bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptin and Amylin Influence the Excitability of Area Postrema Neurons, Possibly via cAMP‐Mediated Mechanisms
Authors: Smith Pauline , Brzezinska Paulina , Hubert Fabien , Maurice Donald , Ferguson Alastair
Published: June 2021

The area postrema (AP), a sensory circumventricular organ with important roles in central autonomic regulation, has been shown to possess amylin and leptin receptors, suggesting a role for the AP in modulating peripheral signals indicating energy status. The present study was undertaken to examine the effects of leptin receptor activation on dissociated AP neurons and to elucidate potential mechanism(s) underlying such effects. Whole cell current clamp recordings demonstrated that leptin (10nM) influenced the excitability of 51% (42/82) of AP neurons. The majority of responsive neurons (62%) exhibited a depolarization (mean Δ MP = 5.3 ± 0.7 mV, n=26) in response to leptin administration, while the remaining affected cells (16/42) hyperpolarized (mean Δ MP = ‐5.96 ± 0.95 mV, n=16). We next examined the responsiveness of single AP neurons to consecutively applied leptin and amylin. Both peptides influenced the same population of AP neurons as 5 of 7 cells that depolarized in response to leptin administration also depolarized in response to amylin (10nM). In order to elucidate the mechanism(s) of leptin and amylin actions in AP, we utilized Fluorescence Resonance Energy Transfer (FRET) to determine the effect of leptin and amylin on cAMP levels in single AP neurons. Both leptin (25nM) and amylin (100nM) elevated cAMP levels in the same dissociated AP neurons to approximately 35% of that induced by a maximal concentration of the adenylate cyclase activator, forskolin (1μM) used in combination with IBMX (100μM). These observations that leptin and amylin influence a subpopulation of AP neurons, possibly mediated by cAMP mechanism, supports a role for the AP as a CNS location at which these circulating signals may act to influence central control of energy balance.
Responses of Lipolysis to a Meal after Physical Activity in Lean and Obese Children
Authors: Yan Huimin , Myers Kimberly , Dubis Gabriel S Dubis , Pierce Joseph , Tanner Chuck , Hickner Robert
Published: June 2021

Purpose

This investigation was conducted to determine whether an acute bout of aerobic exercise would affect lipolytic response to food intake in lean (LN) and obese (OB) children.

Methods

44 children (EX: 16 LN and 28 OB, age 8‐11yr) performed 20 min of aerobic exercise eliciting an average heart rate of 140 beats/min (plus 6 min of warm‐up and cool‐down) between standardized breakfast (~300 kcal) and lunch (~500 kcal) meals, while 21 children (CON: 9 LN and 12 OB, age 8‐11 yr) remained rested between meals. Microdialysis probes were inserted into the subcutaneous abdominal adipose tissue to monitor interstitial glycerol (lipolysis). Changes in interstitial glycerol concentrations were calculated from one‐hour dialysate samples collected before and after ingestion of each meal. Two‐way (meal by adiposity group) ANOVAs were separately performed in EX and CON.

Results

In CON, the lipolytic response to the meal was attenuated after lunch compared to after breakfast regardless of obesity status (main effect of meal, p<0.05; ‐141 ± 44 uM vs. ‐4 ± 24 uM for changes in interstitial glycerol after breakfast and lunch, respectively). In EX, a meal by group interaction was found for changes in interstitial glycerol (p<0.05; LN: ‐416 ± 15 vs. ‐66 ± 58 uM; OB: ‐60 ± 117 vs. ‐152 ± 44 uM for breakfast and lunch, respectively).

Conclusion

The suppressive effect of food intake on lipolysis in subcutaneous abdominal adipose tissue in children may be attenuated after lunch compared to breakfast. The anti‐lipolytic response to food intake can be enhanced with acute exercise in obese children.

Supported by NIH RO1DK071081
Mitochondrial Biogenesis in Response to Exercise or Hydrogen Peroxide Treatment is Not Blunted by Upregulation of Endogenous Antioxidants
Authors: Ehrlicher Sarah , Bruns Danielle , Peelor Frederick , Khademi Shadi , Hamilton Karyn , Miller Benjamin
Published: June 2021

Although prolonged exposure to reactive oxygen species (ROS) may cause cellular damage, beneficial adaptations to exercise including mitochondrial biogenesis may occur, in part, through ROS signaling. Evidence suggests that treatment with an exogenous antioxidant (vitamin C (VitC)) may prevent beneficial redox signaling. A possible alternative strategy to prevent damage while allowing ROS induced signaling is to increase endogenous antioxidants using a Nrf2 activator. We hypothesized that, compared to VitC, treatment with the Nrf2 activator Protandim® (Pro, LifeVantage) would not blunt mitochondrial adaptations to exercise in vivo, or H2O2 treatment in vitro. Deuterium oxide labeling was used to measure mitochondrial protein synthesis (mPS) in vivo, and mPS and breakdown (mPB) in vitro. In vivo, skeletal muscle mPS was significantly greater in high volume runners compared to sedentary controls. This increase in mPS was blunted by VitC but not by Pro. When mPS and mPB were assessed in vitro during H2O2 treatment, Pro did not inhibit mitochondrial responses. From these data, it appears that although treatment with exogenous antioxidants may blunt the positive ROS‐induced mitochondrial responses, treatment with a Nrf2 activator that upregulates endogenous antioxidants does not blunt mitochondrial adaptations.
Centrifuge Size & Operation Effects On Mouse Adaptation To +G
Authors: Fuller Charles , Hoban‐Higgins Tana , Alberts Jeffrey
Published: June 2021

A centrifuge in space provides the only method to create environments between 0‐1G to understand the interactions between biological systems and gravity. Current designs for an on‐board centrifuge are short radius. However, the rotation rate to achieve +G increases as radius decreases, also enhancing irregularities in +G fields produced by centrifuge geometry and operation. Both centrifuge radius and rotation rate play a role in the adaptive response to altered G. This study examined the adaptation profiles of adult female mice to 8 weeks of chronic centrifugation. A rotation rate that would result in a 1.0 G environment in space was used to produce the maximum +G environment for this study. The +G field was either presented immediately upon centrifugation onset (acute) or in 4 equal increases (stepped) at two‐week intervals. Data were collected from: experimental (short 0.67 m diameter), force control (large 4.5 m diameter), cage control (the experimental cage) and vivarium control groups. Each group had an n=12 (4 cages of 3 subjects/group). Data included: biweekly measures of food and water consumption, body mass, body temperature, and video. The centrifuge groups showed the expected adaptation profiles to +G. Both the initial responses and the time to reach the adapted state were affected by the profile of centrifuge operation. The initial decrease in body mass, food consumption and body temperature was greater under the acute compared to the stepped profile. Additionally, the time to adapt was longer in the acute groups. However adaptation levels were similar after 8 weeks of +G. (NASA Grant TO1130JSSC.)
Lower Body Negative Pressure Counters Internal Jugular Vein Engorgement during Simulated Microgravity
Authors: Baird Shannon , Macias Brandon , Hargens Alan
Published: June 2021

Recent ophthalmic evaluations of seven astronauts after six‐month missions to International Space Station (ISS) show unexpected vision problems. A microgravity induced cephalad fluid shift during long‐duration ISS missions may result in cephalic venous congestion. We hypothesized that simulated microgravity would cause internal jugular vein (IJV) engorgement but application of lower‐body negative pressure (LBNP) would mitigate this response. Six normal healthy subjects volunteered for this study (mean age: 23 years) after IRB review. Cross‐sectional area (CSA) of IJV was measured using standard ultrasound procedures. Heart rate and blood pressure were measured using an automatic blood pressure device. Data were collected during the last min of each of the following conditions: sitting (5mins), supine (5mins), head‐down‐tilt (HDT) (5mins), and HDT+LBNP (10mins with LBNP of 25 mmHg). Standard onboard ultrasound software was used to calculate IJV CSA. Data are presented as mean ± SD. RMANOVA was used to determine statistical significance with p<0.05. The IJV CSA significantly increased from the sitting (0.074±0.028 cm2) to the supine (0.602±0.189 cm2) posture, p=0.001. IJV CSA further increased when the subjects were positioned in the HDT position (1.034±0.263 cm2), p=0.002. However, when LBNP was activated during HDT, IJV CSA decreased (0.714±0.289 cm2) and was not significantly different from supine levels (p=0.259). Mean blood pressure and heart rate did not change significantly across all conditions. In conclusion, LBNP during simulated microgravity may reduce cephalad venous congestion. This project was supported by APS Frontiers in Physiology Fellowship (to SLB), NSBRI through NASA NCC 9‐58 (to BRM), and NASA grant NNX13AJ12G (to ARH).
Effect of long‐term voluntary running on bone tissue in mice
Authors: Takamine Yuri , Ichinoseki‐Sekine Noriko , Yoshihara Toshinori , Tsuzuki Takamasa , Machida Shuichi , Naito Hisashi
Published: June 2021

Background

It is well known that exercise has positive effects on bone. It has been reported that voluntary exercise increase bone formation and bone density during growing period. However, effects of more long‐term exercise performed until adult on bone tissue are little known.

Purpose

To investigate the effects of long‐term voluntary wheel running on histological and mechanical properties of bone tissue in mice.

Methods

Four‐week‐old male C57BL/6 mice (n=31) were assigned to 1) a sedentary control (CON) and 2) a voluntary running (VE) groups. Mice in VE group were allowed free access a wheel. Femurs were removed at 4, 28, or 40 weeks of age. Bone volume was measured histologically as the bone volume per tissue volume［BV/TV (%)］, and bone strength was measured using three‐point bending test. Bone strength was normalized to body weight［N/g; maximum breaking force (N) per body weight (g)］.

Results

The body weight in the CON at 4, 28, 40 weeks of age was 18.4±0.5, 30.1±1.3, 29.8±1.4 g, respectively, while it in VE at 28 and 40 weeks of age was 26.7±0.8, and 29.2±2.4 g. BV/TV (%) significantly decreased with aging from 4 to 28 weeks of age in both groups. There were no significant differences in BV/TV (%) between CON and VE. Also, bone strength significantly increase with aging from 4 to 28 weeks of age in both groups. There were no significant differences between CON and VE.

Conclusion

Our data suggests that long‐term voluntary running do not effect on histological and mechanical properties of bone tissue in mice.

This work was supported by grants from the MEXT‐Supported Program for the Strategic Research Foundation at Private Universities (S1101008).
Chemotherapeutic Properties of Factors Released from Contracting Skeletal Muscle: Early “Exercise” versus Fatigue
Authors: Riechman Steven , Davis Amanda , Simpson Tess , Porter Weston , Fluckey James
Published: June 2021

PURPOSE

The purpose of this study is to determine the chemotherapeutic effects of early and late secretions of contracting skeletal muscle on breast cancer cells in vitro. METHODS: MCF7 breast cancer cells were treated with “exercise” perfusate collected from a rat hemicorpus perfusion in which the hindlimb was electrically stimulated via the sciatic nerve. Study groups (n = 3 wells per group x 3 animals) include perfusate collected from quiescent muscle (NoEx), during the first 5 minutes of contraction, Early Exercise (EEx), during the last 5 minutes of contraction, Fatigue (FTG), as well as normal cell culture media (CON) as a control. Cell counts were taken in triplicate on Day 5 after four days of exposure (refreshed media daily, consisting of 10% perfusate). RESULTS: Average percent reduction in cell counts as compared to NoEx controls were 7% for EEx and 20% for FTG perfusate (P < 0.01 and P < 0.05, respectively).

CONCLUSION

These data support evidence for chemotherapeutic factors being released from contracting skeletal muscle which is more strongly associated with fatigue. These results have important implications for the clinical application of exercise in the prevention and treatment of breast cancer. Future studies will include isolation of candidates and determination of mechanisms of action.

This work was funded by the TAMU Cancer Research Center, Cancer Research and Prevention Incentive Grant (SER).
METRNL Rescues Glucose Intolerance and Ameliorates Obesity Phenotype in a Diet‐Induced Model of Obesity
Authors: Saleem Ayesha , Safdar Adeel , Haikalis Maria , Akhtar Mahmood , Haziri Hassan , Tarnopolsky Mark
Published: June 2021

Physical inactivity is a primary modifiable risk factor for obesity and type 2 diabetes (T2D), metabolic diseases that are rampant in both pediatric/adult populations. Endurance exercise has been shown to prevent/attenuate the onset and progression of obesity/T2D. We postulated that exercise mediated these pro‐metabolic effects on distal fat depots and other organs via secretory myokines. Our lab has identified two such myokines: interleukin‐15 (IL‐15) and meteorin‐like protein METRNL. Here we administered these two myokines, singularly and in combination, to deduce the respective potency in treating obesity by utilizing a diet‐induced model of obesity. C57Bl/6 mice were fed high‐fat diet (HFD; 60% kcal from fat) for 6 months until the animals were hyperglycemic and glucose intolerant. Subsequently the animals were divided into HFD control, endurance exercise (15 m/min for 60 min, 5 days/week, END), or given intravenous injections of recombinant METRNL (0.4 ng/kg/day, 3x/week, MET), IL‐15 (25 ng/kg/day, 3x/week), or a combination group of both IL‐15 and METRNL (COMBO) for 8 weeks. Treatment with METRNL or COMBO normalized serum glucose levels, improved glucose tolerance, and reduced body and fat weight, increased pancreas weight as effectively as the mice in END group (all P < 0.05). Inguinal fat analyses showed a marked improvement in gene networks involved in mitochondrial biogenesis and browning of the fat (increased ucp1, prdm16, and cidea expression), and COX activity in END, MET, and COMBO groups in tandem. IL‐15 treatment alone had no effect on these indices. Our data clearly establishes treatment with METRNL as an effective therapeutic approach to counteract diet‐induced obesity. Funded by NSERC and CIHR.
Relationship between Exercise Capacity and Rapid Vasodilator Responses to Single Skeletal Muscle Contractions in Young and Older Adults
Authors: Ganger Charles , Treichler David , Ueda Kenichi , Casey Darren
Published: June 2021

We examined the relationship between exercise capacity (VO2peak) and contraction‐induced rapid vasodilation in skeletal muscle and whether the potential relationship varied by limb (leg vs. arm) and/or age (young vs. older). 33subjects (14 young, 23±1yr; 19 older, 65±1yr) performed single muscle contractions at 10, 20 and 40% of maximum for handgrip and 20, 40, and 60% work rate maximum for knee extension. Blood flow (ml/min) was measured using Doppler ultrasound and vascular conductance was calculated from blood flow and blood pressure (mmHg) and normalized for muscle mass (kg). VO2peak was related to peak contraction‐induced vasodilation in the arm (r=0.32‐0.37, P=0.04‐0.07) and leg (r=0.48 – 0.60, P<0.01) across intensities for all individuals included in the study. However, when separated by age group VO2peak was not related to rapid vasodilation in the arm within either group or in the leg of the young adults. Conversely, older adults demonstrated a strong relationship between VO2peak and peak rapid vasodilation in the leg at the 20% (r=0.68), 40% (r=0.65), and 60% (r=0.75) intensity (P<0.01 for all). Our data suggest that a relationship between VO2peak and contraction‐induced rapid vasodilation exists in the leg of older adults. These relationships may provide insight into the positive effects of exercise training on vascular function and skeletal muscle perfusion in older adults.

Funded by NIH HL‐105467
Mechanisms of Pronounced and Sustained Cutaneous Vasoconstriction During Cryotherapy
Authors: christmas kevin , Patik Jordan , Khoshnevis Sepideh , Diller Kenneth , Brothers R.
Published: June 2021

Cryotherapy is a therapeutic technique using ice or cold water applied to the skin to reduce local blood flow. While beneficial, there are some side effects such as pronounced vasoconstriction and tissue ischemia that is sustained for hours post‐treatment. This study tested the hypothesis that sustained vasoconstriction is mediated by 1) Rho‐kinase and/or 2) elevated oxidative stress. 13 subjects were fitted with a commercially available cryotherapy unit with a water perfused bladder on the lateral portion of the right calf. 3 microdialysis membranes were threaded through the dermis underneath the bladder. One site received lactated ringers (CON), one received the Rho‐Kinase inhibitor Fasudil (FAS), and one received ascorbic acid (VitC). Skin temperature (SkT) and skin vascular conductance (SkVC) was measured at each site. Subjects had 0 °C water perfused through the cryotherapy bladder for 30 min, followed by passive rewarming for 2 hr. SkT fell from 34°C to 17.9°C cold water application across all sites. SkVC was reduced after 30 min of cooling with both FAS and VitC having attenuated vasoconstriction when compared to CON (P<0.05 for both). After 2 hr of passive rewarming SkVC was elevated at the FAS site where as it remained reduced CON and VitC sites (p<0.05). These findings indicate Rho‐kinase and oxidative stress contribute to pronounced vasoconstriction during cryotherapy and that the Rho‐kinase contributes to sustained vasoconstriction during the rewarming period post treatment

Supported by the National Institute of Biomedical Imaging and Bioengineering; R01EB015522 (Multi PI: K.D. Diller & R.M. Brothers).
Microbiological Investigation of Waters of River Moravica during Summer Season 2012
Authors: Rashiti Fahrie , Ismaili Muharrem , Kurteshi Yll , Tuna Lush
Published: June 2021

Water pollution due to chemicals and microbes is one of the serious environmental problems, which has greatly impacted human health. This study was conducted for physiochemical and bacteriological analysis of drinking water of new urban areas ofPeshawarand compared the old historical areas of the city. Present study results had shown that mostly Physio‐chemical and Bacteriological parameters of drinking water were found above the permissible limits of WHO. The results indicate that drinking water is highly vulnerable to bacterial contamination.

Based on achieving results led us to conclude: The waters of MoravicaRiverare high polluted by bacteria at all investigated localities(4).
Post‐Occlusive Reactive Hyperemia: Effects on Skeletal Muscle Capillary Hemodynamics
Authors: Wright J , Ferguson S , Holdsworth C , Colburn T , Fees A , Barstow T , Musch T , Poole D
Published: June 2021

Post‐occlusive reactive hyperemia (PORH) is the commonly accepted non‐invasive diagnostic test used for evaluating endothelial function in humans. In contrast to conduit brachial artery blood flow, little is known regarding capillary hemodynamics during PORH. An understanding of microcirculatory behavior post occlusion is fundamental to interpreting the PORH response. Based on previous results found in humans we tested the hypothesis that mechanically induced ischemia of the spinotrapezius muscle would increase the percentage of capillaries supporting red blood cell (RBC) flux during reperfusion. Capillary hemodynamics were measured via intravital microscopy in young male Sprague‐Dawley rats at baseline (BL), during mechanical feed artery occlusion (OCC, 1 min and 5 min) and subsequent reperfusion. Mean arterial pressure and heart rate were not altered (both P > 0.05). As expected, the percentage of capillaries supporting RBC flux was reduced during 1 min OCC (BL: 90 ± 3, OCC: 2 ± 2%, P < 0.05) and 5 min OCC (BL: 87 ± 3, OCC: 0 ± 0%, P <0.05). At 30 s reperfusion the percentage of capillaries supporting RBC flux decreased relative to BL following 1 min OCC (BL: 90 ± 3%, reperfusion: 85 ± 3%, P < 0.05) and 5 min OCC (BL: 87 ± 3, reperfusion: 29 ± 3%, P < 0.05). Thus during reperfusion the percentage of capillaries supporting RBC flux neither exceeded nor even returned to BL levels up to 5 min post OCC thereby demonstrating impaired capillary function and thus blood‐myocyte O2 and substrate flux. Consequently, the PORH state in the spinotrapezius muscle of the rat differs markedly from physiological contraction‐induced hyperemia found in humans.

Grants: NIH HL‐108328 NIH T32OD011169
Novel Effects of FGF21 and Exercise on Brown Adipose Tissue Inflammation
Authors: Rowles Joe , Fletcher Justin , Zidon Terese , Scroggins Rebecca , Parks Young‐Min , Perfield James , Thyfault John , Rector R , Padilla Jaume , Vieira‐Potter Victoria
Published: June 2021

Fibroblast growth factor 21 (FGF21) is an endocrine hormone known to enhance adipocyte metabolism via “browning” of white adipose tissue (WAT) and activation of brown adipose tissue (BAT). Exercise (EX) increases BAT activity and WAT browning, and has anti‐inflammatory effects in WAT. We investigated the role of FGF21 in AT browning and anti‐inflammatory effects of EX, hypothesizing that such effects of EX in AT are mediated in part through FGF21; further, absence of FGF21 would associate with BAT “whitening,” indicated by increased lipid and inflammatory genes traditionally up in WAT. Male FGF21 knock‐out (KO) and wild‐type (WT) mice fed normal chow were trained (EX) or remained sedentary (SED) for 8 wks creating KOSED, KOEX, WTSED, WTEX groups (n=10/group). By EchoMRI, KO increased, and EX reduced, adiposity (genotype and EX main effects, P<0.05). KO increased BAT mass suggestive of “whitening,” while EX reduced BAT (genotype, EX main effects, P<0.05). KO increased, and EX decreased, BAT markers of inflammation/oxidative stress (leptin, MCP‐1, TNF‐a, and P22Phox; genotype, EX main effects, P<0.05). Macrophage markers (F4/80, CD11c) were also increased in KO and reduced with EX (P<0.05). In both KO and WT, EX reduced Chop and GRP78 suggestive of reduced ER stress. Interestingly, an EX by genotype interaction (P<0.05) was observed such that EX decreased PRDM16 (BAT marker) in KO and increased PRDM16 in WT. These findings reveal a previously unappreciated anti‐inflammatory effect of FGF21 in BAT and suggest anti‐inflammatory/browning effects of EX are influenced by FGF21.
Increased Circulating Angiotensin‐(1‐7) in Transgenic Rats Modulates Lipogenic and Lipolytic Activities in Adipose Tissue
Authors: Moreira Carolina , Cordeiro Letícia , Mario Érica , Chaves Valéria , Santos Robson , Botion Leida
Published: June 2021

We evaluated the lipid metabolism in the adipose tissue of transgenic rats that overexpress an Angiotensin (Ang)‐(1‐7)‐producing fusion protein, TGR(A1‐7)3292 (TGR), which induces a lifetime increase in circulating levels of this peptide. Male Sprague‐Dawley rats, control (C) and TGR, 12‐15 weeks old, were used. After removal of epididymal, mesenteric and retroperitoneal adipose tissues, adiposity index was calculated. Lipogenic and lipoprotein lipase (LPL) activities were evaluated in epididymal adipose tissue and triacylglycerol (TAG) hydrolases activity were analyzed in retroperitoneal adipose tissue. Data are means±SEM (p<0.05). The results obtained in fed rats showed reduction of 35% in visceral adiposity in the TGR group in relation to control. Basal lipogenesis presented reduction in the TGR group when compared to control (TGR:124±14; C:279±36). Insulin‐stimulated lipogenesis was also reduced in the TGR group in relation to control (TGR:229±35; C:414±21). The TGR animals presented decreased LPL activity compared to control (TGR:0.70±0.14; C:1.56±0.18). TAG hydrolases activity was increased in the TGR group in relation to control (TGR:0.79±0.05; C:1.01±0.05). Our data show that increased levels of Ang‐(1‐7) have beneficial effects on the reduction of visceral adiposity, probably by decreasing fatty acids uptake and lipogenic activity in epididymal adipose tissue and by increasing TAG‐hydrolytic activity in retroperitoneal adipose tissue.

Supported by: CNPq, CAPES, FAPEMIG
The ACE2 Sheddase, ADAM17, is Up‐Regulated in Diabetic db/db Mice
Authors: Chodavarapu Harshita , Pedersen Kim , Lazartigues Eric
Published: June 2021

Our previous studies demonstrated loss of ACE2 in the pancreas during progression of diabetes contributes to β‐cell dysfunction and hyperglycemia. The exact mechanism behind the reduction of ACE2 is not known. A sheddase ADAM17 (a disintegrin and metalloprotease 17), can promote cleavage of the ectodomain of ACE2. The aim of this study is to test the role of ADAM17 in the regulation of cellular ACE2 levels in a diabetic mouse model. For this purpose, we used 12 week old db/db (BKS.Cg‐Dock7+/+Lepr/J) mice (n=4/group) and their age‐matched heterozygous controls. Diabetic db/db mice showed hyperglycemia (db/db: 202.8 ±16.6 vs. Control: 85.5 ±10.4 mg/dl p<0.05) and increased body weight (db/db: 46.04 ±0.8 vs. Control: 26.7 ±0.7 g p<0.05). In addition, quantitative RT‐PCR showed 50% reduction (p<0.05) in INS‐2 (Insulin 2) gene expression in isolated pancreatic islets of db/db mice compared to controls. There was no difference in ACE2 and ADAM17 mRNA levels, suggesting that transcriptional regulation of these targets is not glucose sensitive. However, this was associated with a significant increase in ADAM17 activity (db/db: 36.92 ±2.12 vs. Control: 11.4 ±8.2 FU/min/µg protein p<0.05) but no significant difference (p>0.05) in ACE2 activity in the islets of db/db mice. In conclusion, while our results suggests that ADAM17 is up‐regulated in db/db diabetic mice, the role of ACE2 as a potential target remains uncertain. New transgenic mice with beta‐cells specific knockdown of ADAM17 are being developed to clarify the relationship between ACE2 and ADAM17 in type 2 diabetes.

Support: American Heart Association: 12EIA8030004 and 14PRE18830012.
Alcohol and Burn Injury Decreases STAT3 Expression in Intestinal Epithelial Cells
Authors: Hammer A , Li X. , Morris N. , Cannon A , Earley Z. , Kovacs E. , Choudhry M.
Published: June 2021

Gut leakiness and subsequent bacterial translocation can contribute to sepsis and multiple organ failure following ethanol exposure and burn injury. Our laboratory has previously shown alterations in tight junction proteins following combined ethanol and burn injury, however, the mechanism by which this occurs remains largely unexplored. STAT3 has been shown to play a role in proliferation and differentiation of intestinal epithelial cells (IECs), but its role in regulation of tight junction proteins has yet to be established. Therefore, we examined the effect of alcohol and burn injury on STAT3 expression in intestinal epithelial cells. C57BL/6 mice were gavaged with ~3 g/kg ethanol. Four hours after gavage, mice were subjected to a ~12.5% total body surface area dorsal scald burn. One day following injury, IECs were isolated from small intestine and expression of STAT3, occludin, and a subset of claudins were examined. We observed significantly decreased STAT3 expression in ethanol and burn injured mice (p < .01) compared to shams by Western blot. In addition, we found significantly reduced levels of claudin‐1 (75%), claudin‐4 (86%), and occludin (67%) in IECs harvested from mice receiving ethanol and burn injury compared to shams. These findings suggest that alcohol and burn injury results in decreased STAT3 expression, which in turn, may play a role in altered tight junction protein expression following ethanol and burn injury. Supported by R01 AA015731 (MAC), R01 AA015731‐08S1 (MAC), R01 AA012034‐13 (EJK), T32 AA013527 (EJK) and the Dr. Ralph and Marian C. Falk Medical Research Trust (EJK).
Epithelial regeneration after gastric ulceration causes prolonged weakened defenses and altered cell types
Authors: Aihara Eitaro , Matthis Andrea , Montrose Marshall
Published: June 2021

The first clinical presentation of a peptic ulcer tends to occur in mid‐life, but then has a significant rate of recurrence, suggesting that repaired tissues are more susceptible to subsequent damage. Our hypothesis is that even after visual healing from gastric ulceration, the gene profile of the regenerated epithelium is aberrant for extended intervals, increasing susceptibility of the regenerated epithelium to damage and cancer risk. Gastric ulcers were produced by acetic acid topically applied to the exterior of C57BL/6J mice stomachs. A single gavage of 106 H. pylori was performed 30 days after ulcer induction. Gastric ulcers induced by acetic acid visually healed in a time dependent manner, and they cannot be identified macroscopically at 30 days after ulceration. However, using RT‐PCR or immunostaining, the regenerated epithelium at 7 days, 30 days and 4 months is shown to completely lack parietal cells (H‐K ATPase) while the surface epithelial/metastasis marker trefoil factor 2 (TFF2) is upregulated and widely distributed even deep in the gastric gland. On the other hand, at 14 days after H. pylori inoculum, we observed gastric ulcer relapse at the same site where gastric ulcer was induced 30 days prior, although H. pylori did not cause gastric injury in other sites. In conclusion, a macroscopically regenerated epithelium has prolonged abnormal cell distribution with weakened defenses. Funding: NIH RO1 DK54940 (M.H. Montrose).
Attenuation of Claudin‐18 in Mouse Stomach Promotes the Development of Intramucosal Carcinoma
Authors: Zheng Yi , Wu Jessica , Muthupalani Sureshkumar , Wang Yaoyu , Ang Lay‐Hong , Fox James , Hagen Susan
Published: June 2021

Claudin‐18 (cldn18) is a membrane‐spanning tight junction protein responsible for limiting paracellular H+ permeation in stomach. Pro‐inflammatory cytokines, particularly IL‐1β, block cldn18 expression in vitro and cldn18 is attenuated early in human and mouse gastric cancer (GC) development. Our aim was to determine the role of cldn18 in GC development. Methods: Mice with targeted deletion of the cldn18 gene (ko) were obtained and shown to lack both stomach and lung isoforms of cldn18. Neck region cells from uninfected control and ko mice at 7 wk were isolated by laser capture microdissection and the mRNA evaluated by RNA‐Seq followed by genomic pathway analysis. Control and ko mice were sham‐ or infected with H. pylori SS1 at 7 wk and tissues collected 20 and 30 wks later. Histopathology was scored on a 0‐4 scale. Results: By 7 wks of age, uninfected ko mice had limited inflammation but significant atrophy and dysplasia. The most highly upregulated genes in ko mice had in common STAT3 or STAT5 downstream effectors. By 20 wks post sham‐ or H. pylori‐ infection, tissues from ko mice showed intramucosal carcinoma of the corpus irrespective of H. pylori infection status.

Conclusions

Our data suggest that the inflammation‐induced attenuation of cldn18 in GC development may be an independent tumor promoter with downstream effects, at least in part, supported by STAT activation and signaling.

Funding was from the Department of Surgery and the Harvard Catalyst | The Harvard Clinical and Translational Science Center (SJH) and R01CA067463 and R01AI/RR037750 (JGF).
Assessment of Serum Levels of Dehydroepiandrosterone Sulfate (DHEAS) and Estriol (E3) in the 2nd and 3rd Trimesters of Pregnancy, in Some Selected Obstetrics and Gynecological Specialist Hospitals in Port Harcourt: A Measure of Fetal Well‐being
Authors: Anacletus Frank , Onwuka Adaora , Ogwe Geraldine
Published: June 2021

The serum levels of dehydroepiandrosterone sulfate (DHEAS) and Estriol (E3) was investigated in fifty (50) pregnant women in their second and third trimesters of pregnancy in some selected obstetrics and gynecological specialist hospitals in Port Harcourt, as a relevant measure of fetal well‐being. As a routine, the consent of the women was obtained for the test to be performed both for medical and academic purpose. 4‐5ml of blood was collected individually into a labeled lithium heparin bottle, and stored at 40C for up to 24 hours and at – 100C for those samples to be analyzed at a later date. The investigation was carried out using enzyme‐linked immunosorbent assay (ELISA) method with the use of an automated micro plate reader. The data obtained was compared with the standard reference values for DHEAS and E3. The result obtained showed a significant steady increase in dehydroepiandrosterone sulfate as well as that of Estriol when compared to reference values, as the trimester progresses. There was also an observed decrease in DHEAS and E3 as age of the patients increases. Slight increases in the levels of DHEAS and E3 are used to monitor the progress of the pregnancy and they also indicate fetal well‐being
Drug‐associated DNA Damage in HIV‐1 Patients taking HAART
Authors: Jusino Shirley , Godoy Lenin , Albino Elinette , Ortiz Carmen , Hill Martin
Published: June 2021

Despite the reduction in mortality in HIV‐1 positive patients due to the use of highly active antiretroviral therapy (HAART), adverse effects have been associated to long‐term usage. Recently, several studies have demonstrated that HIV‐1 infected patients are under chronic oxidative stress caused by an inflammatory response to the latent infection, or as an adverse effect of the antiretroviral therapy. The aim of this study was to evaluate the DNA damage in peripheral blood mononuclear cells of HIV‐1 patients treated with HAART when compared to healthy donors. Frozen peripheral blood mononuclear cells (PBMCs) obtained from HIV‐1 positive subjects with undetectable viremia and under HAART were used for this study. An aliquot of the thawed cells was used for DNA isolation and the remaining cells were cultured for seven days. Single and double‐strand DNA breaks were evaluated using an alkaline gel electrophoresis (single‐strand gel electrophoresis) and the neutral comet assay, respectively. PBMCs from healthy donors were used as negative controls. EMS (ethylmethane sulfonate) was used as a positive control. Interestingly, the alkaline gel showed higher DNA fragmentation in HIV‐1 positive samples when compared to an HIV‐negative control and to a sample treated with EMS. Additionally, the neutral comet assay showed presence of double‐stranded DNA breaks in HIV‐1 positive cells when compared to a healthy donor sample. The extent of the single or double‐stranded DNA breaks varied among samples depending on type of therapy and the length of time under the therapy. Taken together, our results suggest that HAART may affect the oxidative stress imbalance in HIV‐1 positive patients.
Targeting Disabled‐2 to Stabilize ΔF508‐CFTR
Authors: Chavez Moises , Ayyoubi Sahar , Cihil Kristine , Swiatecka‐Urban Agnes , Barnaby Roxanna , Stanton Bruce , Madden Dean
Published: June 2021

The fatal genetic disease Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. The most common mutation, ΔF508‐CFTR, negatively impacts the processing, gating, and stability of CFTR. Existing combination therapies elicit only modest benefit in ΔF508‐CFTR patients. Potentiators and correctors address the gating and processing defects of ΔF508‐CFTR, respectively, but do not specifically target its stability. Thus, by targeting additional aspects of the mutant CFTR life cycle we may significantly improve CF patient prognosis.
Here, we present evidence for a new class of stabilizers that target CFTR half‐life. The adaptor protein Disabled‐2 (Dab2), is an essential mediator of CFTR endocytosis. The Dab2 Dab homology (DH) domain is required for Dab2 to co‐assemble in complex with CFTR, and our lab has shown that DH peptide inhibitors enhance ΔF508‐CFTR function alone and even more so when combined with correctors and potentiators. We have now used an engineered peptide with high‐affinity for the DH domain as a reporter in a fluorescence anisotropy based high‐throughput screen to identify candidate small‐molecule inhibitors. FRET and NMR are used to further characterize the intermolecular binding between the DH domain and potential stabilizers.
Induction of Peroxiredoxins in Breast Cancer
Authors: Phelan Shelley , Buurma Valerie , Freeman Brendan , Adzenyah Akosua , Pettit Gregory , Heinen Felicitas , Muhlbauer Jillian
Published: June 2021

Cancer cells possess elevated levels of reactive oxygen species (ROS) that can enhance genomic instability and alter redox signaling. Aberrant antioxidant expression has also been recognized in many cancers, suggesting an adaptive response to the cancerous state. Breast cancers express significantly elevated levels of Peroxiredoxins (Prdxs), a family of thiol‐specific antioxidant proteins involved in regulating cell growth and proliferation in many cell types. We previously reported overexpression of many Prdx proteins in the MCF‐7 breast cancer line, as well as their importance in doxorubicin‐resistance in this line. In the present study, we analyzed Prdx levels in several human breast tumor samples, and further investigated possible mechanisms of Prdx regulation in MCF‐7 cells. Comparison of breast tumor and adjacent normal breast tissue from almost 20 patients showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. Sequence analysis revealed potential binding sites for NF‐κB, Nrf2, and AP‐1 in several peroxiredoxin promoters. We found high levels of NF‐κB in MCF‐7 cells, as compared to normal breast epithelial cells. We further showed that a chemo‐resistant MCF‐7 culture generated from 14 days of doxorubicin treatment exhibited markedly decreased NF‐ κB expression. We then used both siRNA and chemical inhibition of NF‐κB to examine the effect on Prdx regulation in these cells and found gene‐specific effects. This investigation will help to elucidate the role of peroxiredoxins in breast cancer cell survival, and the mechanism by which these cells alter Prdx levels to avoid death and achieve chemoresistance.
CB‐5083: A p97 Inhibitor Disrupts Cellular Protein Homeostasis and Shows Potent Anti‐Tumor Effects
Authors: Kumar Brajesh , Rice Julie , Djakovic Stevan , Madriaga Antonett , Wong Steve , Valle Eduardo , Menon Mk , Wang Jinhai , Soly Szerenke , Soriano Ferdie , Yao Bing , Le Moigne Ronan , Zhou Han‐jie , Wustrow David , Rolfe Mark , Anderson Daniel
Published: June 2021

p97 is a homo‐hexameric AAA‐ATPase complex that plays vital roles in protein homeostasis, including ubiquitin‐proteasome mediated protein degradation, endoplasmic reticulum‐associated degradation (ERAD) and autophagy. ATP hydrolysis driven by p97 results in a conformational change within the homo‐hexameric complex that generates mechanical force, utilized for extracting misfolded proteins from the ER and other organelles and chaperoning a subset of those proteins to the proteasome for degradation. Since cancer cells heavily utilize the ubiquitin‐proteasome system, targeting p97 within this pathway is an attractive approach for the development of new cancer therapeutics.
Here we report that CB‐5083 is a potent and specific small molecule inhibitor of p97 as a novel cancer treatment. CB‐5083 is a reversible inhibitor of p97 that binds competitively with ATP to the D2 ATPase site of p97. Mutations in or adjacent to the D2 ATPase binding site resulted in reduced potency of CB‐5083 in biochemical assays, confirming role of CB‐5083 in p97 activity by direct binding to its D2 ATPase site.
CB‐5083 treatment of cells leads to accumulation of poly‐ubiquitinated proteins, retention of ERAD substrates in the ER and generation of irresolvable ER stress that in turn induces a lethal unfolded protein response (UPR). CB‐5083 is orally bioavailable and has good exposure in animal models leading to modulation of key p97‐related pathways. CB‐5083 is now being tested in the clinic as a novel therapeutic for patients with multiple myeloma (MM) and solid tumors.
Identifying Potential Drivers of Differential DNA Methylation Patterns in Breast Cancer Cells
Authors: Fennoy Alexis , Fong Jerry , Andrews Jared , Gasparrini Andrew , Edwards John
Published: June 2021

Global changes in DNA methylation and chromatin modifications are common in breast cancer. Tumors from cancer patients can be categorized based on differential DNA methylation patterns; some of which have been linked to poor prognosis. Correlations between chromatin and DNA methylation patterns are well established in normal cells. However, the interplay between these marks has not been explored in breast cancer cells. We hypothesize that alterations in chromatin modifiers drive global changes in DNA methylation observed in breast tumors. Using breast cancer data from The Cancer Genome Atlas (TCGA) we identified alterations in chromatin modifying proteins that correlate with changes in DNA methylation patterns. We then validated the ability of these proteins to affect DNA methylation. These results will improve our understanding of the mechanism used to establish DNA methylation patterns in breast cancer cells. This research is funded by grant R25HG00668703.
Diallyl Disulfide Inhibits TNF‐alpha Induced CCL2 Release Through MAPK/ERK and NF‐Kappa‐B Signaling
Authors: Bauer David , Mazzio Elizabeth , Soliman Karam , Darling‐Reed Selina
Published: June 2021

TNFα receptors are constitutively overexpressed in tumor cells, correlating to sustain elevated NFκB and monocyte chemotactic protein‐1 (MCP‐1 /CCL2) expression. The elevation of CCL2 evokes aggressive forms of malignant tumors marked by tumor associated macrophage (TAM) recruitment, cell proliferation, invasion and angiogenesis. Previously, we have shown that the organo‐sulfur compound diallyl disulfide (DADS) found in garlic (Allium sativum) attenuates TNFα induced CCL2 production in MDA‐MB‐231 cells. In the current study, we explored the signaling responsible for DADS suppressive effect on TNFα mediated CCL2 release using Signaling Pathway NFκB RT² Profiler™ PCR Arrays, RT‐PCR and evaluation of MAPK/ERK signaling proteins. The data in this study show that TNFα initiates a rise in NFκB mRNA, which is not reversed by DADS. However, TNFα induced heightened expression of IKKe and phosphorylated ERK, corresponding to the rise of CCL2 release, both which were attenuated by DADS. CCL2 induction by TNFα was also lessened by inhibitors of p38 (SB202190) and MEK (U0126) but not JNK (SP 600125), all of which were suppressed by DADS. In conclusion, the obtained results indicate that DADS down regulates TNFα invoked CCL2 production primarily through reduction of IKKE and phosphorylated‐ERK, thereby impairing MAPK/ERK NFκB pathway signaling. Future research will be required to evaluate the effects of DADS on the function and expression of TNFα surface receptors.
Evaluation of The Effects of Organic And Aqueous Extracts of Euphorbia Poissonii on The Gene Expression of BRCA1, BRCA2 and TP53 in MCF‐7 Brest Cancer Cell
Authors: Samuel Titilola , James Babatunde , Onawoga Fatimoh , Negedu Sunday , Adekanle Kehinde , Magbagbeola Olubunmi
Published: June 2021

Euphorbia poissonii (Euphorbiaceae) is a plant native to Northern Nigeria and it is known to possess cytotoxic properties.

The possibility of the crude ethanol extract, hexane, ethyl‐acetate, chloroform, butanol and aqueous fractions to modulate the tumor suppressor genes, BRCA1, BRCA2 and TP53 were investigated. MCF‐7 cell lines were treated with crude ethanolic (CEE), hexane (HF), chloroform(CF), ethyl acetate (EF), butanol (BF) and aqueous (AF) fractions of Euphorbia poissonii and the gene expression of BRCA1, BRCA2 and TP53 were measured using semi‐quantitative RT‐PCR analysis. Results showed down‐regulation of BRCA1, up‐regulation of BRCA2 by CEE, BF, EF, AF and TP53 by HF,CF, EF respectively by the 6 panel fractions. The up‐regulation of BRCA2 and TP53 suggests that the fractions contain bioactive compounds that restored the loss of function of BRCA2 and p53 in the human breast cancer cell line, MCF‐7.
Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members
Authors: Wu Huiyuan , Rong Yongqi , Correia Kristen , Min Jaeki , Morgan James
Published: June 2021

Nna1 (a.k.a. CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1‐6) and is mutated in Purkinje cell degeneration (pcd) mice. Nna1, CCP4 and CCP6 are involved in the posttranslational process of polyglutamylation, where they catalyze the removal of polyglutamate side‐chains. However it is unknown whether these three CCPs share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescue Purkinje cell degeneration in pcd mice, indicating they do not have identical functions. Using biotin‐based synthetic substrates, we establish that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C‐terminal glutamate from substrates with 2 or more glutamates whereas CCP6 requires 4 or more glutamates. CCP4 behaves as a promiscuous glutamase with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher kcat/KM when substrates contained nearby acidic amino acids.
Using a Short Visual‐Text Guide to Allow Ultrasound Self‐Study by Novices
Authors: Benninger Brion , Stringer Rebecca , McLane Lisa
Published: June 2021

Introduction

Ultrasound (US) use in healthcare has grown exponentially. Number of novice users has outpaced trained users available for instruction. There is a need for an efficient teaching method providing autonomy and immediate satisfaction.

Objective of this study was to assess whether novice healthcare students could utilize an US visual‐text guide (VTG) to immediately begin using an US system.

Methods

Literature search was conducted on andragogy of US VTG(s). An US VTG was created using a Fukuda Denshi UF‐760AG US system with classic and Sonivate finger probes. Step‐by‐step photos of the US system in use were compiled with succinct and minimal text instructions supplemented with multi‐media. The US VTG consisted of applying basic US principles to an US system using 3‐4 page segments per probe type. Medical students (yrs. 1‐2) were asked to review the VTG for up to 15 minutes and note their ease of use of the US system with a Likert scale 1‐4. They were allowed to reference the guide during use. Their ability to obtain diagnostic images and use the basic functions of the US system was evaluated by experts in the field.

Literature search revealed no known andragogy studies. After the allotted review time all novices were able to successfully use the VTG to achieve quality images. They reported increased confidence in their US ability compared to pre‐study levels revealed by high Likert scores. A growing gap exists between trained US users and novices in healthcare. This study created an efficient VTG that would not fatigue a user while allowing them to gain valuable probe time thus permitting growth of users independent of trained users.

Conclusion

This study revealed that a novice could begin utilizing an US system using an efficient, VTG.