bluuebunny/crossref_metadata_2025 huggingface dataset:
Tabata style functional exercise increases resting and postprandial fat oxidation but does not reduce triglyceride concentrations
Authors: Pearson Regis , Green Edward S. , Olenick Alyssa A. , Jenkins Nathan T.
Published: April 2020

We evaluated the effect of a high fat meal with and without prior high intensity functional exercise executed in a Tabata style interval pattern on resting and postprandial substrate oxidation, to include blood glucose and triglyceride concentrations in humans. Eleven healthy males completed two trials [Tabata exercise (TE) and non‐exercise control (CON)] in random order separated by 7 days. A two‐day protocol was used in which TE or CON was performed on the first day and a high fat meal was administered ~13 hrs later on the following morning. Power output from the TE session was quantified using a kinematic approach by calculating external work performed per unit of time for each of the 4 exercises (rowing, dumbbell thrusters, kettlebell swings, and burpees). For the meal challenge, respiratory gases and blood samples were taken fasted and at 1, 2, and 3 hrs postprandial. Fat oxidation was significantly higher after TE compared to CON at each time point (P < 0.05). Total fat oxidized, total fat oxidation AUC, and total postprandial fat oxidized all significantly increased after TE (P < 0.05). Carbohydrate oxidation was significantly lower after TE compared to CON at 1 hr postprandial (P < 0.05). Total carbohydrate oxidized, total carbohydrate oxidation AUC, and total postprandial carbohydrate oxidized all significantly decreased after TE (P < 0.05). There were no significant effects of TE on fasting or postprandial glucose or triglyceride concentrations. Functional exercises performed in a high intensity TE pattern enhance fasting and postprandial fat oxidation on the following day with minimal influence on blood triglycerides or glucose levels.

Support or Funding Information

R.C.P. was support by the University of Georgia Graduate School Georgia Research Education Award Traineeship Follows Program
Quantifying Transcription Dynamics of CUP1 gene of S. cerevisiae at the Single‐Molecule Level
Authors: Mehta Gunjan D. , Ball David , Karpova Tatiana
Published: April 2020

Transcription factors (TFs) show rapid and reversible binding to gene promoters to activate transcription in live cells. Transcription often occurs in sporadic bursts, however it is yet poorly understood how the dynamics binding of TFs is coordinated with gene bursting. Gene promoters are generally occupied by the nucleosomes and it is known that the presence of a nucleosome at the gene promoter reduces the accessibility for TF binding. Additionally, nucleosomes are known to evict the TFs from the gene promoters. Hence, chromatin remodeling plays an instrumental role in modulating TF binding. Due to advancements in the Single‐Molecule Imaging approaches, quantification of the transcription parameters such as initiation rate, transcript production time, burst frequency, burst duration and burst size has become achievable. We developed and employed several Single‐Molecule approaches (Single‐Molecule Tracking (SMT), live imaging of CUP1 transcription site (TS) by PP7 stem‐loop approach and smFISH modeling of CUP1 transcription) to quantify the transcription dynamics of the CUP1 gene of S. cerevisiae. In cell population, we observe slow sequential cycles of CUP1 transcription in conditions of the prolonged heavy metal stress. By applying gene bursting model, we extract the dynamics of the transcription parameters within the slow cycle from live TS and smFISH data and correlate these parameters with the dynamics of TF and POLII. The chromatin remodeler RSC speeds up the search process of the TF Ace1p for its Response Elements (RE) at the CUP1 promoter. RSC chromatin remodeler also affects the transcription of CUP1. We investigate the effects of RSC remodeler on the slow cycle of CUP1. Modulation of burst frequency yields more effective and productive response than the modulation of burst amplitude. We are interested in cyclical expression of the genes in stress response.

Support or Funding Information

This work was supported by Intramural Research Program of the National Institutes of Health (NCI, CCR).
Effects of [6]‐Gingerol on β‐amyloid‐induced Cognitive Impairment in Mice
Authors: Bae Hui Jin , Mun Seul Ah , Jang Jung-Hee , Park Gyu Hwan
Published: April 2020

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by disruption of cholinergic neurons and it is likely that promoted inflammation and oxidation is involved in pathogenesis of AD. Consequently, it is worthwhile developing effective phytochemical agents which can reinforce the cholinergic transmission and anti‐inflammatory defense capacity for the prevention and/or treatment of AD. In this study, we tested whether [6]‐gingerol, an active constituent of ginger could reduce β‐amyloid (Aβ)‐induced cognitive impairment in mice. Administration of [6]‐gingerol p.o. (25 mg/kg) significantly alleviated Aβ‐induced cognitive dysfunction as evaluated by Y‐maze and passive avoidance tests. To further elucidate the mechanisms underlying the effects of [6]‐gingerol, the possible involvement of inflammatory mediators were examined. Treatment of [6]‐gingerol reduced the expression of inflammatory enzymes and pro‐inflammatory cytokines such as COX2, IL‐1β, TNFα and iNOS. In addition, [6]‐gingerol decreased the phosphorylation of p66‐Shc which had been increased by Aβ. Taken together these findings suggest that [6]‐gingerol might potentiate anti‐inflammatory cellular defense system and have therapeutic potentials in the management of cognitive impairment in AD.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NFR) grant funded by the Korea government (MSIT)
Detection of Autologous Blood Transfusion by Metabolomics
Authors: Bejder Jacob , Gürdeniz Gözde , Cuparencu Catalina , Hall Frederikke , Gybel-Brask Mikkel , Andersen Andreas Breenfeldt , Dragsted Lars Ove , Secher Niels Henry , Johansson Pär Ingemar , Nordsborg Nikolai Baastrup
Published: April 2020

Autologous blood transfusion (ABT) is illegal for competing athletes but misuse remains difficult to detect. Thus, the development of novel and sensitive methods is warranted. In this study, we explored whether ABT causes a shift of the urine metabolome. The hypothesis was that an untargeted metabolomics analysis of urine is able to identify novel biomarkers sensitive to ABT.

In a randomized, double‐blinded, placebo‐controlled cross over design (3 month wash‐out), twelve trained males donated 900 ml blood or were sham phlebotomized. Four weeks later, a transfusion of the stored red blood cells or a sham transfusion was completed. Urine was collected before phlebotomy and 2 h, 1, 2, 3, 5 and 10 days after transfusion and analyzed by UPLC‐QTOF‐MS.

Models of unique metabolites reflecting ABT was derived by partial least squares regression discriminant analysis. The strongest model appeared 2 h after transfusion (misclassification error: 6.3%, specificity: 98.8%). The remaining time points provided misclassification errors from ~20–50% with specificities of ~50–80%. Metabolite identification revealed secondary di‐2‐ethylhexyl phtalate metabolites as the strongest biomarkers for detection.

In conclusion, untargeted metabolomics of urine identified plasticizers as the strongest metabolites for ABT detection. Future research should validate the proposed models.

Support or Funding Information

The study was funded by Partnership for Clean Competition
Increased Lipopolysaccharide‐Induced Hypothermia in Neurogenic Hypertension is caused by Reduced Hypothalamic PGE2 Production and Increased Heat Loss
Authors: Amorim Mateus R. , Moreira Diego A. , Santos Bruna M. , Ferrari Gustavo D. , Nogueira Jonatas E. , de Deus Júnia L. , Alberici Luciane C. , Branco Luiz G.S.
Published: April 2020

Brain‐mediated thermoregulatory changes in systemic inflammation (SI) induced by lipopolysaccharide (LPS) at a septic‐like model are characterized by hypothermia followed by fever which are associated with inadequate tissue oxygenation, multiple organs dysfunctions, and death. Hypertension is a prevalent chronic disease characterized by autonomic‐induced elevated and sustained blood pressure levels and spontaneously hypertensive rats (SHR) also have abnormal body core temperature (Tb) regulation and metabolic adjustments, by unknown mechanisms. The rationale of the present study was to find out the mechanisms involved in the thermoregulatory changes observed during SI in SHR. We combined Tb and skin temperature (Tsk) analysis, assess prostaglandin (PG) E2 levels in the anteroventral region of the hypothalamus (AVPO), indirect calorimetry, cardiovascular recordings, assays of inflammatory markers and evaluation of oxidative stress status in male Wistar and SHR that receive intravenous administration of LPS (1.5 mg.kg−1) or saline (CEUA #2017.1.585.58.9). As expected, LPS induced hypothermia followed by fever in Wistar whereas in SHR increased hypothermia and no fever were observed. These changes were associated with an increased Tsk in SHR in relation to Wistar (P < 0.05). Oxygen consumption and carbon dioxide production were significantly and similarly reduced in both SHR and Wistar rats during SI (P < 0.05). Interestingly, we measured increased AVPO PGE2 levels in Wistar during SI (P < 0.05), but not in SHR. LPS‐induced drop in MAP was significantly higher in SHR than in Wistar rats. Besides, LPS‐induced plasma and brain cytokines surges were blunted whereas oxidative stress was higher in SHR (P < 0.05). The present data are consistent with the notion that in SHR LPS‐induced SI leads to thermoregulatory changes mediated by the thermo effector mechanisms in the skin but not in energy expenditure and these events are associated with reduced hypothalamic PGE2.

Support or Funding Information

FAPESP, CAPES and CNPq
CYTOTOXIC ACTIVITIES OF SELECTED MOLLUSC SHELLS ALONG THE EASTERN CAPE COAST IN SOUTH AFRICA
Authors: Nakin Motebang Dominic Vincent , Saibu Gbemisola Morounke , Oyedeji Adebola Omowunmi , Piya Viwe , Meyer Mervin
Published: April 2020

Introduction

The application of some natural products from mollusc shells has been known for centuries. Secondary metabolites produced in marine organisms could be the source of bioactive substance and used for cosmetic purposes. Cosmetic potential of mollusc shells has become a critical scientific concern to most researchers around the world. The aim of the present study was to evaluate the cytotoxicity activity of some selected mollusc shells of mussels (Perna perna), oysters (Sacostrea cuculata) and limpets (scutellastra longicosta) collected from Port St Johns in the Eastern Cape coast of South Africa.

Materials and Methods

Human keratinocyte e (HaCaT) and human fibroblast (KMST‐6) cell lines were treated with various concentrations (0.0312, 0.0625, 0.125, 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 mg/ml) of Mollusc shells for 24 hours after which cell proliferation was measured by MTT assay.

Results

The IC50 values could not be obtained for the KMST‐6 treated cells. However, for HaCaT treated cells, there was significantly (p < 0.05) moderate cytotoxicity activity at a concentration range of 0.312‐0.02 mg/ml for all the five extracts.

Discussion and Conclusion

Since the IC50 values could not be obtained for the non‐cancerous (KMST‐6) that implies the safety of the extracts for cosmetic purposes. The moderate cytotoxicity obtained from the cell treated with Human keratinocyte (HaCaT) cell line also make them appropriate candidate for further studies and identification of their bioactive principles. These extracts could also be used as an alternative source for treatment of fungal infections.
Can Interleukin‐8 Alone be Responsible for Regulating Melanoma Growth & Metastasis? In‐vitro studies based on treatments of human melanoma cell lines (BLM and 1205Lu) with sex steroids, IL‐8, curcumin and active vitamin‐D3
Authors: Ramaraj Pandurangan
Published: April 2020

Previous clinical studies indicated that menstruating females were better protected in melanoma than post‐menopausal women and men of any age. But, the mechanism responsible for protection in menstruating females was not investigated. In addition, epidemiological data showed an increased male mortality in melanoma. Our in‐vitro study showed female sex hormone progesterone significantly inhibited human melanoma cell growth. Elisarray showed that progesterone action was mediated by a specific suppression of pro‐inflammatory cytokine IL‐8. So, the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth was checked. AD and T also suppressed cell growth and IL‐8 secretion. But, addition of progesterone (10μM) along with androgens showed an additive effect on the inhibition of melanoma cell growth and IL‐8 suppression. This observation gave the idea of protective function of progesterone in melanoma, which was experimentally observed by a significant decrease in in‐vitro migration and adhesion functions. As steroids (P, AD, T) targeted IL‐8 for their actions, effect of vit‐D3 on melanoma cells was checked. Active form of vitamin‐D3 (25 μM) also targeted IL‐8 and decreased cell growth, suggesting IL‐8 could be regulating melanoma cell growth. In order to check whether IL‐8 regulated melanoma cell growth, IL‐8 was directly added to melanoma cells and endogenous IL‐8 was suppressed by 2 hrs pre‐treatment with curcumin. Addition of IL‐8 (1 ng/ml) to 1205Lu melanoma cells stimulated cell growth to 117%, whereas suppression of IL‐8 by curcumin (100 μM) pre‐treatment decreased cell growth to 24%. Similar results were obtained with BLM cells. This brought IL‐8 into focus as an important molecule regulating melanoma cell growth. Further in‐vitro experiments to restore curcumin (25 μM) pre‐treated melanoma cell growth (71.5%) by the addition of IL‐8 (100 ng/ml) resulted in a near complete restoration of cell growth (95.3%) in 1205Lu melanoma cells, suggesting IL‐8 could be
the molecule regulating melanoma cell growth. Similar results were observed with BLM cells. In addition, in‐vivo conditional expression of IL‐8 in nude mouse by Dr. Singh et al., indicated involvement of IL‐8 in melanoma growth and metastasis.

Conclusion

Both in‐vitro and in‐vivo studies indicated that IL‐8 alone could be responsible for regulating melanoma growth and metastasis. Hence, IL‐8 could be targeted to arrest melanoma growth and metastasis in‐vivo.
Kinetic and Mechanistic Characterization of a Potent and Selective Inhibitor for Human AXL Receptor Tyrosine Kinase
Authors: Chen Yu , Miles Dillon H. , Udyavar Akshata , Cho Sean , Becker Annette , Foley Corinne , Leleti Manmohan R. , Jin Lixia , Young Stephen W. , Powers Jay P. , Walters Matthew J. , Zhao Xiaoning
Published: April 2020

The AXL receptor tyrosine kinase (AXL) is a member of the TAM RTK family comprising TYRO3, AXL and MER. AXL signaling is activated by binding of the growth arrest‐specific protein 6 (Gas6) or vitamin K‐dependent protein S (PROS1), which subsequently induces homodimerization and autophosphorylation, thereby initiating a conventional RTK signaling pathway. Maximal stimulation requires an extracellular lipid, phosphatidylserine (PtdSer).

Activation of AXL regulates an array of signal transduction pathways, including those mediating cell survival, proliferation and migration, as well as promoting an immunosuppressive phenotype within the tumor microenvironment
1,
2. AXL has been shown to be highly expressed in sarcoma, renal cell carcinoma, pancreatic adenocarcinoma and other types of tumors, correlative with poor prognosis in cancer patients. Furthermore, AXL has been shown to facilitate both intrinsic and acquired resistance to multiple anti‐cancer drugs
3, suggesting therapeutic targeting of AXL may be an effective anti‐cancer strategy.

We have developed a potent, reversible and competitive small molecule AXL inhibitor. This inhibitor (Compound 1) inhibits the activity of recombinant human AXL kinase domain with nanomolar potency, as determined by detection of phosphorylated substrate using homogeneous time‐resolved fluorescence (HTRF). Compound 1 is also highly selective against TYRO3, MERTK and MET kinase domains. The compound binds to the intracellular AXL kinase domain with a nanomolar dissociation rate constant measured by monitoring displacement of a competitive fluorescent tracer using bioluminescence resonance energy transfer (BRET). In order to assess the inhibitory effect of the compound on downstream signal transduction, AXL autophosphorylation‐induced SH2 translocation was determined using an AXL PathHunter® assay and phosphorylation of AKT Ser473 induced by AXL activation was also assessed by AlphaLISA.

Support or Funding Information
Characterizing the Function of Anti‐CRISPR protein AcrIIA11
Authors: del Banco Serena Bautista , Kaiser Brett , Forsberg Kevin , Malik Harmit , Stoddard Barry
Published: April 2020

Bacteria and the viruses that infect them are engaged in an evolutionary arms race that has driven the development of bacterial defense mechanisms and phage countermeasures to those defense mechanisms. One instance of this evolutionary competition is the development of the CRISPR‐Cas system, which enables the recognition and destruction of phage DNA. In response to this defense, phages have evolved anti‐CRISPR proteins that inhibit CRISPR‐Cas activity and promote phage infection. It is thought that many anti‐CRISPR proteins exist in nature, but only a handful have been identified and their mechanism of inhibition characterized. The goal of this project is to understand the mechanism by which a recently identified anti‐CRISPR protein, AcrIIA11, inhibits Streptococcus pyogenes Cas9 (SpyCas9). To address this, we have performed in vitro DNA cleavage and protein binding assays with purified components. We have determined the following: AcrIIA11 effectively inhibits Cas9 cleavage at an AcrIIA11: Cas9 ratio of 8:1; AcrIIA11 interacts directly with the Cas9 protein; homologs of AcrIIA11 show a range of ability to inhibit Cas9 activity but do no inhibit as well as AcrIIA11; and the C‐terminus of AcrIIA11 is not required for binding to Cas9. In future studies, we will test AcrIIA11 interaction with a wider range of Cas9 orthologs. From these results, we have gained a broader understanding of how AcrIIA11 interacts with SpyCas9.

Support or Funding Information

Seattle University Summer Undergraduate Fellowship (Peach Foundation)
Heart Rate Variability during Heat Exposure is not Affected by Short‐term Passive Heat Acclimation in Young Healthy Participants
Authors: Gendron Philippe , Gravel Hugo , Barry Hadiatou , Gagnon Daniel
Published: April 2020

INTRODUCTION

Heat acclimation attenuates the increase in heart rate during heat exposure. This heat acclimation‐induced bradycardia has been associated with increased stroke volume due to blood volume expansion. However, exercise‐induced heat acclimation also modulates cardiac parasympathetic activity, as measured through heart rate variability (HRV). Therefore, it is possible that modulation of cardiac autonomic activity could contribute to the effect of heat acclimation on heart rate during heat exposure. The purpose of this study was to determine if HRV during heat exposure is modified by short‐term passive heat acclimation.

METHODS

Fifteen healthy adults (27 ± 5 years, 5 females/10 males) underwent heat exposure in a water‐perfused suit to increase esophageal temperature (Tes) by 1.2°C, before and after a 7‐day controlled hyperthermia protocol. During heat exposure, R‐R interval was measured continuously with a 5‐lead electrocardiogram and used to calculate heart rate (HR), mean normal‐to‐normal intervals (NN), standard deviation of NN intervals (SDNN), root mean square of successive NN interval differences (RMSSD), low‐frequency (LF) and high‐frequency spectral power (HF) and the LF‐to‐HF power ratio (LF/HF). The acclimation protocol consisted of hot water immersion to elevate and subsequently maintain rectal temperature ≥38.6°C for 60 minutes once a day for 7 consecutive days. Variables were compared between the pre and post acclimation visit at baseline and at increases in Tes of 0.6°C and 1.2°C.

RESULTS

Heat acclimation lowered resting Tes (36.81 ± 0.29 to 36.53 ± 0.32°C, P<0.001) and, consequently absolute Tes during heat exposure. When analyzed as a function of the change in Tes from baseline, no difference was observed for HR (P=0.072) at baseline (59 ± 10 vs. 57 ± 9 bpm), +0.6°C (88 ± 12 vs. 86 ± 11 bpm), and +1.2°C (103 ± 13 to 99 ± 12 bpm). Similarly, NN (baseline: 1049 ± 172 vs. 1081 ± 162 ms, +0.6°C: 691 ± 104 vs. 705 ± 100 ms, +1.2°C: 595 ± 74 vs. 600 ± 96 ms, P=0.295), SDNN (baseline: 84 ± 38 vs. 90 ± 37 ms, +0.6°C: 43 ± 15 vs. 45 ± 15 ms, +1.2°C: 28 ± 9 vs. 31 ± 8 ms, P=0.294) and RMSSD (baseline: 83 ± 49 vs. 93 ± 57 ms, +0.6°C: 19 ± 9 vs. 19 ± 14 ms, +1.2°C: 9 ± 4 vs. 9 ± 3 ms, P=0.336) did not differ after heat acclimation. No effect of heat acclimation was also observed for LF (P=0.339), HF (P=0.509) and LF/HF ratio (P=0.449).

CONCLUSIONS

These results suggest that short‐term, passive and isothermic heat acclimation does not modulate cardiac autonomic activity during passive heat exposure in healthy young adults.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada, Canada Foundation for Innovation and Montreal Heart Institute Foundation.
Using an Asparagine Synthetase Inhibitor to Reverse Chemotherapeutic Resistance in Leukemic Cell Lines
Authors: St. Laurent Andrea , Train Tonya Laakko
Published: April 2020

Asparaginase (ASNase) is one of the first‐lines of chemotherapy used in the treatment of acute lymphoblastic leukemia (ALL). It breaks down asparagine in the body forcing the cell to undergo apoptosis. Yet, some patients develop resistance to ASNase treatment. Asparagine is produced in the cell by the enzyme asparagine synthetase (ASNS). One explanation for resistance could be increased ASNS expression leading to the production of more asparagine than can be broken down. Here, we investigated whether using a competitive ASNS inhibitor, albizziine (Alb), to decrease ASNS activity would reverse ASNase resistance in leukemic cell lines. Two human leukemic cell lines with varying ASNS expression were utilized, Jurkat and HL‐60. Real time‐PCR analysis showed an 8.9‐fold higher level of ASNS mRNA in HL‐60 relative to Jurkat. In addition, that HL‐60 was resistant and Jurkat sensitive to ASNase‐induced apoptosis; as determined by staining the cells with Annexin V‐FITC and flow cytometric analysis. After 24 hours, 0.1 IU ASNase treatment resulted in a 43.27% increase in apoptosis in Jurkat cells, but no significant increase in HL‐60 cells. The addition of the ASNS inhibitor, Alb, in combination with ASNase resulted in increased apoptosis in both cell lines. HL‐60 apoptosis increased 6‐fold when treated with a combination of 0.1 IU ASNase and 1mM Alb. Alb also increased Jurkat sensitivity to 0.1 IU ASNase from 43.27% apoptosis with ASNase alone to 67.15% in combination with ASNase. Alb alone did not induce any significant death. These results suggest that the ASNS inhibitor, Alb, can restore sensitivity to ASNase induced death in leukemic cell lines and therefore warrants further investigation into its role in reversing ASNase chemotherapeutic resistance.

Support or Funding Information

This work was supported by the Honors Program at Elon University, Elon Undergraduate Research Program, and Glen Raven grant.
Optimization of a bacterial‐three hybrid assay for discovery and characterization of sRNA‐binding proteins
Authors: Mansky Rachel , Wang Clara , Hegner Courtney , Berry Katherine E.
Published: April 2020

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. Hfq is an RNA chaperone protein that, through RNA interactions on distinct surfaces of its hexameric structure, supports many sRNAs to regulate translation and stability of their mRNA targets. Though Hfq is well characterized in E. coli and some additional model bacteria, Hfq homologs have only been identified in ~50% of sequenced bacteria, leading to speculation that additional RNA chaperone proteins may play important roles in sRNA function in other bacteria. We have recently developed a bacterial three‐hybrid (B3H) assay that detects the binding of E. coli sRNAs with multiple RNA chaperone proteins. This assay couples the transcription of a genetic reporter to the interaction of a DNA bound “bait” RNA (e.g. an sRNA) and an RNAP‐fused “prey” protein (e.g. Hfq). Successful interaction between RNA and protein stabilizes RNA polymerase at a test promoter and activates reporter gene expression. While this assay current detects numerous high‐affinity RNA‐protein interactions, the signal‐to‐noise makes it challenging to detect weaker interactions. In order to lay the foundation to search for new bacterial RNA‐binding proteins, we are using Hfq‐sRNA interactions as a model system to optimize the system, so that weaker RNA‐protein interactions can be reliably detected. Here we will present our efforts to improve the assay’s signal‐to‐noise ratio by adjusting the sequence, concentration, and spacing of different components within the B3H assay. With these improvements, we will work to identify novel protein‐RNA interactions, as well as dissect the binding mechanisms of these interactions.

Support or Funding Information

This work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.
SPARC: Catheter‐Free Wireless Measurement of Feline Bladder Pressures Using Intravesical Sensor
Authors: Majerus Steve J.A. , Panda Binit , Rietsch Anna , Deng Kangli , Hanzlicek Brett , Mrowca James , Damaser Margot S. , Bourbeau Dennis J.
Published: April 2020

Introduction and Objective

In research animal models, catheterization for cystometry requires anesthesia which affects neuro‐urological pathways, or animal restraint, limiting measurement time and social behaviors surrounding natural bladder filling and micturition. To overcome this, we developed a catheter‐free, wireless, intravesical pressure sensor for use in conscious, untethered felines. This study validated sensor function and physiologic outcomes over 4‐week implantations, including (to our knowledge) the first untethered catheter‐free wireless recordings of bladder pressure during bladder filling and voiding.

Methods

Sensor performance was first validated on the bench. Sensors were then encapsulated in layers of medical epoxy and silicone and sterilized by ethylene oxide gas prior to implantation. Implantations were performed on 6 male felines: 2 were sham (no device), 2 implants were inactive devices, and 2 used active devices measuring 18 mm × 12 mm × 5.6 mm. Devices were implanted into the bladder lumen through a 10‐cm detrusor incision, which was stretched on insertion and then sutured. Anesthetized cystometry and urine collection was performed immediately before and after surgery, and at 2 and 4 weeks after implantation. Conscious data collection and behavioral video recordings were performed 3 times/week. Data collection used a small animal‐worn radio harness which received sensor transmissions and logged data to a MicroSD card. Devices were explanted after 4 weeks.

Results

All animals showed a similar decrease in bladder capacity following surgery (sham surgery and implant). All devices remained patent without obstructing the bladder for 4 weeks; one device stopped transmitting at 12 days and one worked through 4 weeks. Wireless pressure data was linearly correlated with spontaneous reflex contractions during anesthetized urodynamics (R2 = 0.96). Conscious data recording was performed on 11 occasions, producing over 200 minutes of catheter‐free pressure recordings. Animals entered and used litter boxes freely while data were recorded to the radio harness. Recorded pressures during voiding were confirmed against simultaneous video recording.

Conclusions

Small wireless, intravesical sensors permitted catheter‐free recordings of bladder pressure in felines for 30 days, including voluntary voiding contractions. Translation of this technology could enable urethrally‐inserted sensors for catheter‐free, wireless ambulatory urodynamics over longer time periods than is feasible with catheters.

Support or Funding Information

This work was funded by the NIH Stimulating Peripheral Activity to Relieve Conditions (SPARC) program, NIH grant number OT2OD023873.
Probing the Molecular Mechanism of ProQ‐RNA Interactions using a Bacterial Three‐hybrid Assay
Authors: Gravel Chandra
Published: April 2020

In bacteria, small RNAs (sRNAs) are known to play important roles in gene regulation. The interactions between sRNAs and mRNAs are often facilitated by protein chaperones. Recently, ProQ has been shown to bind to dozens of E. coli sRNAs and mRNAs, and to facilitate base pairing between the mRNA hupA and sRNA RaiZ with the effect of regulating hupA. These observations have led to the proposal that ProQ may act as a widespread regulator of bacterial gene expression. Our goal has been to understand the molecular mechanisms of ProQ’s interaction with RNAs, mapping the amino acids on ProQ’s surface and nucleotides of RNAs that contribute to binding and regulation. To do this, we have used a bacterial three‐hybrid (B3H) assay to detect ProQ‐RNA interactions genetically. In the B3H assay, ProQ is fused to RNA polymerase (RNAP) and a hybrid RNA containing an sRNA or mRNA of interest is tethered to a DNA sequence upstream of a test promoter. Interaction of ProQ with the RNA stabilizes the binding of RNAP to the test promoter and activates transcription of a reporter gene, lacZ. Using domain truncations, site‐directed mutagenesis, and an unbiased forward genetic screen, we have identified a surface on ProQ’s NTD that we believe to be the primary surface for recognition of these SibB and cspE RNAs in vivo, and have created a genetically guided model for ProQ binding to an RNA duplex. In addition, we are working to probe whether ProQ possesses unique surfaces that contribute to binding of certain sRNAs and/or mRNAs and to determine what aspects of RNA sequence and structure contribute to recognition by ProQ.

Support or Funding Information

This work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.
Chemogenetic Activation of Parasubthalamic nucleus Corticotropin‐Releasing Factor Neurons recapitulates Alcohol Withdrawal‐Related Behavioral Symptomatology
Authors: Okhuarobo Agbonlahor , Kreifeldt Max , de Macedo Giovana Camila , Igbe Ighodaro , Contet Candice
Published: April 2020

Negative reinforcing effects of alcohol, driven by negative affect during withdrawal, are thought to be crucial to drinking escalation in alcohol use disorders. While the brain regions and neurotransmitter signaling affected by chronic alcohol use are well characterized, our understanding of the specific neural circuits driving drinking escalation and negative affect during withdrawal remains limited. Animal studies have highlighted a critical role of corticotropin‐releasing factor (CRF) signaling in the behavioral symptomatology of alcohol dependence. Hence, the present study sought to examine the role of a cluster of CRF neurons located in the parasubthalamic nucleus (PSTN), a small region of the posterior hypothalamus, which has received little attention in behaviors related to alcohol dependence. Adult male Crh‐IRES‐Cre mice were bilaterally injected in the PSTN with a viral vector encoding the excitatory designer receptor hM3D(Gq) fused to the fluorescent reporter mCherry upon Cre recombination. Injection of the hM3D(Gq) agonist clozapine‐N‐oxide (CNO, 1 mg/kg) significantly increased alcohol and saccharin consumption but did not change food and water consumption compared to vehicle. CNO had no effect in mice expressing mCherry only. CNO also increased locomotor and digging activity but did not alter open arm exploration in the elevated plus maze. CNO also decreased grooming duration in the splash test and immobility duration in the tail suspension test. Interestingly, the increased alcohol consumption, digging activity, active coping and reduced grooming all recapitulate phenotypes observed in mice withdrawn from chronic intermittent ethanol vapor inhalation, a model of alcohol dependence. However, CNO injection caused a significant increase in nociceptive thresholds in both the tail immersion and tail pressure tests, which is at odds with the hyperalgesia typically associated with alcohol withdrawal.

The PSTN sends a strong projection to the central amygdala (CeA), a region that plays a critical role in the transition to alcohol dependence. We therefore tested whether the effects of PSTN CRF neuron chemogenetic activation may be mediated by the activation of CeA neurons carrying CRF1 receptors. However, CNO did not affect alcohol drinking, locomotion and digging in Crhr1‐IRES‐Cre mice expressing hM3D(Gq) in the CeA. Combined with our recent finding that PSTN neurons become activated during alcohol withdrawal, our results suggest that PSTN CRF neurons contribute to the behavioral symptomatology of ethanol withdrawal, but CRF1 signaling in the CeA may not be relevant to their mechanism of action.

Support or Funding Information

This work was funded by the following grants from the National Institutes of Health: AA026685 (CC), P60 AA006420 (CC), and University of Benin, Benin City, Nigeria, (AO).
Liver Enzyme Levels of Paracetamol Induced Hepatotoxic Rats Treated with Aqueous Leaf Extract of Artocarpus Altilis
Authors: Nosiri Chidi Ijeoma , Oze Gabriel
Published: April 2020

The aim of this study was to determine the effect of Artocarpus altilis on the liver enzyme levels of paracetamol induced hepatotoxic albino rats. Paracetamol otherwise known as Acetaminophen is widely used as an analgesic and antipyretic drug worldwide. It is commonly abused as it is an over the counter (OTC) drug. N‐Acetyl‐p‐benzoquinone imine (NAPQI), an intermediate product of acetaminophen produced in the presence of cytochrome‐p450 causes hepatic damage and tubular necrosis in the kidney in both humans and experimental animals. In this situation, a large amount of NAPQI is metabolized in the presence of cytochrome P450s, which overwhelms the activity of reduced Glutathione (GSH) leading to hepatotoxicity. Acetaminophen’s clinical and biochemical side effects are well known and it is therefore used as a reference compound to assess the strengths and weaknesses of medicinal plants in ameliorating or preventing its effect in the liver. Twenty‐five (25) albino rats were used hence acclimatized for fourteen days. The experimental animals were divided into five groups of five rats each (control group, negative control group, 250mg/kg, 500mg/kg and Vitamin E). The albino rats were starved for 18 hours before pretreatment with single oral dose of paracetamol (2000mg/kg). Food and water were given ad libitum. They were then administered with the different doses of the leaf extract of Arthocarpus altilis and Vitamin E (an antioxidant) for three (3) days. After two hours of last administration of the extract, they were sacrificed anesthetically using chloroform and blood collected through cardiac puncture. The result showed that there was no significant difference (p≥0.05) between 250mg/kg, 500mg/kg and Vitamin E groups in the concentrations of ALP and ALT. The result also showed that there was a significant (p≤0.05) increase in the levels of serum Alanine Amino Transferase (ALT), Aspartate Amino Transferase (AST) and Alkaline Phosphatase (ALP) in the paracetamol treated groups compared to all the extract treated and control groups. Serum levels of all the liver enzymes were significantly (p≤0.05) reduced in all the treated groups especially the highest dose (500mg/kg) of Artocarpus altilis (breadfruit) leaf extract compared to the negative control. It has been reported that the leaf of Artocarpus altilis possesses in addition to other medicinal properties, an antioxidant potential. This sub‐acute study showed that artocarpus altilis possesses ameliorative effect on the investigated Liver biomarkers since the liver enzyme levels of AST, ALT and ALP reduced by the end of this study
The Role of Vascular Smooth Muscle RhoBTB1 in Hypertension
Authors: Fang Shi , Wu Jing , Silva Sebastiao Donato , Lu Ko-ting , Sigmund Curt D.
Published: April 2020

Rho‐related BTB domain containing protein 1 (RhoBTB1) is a novel transcriptional target of peroxisome proliferator activated receptor gamma (PPARγ). In a GWAS study of over 1 million subjects, RhoBTB1 was identified as an interacting locus for hypertension. However, the physiological and molecular function of RhoBTB1 is not well‐understood. Previous studies suggest that RhoBTB1 in vascular smooth muscle cells (VSMC) mediates the anti‐hypertensive effect of PPARγ. RhoBTB1 expression was decreased in aortas from mice expressing smooth muscle specific dominant negative PPARγ mutation. Genetic complementation of smooth muscle RhoBTB1 reverses the hypertension, vascular dysfunction and arterial stiffness caused by dominant negative PPARγ mutation. In HEK293 cells, RhoBTB1 interacts with phosphodiesterase 5 (PDE5) and increases PDE5 ubiquitination in a Cullin‐3 dependent manner, suggesting that RhoBTB1 might function as a substrate adaptor for Cullin‐3‐RING ubiquitin ligase and mediate PDE5 turn over.

RhoBTB1 expression was also decreased in aortas from mice treated with angiotensin II (Ang‐II, 490ng/kg/min, two weeks). We hypothesized that restoration of VSMC RhoBTB1 will reverse Ang‐II mediated hypertension, vascular dysfunction and arterial stiffness. We treated transgenic mice expressing inducible VSMC specific RhoBTB1 (S‐RhoBTB1 mice) with Ang‐II (490ng/kg/min, six weeks) and activated the transgene with Tamoxifen injections during the third week of Ang‐II infusion. ISM‐Cre mice expressing smooth muscle specific Cre‐recombinase were also treated with Ang‐II and Tamoxifen to serve as control. Blood pressure was measured by radiotelemetry. Ang‐II increased blood pressure in both ISM‐Cre and S‐RhoBTB1 mice to a similar extent. Thus, Ang‐II induced hypertension was not affected by RhoBTB1 transgene expression in VSMC. Carotid artery vasodilation response to acetylcholine and sodium nitroprusside, as well as vasoconstriction response to KCl, serotonin and endothelin‐1 were tested using wire myography. No differences were found in these measures of vascular function between S‐RhoBTB1 mice and ISM‐Cre mice. Arterial stiffness was assessed by pulse wave velocity (PWV). Ang‐II infusion increased PWV to a similar level in ISM‐Cre and S‐RhoBTB1 mice before the transgene induction. PWV of S‐RhoBTB1 mice started to decrease after Tamoxifen whereas PWV of ISM‐Cre mice continued to increase. By the second week after Tamoxifen, RhoBTB1 transgene expression in VSMC significantly attenuated Ang‐II mediated arterial stiffness (ISM‐Cre vs. S‐RhoBTB1 4.21±0.37 vs. 3.17±0.16 mm/ms, p=0.02, n=7–9). This data suggests a potential mechanism where RhoBTB1 improves vascular plasticity independently from blood pressure and is protective against arterial stiffness.

Support or Funding Information

NIH R35 HL144807

AHA 20PRE35120137
Modified MrNV viral capsids with GE11 peptide for binding colorectal cancer cells.
Authors: Grataitong Khwanthana , Weerachatyanukul Wattana , Chakrabandhu Krittalak , Hueber Anne-Odile
Published: April 2020

Virus‐like particles (VLPs) are useful nanoparticles with a great impact on medical nanotechnology. Macrobrachium rosenbergii nodavirus (MrNV), which causes white tail disease (WTD) in giant freshwater prawns, is an icosahedral virus and have 26–27 nm. Recombinant MrNV capsid proteins in Escherichia coli exhibit the self‐assembly of the attractive property of the nanocontainers. Therefore, we hypothesized that modified VLPs either by genetic modification on the C‐terminal domain should enhance specific targeting of VLPs to the destiny of cancer cells. This work thus aims to predict and test the exterior surface of MrNV capsids by genetic modification by the structural modeling and In vitro analysis. We engineered the GE11 peptide to replace the 349–360 residues of the MrNV protruding domain, to insert between the 354 and 355 residues of the MrNV protruding domain and to extend to the C‐terminal of the MrNV capsids, which presumably led the specific binding of modified VLPs to EGFR on cancer cells. However, the site for introducing GE11 peptide into protein still difficult to determine. Therefore, we use the computational method to submit algorithms Phyre2 and Swiss model for the structural prediction. All of the models modified MrNV‐VLPs with GE11 are purified and analyzed by SDS‐PAGE, western blot and TEM analysis. These results lead to an In vitro experiment to enhance binding into EGFR positive cancer cells. This research thus provides a fundamental insight of virus capsid structure and ultimately leads to the development of strategies in drug delivery for carrying therapeutic agents towards the specific host cells.
Regulation of ferroptosis in KRas mutant cancer cells
Authors: Choi Byeong Hyeok , Colon Tania , Jiang Jessica , Lee Eunji , Dai Wei
Published: April 2020

Ferroptosis is a non‐apoptotic, iron‐dependent form of cell death driven primarily by accumulation of iron‐dependent lipid ROS due to GSH depletion or inactivation of glutathione peroxidase 4 (GPX4). GPX4 is the initiator of ferroptosis within the cancer cell. Cancer cells exhibit an increased iron demand compared with normal cells, and thus the iron‐dependency of the cancer cells can be explored to make them more susceptible to iron‐dependent ferroptosis. However, the role of ferroptosis in tumorigenesis remains unclear. KRas mutations are frequently associated with treatment resistance, partly due to defective apoptotic signaling. To date, clinical trials to develop compounds that target mutant Ras proteins have been unsuccessful. Here we showed that erastin treatment, a ferroptosis inducer, reduced cell proliferation, which was correlated with an increase level of cleaved caspase 3 and PARP in KRas mutant lung cancer cells. Interestingly, mutant KRas inhibition by chemical compounds targeting KRas G12C or G12D, coupled with erastin treatment, significantly reduced cell proliferation and promoted cleavege of caspase 3 and PARP‐1. These results suggest that KRas activity may negatively regulate erastin‐induced ferroptosis. To further investigate the regulatory mechanism that mediates erastin‐induced ferroptosis, we determined GPX4 expression after treatment with a mutant KRas inhibitor and/or erastin. We found that GPX4 expression level was not significantly changed by the mutant KRas inhibitor, suggesting that KRas may be targeting GPX4 enzyme activity rather than its expression. Taken together, these results suggest that KRas may play an important role in regulating erastin‐induced ferroptosis.
Increasing Versatility of Small Angle X‐Ray Scattering
Authors: Chakravarthy Srinivas , Hopkins Jesse , Bilsel Osman , Irving Thomas
Published: April 2020

Small Angle X‐ray Scattering (SAXS), a technique initially popular in the bio‐medical community largely due to the relative logistical ease compared to other structural biological techniques has in recent years emerged as a much more versatile biophysical tool that gives unprecedented access to a real time view of structural alterations occurring during critical processes such as protein and nucleic acid folding, enzyme‐substrate binding and other ligand induced conformational changes in biological macromolecules. Developmental efforts spanning the last decade at the Biophysics Collaborative Access Team (BioCAT) located at The Advanced Photon Source (APS), Chicago, have resulted in a time‐resolved SAXS instrument which combines a state‐of‐the‐art synchrotron beamline capable of delivering up to 1013 photons/s with microfluidic mixers that afford access to time regimes ranging from 10s of microseconds to 1.5 seconds. After being made available to the general SAXS user community the time‐resolved SAXS setup has been used to study several interesting systems of biomedical interest, which in turn have led to unprecedented insights. For instance, key aspects of protein‐folding were elucidated using the chaotic flow mixer which gives access to a time regime from ~80μs to ~95ms and mechanistic details of the Insulin Degrading Enzyme (IDE) were revealed using the laminar flow mixer which allows measurement of time‐points from ~5ms to 1.5s. Improved operational efficiency and minimal sample consumption have made these heretofore challenging experiments feasible for a wider user community.

Furthermore, the equilibrium SAXS instrument, which already had an integrated liquid chromatography setup to ensure optimal sample quality, now also has in‐line MALS and DLS detectors making BioCAT a unique facility for comprehensive biophysical characterization of biological macromolecules. Software tools developed in‐house have ensured a high degree of user‐friendliness and we hope to introduce this powerful biophysical tool to a larger community of non‐expert users.

Support or Funding Information

NIGMS ‐ P41 GM103622
Synergistic Correlation between Dietary Patterns, Lifestyle and Hypertension among Medical School Population
Authors: Mhamankar Suyog , Kotak Vikas , Yadav Prashant , Pandey Rajeev , Murthy Guru
Published: April 2020

Hypertension (High blood pressure) is responsible for 7.6 million deaths per annum worldwide (13.5% of the total), more than any other risk factors. Around 54% of stroke and 47% of coronary heart disease are attributable to high BP. The present study was conducted to find out how dietary patterns and lifestyle can affect or cause hypertension and its prevalence among the population on medical school campus. The cross‐sectional study involved a survey of 90 respondents aged 16 to 57 years using the random sampling technique. The results were based majorly on two factors i.e. diet and lifestyle. Considering the analysis on these factors, only 4.5% participants had high cholesterol intake, whereas 16.6% participants take heavy amounts of sugars,3.3% participants had high caffeine intake and 32% of population had high salt intake. Prevalence of smoking was 11.1% and heavy alcohol consumption was 4.07%. Contradictory to the expected results, only 6.7% population was found to be diagnosed by hypertension while 40–45% of people among the selected population were at risk of developing hypertension. 30.7% population did not have any exercise habits which is a risk factor leading to hypertension.

Support or Funding Information

Community Health and Research of Spartan (CHARCOS), Spartan Health Science University School of Medicine, St. Lucia, West Indies