bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance Training Enhances Muscle Function and Hemorheological Properties in Middle‐aged Obese Women
Authors: Seo Jisu , Jung Won‐Sang , Kim Sung‐Woo , Kim Jisu , Park Hun‐Young , Lim Kiwon
Published: May 2022

Purpose

The present study investigated the effects of resistance training for 12‐week on body composition, blood pressure, blood lipid level, cross sectional area (CSA) of muscles, isokinetic muscle function, and hemorheological properties in middle‐aged obese women.

Methods

Thirty obese women (50.79 ± 5.80 years), with no history of any injuries to the musculoskeletal system and no participating any regular exercise program, were included in the present study. All participants were randomly assigned to a control group (CON, n = 15) or an experimental group (EXP, n = 15); but two participants in the CON voluntarily withdrew because of personal reasons, and thirteen participants in the CON finally participated. The EXP underwent resistance training program composed of warm‐up, main resistance exercise (bench press, preacher bench biceps curl, barbell rowing, dumbbell shoulder press, dead lift, barbell squat, seated leg extension, and lying leg curl), and cool‐down. Main resistance exercise was consisted of three sets of 8–10 repetitions (reps) performed with 80% of 1‐RM. The training frequency was 80 min, 3 days per week for 12 weeks. The CON maintained their daily lifestyle without training. All participants underwent body composition (weight, body mass index, lean body mass, fat mass, and %body fat), blood pressure (systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure), blood lipid level (triglyceride, total cholesterol, high‐density lipoprotein cholesterol, and low‐density lipoprotein cholesterol), cross sectional area (CSA) of muscles (quadriceps CSA, hamstring CSA, and total thigh muscle CSA), isokinetic muscle function (peak torque, relative peak torque, mean power, and total work in quadriceps femoris and hamstrings), and hemorheological properties (erythrocyte deformability and erythrocyte aggregation) were measured before and after 12‐week of training.

Results

The EXP showed a significant decrease in fat mass (p = .018) via 12‐week of resistance training. Also, the EXP yielded more significant improved muscle function including peak torque (p < .001), relative peak torque (p < .001), and total work (p = .001) in quadriceps femoris and total work (p = .018) in hamstrings. Regarding hemorheological properties, the EXP showed a significant increase in erythrocyte deformability (p = .001) via 12‐week of resistance training. However, there was no significant change in blood pressure and muscle CSA parameters via 12‐weeks of resistance training.

Conclusion

The present study verified our resistance training program shows greater muscle strength and endurance, decreasing fat mass, and improving hemorheological properties such as erythrocyte deformability.
Dexamethasone ameliorates arthritis through epigenetic and transcriptional regulation of CCL17 production
Authors: Achuthan Adrian
Published: May 2022

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, which leads to poor quality of life due to the debilitating effect of inflammation. Clinical trials in rheumatoid arthritis targeting the cytokine, granulocyte macrophage‐colony stimulating factor (GM‐CSF) are showing promise although its mode of action remains largely unknown. We have previously shown that that GM‐CSF drives CCL17 production via a new interferon regulatory factor 4 (IRF4)‐dependent pathway in human monocytes and mouse macrophages, as well as in vivo. Importantly, in arthritis and pain models IRF4‐regulated CCL17 formation mediates the proinflammatory and algesic actions of GM‐CSF.

Glucocorticoids (GCs) are potent anti‐inflammatory and immunosuppressive agents broadly used in anti‐inflammatory therapy, albeit with adverse side effects associated with long‐term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action on immune cells.

We report here that GM‐CSF‐induced CCL17 expression is inhibited by dexamethasone, a synthetic GC, in human monocytes and mouse macrophages. Moreover, we provide evidence for the first time that dexamethasone suppresses GM‐CSF‐induced IRF4 expression via regulating the expression and activity of JMJD3, which demethylates trimethylated‐H3K27. Further, we demonstrate that dexamethasone suppresses the expression of JMJD3, via the recruitment of GC receptor‐alpha to the negative GC response element in the upstream region of JMJD3 gene. Significantly, we measured elevated levels of CCL17 in synovial fluid from patients with RA compared to healthy controls. Using synovial fluid mononuclear cells from RA patients, we provide molecular evidence for the anti‐inflammatory actions of GCs through epigenetic regulation of IRF4 expression and downstream inhibition of CCL17 production. The delineated pathway potentially provides new therapeutic options for the treatment of inflammatory diseases and their associated pain.
Bioengineered miR‐34a targets MPV17L2 to control cancer cell mitochondrial functions
Authors: Tu Meijuan , Yi Wanrong , Yu Ai‐Xi , Lin Jun , Yu Ai‐Ming
Published: May 2022

MicroRNAs (miRNA or miR) are small noncoding RNAs derived from the genome to control posttranscriptional gene expression critical for various cellular processes. Recently we have invented a novel platform technology that offers high‐yield production of humanized, bioengineered miRNA agents (hBERAs) for research and development. This study is aimed to produce and utilize a novel biologic miR‐34a‐5p (or miR‐34a) molecule, namely hBERA/miR‐34a, to delineate the role of miR‐34a‐5p in the regulation of mitochondrial functions in carcinoma cells. Bioengineered hBERA/miR‐34a was selectively processed to target miR‐34a‐5p in human osteosarcoma and lung cancer cells. The mitochondrial inner membrane protein MPV17 like 2 (MPV17L2) was identified and validated as a new direct target for miR‐34a‐5p. Furthermore, we demonstrated that miR‐34a‐5p‐controlled MPV17L2 protein downregulation significantly inhibited respiratory chain complex I assembly and activities as well as intracellular ATP levels. Consequently, biologic miR‐34a‐5p treatment sharply reduced cancer cell mitochondrial respiration, increased oxidative stress, and elevated apoptotic cell death. These results demonstrate an important role of miR‐34a‐5p‐MPV17L2 pathway in the control of mitochondrial functions in carcinoma cells and support the utility of novel bioengineered miRNA molecules for functional studies.
Long‐term neuroprosthetic hemotherapy treats autonomic dysreflexia after spinal cord injury
Authors: Soriano Jan Elaine , Hudelle Rémi , Squair Jordan W. , Anderson Mark A. , Gautier Matthieu , Mahe Löis , Tso Marcus , Amir Suje , Courtine Gregoire , Phillips Aaron A.
Published: May 2022

Disrupted descending supraspinal control results in a hyper‐excitable sympathetic nervous system connectome after spinal cord injury (SCI). A condition termed “autonomic dysreflexia” (AD) results, which is characterized by severe elevations in blood pressure that are life‐threatening. There are no effective therapies for AD. Long‐term epidural stimulation below the level of the SCI can promote functionally‐relevant plasticity of volitional motor circuits, and this paradigm may prevent the hyper‐excitable sympathetic connectome after SCI.

Here we studied the potential rehabilitative effect of long‐term epidural stimulation, as well as the mechanisms that are responsible for the development of AD. For this experiment, we compared SCI rats that underwent 30 minutes of epidural stimulation 5x/week for 6 weeks to SCI rats receiving normal care. Long‐term epidural stimulation abrogated AD induced by colorectal distension (p<0.0001; Figure 1). Viral tracing of ascending propriospinal neurons was also performed in these groups. Long‐term epidural stimulation normalized the sympathetic connectome as the axons and synapses were significantly reduced compared to SCI rats (p=0.0021; p=0.0127). We next performed chemogenetic silencing of excitatory and inhibitory interneurons, as well as sympathetic preganglionic neurons, in the lower thoracic spinal segments while experimentally inducing AD. Preliminary data indicate that AD is, at least in part, mediated by excitatory interneuronal connections to sympathetic preganglionic neurons.

Long‐term epidural stimulation may be an effective hemotherapy for preventing AD after SCI. This project may provide direction for basic and clinical research to develop treatments for sympathetic nervous system dysfunction after SCI, and thereby improve health and patient quality of life over the long term.
Mobile Gyro Control for Intuitive Manipulation of Virtual Anatomy Specimens
Authors: Lin Tai‐Jiun Terry , Zhang Steve , Jeon Sean , Meleveetil Vedanth , Krebs Claudia
Published: May 2022

The VanVR APP (Vancouver Virtual Reality Anatomy and Pathology Project) is an anatomy educational tool developed as a response to the Covid‐19 pandemic. The web app provided 3D models of scanned prosections to accommodate students with their virtual anatomy lab courses at UBC. The web interface means that interactions with the 3D specimens are controlled by mouse movements, which can be less intuitive and confusing. We have developed a technology that allows students to manipulate and turn the 3D specimens with 6 degrees of freedom using their mobile phones as a controller.

The VanVR APP was created using multiple development tools such as Unity Engine for the overall environment setup and UI implementation, C# scripts for the interactive functionalities, 3D photogrammetry for specimen visualization, MongoDB for database, render Unity built WebGL and deployed the web app on Azure app service. To improve the interactivity and increase the intuitive design of the application, a new functionality of mobile gyro control was implemented using Photon to connect with the 3D specimens in WebGL using generated pairing code to match the web app and the mobile device in a virtual room. The orientation of the phone is transmitted to a spherical model in WebGL, which accordingly transforms the coordinates of any chosen specimen displayed in front of the user. This means that simply by tilting a phone like a gyro controller, users will be able to intuitively mirror how the specimen rotates in the virtual lab with significantly more freedom than the traditional mouse movement control. Along with the functionality of zooming in/out and comparing different models side by side, the app simulates the feeling of handling a specimen physically. In addition, learners can view the documented annotations tailored to each specimen illustrating details of various regions on the model.

The addition of the mobile gyro control gives a more natural way to examine the specimen. This new feature highlights our student‐centred approach with an emphasis on intuitive and accessible design approaches.

The acceleration in the use of virtual technologies during the pandemic has increased the use of related tools and their acceptance in anatomy education. The further development of technologies to improve the student learning experience in AR/VR will certainly broaden accessibility. Some future implementation includes haptic touch feedback on mobile phones which better simulates physical labs, and a mobile AR function that extends the current gyro controller feature, deploying AR rendered specimens assigned from the WebGL end on multiple mobile devices, which can be utilized in a classroom setting.
Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis
Authors: Kundu Tapas K. , Senapati Parijat , Bhattacharya Aditya , Das Sadhan , Dey Suchismita , Sudarshan Deepthi , Shyla G , Vishwakarma Jyoti , Sudevan Surabhi , Ramachandran Ravishankar , Maliekal Tessy T.
Published: May 2022

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation‐dependent transcription from chromatin templates in vitro. Acetylation of NPM1 by p300 has been shown to further enhance its transcription activation potential. Moreover, its total and acetylated pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP‐seq analyses, we provide the first genome‐wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co‐occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high‐throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II‐driven transcription and regulates the expression of genes that promote oral tumorigenesis.
Ethanolic Extract of Calotropis procera Exhibits Antitumor Effects on Human Breast and Colon Cancer Cells via Cell Cycle Arrest
Authors: Abu‐Gharbieh Eman , Bajbouj Khuloud , Shehab Naglaa G. , Elayoty Salma. M. , Abdel‐Rahman Wael M. , Bustanji Yasser , Sinoj Jithna , El‐Huneidi Waseem
Published: May 2022

Calotropis procera is an evergreen, perennial shrub of the family Apocynaceae has been used to treat rheumatism, filariasis, and skin disorders, and its leaf has been used to treat jaundice. The flowers’ extract has been used to treat spleen, liver, and stomach problems. Members of the same plant family are reported to exhibit anticancer potential.

Aim

To assess the flavonoid and phenolic constituents of the plant extract, evaluate the potential antitumor activity of C. procera ethanolic extract and investigate the mechanistic pathway using two different cancer cell lines, namely human breast adenocarcinoma (MCF‐7), human colorectal cancer (HCT‐116) and the cytotoxic effect on a normal human dermal fibroblast (aHDF).

Methods

Total flavonoid and phenolic contents were investigated spectrophotometrically and by HPLC analysis. Cell viability assay, Annexin‐V/PI apoptosis assay, cell cycle analysis and Western blotting were used to evaluate the effect of C. proceraon cytotoxicity, apoptosis, cell cycle arrest and proteins expression profile of the related altered pathways.

Results

Coumaric acid was found to be the principal constituent in the ethanolic extract. Quercetin and hisperdin were the majour flavonoids. The extract demonstrated potential and effective cytotoxic activity against the cancer cell lines, MCF‐7 and HCT‐116, with a minor toxic effect on normal cells. The extract significantly induced apoptosis and cell cycle arrest at the G2/M phase in both cancer cells. Western blot analysis revealed a significant reduction in G2/M regulators, cdk1 and cyclin B1, and the proliferation marker, survivin, protein levels in the treated cells.

Conclusion

This study highlights evidence of the novel antitumor effect of C. procera in breast and colon cancers by altering the progression of the cell cycle.
The Optimized Quantum Dots Mediated Thermometry Reveals the Efficiency of Myosin Extracted from Muscle Mini Bundles
Authors: Li Meishan , Coppo Lucia , Jena Bhanu P. , Larsson Lars
Published: May 2022

The biophysical function of myosin in vitro has been extensively investigated in different motility assays. However, the study of myosin ATPase properties at the fiber level is still limited. In this study we aimed to optimize quantum dots (QD) mediated thermometry to measure the efficiency of myosin extracted from muscle mini bundles. A reduction in fluorescent intensity of QD reflects an increase in temperature caused by the heat release during ATP hydrolysis, denoting the efficiency of the motor protein myosin. The procedure for extracting myosin was modified from the single fiber in vitro motility assay, and the concentration of myosin was represented by the extracted total protein since the ratio of extracted myosin to total protein was constant. Moreover, the efficiency of myosin extracted from preparations containing different isoform compositions revealed that slow myosin has a lower efficiency than fast myosin, i.e., a higher amount of heat was released resulting in faster decay of QD fluorescence intensity. Thus, the optimized QD mediated thermometry provides a new sensitive approach to evaluate myosin function from a thermometrical perspective based on minimal muscle tissue.
First‐in‐class Deubiquitylase Inhibitors Reveal New Enzyme Conformations
Authors: Chandler Francesca , Walden Miriam , Reddy Poli Adi Narayana , Cassel Joel , Bell Lillie , Campbell Lisa , Foglizzo Martina , Sicheri Frank , Salvino Joseph M. , Greenberg Roger A. , Zeqiraj Elton
Published: May 2022

Cells maintain protein homeostasis by adding a small protein, ubiquitin, to regulate a variety of cellular processes, dictating protein activity, localisation or degradation. The addition of ubiquitin, known as ubiquitylation, is a reversible process making it a versatile post‐translational modification aptly suited for cell signalling. Removal of ubiquitin is catalysed by deubiquitylating enzymes, commonly referred to as DUBs. BRCC36 isopeptidase complex (BRISC) is a multi‐protein DUB complex which hydrolyses lysine‐63‐linked ubiquitin chains on Type I interferon receptors (IFNAR1/2), thus regulating interferon‐dependent signalling. Therefore, BRISC‐mediated inflammatory signalling amplification is a promising target for autoimmune disease drug development. We performed a high‐throughput screen to identify small molecules which inhibit BRISC enzymatic activity. Employing an integrative structural biology approach (cryo‐electron microscopy, native mass spectrometry, hydrogen‐deuterium exchange mass spectrometry), complemented with biochemical assays, we have uncovered new enzyme conformations, revealing a remarkable mode of action for BRISC inhibitors. Exploring these key mechanisms will expand current knowledge of inflammatory signalling pathways and establish the use of DUB inhibitors as therapeutics to combat autoimmune disease and hyperactive cytokine signalling.
Blood levels and productivity of lambs according to breed
Authors: Nikitin Georgy , Baimishev Murat , Eremin Sergey , Baimishev Hamidulla
Published: May 2022

The study aimed to determine the correlation between blood and productivity levels according to the breed of lambs. The materials were blood, weight parameters, and amount of wool shorn of lambs. Twenty of them (first group) were lambs of the akzhaik purebred population; animals from the second group (n=20) were two‐breed cross lambs (north‐caucasian and akzhaik breeds), the third group (n=20) were also two‐breed cross lambs (Kuybyshev and akzhaik breeds). The biochemical and morphological examination of blood was researched in the fifth lambs of each group after lambing at the age of four and eight months. The weight was examined in each lamb after lambing and four and eight months by floor scales. The amount of wool shorn was analyzed in each lamb at eight months by the standard methods. There was a higher level of erythrocyte and hemoglobin in the 3d group than in the 1st group (0,55•1012/L and 9,32 g/l accordingly). The total protein and glucose levels of lambs 2d and 3d group at the age of four and eight months were significantly higher than those of the 1st group (2,84 and 4,73 g/l; 0,86 and 1,02 mmol/L). There was a higher weight and the amount of wool shorn of 2d and 3d groups at the age of eight months than in the 1st group (3,04 and 2,74 kg; 0,18 and 0,24 kg, p<0.05). We conclude a significant correlation between blood and productivity level according to the breed of lambs. The higher level of erythrocyte, hemoglobin, total protein, and glucose had a significant correlation with higher productivity of two‐breed cross lambs compared with the same parameters of purebred lambs.
Interaction between CD81 and NCC is associated with LPS induced preeclampsia
Authors: Wang Ping , Wang Weiwan , Jia Yutao , Yu Yanting , Wang Xiaoyan
Published: May 2022

CD81, a member of the tetraspain superfamily, is an important component in the pathogenesis of pregnancy. In order to determine whether there is a connection between CD81 and renal sodium transporters, we studied the interaction of CD81 and Na+‐Cl‐cotransporter (NCC) in vivo and in vitro. A rat model of pregnancy hypertension was built by injecting a small amount of lipopolysaccharide (LPS, 0.5μg/kg in 2ml saline) into the tail vein of pregnant rats on GD 5. On GD 18, BPs (SBP :99.9±1.4 vs 116.9±1.8, DBP :85.9±2.0 vs 94.4±1.8,mmHg, n=7/group) and urine protein (1185±35.0 vs 3550±158.0, μg/day, n=4/group) were higher in LPS ‐rats relative to vehicle‐rats while renal protein abundance of CD81 (186.7±20, % of vehiclel, n=4/group, same as below) and NCC (278.0±53) was increased. CD81 was colocalized and co‐immunoparticipated with NCC, not with NHE3, in rat kidney. The immunofluorescent staining of NCC and CD81 was located in the sub‐cellular membrane and cytoplasm. In order to further explore the relationship between renal CD81 and NCC, we treated mouse kidney distal tubule cells with LPS for 24h to construct an in vitro experimental model. The protein abundances of CD81, and NCC in mDCT cells were increased in the cell groups treated with LPS at concentration of 10, 20, 30 g/mL while LDH and CCK‐8 were not altered. The cd81‐siRNA (32nM) for 48 hrs totally deleted CD81 protein. It also decrease NCC protein(70.9±4.8) expression without changing NCC mRNA(124.9±14) and reversed the increase of NCC(126.0±5.2,86.9±8.8) induced by LPS(10μg/mL) for the last 24 hrs conforming the interaction of CD81 with NCC in vitro. Thus, the interaction between CD81 and NCC are determined in vivo and its role in preeclampsia remains to be studied in future.
Resveratrol Reduces Arterial Stiffness and Improves Functional Capacity in Patients with COPD
Authors: Cobb Kolton , Payne Christian , Lavender Rebekah , Simovic Tijana , Harris Ryan A. , Pollock Jennifer , Baban Babak , Mannino David , Nana‐Sinkam Patrick , Rodriguez Miguelez Paula
Published: May 2022

Introduction

Chronic Obstructive Pulmonary Disease (COPD) is the 3rd leading cause of death worldwide and is strongly associated with the development of cardiovascular disease (CVD). Large artery stiffening is recognized as a major CVD risk factor that contributes to diminished myocardial blood supply and reduced functional capacity, a marker of disease severity in COPD. Elevated oxidative stress plays a pivotal role in the etiology of both COPD and CVD. Apart from the body’s endogenous ability to counteract oxidative stress, the consumption of exogenous antioxidants also enhances the capacity to scavenge free radicals and reduce arterial stiffness. Whether or not antioxidants can reduce arterial stiffness and improve functional capacity in patients with COPD, however, has yet to be elucidated.

Purpose

This study sought to test the hypothesis that oral resveratrol, a powerful antioxidant, will reduce arterial stiffness and improve functional capacity in patients with COPD.

Methods

Ten patients with COPD (GOLD Stage II‐IV) participated in this study and completed 6 weeks of oral resveratrol (500 mg/daily) or placebo (PLC) treatment. Arterial stiffness was evaluated via tonometry (SphygmoCor) before and after RES or PLC to assess augmentation index (AIx), AIx normalized to a heart rate of 75 beats per minute (AIx75), and diastolic pressure time index (DPTI). Functional capacity was measured via six‐minute walk test to evaluate distance walked.

Results

A single dose of RES significantly (p<0.01) improved markers of arterial stiffness (AIx75: ∆RES=‐8±2% vs. ∆PLC=‐4±2%) while increasing myocardial perfusion (DPTI: ∆RES=158±79 mmHg vs. ∆PLC=‐23±18 mmHg p=0.04). No changes (p=0.171) in functional capacity were observed after a single dose of RES or PLC. However, six weeks of RES significantly (p=0.01) reduced AIx75 (∆RES=‐9±3% vs. ∆PLC=‐3±2%), while it improved myocardial perfusion (DPTI: ∆RES=166±85 mmHg vs. ∆PLC=‐36±18 mmHg; p=0.04) and distance walked (∆RES=25±13m vs. ∆PLC=‐2±4m; p=0.04).

Conclusion

For the first time, we have identified that the antioxidant resveratrol reduces markers of arterial stiffness and improves myocardial perfusion in patients with COPD. In addition, six weeks of daily intake of this natural antioxidant also improved functional capacity in this population. Future studies are warranted to investigate the long terms effects of resveratrol in cardiovascular health in patients with COPD.
Excitation Wavelength‐Dependent pKa Extends the pH Sensing Range of Fluorescence Lifetime Sensors
Authors: Tantama Mathew
Published: May 2022

Biological activity is strongly dependent on pH, which may fluctuate within a variety of neutral, alkaline, and acidic local environments. The heterogeneity of tissue and subcellular pH has driven the development of sensors with different pKa values, and a huge assortment of fluorescent sensors have been developed to measure and visualize pH in living cells and tissues. In particular, sensors that report based on fluorescence lifetime are advantageous for quantitation. Here, we apply a theoretical framework to determine how the apparent pKa of lifetime‐base pH sensors depends on fluorescence excitation wavelength. We then demonstrate that theory accurate predicts the behavior of two different fluorescent protein‐base pH sensors. Furthermore, we show that this behavior has great practical value in matching the appropriate detection parameters with the physiological pH range. More broadly, our results show that the versatility of a single lifetime‐based sensor has been significantly underappreciated and our approach provides a means to use a single sensor across a range of pH environments.
Systemic proteomic and metabolomic signature of the SGLT2 inhibitor dapagliflozin
Authors: Billing Anja , Kim Young‐Chul , Vallon Volker , Rinschen Markus
Published: May 2022

Background

Dapagliflozin is a member of a new class of medication called gliflozins indicated for the treatment of diabetes. Gliflozins lower blood glucose levels by inhibition of the sodium glucose co‐transporter 2 (SGLT2), which is predominantly expressed in the S1 segment of renal proximal tubules and responsible for >90% of renal glucose reabsorption. Gliflozins also protect the kidney and heart, which cannot be solely explained by blood glucose lowering. To gain further mechanistic insights, we determined the metabolomic and proteomic signature of dapagliflozin in a murine model of diabetes.

Methods

Male C57BL/6 non‐diabetic wildtype and littermate Ins2 (Akita) diabetic mice, both fed a Western diet, were given dapagliflozin (10 mg/kg diet) or vehicle for one week (4 groups, N=8/group). Blood and urine glucose levels confirmed the expected treatment response. We analyzed metabolome, proteome and phosphoproteome for a range of tissues (kidney, liver, heart, smooth muscle, white adipose tissue) and body fluids (serum, urine, erythrocytes), and extended the analysis to the gut metaproteome. For untargeted metabolomics, analytes were measured by HILIC‐MS/MS and RP‐MS/MS. For proteomics and phosphoproteomics, TMT labelled (TMT16plex) peptides were separated by high pH reverse phase fractionation and measured by LC‐MS/MS including FAIMS (Thermo Scientific Orbitrap Exploris 480 mass spectrometer).

Results

Multi‐layered omics data were obtained comprising 11341 proteins, 14762 phosphorylation sites, and 217 metabolites. In general, dapagliflozin‐induced changes were more distinct in Western diet‐fed non‐diabetic than diabetic mice. Metabolomics revealed effects of dapagliflozin on amino acid, carbohydrate and lipid metabolism with most changes observed in urine, serum and liver. In comparison, dapagliflozin‐induced perturbations on proteome and phosphoproteome level were observed predominantly in kidney and affected ATP/lipid/carbohydrate/nitrogen metabolism, mitochondria, mitophagy, methyl transferases, methyl CpG‐binding, kinases, deacetylases, endosomes, cell junctions and gene expression. Interestingly, also the gut metaproteome was affected by dapagliflozin with significant changes in Bacteroides and Bifidobacterium species.

Conclusion

The SGLT2 inhibitor dapagliflozin induces distinct effects on kidney proteomics, associated with robust metabolomic signatures in plasma, urine and liver, as well as effects on the gut metaproteome. These effects occurred independent of blood glucose levels and support a concept of pleiotropic effects of SGLT2 inhibition that may originate in the kidney but induce wide‐ranged extrarenal metabolic effects and consequences.
A Non‐Human Primate Model of Carfentanil Overdose: Preventing Renarcotization with Superior Countermeasures
Authors: Myers Todd M. , Moffett Mark , Bullock Katherine , Langston Jeffrey
Published: May 2022

We established a physiological and overt intoxication model using African green monkeys (Chlorocebus aethiops sabeus) and a high dose of injected carfentanil. Intoxication is rapid and rescue is typically required within 10 minutes. Naloxone, administered at a 10 mg human‐equivalent dose (HED; 0.355 mg/kg), is immediately effective but is limited by its short duration of action. Specifically, animals typically return to a state of severe respiratory depression within about 2.5 hours, necessitating further medical intervention. Therefore, we studied two novel medical countermeasures: nalmefene and CS‐1131. Nalmefene is a mu‐opioid antagonist with high affinity and a much longer duration of action, but little is known about its efficacy against carfentanil. Therefore, we covered a range of doses from 3‐30 mg estimated HED (based upon allometric scaling) through both the intramuscular and intranasal routes. CS‐1131 is a novel opioid scavenger (created by Clear Scientific, LLC) capable of binding carfentanil stoichiometrically, inactivating the synthetic opioid, and then eliminating the scavenger‐opioid complex through renal excretion. Results show that both countermeasures were superior to naloxone alone and exhibited dose‐dependent efficacy. This research provides strong proof‐of‐concept and sets the stage for additional studies of longer‐lasting opioid antagonists administered through various routes, and for the further development, characterization, and refinement of novel opioid scavenger molecules. This non‐human primate model can be utilized to study potent synthetic opioids, new analogues, and other novel countermeasures in a way that yields predictive results to support human clinical trials.
Development of an Automated, High‐Throughput Assay to Detect Angiotensin AT2‐Receptor Agonistic Activity by Nitric Oxide Measurement In Vitro
Authors: Silva Igor M. S. , Kjærgaard Kenneth , Mortensen Christina , Stage Tore B. , Steckelings Ulrike M.
Published: May 2022

Background

The angiotensin AT2‐receptor (AT2R) is a key component within the protective arm of the renin‐angiotensin system inducing vasodilation by nitric oxide (NO) production. Currently, no high‐throughput assay is available to identify AT2R agonists in vitro, which could explain the low number of AT2R selective ligands in drug development programs.

Objective

To design and validate a high‐throughput method for detection of AT2R activation in vitro based on NO release.

Methods

Human aortic endothelial cells (HAEC) were seeded into 96‐well plates at a density of 5000 cells per well. 48 hours after seeding, cells were loaded for 30 minutes with the NO probe, DAF‐FM diacetate (5µM). Cells were stimulated for 15 minutes with the AT2R‐agonist C21 (1µM) or with classical NO‐releasing agents as positive controls: bradykinin (BK, 0.1µM), acetylcholine (ACh, 1µM) or Ang‐(1‐7) (MasR agonist, 0.1µM). Cells were fixed and fluorescence signals captured by fluorescence microscopy with automated, built‐in image analysis (ImageXpress Pico, Molecular Devices, USA). AT2R‐transfected (AT2R‐CHO) or non‐transfected (NT‐CHO) Chinese hamster ovary cells were cultured and loaded with DAF‐FM diacetate according to the same protocol as HAEC. Concentration‐response curves were obtained for C21, BK, Ang II and Ang‐(1‐5) (0.1nM to 10µM each) using the automated protocol for ImageXpress Pico described above on AT2R‐CHO. To evaluate whether effects of the compounds in AT2R‐CHO were AT2R specific, the response to these compounds at the highest concentration (10µM) was also tested in NT‐CHO.

Results

Using primary HAEC we could show that the automated, high‐throughput workflow was able to detect NO release by AT2R agonists as efficiently as by classical NO‐releasing agents [C21: 16.16 ± 6.84%; Ang‐(1‐7): 25.45 ± 3.37%, BK: 19.30 ± 2.92%; ACh: 24.01 ± 4.24%; one‐way ANOVA followed by Dunnett’s multiple comparison test, p≤0.05 vs control, ≥3 independent experiments for all compounds). In AT2R‐CHO, we detected that both C21 and Ang II (AT2R agonists) increased NO release in a concentration‐dependent manner, while BK had no effect (AT2R‐CHO lacks BK receptors) at any tested concentration. Surprisingly Ang‐(1‐5), a degradation product of Ang II thought to be inactive, induced NO release in a concentration‐dependent manner in AT2R‐CHO. In NT‐CHO, none of the compounds stimulated NO release, indicating that the effects by C21, Ang II and Ang‐(1‐5) in AT2R‐CHO were AT2R specific.

Conclusion

Measurement of NO release from AT2R‐CHO or HAEC by DAF‐FM diacetate in an automated way is suitable as high‐throughput assay for the identification of AT2R‐agonistic compounds in vitro. Application of the assay revealed that Ang‐(1‐5), which has been regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.
Regulation of Mitochondrial Calcium Transport by Caloric Restriction in Rat Kidney
Authors: Serna Julian C. , Amaral Andressa G. , da Silva Camille Caldeira , Munhoz Ana Claudia , Kowaltowski Alicia
Published: May 2022

Caloric restriction (CR) in laboratory rodents prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and retention capacity, preventing Ca2+‐induced mitochondrial permeability transition. Dietary restriction has been demonstrated to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring oxidative phosphorylation, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non‐phosphorylating (state 4) and uncoupled (state 3U) conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced in CR rats, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+‐induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials, or the amounts of the Mitochondrial Calcium Uniporter (MCU). Instead, increased mitochondrial calcium uptake rates correlate with a loss of Mitochondrial Calcium Uptake 2 (MICU2), an MCU modulator, in CR kidneys. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet‐sensitive regulatory role determining mitochondrial calcium homeostasis. Together, our results remark the highly organ‐specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2, and its effects on mitochondrial calcium transport, as a novel and interesting aspect of the metabolic responses to dietary interventions.
Computational and Mechanistic Study of the Therapeutic Potential of Compound Formononetin in Preventing Mast Cell Activation and IgE Production in Food Anaphylaxis
Authors: Musa Ibrahim , Wang Zhen Zhen , Li Xiumin
Published: May 2022

The prevalence of allergic diseases such as Asthma, Rhinitis, Anaphylaxis, Eczema, Urticaria, and Angioedema have been rising dramatically in low‐ and middle‐income countries around the world. According to American College of Allergy, Asthma & Immunology, allergies are the 6th leading cause of chronic illness in the U.S. and more than 50 million Americans suffer from allergies each year. Extensive data suggest that food allergies affect up to 10% of the world population and have been increasing in the last two to three decades. Globally 300 million people have asthma and about 200 to 250 million people suffer from food allergies. Previously we have shown that a small molecule compound, formononetin have shown decreases IgE production from B cells. We hypothesize that formononetin will be a potential candidate for preventing food anaphylaxis in food allergy by regulating molecular pathways that involved in IgE production in B cells, mast cell activation and degranulation. The objective of this study is to reveal potential therapeutic mechanisms of formononetin in prevention and treatment of food anaphylaxis from food allergy, by using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein‐protein interaction analyses and in silico molecular docking to identify gene and protein targets that are regulated in food allergy and mast cells diseases. The targets identified were further validated using qRT‐PCR in B cell multiple myeloma cell line. U266 cells were cultured to 1.0 × 106 cells/mL and then incubated with formononetin at different concentrations of 20 μg/mL for 72 hours. Supernatants were collected for measuring IgE levels by ELISA and cell viability was determined using Trypan blue dye. mRNA expression was determined for the top targets identified and compared with GAPDH. Mining formononetin targets into obtained disease targets uncovers 25 targets for food allergy, 51 targets for IgE diseases and 19 targets for mast cell diseases. This targets where further used for gene ontology, KEGG pathway analysis, CTPD network and protein‐protein interaction. Top regulated KEGG pathways include the primary immunodeficiency pathway, Epstein Barr virus infection pathway, Th17 cell differentiation, PI3K‐Akt pathway. Regulated gene ontology biological processes include B cell proliferation, B cell differentiation, B cell activation and apoptotic process. Formononetin decreased IgE at after 72 hours, P < 0.05 without cytotoxicity. Formononetin decreased mRNA expression of P53, TYK2, CASP8 significantly (P < 0.05) and IgEH very significantly (P < 0.01) and increased the mRNA expression of BCL2, NFKBIA and BTK significantly (P < 0.05) after 72 hours in the culture group compared to the untreated group. These findings suggest that formononetin may be a novel therapeutic candidate for treatment and prevention of IgE/mast cell mediated food allergy and other allergic diseases.
Corynebacterium pseudotuberculosis goat lymphadenitis and mastitis
Authors: Nikitin Georgy , Kudriashov Anatoly , Balabanova Viktoriya
Published: May 2022

In a goat farm, 16 goats showed an increase in the superficial cervical, patellar and superficial inguinal lymph nodes, as well as nodular formations in the udder. After slaughter, these animals were diagnosed with caseous inflammation of the lymph nodes and nodular mastitis. In bacteriological examination, the bacterium Corynebacterium pseudotuberculosis was isolated from the foci of inflammation. The object of the study was 16 goats at the age of two to three years. Samples of organs of three goats (out of 16) served as material for histological and bacteriological research: samples of lymph nodes, lungs and mammary glands, fixed in 10% formalin solution. Histological sections were stained with hematoxylin and eosin. Lymph nodes and abscesses in the udder were used for bacteriological examination. On clinical examination, all 16 goats showed a strong increase in the superficial cervical, patellar and superficial inguinal lymph nodes. The lymph nodes were spherical, 4‐6 cm in diameter, dense, painless. The udder was enlarged and tuberous. During milking, curd particles were found in the milk. In general, the condition of the goats has not changed, the appetite has been preserved. Post‐mortem examination revealed changes in the lymph nodes, mammary gland and lungs. The tissue of the lymph nodes is mostly replaced by soft, cheesy contents of a gray‐white color, enclosed in a thick, dense capsule. The udder contains numerous encapsulated lesions with a diameter of 0.5 to 3.0 cm, containing a thick, white, homogeneous mass. Single dense yellow‐white lesions up to 1 cm in diameter were found in the lungs. In histological sections of the lymph nodes, eosinophilic detritus and dark purple, hematoxylin‐stained fragments of cell nuclei, as well as neutrophils and macrophages were found. In histological sections of the mammary gland, in the alveoli, numerous neutrophils, macrophages and fragments of destroyed cells were found. In the histological sections of the lungs, foci of inflammation with accumulations of leukocytes with a large proportion of macrophages were found, as well as necrotic detritus. Bacteriological examination isolated the bacterium Corynebacterium pseudotuberculosis. It is known that C. pseudotuberculosis, as the causative agent of caseous lymphadenitis, is dangerous both for animals of many species and for humans, causing granulomatous inflammation of the lymph nodes and other organs. Moreover, there have been cases of C. pseudotuberculosis infection in humans through the consumption of raw goat's milk and cheese. These circumstances make the diagnosis of caseous lymphadenitis an important measure in the care of goats, especially during lactation.
Chronic Intermittent Hypoxia Alters Chloride Gradients in Median Preoptic Nucleus (MnPO) Neurons of Rats: Comparing ClopHensorN and Perforated Patch Recording
Authors: Farmer George E. , Little Joel T. , Cunningham J. T.
Published: May 2022

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia associated with sleep apnea. Male rats exposed to CIH exhibit increased sympathetic tone and reactivity and persistent elevations in blood pressure. In MnPO neurons, CIH induces reductions in GABAa inhibition that are, in some neurons, exacerbated to the point of GABAa mediated excitation. The genetically encoded ratiometric Cl‐ sensor, ClopHensorN, was used to monitor the Cl‐ flux of PVN projecting MnPO neurons in normoxic (Norm) and CIH treated rats in response to GABAa receptor activation. In some experiments, ClopHensorN imaging was combined with perforated patch recordings.

Using isoflurane (2‐3%) anesthesia, male Sprague‐Dawley rats (250‐350g) received bilateral infusions (0.4 µL) of a retrograde AAV9‐Cre in the PVN and a DIO‐ClopHensorN in the MnPO. After recovery, rats were subjected to 7 days of CIH (0800‐1600 hr) or normoxia. CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For recordings, rats were anesthetized with isoflurane (2‐3%) and coronal slices (300 µm) containing the MnPO were cut using standard in vitro slice procedures. All cells were tested in standard artificial cerebrospinal fluid for their responses to 10 s focal application of muscimol (100 uM). ClopHensorN images were captured every 3 sec. Cl‐ flux was determined from the ratio of GFP (ex 488 nm, em 500‐550 nm) to tdTomato (ex 594 nm, em 650‐700 nm) fluorescence. For some cells, voltage‐clamp recordings using gramicidin (100 ug/ml in 140 mM K‐gluconate) perforated patch were also collected (in labeled and unlabeled MnPO neurons).

MnPO neurons from CIH treated rats (n = 632), 20.1% showed a decrease in the ratio of GFP to tdTomato fluorescence (n = 127) while 0.3% showed increases in this ratio (n = 2) indicative of Cl‐ efflux. In MnPO neurons from Norm rats (n = 482), 41.9% showed a muscimol dependent decrease in the ratiometric Cl‐ signal (n = 202) with 0 cells showing increases. Muscimol dependent decreases in the Cl‐ signal were reduced in the CIH treated rats suggesting reduced GABAa inhibition. During perforated patch recordings, MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH treated rats show variable mIPSC frequencies and heterogeneous responses to muscimol.

The results demonstrate that CIH alters the Cl‐ flux of PVN projecting MnPO neurons in response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH.
Investigation of Synergistic Combinations of Chemotherapy Drugs for the Treatment of Oral Cancer
Authors: Oby Makayla M. , Del Gaizo Moore Victoria
Published: May 2022

Oral cancer is one of the most common forms of cancer among developing countries but despite current efforts, the survival of oral cancer has remained constant. Current research on oral cancer is centered on clinical studies focused on single drugs or very few combinations of the most common therapeutics historically used to treat oral cancer. However, there is a major gap in the literature about combinations of drug treatments for oral cancer. The goal of this study is to explore combination treatments and assess whether any synergistic effects can be produced. To accomplish this, two oral squamous cell carcinoma (OSCC) cell lines, Cal‐27 and OECM‐1 from the tongue and the oral cavity, respectively, will be used to investigate the efficacy of single drugs as well as combinations of drugs from a wide array of chemotherapy agents currently used to treat oral cancer as well as other forms of cancer. Thus far, Cell Titer Glo assays have been performed in triplicate for both Cal‐27 and OECM‐1 to analyze their viability in the presence of four drugs used to treat other forms of cancer with different mechanisms of action: PD0, a MEK inhibitor, IBET‐762, a BET inhibitor, ABT‐199, a Bcl‐2 inhibitor, and JQ1, a BRD4 inhibitor. GI50 values were gathered from each assay. Cal‐27 presented higher GI50 values than OECM‐1 for IBET‐762 and JQ1 at 1.52 mM and 1.64 mM, respectively. The GI50 values for OECM‐1 treatment with IBET‐762 and JQ1 were lower at 1.07 mM and 1.21 mM, respectively. Both cell lines were found to have similar resistance to PD0, around 1.05 mM, and ABT‐199, around 1.02 mM. High GI50 values across the cell lines indicate that both Cal‐27 and OECM‐1 are resistant to the four drugs that were tested. In the future, Cal‐27 and OECM‐1 will be treated with a panel of 10 drugs currently used clinically to treat oral cancer to gather baseline data to understand how these cell lines react to current treatment. The drugs that will be used include: Cisplatin, 5‐FU, Taxol, Docetaxel, Hydroxyurea, Methotrexate, Bleomycin, Gemcitabine, and Vincristine, all of which interfere with DNA synthesis and cell growth. Then, Cal‐27 and OECM‐1 will be treated with new combinations of drugs currently used to treat oral cancer and drugs not currently used for oral cancer but used to treat other forms of cancer. Some of these drugs include: Gefitinib, a EGFR inhibitor, Venetoclax, a BCL‐2 inhibitor, Irinotecan, a topoisomerase inhibitor, Mitoxantrone, a topoisomerase 2 and PKC inhibitor, and several other drugs. Cellular responses will then be assessed to determine if any combinations are synergistic. The data generated can aid researchers in understanding how different OSCC cells respond to drug combinations, which may lead to more successful clinical treatment options in the future.