by sinoaortic denervation induces left ventricular dysfunction and reduced coronary blood flow in SHR.

Financial support: CAPES
Glia maturation factor levels are elevated in the striatum and substantia nigra in Parkinson's disease
Authors: Zaheer Smita , Thangavel Ramasamy , Zaheer Asgar
Published: June 2021

Glia maturation factor (GMF) is an inflammatory protein, discovered in our laboratory, plays a major role in the pathogenesis of CNS inflammatory diseases. Although GMF has been implicated as a major contributing factor in the pathogenesis of EAE, an animal model of Multiple Sclerosis, there has not been a direct demonstration of GMF expression in human PD brains. In the present study we demonstrate a significant up regulation of GMF expression in human PD brain. Human brain tissues were obtained from autopsy, with PMI less than 6 h, at UI Deeded Body Program. For the comparison, we examined brain tissue from cases with clinically diagnosed and neuropathologically confirmed Parkinson's disease and age‐matched non‐PD controls. Our results show degenerating TH‐ immunostained neurons/ fibers associated with activated microglia and astrocytes in the autopsied brain from PD case. In PD brain, GMF appears to be localized to activated astrocytes and dopaminergic neurons. Additionally, we quantitatively estimated GMF protein by ELISA in different regions of PD brains. We demonstrated a significant increase (over 10 fold) in GMF levels in the substantia nigra and striatum of human PD brains compare to non‐PD. Thus, our results provide strong evidence that under conditions of neurodegeneration in PD the expression of GMF is significantly upregulated. (Supported by VA Merit Review award and NINDS grant NS073670).
Diurnal difference in sympathetic stimulation and microRNA regulation of renin in Schlager hypertensive mice
Authors: Jackson Kristy Larissa , Marques Francine Z , Watson Anna M.D. , Nguyen‐Huu Thu‐Phuc , Morris Brian J , Charchar Fadi J , Davern Pamela J , Head Geoffrey A
Published: June 2021

Schlager BPH/2J mice are hypertensive due to greater contribution of the sympathetic nervous system (SNS) and this could also activate the renin‐angiotensin system (RAS) within the kidney. To determine the contribution of the SNS and peripheral RAS to hypertension, BPH/2J and normotensive BPN/3J mice were pre‐implanted with radio‐telemetry devices to measure blood pressure. The depressor response to ganglion blocker pentolinium in enalaprilat pre‐treated mice, revealed 2‐fold greater SNS contribution to hypertension during the active and inactive period. Depressor responses to enalaprilat were greater in BPH/2J mice, but only during the active period (P=0.01) which was accompanied by 1.6‐fold greater renal renin (Ren1) mRNA (P=0.02) and lower levels of microRNA‐181a (P=0.03), a previously identified regulator of renin. Ren1 mRNA levels were positively associated with depressor responses to pentolinium (P=0.001, r2= 0.99) and tyrosine hydroxylase staining indicated greater renal sympathetic innervation. Thus there is a tonic SNS contribution to hypertension in BPH/2J mice but the RAS contributes predominantly during the active period and less during the inactive period, the opposite of the normal circadian pattern. The mechanism may involve renal hyper‐innervation and enhanced sympathetically‐induced renin synthesis, likely mediated by lower miR‐181a levels. Supported by NHMRC Australia
Angiotensin II (ANG II) slow pressor hypertension enhances NMDA receptor (NMDAR)‐mediated currents and Nox2‐ dependent superoxide (O2‐) production in spinally‐projecting (SP) neurons of hypothalamic paraventricular nucleus (PVN)
Authors: Wang Gang , Davisson Robin L , Pickel Virginia M , Iadecola Costantino
Published: June 2021

Adaptive changes in glutamate (GLU) neurotransmission in the PVN play a role in the increased sympathetic output driving the elevation in blood pressure induced by slow pressor ANG II infusion. We hypothesized that these adaptions alter neuronal production of nitric oxide (NO) and NADPH oxidase (Nox2)‐ derived O2−, resulting in enhanced GLU neurotransmission in SP pre‐sympathetic PVN neurons. Electron microscopic immunolabeling showed a marked increase in the association between Nox2 and the NMDAR NR1 subunits in PVN dendrites of 14 days ANG II‐infused mice (600 ng/kg/min, P<0.01 vs. saline). NMDAR‐mediated O2− production was enhanced in isolated SP PVN neurons of ANG II‐infused mice (P<0.01 vs. saline), an effect blocked by Nox2 inhibitor gp91ds‐tat. NMDAR‐mediated NO production was suppressed in PVN neurons of ANG II infused mice (P<0.01 vs. saline), an effect opposed by gp91ds‐tat. In patch‐clamp recording of SP PVN neurons, NMDAR mediated a larger inward current in ANG II‐infused mice (P<0.01 vs. saline), an effect inhibited by gp91ds‐tat or an NO donor. These findings suggest that slow pressor doses of ANG II increase the association of NR1 with Nox2 in SP PVN neurons, resulting in enhanced production of Nox2‐derived O2− during GLU synaptic activity. The resulting depletion of NO and enhancement of NMDA currents may contribute to the increased sympathetic outflow underlying slow pressor ANG II hypertension. (Supported by NIH HL096571)
Deoxycorticosterone acetate (DOCA)‐salt exacerbates hypertension and vascular dysfunction in mice expressing dominant negative Peroxisome Proliferator‐Activated Receptor‐gamma (PPARG) in smooth muscle
Authors: Ketsawatsomkron Pimonrat , Davis Deborah R , Grobe Justin L , Sigmund Curt D
Published: June 2021

PPARG, a ligand‐activated transcription factor plays a critical role in regulation of blood pressure and vascular function. We hypothesized that smooth muscle PPARG protects against hypertension and resistance vessel dysfunction. Mice expressing dominant negative PPARG (S‐P467L) in smooth muscle or non‐transgenic control (NT) were implanted with DOCA‐salt and allowed ad lib. 0.15 M NaCl for 21 days in addition to chow and tap water. Blood pressure was monitored by telemetry and mesenteric arterial function was assessed by pressurized myograph. At baseline, 24‐hour mean arterial pressure (MAP) was similar between NT and S‐P467L mice, while the transgenic mice were tachycardic. DOCA‐salt increased MAP in all animals, but S‐P467L mice exhibited increased sensitivity (Δ mmHg; S‐P467L n=5, +34.2±7.3 p=0.003 vs baseline; NT n=3, +15.0±8.5 p=0.152 vs baseline). Heart rate was similarly decreased in both groups after DOCA‐salt. Vasorelaxation to acetylcholine was impaired in S‐P467L mesenteric arteries compared to NT at baseline and this effect was further exaggerated after DOCA salt. Vasopressin‐induced constriction was significantly reduced in S‐P467L compared to NT arteries after DOCA‐salt, but the response to KCl and endothelin‐1 did not differ between groups. We conclude that impaired smooth muscle PPARG sensitizes the effects of DOCA‐salt induced hypertension and vascular dysfunction.
Aging exacerbates obesity‐induced cerebromicrovascular injury and cognitive decline in mice
Authors: Tucsek Zsuzsanna , Toth Peter , Sosnowska Danuta , Gautam Tripti , Sonntag William E , Ungvari Zoltan , Csiszar Anna
Published: June 2021

Obesity in the elderly promotes the development of vascular cognitive impairment, which develops in the absence of Alzheimer‐type senile plaques as a result of cerebromicrovascular alterations. To elucidate the mechanisms by which aging exacerbates the deleterious cerebrovascular effects of obesity, young (7 mo) and aged (24 mo) C57/BL6 mice were fed a high fat diet for 5 months. Compared to young obese mice, aged obese mice exhibited impaired pressure‐induced myogenic constriction and flow‐induced constriction of middle cerebral arteries, disruption of the blood‐brain barrier, hippocampal microvascular rarefaction, microglia activation, up‐regulated hippocampal expression of pro‐inflammatory cytokines and chemokines and impaired performance in tests relevant for hippocampally ‐ dependent tasks of learning and memory. Taken together, aging exacerbates obesity‐induced cerebromicrovascular injury and neuroinflammation, which likely promote the development of vascular cognitive impairment in aged obese individuals.
Dietary fructose and glucose alter measures of trabecular bone formation and architecture in growing male rats
Authors: Bass Erica F. , Baile Clifton A. , Lewis Richard D. , Giraudo Silvia Q.
Published: June 2021

This study investigated the effects of dietary glucose and fructose on bone formation, microarchitecture, and strength. Twenty‐four male Sprague‐Dawley rats (60 days old) were randomly assigned to consume a high fructose (n=9; 40% fructose, 10% glucose), high glucose (n=9; 50% glucose), or chow diet (n=6; 44% cornstarch) for 12 weeks. Bone measurements included histology and histomorphometry of trabecular bone in the distal femur and a three‐point bending test of the whole tibia. Epididymal and perirenal fat pads were weighed as a measure of abdominal adiposity. There were no differences in energy intake or body weight between groups. Mineral apposition rates (p=0.005), osteoblast surface (p=0.002), and osteoblast number (p=0.005) were higher in glucose and fructose‐fed rats compared to chow‐fed rats. Despite similar effects on bone formation, trabecular thickness was higher in the fructose group compared to the glucose group (p=0.0016). Differences in cortical bone strength among groups were not observed. Heavier epididymal fat pads (glucose vs. chow; p=0.003) and livers (fructose vs. glucose; p=0.000), combined with variations in bone outcomes with different sugar intakes, suggest a link between energy metabolism and bone that should be explored further as a potential explanation for these findings. This work was supported by Georgia Agricultural Experiment Stations grant GEO00663 Hatch 0217572.
Does Vitamin D add to the effect of 12‐weeks resistance training? A randomized controlled trial on healthy untrained young and elderly men
Authors: Agergaard Jakob , Trøstrup Jeanette , Uth Jacob , Vestergård Jonas , Boesen Anders , Schjerling Peter , Langberg Henning
Published: June 2021

Introduction

Vitamin D (VitD) intake has shown to improve skeletal muscle function in VitD insufficient frail elderly. However, if VitD intake may improve the effect of resistance training (RT) in healthy individuals is unknown.

Material and Methods

20 healthy untrained young (age 22.8 ± 2.0) and 20 elderly (age 67.3 ± 4.3) men were randomized to 16 week daily supplementary intake of either 48 μg VitD + calcium (VitD‐group) or calcium (Placebo‐group), and underwent progressive RT of quadriceps muscle for the last 12 weeks. Pre and post 12 weeks RT cross sectional area (CSA), and isometric strength of quadriceps muscle was measured. Muscle fiber type morphology was analyzed from muscle biopsies taken pre and post training.

Results

VitD intake increased serum 25(OH)D levels in young and elderly to an extent that were different from the respective placebo group. RT increased CSA and isometric strength and improved skeletal muscle morphology of quadriceps muscle. However, no additive effect was seen from VitD intake in young or elderly.

Conclusion

12 weeks of RT altered CSA, strength and fiber type morphology of quadriceps muscle as expected. However, VitD intake did not add to the positive effect of RT.
Angiotensin and AMPA receptor subunit mRNA levels are reduced in NTS catecholaminergic neurons following chronic intermittent hypoxia (CIH)
Authors: Bathina Chandra Sekhar , Rajulapati Anuradha , Mifflin Steve
Published: June 2021

Catecholaminergic A2 neurons in caudal NTS are activated during stressors such as hypoxia. The aim of this study was to assess the effect of CIH on the mRNA expression levels of AT1a, AT1b and AMPA and NMDA receptor subunits in the A2 neurons. AAV vector mediated delivery of green fluorescent protein (GFP) labeled tyrosine hydroxylase promoter (AAV‐GFP‐TH) was used to label A2 neurons. 7 virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). Laser capture microdissection was performed to capture A2 neurons from caudal NTS. Total RNA from these neurons was extracted and mRNA levels assessed by quantitative real time reverse transcription PCR and compared between the control and CIH rats using 2‐ÄÄct method. CIH decreased AT1a (p=0.002; control − 1.08 ± 0.13, n=7; CIH − 0.48 ± 0.07, n= 6) and AMPA receptor subunit GluR2 (p=0.03; control − 1.11 ± 0.24, n=7; CIH‐ 0.52 ± 0.12, n= 6) and increased transcription factor FosB (p=0.03; control − 1.14 ± 0.25, n=7; CIH‐ 1.97 ± 0.25, n= 5) mRNA levels in the A2 neurons. CIH did not alter levels of GluR1, NR1, NR2 or delta FosB. These results indicate that exposure to CIH selectively alters receptor subunit mRNAs in A2 neurons which could influence their responses to ligand. Supported by HL88052.
Time‐dependent muscle‐specific protein oxidation in a mouse model of chronic hypoxia
Authors: O'Halloran Ken , Sheehan David , Lewis Philip
Published: June 2021

Respiratory muscle redox homeostasis is altered by chronic hypoxia (CH). Protein oxidation potentially contributes to respiratory muscle dysfunction in respiratory‐related diseases where hypoxia features. The objective of this study was to determine the type and extent of respiratory and limb muscle protein oxidation temporally in a mouse model of CH.

C57/Bl6J mice were exposed to hypoxia (1, 3 and 6 weeks; 10% FiO2) or normoxia. Following treatment, excised respiratory and limb muscles were homogenised and incubated with carbonyl‐ or thiol‐reactive fluorophores before gel electrophoresis and fluorescence scanning. Optical density (OD) of fluorescence was normalised to total protein, determined by colloidal coomassie staining.

Diaphragm free‐thiol content increased ~9‐fold after 1 week of CH. By 6 weeks, both diaphragm and sternohyoid free‐thiol content were significantly decreased (40% and 80% decrease from control respectively; n=6–8 per group; P=0.0046 and P<0.0001, Student's unpaired t‐test) whereas there was a significant increase in the extensor digitorum longus (232%; P=0.0007) and in soleus (143%; P<0.0001). Diaphragm carbonyl content was unchanged after 1 week of CH, but was increased significantly by 3 weeks and was increased further again after 6 weeks. The extent of carbonylation was different between muscle groups.

We conclude that CH exposure causes differential time‐dependent protein oxidation in respiratory and limb muscles.
Effects of processing on the pollen and nutrient content of honey
Authors: Beals Katherine , Ropa David
Published: June 2021

This pilot study examined the effects of commercial processing on the pollen and nutrient content of honey. From a total of 22, 55‐ gallon drums of honey, 5 random drums were chosen to obtain 5 random samples of raw honey (unblended (UB) raw). The 22 drums were blended and heated at 180°F and 2 additional random samples were selected (blended (B) raw). The blended honey settled at 130°F for 12 hours, was manually skimmed (removing foam and extraneous solids), flash heated to 175°F for 7 minutes, filtered using diatomaceous earth (DE) and flash cooled to 130°F after which two additional samples were taken (blended (B) filtered). All samples were analyzed by outside laboratories (Covance Labs, Madison, WI and ABC Labs, Gainsville, FL) for micronutrient content, antioxidant (AOX) capacity (ORAC) and pollen content. Processing reduced the pollen content of the honey (P<0.05)*, but did not affect the nutrient content (see table) indicating that the micronutrient profile of honey is not related to its pollen content and is not significantly affected by commercial processing. Funded by the National Honey Board
Role of BKCa Channels in NO‐Induced Relaxation of Control and Nitrate Tolerant Isolated Mesenteric Arteries
Authors: Sane Mukta , Shukla Praveen , Sun Chengwen , O'Rourke Stephen
Published: June 2021

The clinical efficacy of nitroglycerin (NTG) is limited by the development of tolerance. The present study was designed to identify the role of BKCa channels in NO‐induced relaxation of control and tolerant arteries. Rats were treated with or without NTG (0.6 mg/hr) patches for 3 days. Isometric tension was recorded in isolated mesenteric arteries and relaxation responses to NTG and sodium nitroprusside (SNP; 10−9 to 10−5 M) were obtained. Nitrate tolerance was evident by marked impairment in responses to NTG and SNP in tissues from rats treated with NTG patches. Iberiotoxin (10−7 M), a selective BKCa‐blocker, inhibited NTG‐ and SNP‐induced relaxations in rings from nontolerant rats; residual responses to NTG and SNP were abolished in rings from tolerant rats. The selective BKCa‐opener, NS1619 (10−9 to 10−6 M), caused relaxation of tolerant and nontolerant arteries; however, tolerant rings were more sensitive than nontolerant rings to relaxation by NS1619. Immunoblot analysis indicated increased BKCa expression in arteries from tolerant animals. The data suggest that NO‐induced relaxation is dependent on BKCa activation in control and tolerant mesenteric arteries. Although BKCa expression is increased in tolerant arteries, responses to NO‐donors, but not BKCa‐openers, are markedly impaired, consistent with defective coupling between NO donors and BKCa activation (Supported by NIH HL098896).
A novel signalling role for NAADP in arterial smooth muscle
Authors: Lim Chloe , Churchill Grant , Galione Antony , Garland Christopher , Dora Kim
Published: June 2021

In arterial smooth muscle, Ca2+ is a pivotal modulator of vascular tone. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizer, releasing Ca2+ from lysosomal‐type acidic stores. NAADP can act as an agonist by binding to two pore channels, with the structural analogue Ned‐19 acting as an antagonist. We investigated the actions of NAADP in arterial smooth muscle using isolated rat mesenteric arteries. In pressurized arteries, confocal imaging of the intrinsic fluorescence of Ned‐19 demonstrated an overlap with a subpopulation of acidic organelles labeled with Lysotracker Green. In arteries mounted on the wire myograph, tone generated with the thromboxane mimetic U46619 was rapidly reversed when exposed to the cell‐permeant acetoxymethylester of NAADP (NAADP‐AM). This relaxation occurred in both endothelium‐intact and ‐denuded preparations, and was sensitive to an inhibitor of large‐conductance K+ channels (BKCa, iberiotoxin). Finally, we used immunohistochemistry in pressurized arteries to characterize the distribution of BKCa channels and lysosomal associated membrane protein (LAMP‐2). In conclusion, these data support a signalling microdomain coupling NAADP‐mediated Ca2+ release from acidic stores to opening of BKCa channels, suggesting a novel potential mechanism for modulation of vascular tone. Supported by the British Heart Foundation and Wellcome Trust, UK.
Directed drug delivery: inhalable magnetic nano‐in‐microparticles for the application of lung cancer
Authors: McBride Amber , Price Dominique , Muttil Pavan
Published: June 2021

Lung cancer, the leading cause of cancer mortality, annually kills 1.4M people. Despite advances in surgery, drug discovery and therapy, lung cancer remains a difficult disease to treat. We propose that targeted inhaled drug delivery to the respiratory tract will facilitate treatment with lower systemic exposure and consequently, reduce side effects. Novel inhalable dry powders will be directed to tumors in the lung airways by a magnetic field gradient concentrating drug at the disease site. Previous studies using inhaled chemotherapeutics showed dose limiting pulmonary toxicity due to the non‐specificity of the delivery mechanism. We believe that targeted inhaled therapies could mitigate this problem. We successfully formulated and characterized the magnetically responsive nano‐in‐microparticles (NIMs) delivery vehicle containing superparamagnetic iron oxide nanoparticles (SPIONs) and doxorubicin for site‐specific pulmonary drug delivery. NIMs were characterized for their size, morphological properties, Fe3O4 loading and drug loading. The hydrodynamic diameter and aerodynamic diameter of NIMs were 1.6 μm and 3.27± 1.69 μm, respectively. SEM images show spherical particles with rough surface morphology. We have shown the ability to direct and retain NIMs in a proof‐of‐concept tube study to mimic human conducting airways with deposition of NIMs in regions experiencing a strong magnetic gradient. We have also administered NIMs in a healthy rat model by pulmonary route to demonstrate NIMs targeting capability, and to characterize their spatial deposition in the lung in vivo. These studies could radically change treatment modalities for lung cancer patients by increasing patient survival rates as well as reducing the side effects observed with current chemotherapies.
Intermittent Hypoxia in the Neonate Leads to an Augmented Stress Response in Adult Rats
Authors: Chintamaneni Kathan , Bruder Eric , Guenther Mitchell A , Raff Hershel
Published: June 2021

Intermittent hypoxia (IH) is used as an animal model of apnea‐induced hypoxia, a common stressor in the premature neonate. Neonatal stressors may have long‐term effects in the adult. We hypothesized that IH in the neonatal rat will lead to significant changes in hypothalamic‐pituitary‐adrenal (HPA) axis function in the adult. We measured baseline AM and PM (circadian rhythm) and stress‐induced plasma ACTH and corticosterone, as well as relevant HPA axis mRNA expression in adult rats exposed to neonatal IH. Rat pups were exposed to normoxia (control) or 6@30‐sec cycles of IH (10% O2 nadir FIO2) over 1 hour daily on postnatal days 2–6. Three months later, basal and stress‐induced HPA axis function was evaluated in adult male rats. Neonates exposed to IH had an augmented adult ACTH response and a prolonged corticosterone response to restraint stress. Exposure to neonatal IH did not alter the HPA axis circadian rhythm in adult rats. Pituitary Pomc, Crhr1, GR, MR, Avpr1b, and Hif1a mRNAs were decreased in adults exposed to neonatal IH; HPA axis–specific hypothalamic and adrenal mRNAs were unchanged. We conclude that exposure to neonatal IH programs the adult HPA axis to hyper‐respond to acute stress.
Acute skeletal muscle injury induces temporal changes in NF‐kB activation and MCP‐1 secretion in C2C12 myotube cultures
Authors: O'Fallon Kevin S , LaBarbera Katherine , Schwartz Lawrence M , Clarkson Priscilla M
Published: June 2021

Nuclear factor kappa‐beta (NF‐κB) and monocyte chemoattractant protein‐1 (MCP‐1) have been shown to regulate the inflammatory response to skeletal muscle injury. To determine the temporal responses of muscle cell‐derived NF‐κB and MCP‐1 following injury we utilized an acute scrape‐injury model in C2C12 myotube cultures. Culture medium (CM) and total protein were collected from control and injured cultures at baseline and 1h, 3h, 6h, 12h, and 24h post‐injury. Nuclear NF‐κB/p65 DNA binding activity and MCP‐1 protein in CM were quantified via ELISA. After injury p65 DNA binding activity decreased at 12h (0.6 fold, p<0.05) and MCP‐1 protein in CM increased at 6h (2.4 fold, p<0.02) and 24h (2.7 fold, p<0.001) relative to controls. The change in p65 activation relative to controls was not correlated to the change in MCP‐1 protein, relative to controls. Acute injury does not induce p65 activation in myotube cultures. Conversely, the suppression of p65 binding at 12h may function to limit myoblast proliferation and promote myoblast fusion to injured myotubes. Muscle‐derived MCP‐1 secretion increases within 6h but is not related to NFkB activation during the first 24h of wound healing.
Norepinephrine Accelerates Diaphragm Fatigue In Vitro
Authors: Frye Gregory Scott , Ferreira Leonardo F.
Published: June 2021

Sympathetic nervous activity is increased during exercise, chronic diseases, aging, and psychological stress. Muscle sympathetic nerve endings release norepinephrine, which stimulates primarily alpha‐adrenergic receptors, to regulate vascular conductance and blood pressure. However, little is known about norepinephrine effects on skeletal muscle function that are independent of blood flow. To address this issue, we assessed the contractile properties of diaphragm bundles exposed to buffer solution (control; n = 5) norepinephrine (NE, 100 μM; n = 3) for 60 minutes in vitro (i.e., free of blood flow). After the equilibration period, we determined the diaphragm isometric force‐frequency relationship and fatigue characteristics. The relationship between specific force (N/cm2) and stimulus frequency of diaphragm bundles was unchanged by NE. However, NE accelerated fatigue in vitro. Specifically, NE‐treated muscles generated lower forces than control (P< 0.05) from 45 s until the end of the fatigue trial (300 s). The rate of force development and relaxation were also slower in NE‐treated muscles during the fatigue trial. In subsequent studies, we found that the α1‐adrenergic receptor agonist phenylephrine (PE, 100 μM; n = 3) exerted effects nearly identical to those of norepinephrine. Our preliminary studies suggest that norepinephrine accelerates skeletal muscle fatigue through activation of α1‐adrenergic receptors, and these responses are independent of blood flow.
Intestinal inflammation induces functional Na+/H+ exchanger 3 defect via downregulation of PDZ‐domain adaptor protein PDZK1 (NHERF3)
Authors: Seidler Ursula Elisabeth , Yeruva Sunil , Chodisetti Giriprakash , Luo Min , Hagen Susan , He Peijan , Yun Chris C.
Published: June 2021

Intestinal inflammation results in loss of sodium absorptive function and diarrhea. The present study investigates a potential causal relationship between the inflammation‐induced reduction in expression levels of the PDZ‐adaptor protein PDZK1 and decreased functional activity of its binding partner Na+/H+ exchanger NHE3. Expression of PDZK1 mRNA and protein was found strongly decreased in inflamed intestinal mucosa of murine colitis models, in colonic mucosa of patients with active ulcerative colitis, and in Caco‐2bbe cells treated with Th1 proinflammatory cytokines. Acid‐activated NHE3 transport activity as well as its inhibition by forskolin (FSK) was assessed fluorometrically in microdissected murine and human BCECF‐loaded ileal villi and colonic crypts. The decrease in acid‐activated NHE3 activity and loss of FSK inhibition was similar in PDZK1 +/− enterocytes, in which PDZK1 protein content was reduced by >; 50%, than in inflamed enterocytes, suggesting a causal relationship between PDZK1 downregulation and NHE3 dysfunction, independent of inflammation. Likewise, ShRNA‐mediated PDZK1 knockdown in Caco2bbe resulted in a significant decrease in NHE3 membrane abundance, transport activity and FSK inhibition. We conclude that the marked decrease in the PDZ‐adaptor protein PDZK1 in inflamed murine and human enterocytes is causally related to inflammation‐associated NHE3 dysfunction.
Designing effective student presentation assignments that include thoughtful peer assessment
Authors: Miller Sue Ann
Published: June 2021

Investigating and presenting anatomical topics supplements learning by dissection. In addition to dissecting, students in Biology 222, Anatomy with Histology, present information and materials 6 times during a 1‐semester course. Two presentations involve dissected material and require coordinating presentation with at least 1 lab partner. Two are individual short oral presentations that require research about historical use of anatomical terms and eponyms. Two class discussions follow separate readings and require an assessment statement and consideration of associated ethical issues. Guidelines in the assignments assist preparation of good and effective presentations, and peer assessment is part the evaluation of most of these presentations. This report will address design of effective student presentation assignments including ways to obtain honest and thoughtful peer assessment as part of the evaluation.
Interactive video animations as a study guide for the human skeleton: 1. The scapula
Authors: Casey Gregory Patrick , Oliver Peter David , Cork Robert John
Published: June 2021

We have started to develop a series of teaching modules on the human skeleton. Each of these modules will have three components, a short instructional video, interactive Flash animations, and a self‐testing unit. The first module in this ‘Interactive Atlas of the Human Skeleton” is about the Scapula. The narrated video highlights the general orientation of the scapula and introduces the key bony landmarks to the new student. The interactive animations allow the student to rotate the scapula and locate all of the landmarks and the sites of muscle attachment. The animations can be used as a reference resource with the labels turned on, or can be viewed in self‐testing mode with labels that pop‐up when the mouse is scrolled over a region. The self‐testing aspect of the module can be expanded further by adding more Flash animations with ‘drag‐and‐drop’ labeling of the images.

These teaching modules will be implemented as part of the undergraduate ‘Human Anatomy’ course to give to the nursing students at LSUHSC where it will replace existing anatomy videos and printed ‘bone worksheets’. The student's performance on scapula related exam questions, with and without these modules will be compared.

Grant Funding Source: LSUHSC ‐ Department of Cell Biology & Anatomy
Using Ultra‐Deep miRNA sequencing for identification of possible new biomarkers in endometriosis patients
Authors: Rozenchan Patricia Bortman , de Carvalho Cristina Valeta , Gonçalves Giovana Aparecida , Invitti Adriana Luckow , Parreira Rafael Martins , Ramos Camila Lopes , Bettoni Fabiana , Parmigiani Raphael Bessa , Camargo Anamaria Aranha , Schor Eduardo , da Silva Ismael Dale Cotrim Guerreiro
Published: June 2021

Endometriosis seems to be a precursor of epithelial ovarian cancer, especially clear cell and endometrioid adenocarcinomas. The features shared by endometriosis and cancer include the ability to evade apoptosis, the stem cell‐like ability and angiogenic potential. As such characteristics are encoded by the cell's genetic constitution; acquired mutations are responsible for the malignant transformation of endometriosis. So, a number of differences between endometriosis and cancer are found at the molecular level and miRNAs are recognized as key regulators of gene expression. Besides, aberrant expression of these small RNAs has been linked to human disease, including those related to estrogen‐dependent gynecological disorders and cancer. Total RNA of 09 type IV endometriosis patients, as well as, 07 normal endometrioid tissue was extracted, enriched for small RNAs and used for microRNA expression analysis on SOLiD4 sequencer. Sequences were mapped against the miRBase using CLC Genomics Workbench 6.0.1. An average of 5 million reads per sample was mapped on known miRNAs. After mapping and normalization of reads, we used EdgeR to identify differently expressed miRNAs, those with p<0.01 and FDR < 0.05. With this methodology we could obtain differentially expressed miRNAs between the samples which indicates that this approach is feasible for new biomarkers search.
Sustained delivery of steroid and protein hormones by ceramic delivery system in human plasma
Authors: Benghuzzi Hamed A , Tucci Michelle
Published: June 2021

The major objective of this study was to investigate the effectivenees of Tricalcium phosphate drug delivery system (TCP) to release steroid and protein hormones at sustained levels for long duration. A total of 70 TCP capsules were fabricated following standard lab protocols. The capsules were randomely divided into seven groups of 10 each. Each capsule in groups 1–6 were loaded with Testosterone (40mg), Estrogen (40mg), Progesterone (40mg), insulin (40mh), Thyroid Stimulating Hormone (40mg), and Insulin‐like Growth Factor‐1 (40mg); repecrively. Group 7 capsules were left empty and served as a control group. The capsules were sealed and suspended in serum bottles filled with 100 mls of human plasma. Samples were collected every three hours for first 24 hours and daily thereafter. Data collected from this study demonstrated that TCP capsules were cabable of releasing steroid and protein hormones for 26 days at different sustained levels. The results suggest that physiochemical characteristics of hormones have to be taken in consideration in developing ceramic drug delivery system.
NK1 receptors in the paraventricular nucleus are not involved in the regulation of the cardiovascular responses induced by chemoreflex stimulation
Authors: da Silva Nathalia Teodoro , Haibara Andrea Siqueira
Published: June 2021

Studies have shown that paraventricular nucleus (PVN) is involved in the control of the simpathoexcitatory component of the chemoreflex. However the receptors involved in the neurotransmission of this reflex still are not known. Studies have shown that glutamate, GABA or nitric oxide receptors in the PVN are not involved in the chemoreflex responses. This study evaluated the effect of blockade of substance P receptors (NK1 receptors) in the PVN on the cardiovascular responses of the chemoreflex induced by intravenous injection of potassium cyanide (KCN) in conscious rats. Bilateral microinjection of CP 99,994, NK1 receptor antagonist, into the PVN produced no significant changes on the pressor response (+39 ± 3 vs +34 ± 4 mmHg) and bradycardia (−225 ± 15 vs −207 ± 21 bpm) of the chemoreflex. Furthermore, the bilateral microinjection of CP 99,994 also produced no changes on baseline cardiovascular parameters (114 ± 4 vs 117 ± 4 mmHg; 390 ± 11 vs 388 ± 14 bpm). These results indicate that NK1 receptors in the PVN are not involved in the regulation of the cardiovascular parameters during baseline or chemoreflex stimulation. Support: INCT‐CNPq/ CAPES/ FAPEMIG.
Hepatocyte‐specific GOLM1 Knockout Mice and Human Hepatocellular Carcinoma Cell Lines Provide Insights into Potential Roles for the GOLM1 HCC Serum Biomarker
Authors: Arora Gunisha , Fimmel Claus , Wright Lorinda , Bode Barrie
Published: June 2021

Golgi Protein – 73kDa (GP73/GOLM1) is a resident cis‐Golgi Type II membrane protein that is nearly undetectable in normal hepatocytes, but whose expression increases in hepatocellular carcinoma (HCC). Serum GOLM1 levels increase in patients with chronic hepatitis C infection and HCC, yet its biological role in the liver and in HCC pathogenesis remains unclear. Therefore, liver‐specific GOLM1 null mice were created using the Cre‐loxP system, with floxed GOLM1 gene exon 3 and Cre recombinase driven by the albumin promoter. Mouse genotypes were confirmed by PCR analysis. GOLM1Fl/Fl/Cre(+) animals showed no obvious biological phenotype compared to their Cre(‐) littermates, exhibited normal growth, behaviors, and mated successfully, suggesting that hepatic GOLM1 is not vital for normal physiological function. Examination of GOLM1 expression in 14 human HCC cell lines revealed that GOLM1 is consistently expressed in mesenchymal cells but not in most epithelial cell lines. Cell invasion assays indicated that mesenchymal HCC with high GOLM1 expression were more invasive suggesting a potential role for GOLM1 in metastasis – a role currently being tested by silencing in invasive mesenchymal HCC cells and over‐expression in non‐invasive epithelial HCC cell lines. Collectively the results suggest that the mice will serve as a valuable tool to investigate the role of GOLM1 in HCC development, and that enhanced GOLM1 expression is linked to post‐epithelial‐mesenchymal transition (EMT) cellular behavior like metastasis. Support: NIU College of Liberal Arts and Sciences
Antral and Duodenal Myoelectric Activity Changes Around the Day, Effect of Obestatin and Ghrelin in Conscious Suckling Piglets
Authors: Woliński Jarosław , Słupecka Monika , Ochniewicz Piotr
Published: June 2021

Myoelectrical activity of the GI tract during early postnatal period in mammals is still little known. The present study aims to examine the pre ‐ and postprandial patterns of GI tract myoelectric activity during suckling period and impact intra gastric ghrelin and obestatin admission (15μg/kg b. wt., every 8 hours). Another objective was to estimate the postsurgical time of MMC reemergence. Conscious suckling piglets (7 days old, n=10) were fed every 75minutes by artificial sow system in laboratory condition. During right–side laparotomy platinum bipolar electrodes were sutured on the antrum, duodenum and jejunum. Electrodes were connected to a telemetry implant (DSI, USA) fixed extraperitoneally in a pocket made between the abdominal muscles. The recording was begun immediately after surgery. Signal acquisition was carried out by an RMC ‐ 1 receiver coupled to a DL10 analogue output (DSI, USA). Signal was filtered (hi cut–off 50Hz, low cut–off 10Hz) and amplified. PowerLab/4e and a computer outfitted with LabChart software (ADInstruments) were used to record and analyze the data. Mean values of amplitude and frequency described the pre‐ and postprandial patterns present. Root mean square and mean power frequency values were also extracted from the data. Values were tested with the Student's t – test.

The first full MMC cycle was recorded 28±5 hours after the surgery in all animals involved in the experiment. No relevant differences were observed between pre‐ and postprandial patterns of antral and duodenal myoelectrical activity after the admission of obestatin and ghrelin.

Project of National Science Centre Poland 2012/05/B/NZ9/00901.