bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of eNOS in Endothelial Cells Promotes Proliferation
Authors: Bu Shuhan , Nguyen Hien C. , Nikfarjam Sepideh , Michels David C. , Rasheed Berk , Ramesh Sauraish , Patel Darshil , Patel Margi , Singh Shweta , Frisbee Jefferson , Singh Krishna
Published: May 2022

Background

Endothelial cells help maintain vascular homeostasis mainly by balancing interplay between vasorelaxation and vasoconstriction via regulating availability of Nitric Oxide (NO). Endothelial nitric oxide synthase (eNOS) is one of three NOS isoforms that catalyses the synthesis of NO to regulate endothelial function. To date, however, eNOS’s role in endothelial cell proliferation, remains unclear.

Methods and Results

To gain a better understanding about eNOS and cell proliferation, we genetically inhibited eNOS viasilencing by transfecting cultured human umbilical vein endothelial cells with sieNOS or scrambled control. Successful eNOS silencing was confirmed at transcript and protein levels, and then endothelial cell proliferation was evaluated. Surprisingly, loss of eNOS significantly induced endothelial cell proliferation, concomitant with significant downregulation of both cell cycle inhibitor p21 and cell proliferation antigen Ki‐67. To confirm the specificity of eNOS silencer, we used another sieNOS molecule, which also showed the similar results as endothelial proliferation was significantly induced in eNOS‐silenced endothelial cells. Later, to demonstrate that the induced endothelial cell proliferation is due to eNOS inhibition, we pharmacologically inhibited eNOS by treating endothelial cells with L‐NAME. Contrastingly, L‐NAME‐treatment significantly inhibited endothelial cell proliferation in a dose‐dependent manner.

Conclusion

Our findings, for the first time, indicate that eNOS regulate endothelial function by directly controlling endothelial cell proliferation. Observed inhibition of endothelial cell proliferation by L‐NAME, may be due to its non‐specific effects. The findings also indicate that eNOS and L‐NAME might employ different pathways regulating endothelial cell proliferation warranting further investigation.
Deep Learning‐Based Identification of Microglial Cellular Populations in Glioblastoma Patients
Authors: Wang Wesley , Tugaoen Jonah Domingo C. , Otero José J.
Published: May 2022

Glioblastomas (GBMs) are primary brain malignancies with poor overall survival; however, disease sequelae following primary treatment is highly variable in nature—leading to variation in therapy response and overall survival. Recent discoveries characterizing the underlying GBM microenvironment evidence dynamic responses in both residential and recruited immune cell populations which may in fact drive these clinical observations. To validate these finding and evaluate the potential role of immune cells in differential survival, we analyzed recurrent GBM RNAseq data from the Chinese Glioma Genome Atlas and subsequently performed unsupervised clustering through k‐means to define 3 sub‐clusters of recurrent GBM with differential overall survival (p = 0.019). Subsequently, to evaluate the role of immune cells in this paradigm, we applied Weighted Gene Correlation Network analysis to our findings which indicate modulation of immune response as a key driver of this differential clinical pattern. Translating these findings to actionable clinical utilization in neuropathology is however challenged by poor signal‐to‐noise ratios during immuno‐staining and complex arborization of microglial processes. We thus present a convolutional neural network workflow using a UNET framework in python which can mitigate these challenges and successfully annotate diagnostic pathology slides from real glioblastoma patients treated at Ohio State with 80% accuracy—with attribution of lowered accuracy due to annotation error. Using this approach, we plan to subsequently annotate additional histology slides and utilize the morphologic variation within our segmented microglial population to discern post‐surgical response patterns in glioblastoma patients.
A Non‐Invasive Method to Estimate Pulmonary Oxygen Transfer Rate
Authors: Shukla Pooja Rajesh , Limberg Jacqueline K. , Jacob Dain W. , Tan Jinglu
Published: May 2022

Objective

Ineffective transfer of oxygen from the atmospheric air to the circulation is associated with low cardiorespiratory fitness and increased disease risk. Unfortunately, measuring the efficiency of gas exchange requires invasive and expensive procedures. We first sought to develop a parameter (Do) that reflects the capacity to move oxygen from air to the arterial circulation using non‐invasive methods. We then tested the utility of Do to detect changes in pulmonary oxygen transfer rate under differing conditions of oxygen supply and demand. Last, we compared Do with the Oxygen Deficit (OD) method, a non‐invasive way to estimate gas‐exchange impairment.

Methods

Heart rate (ECG), tidal volume (pneumotachometer), respiratory rate (pneumobelt), and oxygen saturation (finger pulse oximeter) were measured in 25 young healthy adult participants (13 males, 12 females; 25 ± 4 years of age). Participants completed 4 trials: 1) Resting normoxia, 2) Steady‐state hypoxia (80% SpO2, SSH), 3) Cold pressor test (CPT, to increase oxygen demand), and 4) SSH + CPT. Do was derived by engineering analysis considering the biophysics of the 3 major process essential for effective oxygen transfer (i.e., ventilation, alveolar gas exchange, hemoglobin binding). Do was estimated for each condition and results were compared with the OD method.

Results

Doincreased during SSH (5.13±1.07×10‐2L/min/kPa) and CPT (2.91±1.03 ×10‐2L/min/kPa) when compared to resting normoxia (2.24±0.66×10‐2 L/min/kPa, p<0.05). Dowas further increased during SSH+CPT (5.71±1.52×10‐2L/min/kPa) when compared to SSH alone (p<0.05). Applicability of the OD method was limited to SpO2<95%; thus OD could only be calculated in 40% of participants (n=10). OD was lower during SSH (2.18±0.63 kPa) and CPT (2.48±1.56 kPa) versus normoxia (3.15±1.3 kPa, p<0.05). In contrast, OD increased during SSH+CPT (2.42±0.59 kPa) when compared to SSH alone (p<0.05).

Conclusion

Application of the novel parameter, Donot only showed the ability to respond to changes in supply and demand of oxygen but exhibited advantages over a conventional measure (OD). Specifically, Do showed consistent effect of CPT under normoxic and hypoxic conditions, whereas: 1) OD could only be determined in 40% of study participants, and 2) OD exhibited a paradoxical increase during SSH+CPT, despite a decrease during SSH and/or CPT alone. Future studies seek to test the utility of Do in cardiopulmonary patients.
Exploring the Combined Effect of High‐Fat High‐Sugar Diets and Volumetric Muscle Injury on Skeletal Muscle Metabolic and Contractile Function in Mice
Authors: Heo Jun‐Won , Schifino Albi , McFaline‐Figueroa Jennifer , Greising Sarah , Call Jarrod
Published: May 2022

INTRODUCTION

Volumetric muscle loss (VML) injuries due to trauma or surgery cannot fully recover muscle mass and function, resulting in long‐term disability. Western diets, i.e., high‐fat, high‐sugar (HFHS) diets, promote metabolic stress and are associated with impaired skeletal muscle contractile function. The objective of this study was to investigate the extent to which a HFHS diet affected the pathophysiology following a VML injury as diet and disability may influence the effectiveness of rehabilitation. To determine the combined effects of HFHS diet and VML injury on skeletal muscle, metabolic and muscle contractile function was assessed. We hypothesized that a HFHS diet would worsen skeletal muscle metabolic and contractile function after VML injury.

METHODS

Male C57BL/6 mice (N=23) were randomly divided into four groups: Normal Chow (NC), High‐Fat High‐Sugar (HFHS), Volumetric Muscle Loss (VML), Volumetric Muscle Loss plus High‐Fat High‐Sugar (VML+HFHS). At age 12 weeks, VML animals underwent unilateral VML surgery to the ankle plantarflexors (gastrocnemius, plantaris, soleus muscles). HFHS groups received 60% high‐fat diet and 11% high‐fructose corn syrup for 8 weeks ad libitum. Oxygen respiration and electron conductance of mitochondria within permeabilized gastrocnemius muscle fibers and peak‐isometric torque of the ankle plantarflexors were measured to assess skeletal muscle metabolic and contractile function, respectively. Data were analyzed using a two‐way ANOVA to detect significant interactions between diet and injury, or in the absence of a significant interaction, the main effects of diet or injury.

RESULTS

Independent of injury, mice on HFHS diet had 51% greater body mass compared to mice on NC (p<0.001). Additionally, mice on HFHS diet had less endurance capacity on a treadmill test compared to NC, independent of injury (77% less distance covered, p<0.001). Peak‐isometric torque was 6% less in HFHS mice, independent of injury (p=0.011) and peak‐isometric torque was 20% less in VML‐injured mice, independent of diet (p<0.01). There was a significant interaction between diet and injury for permeabilized muscle fiber oxygen respiration (p=0.016) where NC mice had greater oxygen respiration compared to HFHS, VML, and VML+HFMS mice. Electron conductance was 32% less in mice on HFHS diet, independent of injury (p<0.001) and 20% less in VML‐injured mice, independent of diet (p<0.001).

CONCLUSION

The current study indicates that both HFHS diet and VML injury have detrimental consequences on skeletal muscle contractile and metabolic function. Ongoing and future research is needed to determine the extent to which HFHS diets influence skeletal muscle plasticity and if diet impacts the effectiveness of rehabilitation strategies for VML‐injured skeletal muscle.
Anatomical Study of the Innervation of the Tibialis Anterior Muscle and its Correlation with Miofascial Trigger Points: Preliminary Results
Authors: Akamatsu Flávia E. , de Alencar Cesar R. , Hojaij Flávio , Andrade Mauro , Jacomo Alfredo L.
Published: May 2022

Background

Myofascial syndrome is a common cause of chronic musculoskeletal pain. It is associated with movement restriction and is characterized by myofascial pain syndrome (MPS). Myofascial trigger points (MTPs) represent tender areas of the muscle where painful symptoms are elicited whenever stimulated. Although pathophysiology of MTPs is unclear, some authors report that they coincide with motor plates at the innervation zone (IZ). Trigger points of the tibialis anterior muscle are located at the anterior third of the muscle.

Objective

Our study aimed to describe the innervation of the tibialis anterior muscle and relate it to clinically described MTPs.

Methods

Tibial anterior muscles from five cadavers were dissected from their origins to observe the exact point where the deep fibular nerve penetrates the muscle belly. The entry points were mapped using a Cartesian graph. Statistical analysis was performed with Poisson marginal distribution and identity linkage function followed by Bonferroni multiple comparisons. Significance was set in p <0.05.

Results

Areas I and II had most of the deep fibular nerve branches, with a mean of 5 sites (42%) and 4 sites (33%) respectively. Area 3 and 4 had a mean of 2 (17%) and one (8%) respectively.

Conclusion

In accordance with the clinical literature, the branches of the deep fibular nerve in the tibialis anterior muscle seem to correspond to the described areas of the MTPs. Anatomical correlations between MTPs may be a useful tool for a better understanding of the physiopathology of these disorders and may provide a rational basis for their treatment.
Administration of Estradiol in Young Adulthood in the Male Sprague Dawley Rat to Mimic Female Physiological Levels is Associated with a Reduction in Body Weight, Lean Mass and Testicular Size
Authors: Alexander Barbara T. , Rawls Adam Z. , Cockrell Kathy , Davenport Kacey , Ojeda Norma B. , Reckelhoff Jane F. , Yanes Cardozo Licy L. , Shawky Noha M.
Published: May 2022

Transgender females are biological males at birth but identify as female by gender. The prevalence of the transgender population is increasing, yet the burden of increased cardiovascular (CV) risk is unclear. Sex is an independent predictor of CV risk but the role of sex hormones in mediating CV disease in the transgender population is unknown. Gender affirming therapy (GAT) in transfemales (male to female) involves administration of estradiol with a goal to achieve the female physiological range of estrogen. The aim of this study was to optimize estradiol levels in a rat model of GAT. Male Sprague Dawley (SD) rats were implanted with silastic capsules to deliver 17‐beta estradiol benzoate (E2) at doses of 2.5 (Low), 5 (Medium) and 7.5 (High) mg/day versus empty pellet (EP) starting at 13 weeks of age (n=4/group). At 16 weeks of age, E2 was determined by ELISA, lean and fat mass were measured via EchoMRI, and creatinine, BUN, lipids and liver enzymes were quantitated by Chemistry Analyzer. The different doses of E2 were compared to EP using unpaired t test (GraphPad) with p<0.05 considered significant. All doses of E2 caused significant increases in circulating estradiol versus EP (Table, p<0.001) that were equivalent to estradiol levels in age‐matched female SD rats as previously reported by our laboratory (PMID: 17724277). Food intake, body weight, and lean mass, were significantly reduced after 3 weeks of E2 compared to EP (Table, p<0.001). Administration of E2 was also associated with a significant reduction in testicular weight versus EP (Table, p<0.001). To conclude, optimization of E2 levels in the male rat is critical to the development of a rodent model that mimics GAT in male to female transgender. The use of a transfemale rodent model will allow study of the pathophysiology of increased CV risk associated with GAT across the lifespan.
Anatomical Study of the Innervation of the Fibularis Longus (FL) Muscle and its Correlation with Miofascial Trigger Points: Preliminary Results
Authors: Park Lucas Y. , Akamatsu Flávia E. , Hojaij Flávio , Andrade Mauro , Jacomo Alfredo L.
Published: May 2022

Background

Myofascial syndrome is a common cause of chronic musculoskeletal pain and results in dysfunction of the affected muscles. It is characterized by myofascial trigger points (MTPs) which are clinically identified by palpation of a muscle or fascial taut band. Previous publications have found MTPs to coincide with neuromuscular junctions at the innervation zone (IZ). The fibularis longus syndrome is one of the most frequent myofascial syndrome of the lower limbs and occurs due to a wide variety of etiologies, most commonly, overuse and ankle sprain. Referred pain and tenderness caused by the trigger points of the FL muscle are located above, behind and below the lateral malleolus and may extend along the lateral aspect of the foot and leg.

Objective

Our study aimed to correlate the innervation of the FL muscle to clinically described MTPs.

Methods

FL muscles from five cadavers were dissected to observe the exact point where nerve fibers penetrated the muscle belly. As muscle size varies among individuals, we describe the relative entry point of the nerve into the muscle by defining four different areas in the muscle belly: two superior (I, II) and two inferior (III and IV). Statistical analysis of anatomical data was obtained by Poisson distribution and logarithm link function followed by Bonferroni multiple comparisons.

Results

We found a mean of 12 branches of the superficial fibular nerve to the FL muscle. All of them were located into the areas I (67%) and II (33%). No branches were observed in areas III and IV. Our findings corresponded to previously described MTPs.

Conclusion

Anatomical correlation between MTPs may be a useful tool for a better understanding of the physiopathology of these disorders and may provide a rational basis for their treatment.
What does the Historical Record Say about the Chances of Surviving an Escape from a Disabled Submarine?
Authors: Fothergill David M. , Whybourn Leslie A.
Published: May 2022

Introduction

Despite US Navy and international Disabled Submarine (DISSUB) rescue capabilities, escape remains an important means of DISSUB crew survival as the crew may be forced to conduct an escape before rescue assets arrive on site due to adverse conditions onboard or operational or engineering constraints that may render rescue impossible. The main aims of the current study were to conduct an analysis of historical DISSUB records to describe the likely biomedical evolution of crew survivability following the disabling of a submarine in shallow waters, in order to develop a casualty estimate and define the main risks to escapee survival.

Methods

From unclassified historical and scientific publications, we collated and categorized international DISSUB events in which escape was effected or attempted by any part of the crew, dating from the earliest documented DISSUB escape in 1851 until September 2019. Both non‐combat and combat sinkings were included. We also reviewed at‐sea submarine escape trial experience and medical incidents from US Navy pressurized submarine escape training records to estimate air embolism and extra‐alveolar air casualties and fatalities during escape training. Our analysis identified the casualty risk for DISSUB survivors for the following three phases: (1) survival in the submarine before escape, (2) survival during the escape ascent, and (3) survival on the surface.

Results & Conclusions

Using historical data from a total of 437 attempted DISSUB escapes that included 37 accidental and combat submarine sinkings, the following observations were made:

The largest number of casualties occurred during the onboard survival phase due to atmospheric toxicity.

72% of the submariners who attempted an escape survived the escape.

Escapes have been successful from a DISSUB as deep as 240 feet sea water (fsw) and experimentally during at sea submarine escape trials from depths down to 600 fsw.

All historical escapes were effected within the current 72‐hour time to first rescue timeframe, with the majority occurring within 4 hours of DISSUB onset.

The majority of escapees who survived were recovered from the water within 60 min.

Early rescue surface response mobilization and detailed crew knowledge of escape equipment, procedures, and the physiological phenomena encountered under increased and changing pressures are essential to enhance escapee survivability.

US Navy submarine escape training records indicated very low risk (<0.5%) of pulmonary over inflation syndrome or arterial gas embolism and no fatalities during the past 11 years of pressurized escape training.
BPA and BPS increase oxidative stress inducing a decrease in physical fitness and dyslipidemia in young adults
Authors: dos Santos Julia M. , Jones Jessica N. , Sine Tyler , Senador Daniela , Metcalf Amanda A. , Haney Andrea C.
Published: May 2022

Objectives

Bisphenol‐A and ‐S (BPA and BPS, respectively) exposure occurs primarily due to leaching of these chemicals from the packaging of industrialized food. BPA and BPS intake is indicative of unhealthy lifestyle choices. Levels of BPA and BPS in human specimens have been closely associated with dyslipidemia and insulin resistance. Physical exercise has been shown to attenuate insulin resistance and the development of type 2 diabetes (T2D). The aim of this study was to investigate the relationship between lipid profiles, levels of BPA, BPS, oxidative stress, and their influence on glucose uptake induced by insulin and aerobic exercise.

Methods

Healthy young adults (age 23.5±2 years old) participated in 2 separate testing days. In both days, subject’s blood glucose (BG) was measured before 50g of maltodextrin consumption and following 30‐, 60‐, and 75‐minutes. On the “Exercise” testing day, subjects ran for 30‐minutes, while on the “Rest” day, they remained seated. Urinary BPA, BPS, and oxidative stress (OS), lipid profile, as well as body fat were also assessed. Distance ran was used to assess physical fitness.

Results

The exercise condition produced a decrease in BG during the 30‐60‐minute protocol interval when compared to the resting day. The decrease of BG in the resting and exercise days were correlated with body fat (r=0.5 and 0.51, respectively). Levels of BPA and OS were negatively associated with decreased physical fitness (r=‐0.45 and ‐0.5, respectively), while BPS and OS were associated with increased triglycerides (TG) levels (r=0.5 for both). Levels of OS were positively associated with levels of BPA and BPS (r=0.62 and 0.82), respectively.

Discussion

Concentration of BPA and BPS in urine may not directly affect glucose transport in healthy young adults. BPA and BPS might induce increase in OS and further TG affecting physical fitness and potentially mediating changes linked to the development of T2D.
Molecular Basis for Membrane Binding and Lipolysis Activation by ABHD5
Authors: Wu Lingshuang , Sokolova Vladyslava , Tan Dongyan , Airola Michael
Published: May 2022

Triglycerides are the main reservoir for long‐term energy storage in humans and are stored intracellularly in lipid droplets. Abnormal triglyceride metabolism is a key factor leading to obesity, diabetes, fatty liver disease and cardiovascular disease. When energy is needed, triglycerides are broken down in a metabolic pathway called lipolysis. Intracellular lipolysis is initiated by adipose triglyceride lipase (ATGL), which hydrolyzes triacylglycerol (TAG) to make diacylglycerol and a free fatty acid. Alpha‐beta hydrolase domain‐containing 5 (ABHD5) is a highly conserved regulator of ATGL‐mediated lipolysis that increases ATGL activity and is necessary to maintain lipid homeostasis. Consistently, mutations in ABHD5 cause Chanarin‐Dorfman syndrome, which leads to excessive accumulation of triglycerides. Despite over two decades of studies using biochemical approaches to mouse models, the structure of ABHD5 and molecular mechanisms of regulation remained uncharacterized. To understand the mechanism, we purified full‐length human ABHD5, visualized its self‐assembled, multiple oligomeric state in the absence of detergents by cryo‐EM, and generated preliminary crystals of detergent‐solubilized, monomeric ABHD5. We found ABHD5 is phosphorylated in vitro by PKA and recruited to membranes by specific lipids. We also purified the full‐length human ATGL. To further investigate the structural and regulation mechanisms of ABHD5 and ATGL, we will utilize hydrogen deuterium exchange mass spectrometry to map ATGL and ABHD5 protein‐protein and protein‐membrane interactions, biochemical experiments to study their co‐activation mechanism and membrane interaction. This study will provide a deeper understanding for the clinically relevant mutations, membrane recruitment and lipolysis activation of these key players in lipid metabolism.
Quantitative Analysis of Protein Bioconjugation in Cells with an Expanded Genetic Code
Authors: Shade Olivia , Ryan Amy , Deiters Alexander
Published: May 2022

Protein bioconjugation – specific covalent bond formation between a protein of interest (POI) and a label – has a major impact on protein imaging, protein function studies, and drug discovery. Ideal protein bioconjugation should be fast, quantitative, one step with no off‐target binding, and have minimal impact on protein function, no matter the complexity of the biological environment. The impressive selectivity and kinetics of bioorthogonal reactions, specifically the catalyst‐free inverse‐electron demand Diels‐Alder (IEDDA) cycloaddition of tetrazines and strained alkene dienophiles, make IEDDA‐reactive unnatural amino acids an exceptional tool for protein bioconjugation via genetic code expansion. Analyses of these bioconjugation reactions in live cells has been mostly limited to fluorophore labeling, and thus is rarely quantitative. Self‐labeling enzymes, like HaloTag, have been used for protein isolation and purification, studies of protein‐protein interactions, and in vivo and in vitro imaging. The excellent permeability and low reactivity of the chloroalkane substrate of HaloTag yields bioconjugation detection independent of ligand permeability. The objective of this research is to utilize the HaloTag technology in combination with genetic code expansion to develop a generalized method for quantification of intracellular bioconjugation to any POI at defined sites. We hypothesize that bioconjugation efficiency could be assessed through site‐specific installation of a reactive handle on a POI, followed by ligand‐mediated protein labeling via ternary complex formation after cell lysis and exposure to HaloTag. Here we describe the optimization of concentrations, reaction times, and reaction partners for bioconjugation and pulldown for POIs expressing different IEDDA reactive surface residues. Additionally, we describe the attempted application of this method for labeling protein targets, such as kinases and nuclear receptors. With this new methodology, we have demonstrated quantitative assessment of protein bioconjugation reactions in live cells.
A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D‐dopachrome tautomerase
Authors: Chen Emily , Shah Dilip , Bhandari Vineet , Lisi George P.
Published: May 2022

The human macrophage migration inhibitory factor (MIF) superfamily consists of MIF and D‐dopachrome tautomerase (also called MIF‐2). Both MIF proteins have critical innate and adaptive immunomodulatory functions in regulating differentiation, expression of pro‐inflammatory cytokines, and activation and recruitment of immune cells. They are broadly and constitutively expressed, but elevated levels of MIF family proteins are upregulated in various inflammatory diseases such as asthma, acute respiratory distress syndrome (ARDS) and arthritis, and antibody neutralization of MIF decreased inflammatory symptoms in murine models. While their potential as drug targets has led to the creation of a few commercial inhibitors of MIF, the efficacy of targeting MIF proteins as a therapeutic is currently obstructed by a lack of molecular level detail about their structures and dynamics and how those properties impact disease‐state activity.

Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. The MIF homolog DDT (MIF‐2) has low sequence identity with MIF (35%) though they overlap in multiple functions, including activation of the CD74 receptor, and share a nearly identical tertiary structure. This prompted the question of whether this dynamic regulatory network is conserved within the superfamily. We identify a comparable allosteric network in MIF‐2, connecting the catalytic site to CD74‐activating residues, showing with solution nuclear magnetic resonance (NMR) spectroscopy that dynamic communication is preserved in MIF‐2 despite differences in primary sequence. Disruption of the allosteric relay by mutagenesis also attenuates MIF‐2 enzymatic activity in vitro and biological CD74 signaling in vivo, highlighting amino acids in this pathway as trigger residues for potential functional control of the protein.
Inhibition or deletion of Adenosine A2Areceptor enhances acetylcholine‐induced vascular response: role of angiotensin‐II in A2AAR‐/‐ vs. C57Bl/6 mice
Authors: Agba Stephanie O. , Hanif Ahmad , Ledent Catherine , Stephen Tilley L. , Nayeem Mohammed A.
Published: May 2022

In previous studies, we showed that adenosine‐induced vascular relaxation was reduced in adenosine A2A receptor (A2AAR)‐null (A2AAR‐/‐) or A2AAR‐inhibited C57Bl/6 mice. However, it is unknown the acetylcholine‐induced vascular response in A2AAR‐/‐or A2AAR‐inhibited C57Bl/6 mice; therefore, we hypothesized that the acetylcholine enhances endothelial‐dependent vascular relaxation in A2AAR‐gene deleted (A2AAR‐/‐) or inhibited C57Bl/6 mice compared to their respective controls. Acetylcholine‐induced dose dependent vascular response was tested with SCH58261 (A2AAR‐antagonist) in C57Bl/6 vs. non‐treated C57Bl/6 mice and angiotensin‐II (Ang‐II) in C57Bl/6 vs. non‐treated C57Bl/6 mice, Ang‐II treated A2AAR‐/‐ vs. non‐treated A2AAR‐/‐ mice and Ang‐II treated A2AAR‐/‐ vs. Ang‐II treated C57Bl/6 mice. In C57Bl/6 mice, SCH58261 (1µM) increased in acetylcholine‐induced dose‐dependent vascular relaxation compared to non‐treated C57Bl/6 mice. Similarly, in A2AAR‐/‐ mice, acetylcholine enhanced dose‐dependent vascular relaxation compared to C57Bl/6 mice. However, acetylcholine‐induced dose‐dependent vascular relaxation was reduced with angiotensin‐II (Ang‐II,1µM) in C57Bl/6 compared to non‐treated C57Bl/6 mice and acetylcholine‐induced dose‐dependent vascular relaxation was reduced with Ang‐II (1µM) in C57Bl/6 compared to A2AAR‐/‐ treated mice. Our data suggest that the acetylcholine dose‐dependent vascular relaxation is endothelial dependent and is enhanced in the absence or inhibition of A2AAR unlike adenosine dose‐dependent vascular relaxation in mice.
Pharmacological reactivation of RBL2/p130 could be an effective antitumoral strategy for malignant pleural mesothelioma
Authors: Costa Aurora , Forte Iris Maria , Ventura Elisa , Pentimalli Francesca , Giordano Antonio
Published: May 2022

Malignant mesothelioma (MM) is an aggressive tumor developing from the mesothelium covering the body cavities. The most common MM type affects the pleura surrounding the lungs. MM is mainly associated with asbestos exposure. At present, no curative modalities are effective against MM exist. The molecular mechanisms underlying MM development seem to depend mostly on the inactivation of tumor suppressors, among which also the dysfunction of the retinoblastoma (RB) protein pathway that is a hallmark of cancers. We previously demonstrated that RBL2/p130 is a direct AKT target and its reactivation following AKT inhibition mediates apoptosis in MM.

So, we hypothesize that concomitant inhibition of CDK and AKT activity, both converging on the reactivation of RBL2/p130 tumor suppressor role, synergize in counteracting MM.

We first assessed, through an MTS assay, the effect of three new generation CDK inhibitors (CDKi), palbociclib, ribociclib, abemaciclib, on the cell viability of MM (NCI‐H28, IST‐MES TWO, NCI‐H2452, MMB, MSTO‐211H, NCI‐H2052) and immortalized human normal mesothelial (MET‐5A) cells at 72h. Then, to verify whether CDKi exerted long‐term cell growth inhibition, we performed clonogenic assays upon treatment of all the MM All the MM and mesothelial cell lines. Finally, we also examined, by MTS, the possible synergistic effects of abemaciclib in combination with an AKT inhibitor (AKT1/2 VIII).

Our preliminary data show that ribociclib and palbociclib only moderately reduce viability of the MPM cells, whereas abemaciclib shows cytotoxic effects at very low doses in all MM cell lines. Abemaciclib treatment induces a dose‐dependent cytotoxic effect in all the MM cell lines without meaningfully affecting the normal mesothelial cells. Interestingly, abemaciclib also induced a cytotoxic effect in the most aggressive histotype, the sarcomatoid NCI‐H2052 cells. To verify whether abemaciclib exerted long‐term cell growth inhibition, we performed clonogenic assays upon treatment with IC50 of abemaciclib and found that the CDKi dramatically reduced colony formation in all the MM cell lines. We also examined, by MTS, the possible synergistic effects of abemaciclib in combination with a kinase AKT inhibitor (iAKT1/2 VIII), so we treated the MM cell lines for 72 h with the two agents, both alone and in combination, at five different concentrations, in a constant ratio. The cell viability data were evaluated through the Chou–Talalay method and showed a strong synergism revealing a combination index (CI) values < 1 for all cell.

Although reactivating oncosoppressor genes is difficult to achieve because they are hardly ‘druggable’, our preliminary findings will provide the rationale for pharmacological strategies able to unleash RBL2/p130 tumor suppressor potential. The pharmacological reactivation of RBL2/p130 could be an effective strategy against MM, which can be rapidly implemented in the clinical practice.
The Influence of Ovariectomy on Performance and Thermoregulation During Exertional Heat Stroke in Mice
Authors: Garcia Christian K. , Renteria Liliana I. , Leite‐Santos Gabriel , Valera Isela , Pindado Jose , Hickman Corrinne , Carta Jillian , Ward Jermaine R. , Ward Shauna M. , Leon Lisa R. , Laitano Orlando
Published: May 2022

Background

Exertional heat stroke (EHS) is a condition characterized by central nervous system dysfunction and multi‐organ injury in hyperthermic victims. Most studies addressing EHS pathophysiology are performed in males. This is a concern since there is an increasing participation of females in sports, active military service, and sex‐dependent susceptibilities to EHS. Preclinical data suggest that female mice outperform male mice regarding distance ran, speed, and time to collapse in a model of EHS. Importantly, differences in body mass between males and females do not explain these contrasting heat tolerances. Whether the female sex hormones could account for the superior performance in the heat remains unknown. Our goal was to determine the influence of female sex hormones on performance and thermoregulatory parameters in a preclinical model of EHS.

Methods

We performed ovariectomy (OVX, n=8) or sham (n=8) surgeries in young adult (18 weeks old) female C57/BL6 mice. Ovariectomy was performed by the bilateral removal of the ovaries. To allow for real time monitoring of core temperature during the entire experiment, all mice were implanted with telemetry emitters at the time of ovariectomy/sham surgeries. Upon recovery from surgeries and training (~6 weeks), all mice underwent the EHS protocol in an environmental chamber set at 37.5℃ and 40% relative humidity. Mice exercised on a running wheel until experiencing loss of consciousness.

Results

Ovariectomy increased body mass by 10% at the time of EHS (Sham = 25 ± 1 vs OVX = 28 ± 2 g, P<0.05), resulted in 35% shorter distance (Sham = 753 ± 189 vs. 490 ± 87 m, P<0.05), 25% shorter time to collapse (Sham = 126 ± 21 vs. OVX = 95 ± 12 min, P<0.05), and 15% lower final speed (Sham = 9 ± 1 vs. OVX = 7.5 ± 1 m/min, P < 0.05).

Conclusion

Ovariectomy negatively impacts tolerance to EHS. Ovariectomized mice not only displayed a marked decrease in performance during the EHS trial but also gained more mass than their controls. This suggests that female sex hormones are required for a greater tolerance against EHS.
A Critical Role for Pannexin 1 in Heart Failure Induced by Acute and Chronic Isoproterenol Administration
Authors: Pavelec Caitlin M. , Wolf Matthew , Yeudall Scott , Upchurch Clint , Leitinger Norbert
Published: May 2022

Pannexin 1 (Panx1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation and blood pressure regulation. Panx1 channel function is inhibited by spironolactone, a frontline therapy for heart failure patients. Despite this, the function of Panx1 in cardiomyocytes and a possible role in heart failure has not yet been studied. To investigate if Panx1 is involved in the development of cardiac dysfunction and fibrosis, we use a model of chronic adrenergic stimulation to induce heart failure in two novel mouse models. We have generated mice with constitutive deletion of Panx1 in cardiomyocytes by crossing mice expressing loxP‐flanked alleles of Panx1 to mice expressing Cre recombinase under the control of the cardiomyocyte specific MyHC6 promoter (Panx1MyHC6‐Cre). Using the same approach with mice expressing the tamoxifen‐inducible MerCreMer system under the control of the Tnnt2 promoter we generated mice with tamoxifen‐inducible deletion of Panx1 in cardiomyocytes (Panx1Tnnt2‐MerCreMer). Constitutive deletion of Panx1 in cardiomyocytes had no effect on heart function at baseline as measured by echocardiography. However, after acute isoproterenol treatment (15 mg/kg/day, i.p.), Panx1MyHC6‐Cre mice were protected from systolic and diastolic dysfunction and cardiac hypertrophy. In vitro, treatment of H9c2 cardiomyocytes with isoproterenol led to Panx1‐dependent release of ATP. Furthermore, siRNA‐mediated knock down of Panx1 in H9c2 cells attenuated basal and maximal extracellular acidification rate (a measure of glycolysis) after 24 hours of isoproterenol treatment, indicating a role for Panx1 in regulation of cardiomyocyte metabolism. Taken together, these data demonstrates that Panx1 deficiency protects against isoproterenol‐induced cardiac dysfunction and pathological glycolytic shift in stressed cardiomyocytes. Further elucidating the role of Panx1 in cardiomyocytes in regulating cardiac function and fibrosis will identify a novel therapeutic target to prevent heart failure.
Assessment of Ferrous Sulfate Contribution to Oxidative Stress in Post‐bariatric Surgery Patients with Iron Deficiency
Authors: Babayev Maksat , Silveyra Patricia , Klaunig James E. , Gletsu Miller Nana A.
Published: May 2022

Obesity is a global public health problem with numbers of affected people increasing despite the policy efforts1‐3 Bariatric surgery is the common treatment for patients with severe obesity, with increasing number of patients undergoing the surgery (252,000 in 2018 to 256,000 in 2019).4 Unfortunately, 10‐60% of patients develop iron deficiency after the bariatric surgery.5, 6 High‐dose oral iron (i.e., 150‐200 mg of non‐heme iron supplement such as FeSO47) is a recommended treatment for patients with iron deficiency anemia (IDA) in post‐bariatric surgery patients.7 Studies show that oxidative stress (OxS) is an adverse effect of high dose oral iron therapy8, therefore we hypothesized that patients undergoing supplementation with high dose FeSO4 would exhibit increases in systemic oxidative stress. We tested our hypothesis with a secondary analysis using archived plasma specimens from a randomized, single‐blind study that included an arm that underwent treatment with high dose oral FeSO4and a comparison group that underwent a novel heme iron supplement (heme‐iron‐polypeptide, HIP). Participants were women who had developed IDA after bariatric surgery and findings of the study demonstrated superiority of high dose FeSO4 for 8 weeks for improving iron status.9 At baseline, the mean ± standard deviation for age, BMI, and years since surgery of the sample was 39.2 ± 8.7 years, 31.4 ± 7.2 kg/m2, and 6.9 ± 3.1 years, respectively. We used plasma from participants without obesity or bariatric surgery as a control group. We measured malondialdehyde (MDA), an established biomarker of oxidative stress (Abcam, UK). The two‐way ANOVA (IBM SPSS 27) showed no significant difference in mean MDA levels. Mean MDA values for FeSO4, HIP, and control group were 65.4 ± 38.3, 98.70 ± 63.4, and 84.9 ± 60.7 pmol/mL, respectively (all p>0.05).

Conclusions

Our findings of no difference in change in oxidative stress between groups does not support our hypothesis that high dose oral iron increases OxS. Limitations of our study are the small sample size and future clinical studies need to determine the potential of oxidative following high dose oral iron therapy in bariatric surgery patients with IDA.
The effects of dietary nitrate supplementation in post‐menopausal women: Does timing matter?
Authors: Delgado Jocelyn M. , Gosalia Jigar , Studinski Matthew , Somani Yasina , Proctor David
Published: May 2022

There is a need for low risk, non‐estrogen therapies for cardiovascular disease prevention in post‐menopausal women, a population expected to reach 1.2 billion worldwide by 2030. Consumption of beetroot juice and other nitrate‐rich products (green leafy vegetables, supplements) is a promising non‐pharmaceutical strategy for increasing circulating nitric oxide levels and improving vascular function in post‐menopausal women. Whether time since menopause (reproductive age) influences the vasoprotective effects (arterial stiffness and endothelial function) of dietary nitrate supplementation has not been established.

PURPOSE

To determine the vascular protective mechanisms and efficacy of a nitric oxide boosting supplement (beetroot juice) in women at two post‐ menopausal stages (early‐ vs late‐post menopause).

METHODS

Early‐ (1‐6 years following their final menstrual cycle, n=5) and late‐ (n=10) postmenopausal women consumed nitrate‐rich (300 mg/70 mL) and nitrate‐depleted beet root juice acutely and for 7 days. Percent brachial artery flow‐mediated dilation (FMD) was measured at baseline, acutely (90 minutes post‐BRJ consumption), and 15‐minutes and 30‐minutes after 20‐minute upper arm ischemia‐reperfusion (IR) injury for each drink. Brachial‐ankle pulse wave velocity (baPWV) was measured pre‐ and post‐ 7‐day supplementation.

RESULTS

Due to the nature of this double‐blind trial, data presented are from the baseline (pre‐juice consumption) visit only. Significant (p<0.05) group differences were observed for baseline PWV (early = 1158+115 cm/s; late = 1469+296 cm/s) and FMD at the 30‐minute post‐IR injury timepoint (early = 8.4+4.6%; late = 5.8+2.7%). No group differences were observed in FMD responses at rest (p=0.2) or 15 minutes post‐IR injury (p=0.4).

CONCLUSION

These preliminary results suggest that time since menopause negatively influences arterial stiffness and may prolong recovery from endothelial IR injury in women.
Search and investigation of potential peptide agents of interaction between human organism and its microbiome
Authors: Arapidi Georgij P. , Urban Anatoly , Shender Victoria , Kuznetsov Alexandr , Ivanova Olga , Lopuhov Leonid , Laikov Alexander , Sharova Nina , Nikonova Margarita , Mitin Alexandr , Martinov Alexandr , Grigorieva Tatiana , Ilina Elena , Ivanov Vadim T. , Govorun Vadim M.
Published: May 2022

Symbiotic interaction between the human body and its microbiota is an important issue of modern biomedicine and personalized medicine. However, little is known on molecular mechanisms of that relationship. Bearing in mind the ubiquitous participation of peptides in biomolecular interactions and regulatory processes we attempted direct search of blood peptides originated from microbial proteins. LC‐MS/MS analysis was carried out of blood serum and plasma samples taken from 20 healthy donors on Q Exactive HF‐X Hybrid Quadrupole‐Orbitrap mass‐spectrometer. Sample preparation was carried out based on our previously developed method of peptide desorption from the surface of major blood plasma proteins followed by standard chromatographic steps. Human microbiota protein sequences were taken from NIH Human Microbiome Project.

As a result, out of 13,625 identified peptides 912 were unique fragments of microbial precursors, which is about 6.69% of the total amount of detected bloodstream peptides. In 30 cases peptide identification was confirmed by mass‐spectral study of individual synthetic samples. Absolute quantification by the mass‐spectrometric method of multiple reaction monitoring (MRM) confirmed the presence of bacterial peptides in plasma and serum in the range of approximately 0.1 nM to 1 uM, which is comparable to physiologically significant hormone concentrations in human blood in normal conditions. The abundance of microbiota peptides reaches its maximum 5h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin — the main proteases of the gastrointestinal tract.

The isolated fraction of peripheral blood mononuclear cells showed increase secretion of proinflammatory cytokines, colony stimulating factors and chemoattractants as the response to the addition of some of the identified microbiotic peptides. Such peptides — identified both in the plasma and in the serum — have properties of resistance to fibrinolysis, since the pool of peptides is preserved after passing through the digestive tract. The physicochemical properties of the identified bacterial peptides are consistent with those required for the selective permeability of mucosal barriers. Our approach to the identification of microbiota peptides in the blood serum and plasma may be useful for determining the microbiota composition of hard‐to‐reach intestinal areas, such as the small intestine, and for monitoring the permeability of the intestinal mucosal barrier.
Exploring the Neuroprotective Role of Myo/Nog Cells in a Glaucomatous Mouse
Authors: Cuff Evan
Published: May 2022

Introduction

Myo/Nog cells express MyoD, Noggin and brain‐specific angiogenesis inhibitor 1 (BAI1). They are critical for eye development and respond to injury and stress in the neonatal and adult retina. Their depletion in the embryo results in eye and retinal malformations. Myo/Nog cells increase in number and migrate to areas of injury in response to stress. Cell death in the retina increases with Myo/Nog cell depletion and decreases with their addition to the vitreous. Here we examine the effects and locations of endogenous and exogenous Myo/Nog cells in mice eyes following the induction of glaucoma.

Methods

The anterior chamber of the right eye of C57BL/6J mice was injected with magnetic microbeads and guided to the iridocorneal angle using a handheld magnet, preventing the outflow of aqueous. Intraocular pressure was measured in both eyes following microbead injection on Days 0, 4, 7, 14, D21, and 28. On D7, three of the four groups were given bilateral injections into the anterior chamber of either PBS, unsorted brain cells, or Myo/Nog cells isolated from the brain. The fourth group did not receive any follow‐up injections. Eyes were collected on D32 and labeled with an antibody to BAI1 to locate Myo/Nog cells. H&E staining was used to count living cells in the retinal ganglion layer (RGC) and measure the thickness of the nerve fiber layer and inner nuclear layer (NFL:INL).

Results

Endogenous and exogenous Myo/Nog cells increased in glaucomatous eyes around areas of stress, including the cornea, trabecular meshwork, ciliary body and retina. The highest concentration of Myo/Nog cells was present around the RGC layer and cornea. Some Myo/Nog cells had phagocytized microbeads. Glaucomatous eyes treated with Myo/Nog cells displayed a lower concentration of endogenous Myo/Nog cells when compared to PBS and mixed population treatment groups. Addition of Myo/Nog cells also decreased loss of RGC cells and increased NFL:INL thickness in glaucomatous eyes compared to the control groups.

Conclusion

Consistent with other types of ocular injury, Myo/Nog cells accumulate in areas of stress following damage from increased intraocular pressure. Addition of Myo/Nog cells was neuroprotective in the glaucomatous retina.
Structural characterization of the Long Non‐Coding RNA SChLAP1
Authors: Oh Mihyun , Somarowthu Srinivas
Published: May 2022

Long non‐coding RNAs (lncRNAs) emerged as key players in a diverse range of cellular processes and disease malignancies including cancer. The lncRNA SChLAP1(second chromosome locus associated with prostate‐1) has been shown to be overexpressed and highly associated with the aggressive nature and poor prognosis in a subset of prostate cancers. SchLAP1is recognized to function, in part, by antagonizing the tumor suppressive SWI/SNF complex. However, the details of this mechanism remain only partially understood. As the name suggests, lncRNAs are long and present exceptional structural complexity. The structural diversity of lncRNAs allows them to mediate various biological functions in collaboration with proteins and nucleic acids. In this study, we determined the secondary structure of SchLAP1utilizing chemical probing methods. Our structural model proved that SchLAP1possesses distinct structural domains that may contribute to its function. In conclusion, for the first time, we determined the secondary structural map of SChLAP1, which will serve as a guide to understand its mechanism of action at the molecular level.