bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exaggerated Store‐Operated Calcium Entry: A Mechanism for High Glucose‐induced Podocyte Injury and Mitochondria Damage
Authors: Tao Yu , Chaudhari Sarika , Yazdizadeh Shotorbani Parisa , Ma Rong 
Published: May 2022

Purpose and hypothesisPodocyte injury induced by hyperglycemia is the key factor contributing to proteinuria and kidney dysfunction in diabetic nephropathy (DN). Accumulating evidence suggests that mitochondria dysfunction leads to DN‐associated podocyte injury. However, the mechanism of podocyte mitochondria injury by high glucose (HG) is poorly understood. Store‐operated calcium entry (SOCE) is essential for multiple cell functions in both excitable and non‐excitable cells. However, the role of this Ca2+ signaling in podocytopathy remains unknown. The present study was carried out to test the hypothesis that enhanced SOCE mediated HG‐induced podocyte injury and mitochondria damage.MethodsAll experiments were carried out using cultured immortalized human podocytes. The conventional whole‐cell patch‐clamp was performed to measure Orai1 channel currents. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and STIM1. Calcium imaging was used to evaluate SOCE. Confocal microscopy was used to visualize podocyte actin arrangement. Mitochondrial function was evaluated by measuring: 1) the mitochondria membrane potential (MMP) using TMRE fluorescence; 2) generation of mitochondria reactive oxygen species (ROS) using MitoSox Red Mitochondrial Superoxide Indicator; 3) ATP production using ATP assay kit. In addition, CRISPR‐CAS 9 approach was used to delete Orai1 in podocytes.ResultsThapsigargin (TG, 1 µM), the activator of Orai1 channel induced robust inward currents which were blocked by La3+ (2 µM), an inhibitor of Orai1 channel. Ang II (1 µM) also evoked inward currents, which were significantly blunted by BTP2 (10 µM), an Orai1 channel blocker. Orai1 protein abundance significantly increased by HG (25 mM) treatment for the time periods ranging from 2 to 12 h. This HG induced Orai1 increase was dose‐dependent. Consistently, Ca2+ imaging experiment showed that HG treatment for 12 h significantly increased podocyte SOCE. Thus, the biochemical and functional data suggested an enhancement of SOCE by HG. Furthermore, HG treatment resulted in podocyte cytoskeleton rearrangement by formation of cortical F‐actin, an indication of podocyte injury. This HG response was significantly blunted by BTP2 and Orai1 CRISPR‐CAS 9 lentivirus. Moreover, HG treatment significantly decreased podocyte MMP, ATP production and increased mitochondrial ROS generation, all of which were significantly inhibited by BTP2. Furthermore, activation of SOCE by TG significantly increased mitochondrial ROS generation, which was prevented by pre‐treatment of BTP2.ConclusionEnhanced SOCE contributed to HG induced podocyte injury and mitochondrial dysfunction.
Development and validation of a novel UPLC‐MS/MS method for the simultaneous quantification of mycophenolic mofetil and its major metabolites in rat plasma and liver S9 hydrolysis assay in rats
Authors: Etim Imoh , Du Ting , Sunsong Robin , Siddiqui Nyma , Gao Song 
Published: May 2022

A sensitive liquid chromatography‐tandem mass spectrometry method was developed and validated for the simultaneous quantification of mycophenolic mofetil (MMF) and its major metabolites, mycophenolic acid (MPA), mycophenolic acyl glucuronide (AcMPAG), and mycophenolic glucuronide (MPAG), in rat plasma. Upon administration, mycophenolic mofetil (MMF), a prodrug, undergoes extensive metabolism in the liver. Following hydrolyzation, the active metabolite MPA is produced. MPA can then be further metabolized to produce its glucuronide conjugated metabolites, AcMPAG and MPAG. This method can simultaneously determine both the parent compound and its major metabolites within biological samples. In addition, liver S9 fraction hydrolysis studies of MMF results in the production of MPA, demonstrating that the parent drug is first hydrolyzed within the liver. A novel UPLC method was developed to simultaneously quantify both compounds following S9 liver enzyme hydrolysis.A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Ultra Biphenyl 5µm column (100 × 2.1mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. Additionally, to determine the hydrolysis rate, different concentrations of MMF were incubated with liver S9 fraction (0.5µg/ml final concentration) for 30 mins, after which the reaction was terminated using 6% formic acid in acetonitrile. Samples were prepared and MPA concentrations were determined in the reaction system using UPLC for detection and quantification.The method was linear in the range of 9.77nM – 5000nM with correlation coefficient values > 0.99 for all components. The method has been shown to be reproducible, with intra‐ and inter‐day accuracy and precision ±12.3% of nominal values, for all analytes. The average extraction recovery rates for MMF, MPA, AcMPAG, and MPAG were 91.3%, 97.9%, 98.4%, and 90.5%, respectively. Matrix effect was in the acceptable range (<15%). The analytes in plasma were found to be stable under bench –top, freeze‐thaw, and storage (‐4°C) conditions. The metabolic studies showed that mycophenolic mofetil can be rapidly hydrolyzed by rat liver S9 fraction with Vmax and Km values of 0.66±0.78 nmol/min/µg and 80.06±123.1µM respectively.This novel UPLC‐MS/MS method can comprehensively evaluate and quantify the concentration of both mycophenolic mofetil and its major metabolites, mycophenolic acid, mycophenolic acyl glucuronide, and mycophenolic glucuronide in biological samples. This method can be used as a tool to further investigate and understand the extensive metabolism of mycophenolic mofetil in clinical practice.
3D‐Printing Offers Dry Replication and New Insight into Unique Museum Specimens
Authors: Streicher Johannes , Reisinger Andreas , Warnung Lukas , Pahr Dieter 
Published: May 2022

Students dissection classes again and again reveal unique anatomical variations and pathologies, which are usually preserved and stored in fluid. Due to their unique nature and hazardous conservation fluids, they neither ever leave their display case again nor are handed over to students at the dissection lab.Thus the present study aims at routinely generating solid, dry, and numerically unlimited 3D‐replica of such unique specimens.We have selected a particularely severe case of Mönckebergs aortic valve sclerosis and a transcatheter aortic valve implantation (TAVI)‐treated valve to develop a standard scanning, segmentation and multicolor 3D‐printing protocol for soft and hard tissue specimens.Subsequent to anatomical dissection of the appropriate valve segment from the ventricular outlet and the ascending aorta, scanning was performed on a Skyscan 1173 micro‐CT at a x‐y‐z resolution of 30µm. Soft tissue, calcified tissue, and the TAVI's wire cage were segmented separately using the software packages Slicer and Medtool. For 3D‐printing, the Prusa MK3S filament printer and the Prusa SL 1 resin printer were used.The overall time expense for scanning (40 minutes), data processing and segmentation (about 16 hours) was around two to three working days per model. The 3D‐printing took from 5 hours (single‐color resin printing at a scale of 1:1) up to 4 days (triple‐color filament printing at a scale of 3:1).The generated display models range from real size, opaque surface models to semi‐vitreous visualizations of the calcification pattern and the implanted TAVI.Taken together, after a substantial time expenditure for initial data processing and modeling, with small time requirements not only hands on replica for teaching can be easily reproduced at different scales without limitation by specimen durability or chemical hazards. Additional impact is achieved by the ability to highlight and analyze internal structures in situ, making laborious, traditional whole mount staining and ‐clearing dispensable. In a next step, the adaption of colors and material characteristics will approximate the haptic experience to the real specimen.
COVID‐19: Implications Regarding the Properties and Behaviors of the Coronavirus Disease
Authors: Chen Mark H. , Tulp Orien L. , Einstein George P. 
Published: May 2022

The novel coronavirus SARS‐Cov‐2 or COVID‐19 became a global pandemic and currently few medically approved curative treatments exist. SARS‐Cov‐2 acts similarly to SARS‐CoV‐1 from where it may have evolved. The COVID‐19 virus can survive ~3 hours in air and < 72 hours on distinct surfaces. COVID‐19 mutates by introducing sequence errors in the host’s RNA genome or by modifying proteins and enzymes. Vaccination, including booster shots, social distancing and isolation are the most generally practiced guidelines in the global management of COVID‐19. Globally the most frequently utilized pharmacologic treatments include ivermectin, hydroxychloroquine, glucocorticoids, the anti‐viral agent remdesivir, monoclonal antibodies, and convalescent plasma in combination with an antimicrobial agent such as azithromycin to minimize secondary microbial infection and nutritional supplementation (vitamins C, D3, and zinc) to enhance cellular immune responses and are included in the routine protocols of some emergency rooms. COVID‐19 viral transmission occurs via respiratory microdroplets, by inhaling COVID‐19 laden airborne particles and contact with contaminated surfaces on which these droplets have been deposited. SARS‐CoV‐2 targets ACE2 receptors in the upper and lower respiratory tracts in addition to the heart, brain, and gastrointestinal tract, and may cause thromboses in the liver, heart, and kidney.Significant risk factors for the severity of COVID‐19 infection include advanced age and pre‐existing comorbid conditions obesity, hypertension, cardiovascular disease, diabetes, and compromised immune status, which all correlate with greater mortality. Outcome‐effecting factors include viral load, viral mutations, and pre‐existing conditions. Since its origination, genomic studies of the virus have identified numerous variants which have become regionally prevalent in different countries. Infective factors, comorbidities and viral load strongly affect outcomes; patients infected with the greatest viral load showed a higher mortality. It is opined that the number of deaths attributed to COVID‐19 may be inaccurate due to errors in diagnosing and reporting, since other similar illnesses may exhibit similar symptoms.Future research should focus on prevention practices, comorbidities, genetic prevalence, reliable systematic and consistency in country‐by‐country testing and reporting procedures, further scrutiny regarding the efficacy of current vaccines and protocols, and the pursuit for innovative therapies for Coronaviruses and variants including biophotonics and exploration of emerging bioenergetic, nutritional, pharmacological, immunotherapeutic and vaccination‐preventive applications for eradication of COVID‐19.Refs: 1. Cheng, RZ. (2020a). Med Drug Disc, 5, 100028; 2. Shankar, AH, & Prasad, AS. (1998). ACJN, 68(2), 447S‐463S; 3. Petrilli, CM, Jones, SA, et al. (2020); medRxiv; 4.van Doremalen, N, Bushmaker, T et al. (2020). NEJM, 382(16), 1564‐1567
Activation of the Soluble Guanylate Cyclase Increases 26S Proteasome Activities and Protects against Proteotoxicity in Cardiomyocytes
Authors: Cai Mingqi , Wang Xuejun 
Published: May 2022

BackgroundThe ubiquitin‐proteasome system (UPS) mediates the selective degradation of unwanted and unneeded proteins in the cell, playing crucial roles in protein quality control and diverse other cellular processes. Proteasome malfunction and proteotoxicity are implicated in the genesis of a large subset of heart diseases. Our laboratory has previously discovered that activation of the cGMP‐dependent protein kinase (PKG) primes the proteasome. Here we test the hypothesis that by increasing cGMP and PKG activity, soluble guanylate cyclase (sGC) activation activates the proteasome and thereby protects against proteotoxicity in cardiomyocytes.Methods and ResultsCompared with vehicle control (DMSO), treatment with an sGC activator (cinaciguat) in cultured neonatal rat cardiomyocytes (NRCMs) led to increases in the levels of Ser239‐phosphorylated vasodilator‐stimulated phosphoprotein (VASP) (2.17±0.07 vs. 1.0±0.20, p<0.0001), indicative of PKG activation. We then used adenoviral vectors expressing degron CL1‐fused green fluorescent proteins (GFPu, a proven UPS substrate) or red fluorescent proteins (RFP, a stable protein control) as a reporter system for UPS performance. Compared with DMSO, cinaciguat treatment reduced the GFPu/RFP ratio (0.59±0.06 vs. 1.0±0.18, p=0.0135) as revealed by western blotting, and shortened GFPu half‐life in cultured NRCMs as revealed by cycloheximide chase assays. Cinaciguat treatment decreased Thr41/Ser45‐phosphorylated β‐catenin, an endogenous substrate of the UPS (0.60±0.12 vs. 1.0±0.08, p=0.0014). These data demonstrate that sGC activation enhances UPS performance. Moreover, in‐gel proteasome activity assays followed by western blot analyses reveal that cinaciguat treatment increased the chymotrypsin like activities of 26S proteasomes (1.52±0.17 vs. 1.0±0.09, p=0.0019) without changing 26S proteasome assembly, showing that sGC activation enhances UPS performance via activating 26S proteasomes. We next used NRCMs with adenovirus‐mediated overexpression of a human‐disease‐linked misfolded protein CryABR120Gas a cell model of cardiac proteotoxicity. CryABR120G overexpression‐induced increases in LDH leakage and PKCδ cleavage (both indicative of cell injury/death) were significantly attenuated by cinaciguat treatment (LDH in media: 0.71±0.20 vs.1.44±0.26, p<0.0001); further MTT assays consistently showed cinaciguat promoted NRCMs viability (1.32±0.11 vs. 1.02±0.05, p<0.0001).ConclusionsActivation of sGC with cinaciguat increases PKG activity, activates the 26S proteasome, facilitates UPS functioning, and thereby protects against proteotoxicity in cardiomyocytes.
Cardiac Adjustments During a Very Hot and Dry Heatwave Simulation in Older Adults
Authors: Watso Joseph C. , Belval Luke N. , Foster Josh , Cimino Frank A. , Orth Bonnie D. , MacNamara James P. , Sarma Satyam , Crandall Craig G. 
Published: May 2022

Heatwaves are the leading cause of death among natural disasters in the United States and are predicted to increase in frequency, duration, and severity. During heatwaves, older adults (over 65 years old) have disproportionally greater morbidity and mortality, primarily from cardiovascular complications. However, it is unknown whether a very hot and dry heat wave simulation would impair cardiac function in older adults.PurposeWe tested the hypothesis that three hours of exposure to a very hot and dry heat wave simulation would increase left‐ventricular contractility (s’) in older adults.MethodsWe assessed internal body temperature (ingestible telemetric pill and/or rectal thermistor), mean skin temperature (thermocouples), cardiac function (echocardiography), limb blood flow (brachial artery sonography) in eight healthy older adults (5 Female/3 Male; 70 ± 4 years; 26 ± 3 kg•m‐2) while they were exposed to a simulation of the 2018 Los Angeles heatwave for three hours. We measured hemodynamics in a temperate environment (~23 ºC) before repeating these measures after three hours of heat exposure in an environmental chamber (46 ± 1 ºC, 17 ± 1 % relative humidity). For ecological validity, participants performed seven bouts of exercise (3 METS for 5 minutes) dispersed across the three‐hour‐long heat exposure to simulate activities of daily living. Additionally, participants consumed 9 mL of 15 ºC (tap‐temperature) water per kilogram of body mass dispersed throughout the trial. We compared values before (BASE) or at the start (START) of heating with values obtained at the end of heating (END) using a Wilcoxon test for propagation velocity, limb blood flow and vascular conductance, and paired, two‐tailed t‐tests for all other variables.ResultsInternal body temperature (START: 36.8 ± 0.4, END: 38.2 ± 0.5 ºC, p<0.01) and mean skin temperature (START: 35.5 ± 1.3, END: 37.3 ± 0.7 ºC, p=0.02) increased during heating. Heart rate increased (BASE: 73 ± 19, END: 99 ± 40 bpm, p=0.04) and mean arterial pressure decreased (BASE: 90 ± 14, END: 77 ± 11 mmHg, p=0.03) during heating. Combined [septal & lateral] s’ (BASE: 8.2 ± 1.4, END: 11.3 ± 1.5 cm/s, p<0.01) increased during heating, for which changes from BASE to END were moderately, but non‐significantly, related to Δ internal body temperature (R2= 0.25, p= 0.21). Indices of left ventricular diastolic function did not change during heating (p>0.24 for E, A, E/A, combined e’, E/combined e’, propagation velocity, & iso‐volumetric relaxation time). Limb blood flow and vascular conductance increased ~four‐fold during heating (p<0.02 for both).ConclusionThese preliminary data suggest that older adults increase left‐ventricular contractility to maintain stroke volume, potentially to compensate for increased limb vascular conductance, during three hours of exposure to a very hot and dry heatwave simulation.
A High throughput bioluminescent assay to monitor the activity of COVID‐19 Methyltransferases Capping Enzymes
Authors: Goueli Said A. , Hsiao Kevin A. 
Published: May 2022

The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐Cov‐2) was first reported in Wuhan, China in late 2019 resulting in infection of over 259 million and death of over 5.17 world‐wide as of November 2021. In the USA, the number of diagnosed individuals reached 48 million with 92,000 Death cases. Despite the lower pathogenicity compared to SARS‐CoV and MERS‐CoV, the high infection rate of SARS‐CoV‐2 results in large numbers of hospitalizations and deaths worldwide.The SARS‐Cov‐2 is an enveloped virus and belongs to the beta coronavirus family containing a large and complex positive–sense single stranded RNA genome (~30kb). It binds to human ACE2 receptor with high affinity via its spike protein (S) and membrane fusion leading to the release of its genomic RNA that replicates and produce higher number of sub‐genomic RNAs. The viral RNA is then capped by nsp 14 and nsp10/16, two nonstructural proteins that catalyze the methylation of RNA to limit its degradation by cellular 5’‐3’ exonuclease and evade host immune system. These enzymes play critical role in translation of the viral RNA and its replication and thus leading to viral survival.We have used a bioluminescent high throughput enzyme assay for monitoring the activity of these enzymes in order to develop novel inhibitors which can be advanced for therapeutic treatment. We show that we can determine the activity of nanogram amounts of these enzymes and test the activity of these enzymes using different inhibitors, some of which are competitive with S‐adenosyl methionine the canonical substrate for these enzymes. We also show the effect of inhibitors that compete with the oligonucleotide substrate. Finally, we tested the effect of a peptide modulator of COVID‐2 on the activity of both nsp 10/16 and nsp 14. The assay is simple, homogenous, fast, and bioluminescent which provides a very robust platform for testing novel compounds on the viral methyltransferases leading to the generation of new drugs towards inhibiting the replication of COVID‐19.
Integrative therapies for amyotrophic lateral sclerosis disease using dynamic physiological systems
Authors: Nnake Ijeoma , Tulp Orien L. , Einstein George P. 
Published: May 2022

BackgroundAmyotrophic Lateral Sclerosis (ALS) is a fatal motor neuron condition that is characterized by progressive loss of upper and lower motor neurons at the spinal or bulbar level with an incidence of about 1/100,000. ALS is categorized in two forms. The most common form is sporadic (90–95% of cases) which has no obvious genetically inherited component. The familial type of ALS (FAL5, represents ~10% of cases) is due to associated genetic dominant inheritance factors. The first onset of symptoms of ALS usually occurs between the ages of 50 and 65. The pathogenesis of ALS is unknown. There are two approved medications for ALS treatment: riluzole and edaravone, both of which have many reported side effects. ALS patients and their family members seek an effective therapy for treating the symptoms and/or delaying the progression of ALS.MethodA comprehensive literature search consisting of 12 peer reviewed studies was conducted using PubMed, Medline and Nature review from inception to 2019, and meta‐analysis used to evaluate the findings. Amyotrophic Lateral Sclerosis diagnosis was determined using El Escorial criteria. Exposures to agriculture chemicals, heavy metals, electrical magnetic fields, types of diet, and physical activity were all examined for association with ALS. Also, use of integrative therapies (IT) were evaluated among the treatment options reviewed.ResultsThe results of the study did not indicate that factors of diet, smoking or cardiovascular disease increase the risk of developing ALS. Vitamins and Chinese herbal decoctions, Chinese herbal compounds, massage therapy, and acupuncture were the 5 most commonly used IT reported. In the meta‐analysis reviewed, the most common reason for using IT was to treat weakness and fatigue in males (68.72% of males were IT users) and muscle atrophy in females (66.35% of females were IT users). Most of the effects of the IT therapies reported were improvements in subjective symptoms of the disorder.ConclusionThis study could not identify smoking, physical activity, environmental exposure, occupational factors, or dietary habits as risk factors or protective factors for the onset or progression of ALS. ALS has multiple pathological mechanisms. Since integrative therapy contains various components which display multiple effects, it would be helpful in alleviating the pathological events affecting patients with ALS, thereby increasing the quality of life for the individuals and families of patients. However, more research must be performed regarding the efficacy and safety of integrative therapy or complementary and alternative therapy and clinical studies will be required to demonstrate the complimentary beneficial effects of integrative therapies in the management and treatment of ALS.