bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of OJT007 and OJT008 as Inhibitors of Methionine Aminopeptidases from Mycobacterium tuberculosis
Authors: Kaur Manvir , Onyenaka Collins , Voufo Marie Toukam , Nigro Maria Rincon , Liang Dong , Olaleye Omonike Arike
Published: June 2021

Despite recent advances in the diagnosis of Tuberculosis (TB), the therapeutic management of this ancient disease is still challenging. Mycobacterium tuberculosis, the causative infectious agent of TB, is the number one cause of death from infectious diseases in the world. According to World Health Organization TB report, globally, 558, 000 people developed multi‐drug resistance TB that was resistant to the most efficacious first‐line drug, rifampicin. The emergence of multi‐drug and extensively drug resistance strains of M. tuberculosis has made the requirement for the development of novel anti‐TB agents imperative. Our research focuses on targeting Methionine Aminopeptidase (MetAP) an essential protein for growth of M. tuberculosis. MetAP is a dinuclear metalloprotease that catalyzes the process of removal of N‐terminal methionine during protein synthesis. N‐terminal methionine excision from peptides and proteins, is a universally conserved process required for stability, localization, biologic maturation and post‐translational modifications of some proteins. This essential role of MetAPs makes this enzyme an attractive target for the development of new therapeutic agents for treatment of TB. Our hypothesis is that, the development of potent and selective mycobacterial MetAP inhibitors may serve as therapeutic agents against infections caused by M. tuberculosis. Here, we report the characterization and pre‐clinical development of two novel structurally diverse small molecules (OJT007 and OJT008), as inhibitors of M. tuberculosis MetAP with potent activity against M. tuberculosis in the low micromolar range in vitro. These results suggest that OJT007 and OJT008 are potential lead compounds for the development of novel small molecules for the therapeutic management of TB. This target‐based approach has demonstrated the utility of MetAPs as promising antimycobacterial targets. Furthermore, we have developed optimal co‐solvent formulations for both OJT007 and OJT008. Preliminary in vivo pharmacokinetic studies for both compounds are in progress. The discovery of new lead compounds to the diminishing anti‐TB pipeline could have potential impact in accelerating the development of new drug candidates for treatment of drug‐sensitive and drug‐resistant TB. In addition, OJT007 and OJT008 may serve as novel chemical probes for understanding the essential role of N‐terminal methionine excision in TB biology.

Support or Funding Information

This research was supported in part, by research infrastructure support from RCMI grant number 2G12MD007605‐22A1 from the NIMHD/NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficits in Motor Function After Repeated Exertional Heat Stroke Exposure in Female Mice
Authors: Alzahrani Jamal Mohammed , Murray Kevin O , Sheikh Laila , Garcia Christian K , Robinson Gerard P , Laitano Orlando , Clanton Thomas L
Published: June 2021

Exertional Heat Stroke (EHS) is a life‐threatening illness that can result in a number of negative health outcomes. Once exposed to EHS, patients are considered to be at greater risk of subsequent EHS. In this study we hypothesized that a single EHS exposure would result in greater susceptibility to a second EHS, and that the consequences of EHS on skeletal muscle/motor function would be more profound. Twelve female mice were exposed to an initial EHS, allowed to rest for 2 weeks and then exposed to a second EHS and followed for 1 week. All mice reached symptom limitation (loss of consciousness) at lower maximum core temperatures (Tc, max) in their second EHS. Mice also had lower durations of unconsciousness after the second EHS. We evaluated motor function, before and after the EHS using behavior assessments including trunk curl, horizontal rotation response, catalepsy and a ‘weight test’ protocol. Mice showed a significant decrease in the strength (P = 0.0182) after the second EHS, as measured by the weight test protocol, an impaired rotation response (P = 0.0061), catalepsy (P = 0.0398) and trunk curl (P = .0001). These measures indicate reductions in strength and/or motor coordination. In conclusion, we identified that exposure to EHS increases the susceptibility to a second EHS with worsening outcomes in motor function after the second EHS. These data suggest that following a single exposure to EHS there is a prolonged period of increased vulnerability to EHS and greater potential risk of deficits in organ function.

Support or Funding Information

Supported by the BK and Betty Stevens Endowment and a Fellowship from King Saud University, Saudi Arabia.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Alpha‐glucosidase Inhibition by Alcoholic Extract of Garcinia Kola
Authors: Obih Patience O , Obih John‐Clifford A , Nwogu Ivy
Published: June 2021

Diabetes mellitus is a serious chronic disorder and the 7th leading cause of death in the United States of America. It is a metabolic disorder where the body cannot properly metabolize food for energy. It is a chronic condition that is dramatically increasing every year. Recently, more individuals with diabetes are interested in alternative strategies for treating diabetes such as herbal remedies. The objective of this study was to investigate the alpha‐glucosidase inhibitory potential of alcoholic extracts of Garcinia Kola (Bitter Kola) in regulating post‐prandial glucose levels in diabetes. Garcinia Kola, known in Africa as “Wonder plant” was selected for this study because of its numerous use cited in literqture. The in vitro assay was carried out in a 96‐well microplate. The reaction mixture consisted of 2.5 μL of various dilutions of Garcinia extract, 122.5μl of 0.2M phosphate buffer and 62.5 μL of 5mM para‐nitrophenyl‐α‐D‐glucopyranoside. The mixture was pre‐incubated at 37 °C for 5 min followed by the addition of 62.5 μL of α‐glucosidase solution. The mixture was then incubated for an additional 10 minutes and analyzed with a ThermoScientific® Multiskan Spectrophometer at an absorbance of 400nm. Acarbose was used as a positive control of α‐glucosidase inhibitor. From our observation, Garcinia Kola produced inhibition of alpha‐glucosidase, an indication that it may be useful in reducing hyperglycemia.

Support or Funding Information

Center of Excellence/BUILD, Xavier University of Louisiana

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antidiabetic Activity of Root Tubers of Cyanotis tuberosa in Streptozotocin Induced Diabetic Rats
Authors: apparao chippada , Vedasree N , Peddanna Kotha , Rajasekhar Allagadda , Badri Kameswara Rao
Published: June 2021

Diabetes mellitus arising due to insufficient insulin production or its action or both is characterized by hyperglycemia. According to WHO it ranks as the third leading cause of death. Presently available antidiabetic drugs have one or more side effects. There is a need for new antidiabetic agents that will have therapeutic effect with fewer side effects. Cyanotis tuberosa is traditionally used as ethno medicine for curing several ailments in India. The objective of this study was to evaluate the antihyperglycemic activity of root tubers of Cyanosis tuberosa (Commelinaceae) in STZ induced diabetic rats. Different doses of aqueous suspension and different solvent extracts (made by soxhlation) were given to the Streptozotocin induced diabetic rats orally by using oral gavage. After treatment aqueous suspension at the dose of 250 mg/kg b.w has markedly (69%) decreased the fasting blood glucose levels after 6 hours of treatment. Whereas among the solvents extracts hexane extract at the dose of 750mg/kg b.w has produced highest (87%) anti hyperglycemic activity than the other solvent extracts and it is higher than that of the standard drug glibenclamide (20 mg/kg b.w). No hypoglycemic effects were observed in the diabetic rats after the treatment. And Cyanosis tuberosa improves the oral glucose tolerance of diabetic rats when given the hexane extract at the dose of 750mg/kg b.w. Phytochemical analysis of the hexane extract revealed the presence of bioactive secondary metabolites such as flavonoids, tannins, saponins, terpenoids, phenolics, and triterpenes which are responsible for antihyperglycemic activity. In conclusion our findings suggest that the hexane extract of Cyanotis tuberosa can decrease the hyperglycemia in diabetic rats most efficiently without any side effects.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Cardiolipin on Vascular Smooth Muscle Cell Dedifferentiation and Migration
Authors: Galambo Deema , Bergdahl Andreas
Published: June 2021

PURPOSE

The initial phases of atherosclerosis involve the transition of smooth muscle cells (SMCs) to a synthetic, dedifferentiated phenotype, which is associated with alterations in receptor, ion channel and contractile filament composition. The late, irreversible atherosclerosis stages are characterized by SMC migration and subsequent formation of a ‘neointima.’ This leads to blood flow obstruction which, when destined to the cardiomyocytes, induces hypoxic conditions and myocardial infarction. Apoptotic cardiomyocytes release a phospholipid called cardiolipin (CL) into the systemic circulation. Previously, we have shown that physiological concentrations of CL inhibit endothelial cell migration. The objective of this project was to investigate the impact of CL on SMCs, specifically addressing cellular dedifferentiation and migration.

METHODS

For this study, we used adult, male C57Bl/6 mice. Aortas from these mice were extracted and incubated in Dulbecco's modification of Eagle medium containing physiological CL concentrations (1 μM and 10 μM) at 37°C for 48 hours. We subsequently quantified contractile proteins (such as smooth muscle α‐actin and calponin) using immunoblotting to determine whether CL affects SMC dedifferentiation. These concentrations were also used to investigate the migration of SMCs using a migration assay. For this, we mimicked wounds by scraping off the cell culture plates' diameters to create cell‐free gaps. The plates were then incubated at 37°C for six days, during which the width of the gaps was measured every 48 hours under a light microscope.

RESULTS

The immunoblotting results demonstrated a significant drop in calponin in a dose‐dependent manner with increasing CL concentrations, while the other de/differentiation markers remained unchanged. The migration assay revealed that the gap width was significantly narrower in control plates compared to CL‐treated plates.

CONCLUSIONS

Our results suggest that the effect of physiological concentrations of CL on SMCs is calponin‐specific and does not initiate dedifferentiation within 48 hours. The significantly wider gaps observed in the CL‐treated plates compared to the control plates indicate the potent effect CL has on inhibiting SMC migration. Since SMC migration is considered the key step in the atherogenic progression, our migration assay data suggest that, in a physiological context, CL has the potential to arrest the progression of the disease into its irreversible stages. Thus, we hypothesize the potential involvement of CL in developing anti‐atherosclerosis therapeutics.

Support or Funding Information

Canadian Institute of Health Research (CIHR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Buyang Huanwu Decoction on Renal Injury in Spontaneously Hypertensive Rats
Authors: Wang Youping , Xu Hui , Wang Xiaoxiao , Cui Lin , Liu Suxiao , Zhang Songjiang
Published: June 2021

Hypertension is an independent risk factor contributing to chronic kidney disease. Buyang Huanwu Decoction (BHD) is a popular traditional Chinese medicine prescription for the treatment of stroke for centuries. Recent studies reported that the use of BHD had been extended to treat various kinds of disorders following traditional Chinese medicine syndrome theory of Treating Different Diseases with the Same Method. Therefore, this study aimed to determine the effects of BHD on renal injury in spontaneously hypertensive rats (SHR). Treatment with BHD at the dose of 12.6 g/kg/day by gavage for 5 weeks had no effect on the increased systolic blood pressure and renal hypertrophy in 17‐week‐old SHR. However, BHD treatment obviously reduced urinary albumin excretion compared with the control SHR (P<0.05). Periodic acid‐Schiff and Masson's trichrome staining showed that obvious glomerulosclerosis and tubulointerstitial injury were found in the kidney in SHR and reduced by BHD treatment (P<0.05). Consistent with the results, renal collagen level determined using hydroxyproline assay was higher in SHR and attenuated by BHD treatment (P<0.05). In addition, the renal monocyte/macrophage infiltration was seen in SHR and inhibited by BHD treatment (P<0.05). Our data showed that treatment with BHD attenuated renal damage in SHR independently of their effects on blood pressure. The results indicate that BHD exhibits a protective effect on renal damage in SHR. This work was supported in part by the National Natural Science Foundation of China (No. 81673734).

Support or Funding Information

This work was supported in part by the National Natural Science Foundation of China (No. 81673734).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gomisin M2 ameliorates imiquimod‐induced psoriasis‐like skin inflammation via the inhibition of mitogen‐activated protein kinase
Authors: Kim Namkyung , Kang Jinjoo , Kim Hyemi , Kim Sang‐Hyun
Published: June 2021

Psoriasis (PS) is a chronic inflammatory skin disease with complex pathogenesis. Gomisin M2 (GM2) is a natural product extracted from Schisandra chinensis (Turcz.) Baill. In this study, we aimed to investigate the pharmacological effects of GM2 on PS‐like skin inflammation. For induction of PS‐like skin inflammation, imiquimod (IMQ) cream topically applied to the back skin of female C57BL/6J mice for 7 consecutive days. Oral administration of GM2 (0.1, 1 or 10 mg/kg) reduced the symptoms of PS‐like skin inflammation based on Psoriasis Area and Severity Index and histological observation. GM2 reduced serum levels of tumor necrosis factor (TNF)‐α, IgG2a, and myeloperoxidase. It also decreased MPO‐associated cell infiltration in skin tissue in a dose‐dependent manner. Furthermore, GM2 decreased production of Th1 and Th17‐related cytokines such as TNF‐α, interferon (IFN)‐γ, interleukin (IL)‐8, IL‐1β and IL‐17A in the back skin. For in vitro, we investigated the pharmacological effects of GM2 on TNF‐α and IFN‐γ stimulated keratinocytes (HaCaT). Pre‐treatment of GM2 (0.1, 1, or 10 μM) alleviated PS‐associated cytokines such as TNF‐α, IL‐1β, IL‐6, IL‐8, and Chemokine (C‐C motif) ligand 17. GM2 inhibited phosphorylation to mitogen‐activated protein kinase in the activated HaCaT. Our study reveals that GM2 possesses anti‐inflammatory effect on PS. Based on our results, we propose that GM2 could be a potential therapeutic candidate on PS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Omega‐3 Polyunsaturated Fatty‐Acid Supplementation on Neuropathic Pain Symptoms and Sphingosine Levels in Mexican‐Americans with Type 2 Diabetes
Authors: Duran Alfonso , Salto Lorena , Camara Justin , Basu Anamika , Paquien Yvette , Beeson Lawrence , Firek Anthony , Cordero‐MacIntyre Zaida , De Leon Marino
Published: June 2021

Purpose

To determine whether dietary supplementation with omega‐3 polyunsaturated fatty acids (PUFAs) reduces neuropathic pain symptoms in Mexican‐Americans with type 2 diabetes.

Methods

Forty volunteers with type 2 diabetes enrolled in the “En Balance‐PLUS” program, which provided weekly nutrition–diabetes education and daily supplementation with 1,000 mg docosahexaenoic acid (DHA)–200 mg eicosapentaenoic acid over 3 months. The study assessed self‐reported neuropathic pain symptoms pre/postintervention using the short‐form McGill Pain Questionnaire (SF‐MPQ), monitored clinical laboratory values at baseline and 3 months, and performed baseline and 3‐month metabolomic analysis of plasma samples.

Results

A total of 26 participants self‐reported neuropathic pain symptoms at baseline. After 3 months of omega‐3 PUFA supplementation, participants reported significant improvement in SF‐MPQ scores (sensory, affective, and visual analogue scale; P<0.001, P=0.012, and P<0.001, respectively). Untargeted metabolomic analysis revealed that participants in the moderate–high SF‐MPQ group had the highest relative plasma sphingosine levels at baseline compared to the low SF‐MPQ group (P=0.0127) and the nonpain group (P=0.0444). Omega‐3 PUFA supplementation increased plasma DHA and reduced plasma sphingosine levels in participants reporting neuropathic pain symptoms (P<0.001 and P<0.001, respectively). Increased plasma DHA levels significantly correlated with improved SF‐MPQ sensory scores (r=0.425, P=0.030). Improved SF‐MPQ scores, however, did not correlate with clinical/laboratory parameters.

Conclusion

The data suggest that omega‐3 PUFAs dietary supplementation may reduce neuropathic pain symptoms in individuals with type 2 diabetes and correlates with sphingosine levels in the plasma.

Support or Funding Information

This study was funded by NIH grants 5P20MD006988 and 5R25GM060507. The content of this article is solely the responsibility of the authors, and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Isometric Handgrip Exercise on Diastolic Function in Pediatric Heart Transplant Recipients
Authors: Tomczak Corey R , Blushke Corey R , Lahti Dana S , Kosokowsky Kylee , Iqbal Ramlah M , Singh Shreya , Boyes Natasha G , Erlandson Marta C , Wright Kristi , Pharis Scott , Pockett Charissa
Published: June 2021

Pediatric heart transplant recipients (HTR) have reduced exercise tolerance and diastolic dysfunction at rest despite normal ejection fraction. In adult HTR the impaired diastolic function is related to peak exercise tolerance, but this relationship is poorly understood in pediatric HTR. To determine the effects of low‐grade exercise stress on diastolic function in pediatric HTR, we tested the hypothesis that diastolic function would be augmented during isometric handgrip exercise.

Methods

Seven pediatric HTRs were studied (mean±SD age: 10±3 yrs; sex: 3F, 4M; time since transplant: 5.4±2.1 yrs; left ventricular mass: 68±19 g/m2; end‐diastolic volume: 42±6 mL; end‐systolic volume: 12±2 mL; ejection fraction: 71±9 %). Pulsed Doppler was used to quantify early (E) and late (A) diastolic inflow velocities, deceleration time and slope. Tissue Doppler imaging of the lateral mitral annulus was used to assess velocity during early diastole (e′) and late diastole (a′). Left ventricular filling pressure responses were estimated using the early diastolic mitral valve inflow velocity/early diastolic lateral wall tissue velocity ratio (E/e′). Resting diastolic assessment was performed followed by 2 min of isometric handgrip exercise at 30% maximal voluntary contraction with diastolic function re‐assessed during the last 30 s of exercise.

Results

Heart rate (92±10 to 105±13 bpm; P=0.01) and mean arterial pressure (73±7 to 90±21 mmHg; P=0.035) increased from rest to exercise. There was no significant change in E/A (2.2±0.8 to 2.2±1.1; P=0.831), deceleration time (0.183±0.031 to 0.173±0.035 s; P=0.529), deceleration slope (613±152 to 619±169 cm/s2; P=0.926), e′/a′ (2.8±0.7 to 2.5±1.2 cm/s2; P=0.433), or E/e′ (7.02±1.37 to 7.13±1.71; P=0.709) from rest to exercise.

Conclusion

Pediatric HTR have diastolic dysfunction at rest that is not augmented during low‐grade handgrip exercise stress.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estradiol Decreases MAPKs phosphorylation in Response to Angiotensin II by Increasing MKP‐1 in the Hypothalamus, but not in the Lamina Terminalis
Authors: Almeida‐Pereira Gislaine , Vilhena‐Franco Tatiane , Coletti Ricardo , Cognuck Susana , Silva Hellen , Elias Lucila , Antunes‐Rodrigues Jose
Published: June 2021

Introduction

The angiotensin II (ANGII) plays an important role in the control of body fluids and estradiol (E2) modulates several actions of ANGII in the brain. Recently, our group showed that ERK1/2, JNK and p38MAPK are involved in sodium intake induced by ANGII in female rats. On the other hand, ANGII‐induced water intake requires JNK and p38MAPK, but not ERK1/2. In regarding to neurohypohysial hormone release, our group showed that ERK1/2 is involved in vasopressin (AVP) release but not in oxytocin (OT) release induced by ANGII. While ANGII‐induced OT release requires p38MAPK signaling. Therefore, herein the aim of study was evaluated whether E2 modulates MAPKs phosphorylation in response to ANGII as well MAPK phosphatase 1 (MKP‐1) which is responsible for inhibiting all the MAPKs (ERK1/2, JNK and p38MAPK) in the paraventricular (PVN) and supraoptic nuclei (SON) of hypothalamus, and in structures of lamina terminalis.

Materials and Methods

Wistar rats (~250g) were submitted to ovariectomy (OVX) and on the following day they were treated with estradiol cypionate (10μg/rat, sc, OVX+E2) or vehicle (corn oil, 0.1mL/rat, sc) for eight days. On the eighth day, the rats received an icv (lateral ventricle) injection of ANGII (25ng/2μL/rat) or vehicle (0.9% saline, 2μL/rat). After ten min of ANGII injection the animals were decapitated for brain collection for ERK1/2, JNK and p38MAPK total and phosphorylated, MKP‐1 and β‐Actin analyses by western blot. Data were analyzed using ANOVA two‐way, followed by Newman‐Keuls post‐test and the level of significance was set at 5%.

Results

The treatment with E2 attenuated JNK and p38MAPK phosphorylation in response to ANGII in the lamina terminalis (E2 x ANGII interaction: F1,17=19.00, p<0.001; E2 x ANGII interaction: F1,29=6.1, p<0.05, respectively). In regarding to PVN and SON, it was observed that E2 attenuated ERK1/2 and p38MAPK phosphorylation in response to ANGII (E2 x ANGII interaction: F1,29=7.91, p<0.01; F1,26=4.1, p<0.05, respectively). E2, but not ANGII alone, was able to increase MKP‐1 expression in the PVN and SON (E2: F1,18=24.29, p<0.001), but not in the lamina terminalis.

Conclusion

These results suggest that E2 dephosphorylates ERK1/2 and p38MAPK in response to ANGII by increasing MKP‐1 in the PVN and SON, but not in structures of lamina terminalis, such as subfornical organ, median preoptic nucleus and organum vasculosum of the lamina terminalis. E2 is able to dephosphorylate JNK and p38MAPK in the lamina terminalis in response to ANGII, however this effect does not involve MKP‐1.

Support or Funding Information

Financial support: FAPESP, CNPq and CAPES.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CGRP stimulates profibrogenic growth factors in kidney proximal tubular cells
Authors: Kim Jinu
Published: June 2021

Kidney denervation prevents the development of tubulointerstitial fibrosis, but local infusion of calcitonin gene‐related peptide (CGRP) into the denervated kidneys upregulates profibrogenic growth factors and restores the fibrotic feature. However, it is not clear how CGRP contributes to the upregulation of profibrogenic factors.

Both human HK‐2 and pig LLC‐PK1 kidney proximal tubular cells undergoing a 6 hours exposure to CGRP were treated with CGRP receptor antagonist (CGRP8–37), a specific protein kinase C (PKC) inhibitor chelerythrine, or a potent JNK inhibitor SP600125. Levels of transforming growth factor‐β1 (TGF‐β1) production and PKC activity were measured by enzyme‐linked immunosorbent assay. Western blot analysis performed to determine the protein levels of connective tissue growth factor (CTGF) expression and c‐Jun N‐terminal protein kinase (JNK) phosphorylation.

Administration of 1 nM CGRP significantly increased the levels of TGF‐β1 production and CTGF expression at 6 and 24 hours after the onset. The exogenous CGRP also increased their protein levels in the incubation media, indicating release of their proteins from the cells. Treatment with 100 nM CGRP8–37 immediately after the onset significantly inhibited the increase in intracellular and released protein levels of TGF‐β1 and CTGF during CGRP exposure. Furthermore, treatment with 1 thru 10 μM chelerythrine markedly reduced the upregulation and release of TGF‐β1 and CTGF after 6 hours exposure to CGRP. Finally, inhibition of JNK phosphorylation using 1 μM SP600125 prevented the increase in TGF‐β1 and CTGF upregulation and release after 6 hours exposure to CGRP.

Exogenous CGRP induces the upregulation and secretion of profibrogenic TGF‐β1 and CTGF proteins through CGRP receptor/PKC/JNK signaling pathway in proximal tubular cells, and this pathway might be a cause of triggering inflammation cascade and tubulointerstitial fibrosis.

Support or Funding Information

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF‐2016R1C1B2012080).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Microvascular Rarefaction Accompanies Interstitial Fibrosis and Tubular Damage in One Kidney‐Deoxycorticosterone Acetate‐Salt (1K/DOCA/salt)‐Dependent Hypertension
Authors: Saini Narbada , Thomas Jordyn , Huuskes Brooke , Sharma Prerna , Sobey Christopher , Vinh Antony , Drummond Grant
Published: June 2021

High dietary salt intake is a risk factor for hypertension and chronic kidney disease. However, the mechanisms involved remain poorly understood. Microvascular rarefaction ‐ defined as the loss of arterioles and capillaries within a tissue ‐ has the potential to increase total peripheral vascular resistance and cause local hypoxia and tissue damage. Here we utilised one kidney‐deoxycorticosterone acetate‐salt (1K/DOCA/salt)‐treated mice to investigate whether the development of hypertension and renal damage are associated with microvascular rarefaction. C57BL6/J mice were uninephrectomised and treated for 21 d with DOCA (2.4 mg/day, s.c.) and 0.9% NaCl in the drinking water. Normotensive controls were uninephrectomized and received a placebo pellet and normal drinking water (1K/placebo). Blood pressure (BP) was measured via tail cuff; renal vascular density was assessed immunohistochemically as the percentage area of CD31‐positive staining; renal collagen was detected with picrosirius red; and hematoxylin and eosin were used to visualise renal inflammation and histopathology. 1K/DOCA/salt‐treated mice had higher BPs (160±4 vs. 126±4 mmHg; n≥7; P≤0.0001) and renal weights (321±16 vs. 251±13 mg; n≥7; P<0.05) than 1K/placebo mice. 1K/DOCA/salt‐treated mice also displayed marked renal fibrosis, inflammation and damage as evidenced by a 10‐fold increase in interstitial collagen (n≥7; P<0.05), the presence of inflammatory cells, tubular dilatation and atrophy, and a loss of epithelial brush borders. Importantly, 1K/DOCA/salt‐treatment was associated with a 50% reduction in microvascular density (n≥7; P<0.05), mainly due to a loss of peritubular capillaries. Correlation analyses revealed a trend for an inverse association of peritubular capillary density with collagen deposition (n≥7; r= −0.7058; P= 0.117), but not with BP or inflammatory cell infiltration. Low magnification analysis of kidney sections revealed that peritubular capillary loss occurred in bands emanating from the arcuate arteries. Moreover, collagen deposition, inflammatory cell infiltration and tubular atrophy displayed a similar banding pattern. In summary, these studies reveal a likely association between microvascular rarefaction and fibrosis during the development of 1K/DOCA/salt‐dependent hypertension and renal damage. Future studies will assess whether strategies aimed at maintaining renal microvascular density can minimise renal fibrosis and damage.

Support or Funding Information

The work was funded by the National Health and Medical Research Council of Australia (GNT1143674)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renin Gene Editing in Zebrafish
Authors: Mullins Linda Jane , Hoffmann Scott , Rider Sebastien , Bradley Mark , Mullins John
Published: June 2021

Recent studies in zebrafish show that the renin‐angiotensin system (RAS) is highly conserved across vertebrates. In common with mammals, the zebrafish kidney contains specialised renin‐expressing mural cells throughout the kidney micro‐vasculature that contain acidic granules indicative of the processing and regulated secretion of active renin. Zebrafish are a unique model organism suitable for developmental studies and due to the translucent nature of zebrafish larvae, the functional embryonic pronephros is readily accessible for high‐resolution in vivo imaging and high‐throughput genetic and chemical screens. To investigate the role of renin and the RAS in zebrafish we designed guide RNAs to target CRISPR‐Cas9 to renin exon 2. We identified an 8bp deletion resulting in a frameshift mutation and early termination of renin transcription. Adult ren−/− knockouts, identified by sequencing, were phenotypically screened using the automated screening system (VAST), which permits the imaging of large numbers of live fish with reproducible precision. Knockout fish are viable but exhibit delayed growth during early stages of development. Imaging revealed a delayed appearance and enlargement of the swim bladder and reduced fish length was indicative of an overall developmental delay. The knockout fish were further crossed with already existing renal reporter lines to investigate further phenotypes at a cellular level. When ren−/− knockout fish were crossed with wt1b:GFP fish a delay in glomerular fusion of the pronephric kidney was observed, whilst a cross of ren−/− knockout with ren:RFP fish revealed a dramatic increase in renin cell number along the renal vasculature, suggesting a continued but non‐critical requirement for renin in the adult fish.

Support or Funding Information

BHF Centre of Research Excellence Award EPSRC/MRC CDT (Optima) Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pre‐transcriptional fibrotic factor alterations do not contribute to high fat diet associated renal fibrosis in Dahl salt sensitive male rats
Authors: Fernandes Roxanne , Galligan James J , Fink Gregory D , Xu Hui
Published: June 2021

We previously reported that high fat diet (HFD) feeding for 24 weeks promotes hypertension similarly in male and female Dahl salt‐sensitive rats. However, HFD fed males exhibited more severe renal inflammation, fibrosis and injury than females. The underlying mechanisms responsible for sex differences in HFD associated renal fibrosis/injury are not fully understood. It is well established that extracellular matrix (ECM) remodeling is the hallmark of hypertension associated renal fibrosis. Proteins involved in ECM remodeling are key mediators in the fibrogenic process. Therefore, we determined if HFD increases ECM remodeling resulting in renal fibrosis and if sex differences of ECM remodeling in response to HFD contribute to hypertension‐associated renal fibrosis/injury in males. Renal cortex was collected from male and female rats fed a control diet (CD, 10% kcal fat) or a high fat diet (60% kcal fat) for 24 weeks. mRNA expression of fibrosis markers in the renal cortex was assessed, which included transforming growth factor (TGF), smooth muscle actin (SMA), cysteine‐rich angiogenic inducer 61 (CYRG1), connective tissue growth factor (CTGF), collagen (COL1A1, COL3A1, COL1A2), and matrix metalloproteinases (MMP9, MMP14). We found that only MMP9 mRNA levels were significantly elevated in HFD fed females compared to CD fed females. However, MM9 mRNA expression was similar in HFD fed and CD fed males. HFD feeding did not change expression of the other fibrosis markers in males or females. We next determined if elevated MMP9 mRNA levels in HFD females were translated into increased total MMP9 protein and/or increased MMP9 activity. MMP9 activity was determined by zymograph assay. We found that the total MMP9 protein expression and the activity were similar in HFD fed males and females. Our studies suggest that it is unlikely that HFD causes pre‐transcriptional alterations in the fibrosis signaling pathway. HFD associated post‐transcriptional alterations may contribute to renal fibrosis in males, but the underlying mechanisms are still undetermined. In addition, the role of ECM remodeling in HFD associated renal fibrosis needs to be assessed prior to the onset of renal fibrosis.

Support or Funding Information

Supported by NHLBI 2P01HL070687 for G.D.F, J.J.G. and H.X

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of NanoString Technology for Detection and Genotyping of Human Papillomavirus (HPV)
Authors: Patel Sonya , Unger Elizabeth , Rajeevan Mangalathu
Published: June 2021

Objective

To date, CDC's HPV vaccine effectiveness studies have relied on one highly quality‐controlled commercial assay that uses consensus PCR of the HPV L1 gene followed by type‐specific hybridization. However, the reporting process is complex and additional testing is needed to resolve ambiguous results. Alternative assays with increased automation, faster turnaround and digital readout are needed.

Methods

We evaluated NanoString technology as a method for digital readout of the L1 gene for 48 HPV types in a single reaction. Unique probe pairs targeting the L1 and globin genes were designed in collaboration with NanoString. Forty residual DNA extracts from epidemiologic specimens and eight controls were tested directly (no amplification step) as well as after L1 consensus PCR. After overnight hybridization, type‐specific hybridization signals from scans were normalized and background assessed per the recommendations of NanoString. A positive result was defined as specimen‐normalized counts greater than the calculated sample specific background (average+2SD).

Results

The NanoString assay produces a direct unambiguous readout of type‐specific results directly populating a database without manipulation. HPV detection in plasmid controls was successful only at the highest copy number tested (10,000 copies) and comparison of results of prior method and NanoString in residual extracts showed good type‐specific agreement (k=0.621) but lower sensitivity (65%) of NanoString. With PGMY primer L1 amplification, 45 cycles, hybridizing the highly diluted product gave almost‐perfect type‐specific agreement (k=0.862) between NanoString and prior method. Relative to prior results, NanoString had 82% sensitivity and 99% specificity. Initial evaluation of reduced amplification cycles suggest that sensitivity is maintained with only 15 cycles.

Conclusions

This proof‐of‐principle study demonstrates the potential of using the NanoString platform for highly multiplexed, high throughput and digital genotyping of HPV with a low number of PCR cycles. The platform improves the reliability of data management. NanoString technology is a candidate to improve the quality and reduce the cost of surveillance in studies of HPV and other infectious diseases.

Support or Funding Information

Source of research support: Centers for Disease Control and Prevention

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of Artificial Sweeteners on Insulin, Body Composition and Drink Choice in Mice
Authors: Provost Terri , Bortiatynski Garrett , Scalise Anthony , Smith Quinaya , Twitchell Brielynn
Published: June 2021

The average American consumes almost 42 gallons of artificially sweetened beverages per year and about 66 pounds of sucrose. Although the USFDA supports that aspartame and stevia are safe for consumption, both nutritive and non‐nutritive sweeteners have been shown to negatively impact health. Sucrose causes addictive behaviors as well as excessive release of dopamine. Our study was designed to determine the behaviors and physiological changes in mice associated with consumption of aspartame and stevia when compared with sucrose. We exposed mice to untainted water or water containing 0.2% aspartame, 0.2% stevia, or 0.2% sucrose for 8 weeks, then provided the animals with a two‐bottle, treated and untreated, choice test for an additional week. After 9 weeks of treatment, mice were sacrificed and body composition, blood glucose, and serum insulin were determined. Stevia animals gained significantly more body weight than all other groups (p<0.05) and consumed significantly more food than controls (p<0.05). All treatment animals consumed significantly more fluid than control animals (p<0.05). When provided with the two‐bottle choice, the treated animals chose the sweetened drink significantly more than untreated water (p<0.05), while the control animals consumed water equally from both bottles. Aspartame treated animals had significantly heavier relative heart weights (p<0.05). We found no significant difference in relative liver weights, blood glucose or serum insulin concentrations in this short term test. These results are consistent with previous studies reporting addictive behavior with sucrose consumption. Current work is focused on the presence of brain lesions and dopamine release with exposure to sugar versus substitutes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotinate supplements slow onset and severity of symptoms in the monosodium iodoacetate (MIA) rat model for osteoarthritis (OA)
Authors: Bellamine Aouatef
Published: June 2021

High doses of niacinamide have been used for the treatment of rheumatoid arthritis in humans. However, it is unclear if nicotinate supplementation can slow the onset of osteoarthritis. To assess this possibility, we supplemented Sprague Dawley rats with niacin (80 mg/kg) or niacinamide (400 mg/kg) then induced arthritis using a single intra‐articular injection of mono‐iodoacetate (MIA). Three MIA concentrations (0.5 mg, 1.0 mg and 2.0 mg) were used to simulate mild, moderate and severe OA disease levels. Changes in levels of pain and inflammation were assessed via dynamic weight bearing (DWB) and joint diameter, respectively. Supplementation with niacinamide, but not niacin, led to a significant decrease in knee joint diameter in both the 0.5 mg and the 1.0 mg MIA cohorts, with relatively better effects in the 0.5 mg MIA group presenting with nearly complete reversal of disease at day 14. Nicotinate treatment did not improve the DWB characteristics of these animals. Blinded histopathological examinations showed that niacinamide's favorable clinical outcomes were correlated mostly in the 0.5 mg MIA group. A second, dose‐response study, using the 1.0 mg MIA model, found that supplementation with niacinamide at 130 mg/kg, and niacin at 10 mg/kg, also yielded beneficial outcomes. These results indicate that supplementation with niacinamide may alleviate some OA symptoms in a relative short period of time.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Resistance Exercise on Bones in Calcium‐deficient Rats
Authors: Hirakawa Tomoe , Fujii Takako , Matsumoto Kinuyo , Okamura Koji
Published: June 2021

Resistance exercise has been reported to enhance the reuse of iron in the body (Fujii T et al. Biol Trace Elem Res. 2014). Exercise increases the calcium absorption rate and bone mass. However, aerobic exercise reportedly does not lead to any improvements in bone strength in calcium‐deficient rats (Senge et al. J Physiol Soc Japan. 2003), although the effects of resistance exercise on bone in these animals are unclear. Therefore, we investigated the effect of resistance exercise on bones in calcium‐deficient rats. Twenty‐one male Sprague‐Dawley rats (4 weeks old) were divided into a calcium‐sufficient diet group (5 g calcium/kg diet) and a calcium‐deficient diet group (1.67 g calcium/kg diet), and half of the rats in both groups were subjected to climbing exercise training (5 minutes × 6 sets, 5 minutes of rest between sets) every other day for 3 days a week. The rats were euthanized after four weeks of the treatment. There were no significant differences in the blood calcium concentration among the four groups. The body weight and the weight of the flexor hallucis longus muscle, soleus muscle and kidneys were significantly higher in the exercise groups than in the non‐exercise groups, while no significant differences in these values were found between the calcium‐sufficient and calcium‐deficient diet groups. The weight, center width, rupture strength and stiffness of the femur were significantly higher in the calcium‐sufficient group than in the calcium‐deficient group, whereas no significant effect of exercise was observed. Resistance exercise enlarged the skeletal muscle in calcium‐deficient rats; however, this form of exercise showed no marked effect on bone parameters.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Protein Diet‐Induced Changes in Body Composition and Metabolic Flexibility in Women: Feasibility of a Novel Template‐based Intervention Delivery Method
Authors: Olenick Alyssa , Niersbach Abigail , Pearson Regis C. , Green Edward S. , Jenkins Nathan T.
Published: June 2021

Current recommended dietary allowance for protein is 0.8 g/kg/d. Recently, research has posited that this value is insufficient for resistance‐trained individuals and inadequate for the retention of lean mass while on a fat loss diet. Studies support an intake of nearly double the RDA, i.e. ~1.4–2.0 g/kg/day, but effective implementation strategies for achieving this higher protein intake have not been identified. Additionally, metabolic flexibility is the ability to adjust substrate oxidation according to nutrient availability and may be reflective of metabolic health. Whether a higher protein, caloric deficit diet improves metabolic flexibility in resistance‐trained women is not known.

PURPOSE

To determine the effects of a novel high protein diet template on body composition and metabolic flexibility during an 8‐wk intervention in recreationally resistance trained females.

METHODS

Recreationally resistance‐trained females (n=7, Age=21.7±2.1 yrs) were underwent baseline and post 8‐wk intervention dual‐energy X‐ray absorptiometry scans, anthropometrics and an acute high fat meal challenge. Baseline (T1) fasting glucose, resting energy expenditure (REE), and lipid (LO) and carbohydrate oxidation (CO) were measured. Participants then consumed a high fat meal (1127±132 kcal; 77±9 g fat) and glucose, REE, LO and CO measurements were taken at 60 (T2), 120 (T3) and 180 (T4) minutes postprandial. The meals contained 61% fat/29% carbohydrate/10% protein and were standardized to participants' body surface area (BSA). Participants were instructed how to follow a high‐protein dietary template (1.5–1.6 g/kg/day) and asked to maintain their resistance training volume and intensity throughout the study. The template was a PDF document containing specific instructions on amounts of lean proteins, vegetables, carbohydrates, and fats to be consumed at each meal throughout the day.

RESULTS

Significant decreases were observed in % body fat (pre: 30.7±3.9%, post: 28.3±3.0%; p=0.010), waist circumference (pre: 77.0±4.7 cm, post: 74.4±5.2 cm; p=0.030), and waist‐to‐hip ratio (pre: 0.77±0.04, post: 0.76±0.04; p=0.050), with total body weight (pre: 67.5±6.7 kg, post: 65.5±5.3 kg; p = 0.076) and fat mass (pre: 19.7±3.7 kg, post: 18.6±3.0 kg; p=0.062) approaching significance. There were no significant changes in bone density, lean mass, fasting glucose, REE, LO, or CO.

CONCLUSIONS

A high protein diet leads to favorable improvements in fat mass, while preserving both lean mass and bone density in resistance trained females, without affecting metabolic flexibility. Our study also supports the feasibility of delivering a dietary intervention in a user‐friendly diet template format.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.