bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combination Effect of Tofacitinib and Berberine for the Treatment of Dextran Sulfate Sodium‐Induced Chronic Colitis in C57BL/6 Mice
Authors: Li Yanhong , Tang Huazheng 
Published: April 2020

Background and AimsUlcerative colitis (UC) is a chronic inflammatory disease of the colon with a relapsing–remitting pattern that belongs to autoimmune disease (AID). The JAK inhibitor tofacitinib is a newly (2018.05) FDA approved drug for UC which is a drug previously used to treat another AID, rheumatoid arthritis. A higher dose of tofacitinib has been associated with a safety warning for increased lipid levels as well as serious infections and malignancy. Berberine has proved to be a promising drug candidate for the treatment of UC and shows activity in hyperlipidemia, bacteria; infections and cancer. We hypothesized that a balance between clinical benefit and side effects might be attained by using a combination of tofacitinib and berberine‐‐with reduced doses of both drugs. We first investigated the preclinical activity of reduced doses of tofacitinib plus berberine in experimental colitis in the mouse.MethodsTofacitinib (30 mg/kg/day), berberine (40 mg/kg/day) and reduced doses of tofacitinib (15 mg/kg/day) plus berberine (20 mg/kg/day) were administered orally for 14 and 30 days to C57BL/6 mice with colitis induced by two schedules of three cycles of 2% dextran sulfate sodium (DSS). Disease severity, colon length, colon and spleen swelling and cytokine expression in colon tissue as well as histology of major organs were examined.ResultsThe results showed that compared to the two drugs alone, half reduced doses of tofacitinib plus berberine showed a comparable effect in ameliorating DSS‐induced colitis severity and colon shortening. Co‐treatment showed a greater antagonism of DSS induced colon and spleen swelling when compared to either drug alone. Similar effects were observed in cytokine levels in the colon. In addition, no potential toxicity to major organs was observed with this combination therapy.ConclusionsOur studies provide a preclinical rationale for the combination of reduced dosage tofacitinib and berberine as a possible clinical therapeutic strategy for the treatment of UC.Support or Funding InformationThis work was funded by the Research Foundation of South China University of Technology (x2yxD2182200), the Natural Science Foundation of Guangdong Province (2018A030310388), Guangdong Medical Science Foundation (A2018363), and China Scholarship Council (Grant No. CSC201806155103).
Localization of NUCB2/Nesfatin‐3/Nesfatin‐1 in Normal and Inflamed Human and Mouse Lungs, and Human Neutrophils
Authors: Singh Baljit , Hui Jasmine , Singh Atul K. 
Published: April 2020

Nesfatin‐1 regulates hunger and fat storage and is produced in the hypothalamus of mammals. Nucleobindin2 (NUCB2)/nesfatin‐1 protein expression in human plasma positively correlates with expression of pro‐inflammatory cytokines in patients with chronic obstructive pulmonary disease (COPD) suggesting its potential role in lung inflammation. However, we still don’t precisely know the expression of NUCB2/nesfatin‐1 in lungs and neutrophils. In this study, the expression of NUCB2/nesfatin‐3/nesfatin‐1 in normal and inflamed human and mouse lungs and neutrophils were examined with light and electron microscopic immunocytochemistry. Results from light microscopy showed that the localization of NUCB2/nesfatin‐1 in the pulmonary epithelium, alveolar septa, vascular endothelium and various immune cells does not change with inflammatory status. Electron microscopy revealed constitutive localization within the nucleus and cytoplasm of the aforementioned cells. Further, NUCB2/nesfatin‐1 accumulated within 0.5μm of the plasma membrane in human neutrophils following 90mins of 1ng/mL LPS stimulation. NUCB2/nesfatin‐3 was also found to localize in euchromatic portions of neutrophilic nuclei at 5 times the mean concentration compared to heterochromatin. Finally, our results indicate that NUCB2/nesfatin‐3 is predominantly cytoplasmic as it localizes at 2 times the concentration in neutrophilic cytoplasm compared to nucleus. Our study is the first to detail the localization of NUCB2/nesfatin‐1/nesfatin‐3 in lungs and neutrophils, and also implicate their potential role in transcriptional regulation.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada
The Plasminogen Receptor, Plg‐RKT, Regulates Inflammation and Fibrinolysis During Wound Healing
Authors: Miles Lindsey A. , Ny Lina , Holmberg Sandra , Baik Nagyung A. , Bäckman Assar , Broden Jessica , Parmer Robert J. , Wilczynska Malgorzata , Ny Tor 
Published: April 2020

Plasminogen is a key regulatory protein that functions to both promote fibrinolysis and to potentiate the inflammatory response during wound healing. Here we have tested the hypothesis that the novel plasminogen receptor, Plg‐RKT, regulates distinct steps in the wound healing process.MethodFull‐thickness standardized burn wounds (1 cm in diameter) were induced in mice and the wound area was quantified at different time points. Wound tissue was subjected to qPCR and ELISA for cytokine expression and immunohistochemistry and Western blotting for fibrin deposition and leukocyte recruitment.ResultsFrom day 9 after injury, healing in Plg‐RKT−/− mice was significantly delayed compared with healing in Plg‐RKT+/+ littermates. Expression of cytokines, IL‐1β, CCL‐20, TNFα, and IL‐10 was dysregulated in Plg‐RKT−/− tissue although no genotype‐dependent differences in recruitment of macrophages or neutrophils to the wound area were detected. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase (days 9 and 10) was observed in wound tissue from mPlg‐RKT−/− mice in which Plg‐RKT was specifically deleted in myeloid cells. Regarding fibrinolysis, fibrin clearance was significantly impaired in Plg‐RKT−/− wound tissue. Genetic reduction of fibrinogen levels of Plg‐RKT−/− mice to 50% completely abrogated the effect of Plg‐RKT deletion on healing of burn wounds and, remarkably, effects of Plg‐RKT deletion on cytokine expression were modulated by the reduction in fibrinogen levels. In conclusion, Plg‐RKT plays a key role in wound healing by regulating both fibrinolysis and the inflammatory response and fibrin plays a role in regulating Plg‐RKT function on myeloid cells.Support or Funding InformationUnited States National Institutes of Health grants HL 081046 to LAM and HL 149511 to LAM and RJP and Merit Review Award #5I01BX002026 from the U.S. Department of Veterans Affairs to RJP.
Role of PLA2R1 and sPLA2 on Drug Release and Uptake of Liposome Nanoparticles in Prostate Cancer
Authors: Alnaim Ahmed Saad , Arnold Robert , Eggert Matthew , Nie Ben , Jasper Shanese , Davis Joshua , Panizzi Peter , Cummings Brian , Quach Nhat 
Published: April 2020

Exploiting differences in tumor pathophysiology can enhance the delivery and antitumor activity of chemotherapeutics encapsulated in drug carriers. Long‐circulating nanoparticulate drug carriers, such as pegylated‐sterically‐stabilized liposomes (SSL), can stably entrap drug, alter drug disposition, improve antitumor activity and minimize toxicity. However, control of their drug‐release kinetics has limited their clinical potential. Secretory phospholipases A2 (sPLA2) are over expressed and excreted in a variety of tumors, e.g., up to 22‐fold in prostate. These enzymes degrade phospholipids preferentially and have been hypothesized as targets to control drug release from lipid‐based nanoparticles, such as liposomes. While they have shown activity in preclinical models, the clinical performance of these formulations has been limited. The goal of these studies was to gain insights into how sPLA2‐targeted liposome formulation (SPRL) alter drug release and uptake, specifically examining the role of sPLA2 and its membrane receptor (PLA2R1). Studies were performed using a metastatic‐derived human prostate adenocarcinoma cells (PC‐3) and a PLA2R1 knock‐down variant (PC‐3‐PLA2R‐KD) while varying supplementation of sPLA2 enzyme isoforms (IIA, X, V). Liposomes (SSL & SPRL) were made as described previously and included DiR, a non‐exchangeable near‐infrared fluorescent dye. In vitro uptake and deposition of various formulations was determined using flow cytometry and fluorescence microscopy by measuring DiR and doxorubicin (fluorescent anticancer agent) entrapped within some of the formulations. Biodistribution and circulation times were determined non‐invasively on an IVIS Lumina XRMS and iThera Multispectral Optoacoustic Tomography (MSOT) systems in Athymic mouse xenograft models of prostate cancer. Liposomes formulations of SPRL and SSL were prepared at size of 120±1 and 118±2 nm. Circulation half‐life and tumor deposition was similar for both formulations, suggesting the enhanced pharmacological activity is related to release/uptake after drug extravasation. In‐vitro studies showed that PC‐3‐PLA2R‐KD cells had greater DiR fluorescence of both formulations than wild PC‐3 after 48 hr. Moreover, SPRL had greater uptake than SSL formulation (p<0.05) beyond 24 hr. In‐vivo studies showed greater uptake of SSL and SPRL in the PC‐3‐PLA2R‐KD tumors. Overall these data suggest that PLA2R1 plays an important role in the performance of SSL and SPRL formulations. Further studies are needed to gain insights into the clinical significance and to determine if presence of PLA2R1 can be used to identify specific patient populations that would best respond to treatment with SPRL.Support or Funding InformationNIH/NIBIB 1R01EB016100‐01(RDA/BSC), 1R21EB008153 (RDA/BSC), and AU Research Initiative in Cancer “AURIC”.
GPR139 Signals Through Gq/11 to Oppose Mu Opioid Receptor Signaling
Authors: Stoveken Hannah M. , Zucca Stefano , Masuho Ikuo , Wang Dandan , Dao Maria , Grill Brock , Martemyanov Kirill A. 
Published: April 2020

Signaling through G protein coupled receptors (GPCRs) is highly regulated and shaped not only by how and when a GPCR engages its ligand, but also through complex and varied protein interactions at the level of the receptor and at downstream effectors. Opioid receptors are an excellent example of a GPCR system that is finely regulated in the central nervous system. The mu opioid receptor is the primary receptor that modulates acute pain responses and has been heavily targeted therapeutically for its anti‐nociceptive properties. The duration and extent of endogenous MOR signaling is modulated by available endogenous opioid peptides, by β‐arrrestin recruitment and subsequent receptor desensitization, by regulator of G protein signaling (RGS) control of Gαo activation, and by interaction with other GPCRs that may positively or negatively alter signaling. Using an unbiased forward genetic screen in MOR‐expressing c. elegans, we uncovered another possible regulator of MOR signaling: the orphan GPCR GPR139. Studies using GPR139 knockout mice demonstrate hypersensitivity to opioid analgesics across multiple measures of antinociception. However, the exact molecular mechanism(s) of how GPR139 activation and signaling can modulate MOR downstream signaling is unknown. We ran a battery of in vitro signaling assays to determine how GPR139 signals in cells and how that could negatively modulate MOR signaling. GPR139 G protein coupling was tested using every G alpha protein and was found to couple to both Gq/11 and Gi/o family of G proteins. We examined the effect of GPR139 on multiple parameters of MOR signaling and trafficking and found that GPR139‐Gq signals in an opposing manner to MOR‐Go signaling at multiple downstream effectors in vitro. Further, MOR‐mediated changes in habenular neuron firing are blunted by dynamic GPR139 activation and can be rescued by Gαq inhibition. Overall, this research examines the molecular underpinnings of GPR139 negative regulation of MOR, explores mechanisms of GPCR cross‐regulation, and evaluates the possibility of therapeutically targeting GPR139 to increase the safety and efficacy of opioids.Support or Funding InformationNational Institute on Drug Abuse (NIDA) NRSA F32DA047771 to HMSNational Institute on Drug Abuse (NIDA) DA042746 to KAM
Profiling of Inflammatory Biomarkers and Coagulation Factors in End‐Stage Renal Disease
Authors: Bontekoe Emily , Grewal Arjun , Bansal Vinod , Tahar Meriam Ben Hadj , Hoppensteadt Debra , Fareed Jawed 
Published: April 2020

BackgroundEarly diagnosis of chronic kidney disease (CKD) is necessary in preventing complications that develop into kidney failure and vascular complications. Currently, the detection of early stages of CKD is lacking predictive values, delaying the application of specific diagnostics and therapeutic interventions. End‐stage renal disease (ESRD) patients are at risk for cardiovascular complications and vascular thrombosis, exhibiting numerous coagulation and fibrinolytic abnormalities.ObjectivesProfiling inflammatory biomarkers and coagulation factors in ESRD patients may allow early onset detection and severity of kidney damage, furthering the understanding of the pathogenesis of this disease.Materials & MethodsCitrated blood samples were collected from 95 confirmed ESRD patients undergoing maintenance hemodialysis under an IRB approved protocol. Biomarkers of inflammation and coagulation, including PAI‐1, TAFI, CRP, MMP‐9, MP, D‐Dimer, Factor VII, IX, X, and XIII were measured using commercially available ELISA methods. Normal controls were comprised of commercially available male and female citrated plasma (George King Biomedical). All results were compiled as mean ± SEM, and percent change in comparison to normal human plasma.ResultsBiomarkers including CRP (7ug/ml ± 0.6, 183%), MMP‐9 (88ng/ml ±8, 58%) D‐Dimer (1059ng/ml±138), Factor IX (124%±3, 23%), and Factor XIII (104%±1, 27%) showed significant increase when compared to normal plasma. Elevated CRP indicates severe inflammatory response and elevated D‐Dimer indicates endogenous fibrin formation. MP and PAI‐1 suggest endothelial dysfunction.DiscussionThese results indicate that the upregulation of inflammatory responses and hemostatic defects contribute to the multifactorial pathophysiology of ESRD. Profiling of these biomarkers may be helpful in the prognostic management of these patients.
CDK5 promotes ventricular arrhythmogenesis through phosphorylation of N‐type calcium channels in cardiac sympathetic postganglionic neurons
Authors: Zhang Dongze , Tu Huiyin , Hu Wenfeng , Wadman Michael C. , Li Yulong 
Published: April 2020

Cardiac sympathetic overactivation is an important trigger of fatal ventricular arrhythmias in patients with chronic heart failure (CHF). Our previous study found that increased N‐type calcium (Cav2.2) currents in cardiac sympathetic postganglionic (CSP) neurons contributes to cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF rats. Cyclin‐dependent kinase 5 (CDK5), a proline‐directed serine/threonine kinase, has been reported to play an important role in regulation of calcium influx and neurotransmitter release through phosphorylation of Cav2.2α subunit. Our current study aims to investigate whether CDK5 induces cardiac sympathetic overactivation and increases the susceptibility to ventricular arrhythmias via phosphorylation of Cav2.2α in CSP neurons. Lentiviral‐CDK5 cDNA (1×107 TU/ml, 2μl) was in vivo transfected into bilateral stellate ganglia (SG) in sham rats. Nontransfected sham rats served as controls. Final experiments were performed at 9 days after gene transfection. Western blot data demonstrated that gene transfection with lentiviral‐CDK5 cDNA into SGs significantly increased protein expression of CDK5 in CSP neurons in sham rats, compared with nontransfected sham rats. CDK5 cDNA transfection in SGs increased phosphorylation level of Cav 2.2α in CSP neurons from sham rat. The power spectral analysis of heart rate variability in conscious rats demonstrated that overexpression of CDK5 in CSP neurons increased low frequency power and ratio of low frequency to high frequency (index of cardiac sympathetic activation). Additionally, transfection of CDK5 cDNA into CSP neurons markedly increased cardiac sympathetic nerve activity in anesthetized sham rats (45.3±6.4% in sham+CDK5 group vs. 29.2±2.1% in sham group). ECG data obtained from 24‐hour continuous telemetry ECG recording revealed that overexpression of CDK5 in CSP neurons induced heterogeneity of ventricular electrical activities in conscious sham rats, as evidenced by CDK5 cDNA‐elongated ventricular arrhythmia‐related ECG markers including QT interval, QT dispersion, and T‐peak to T‐end interval. More importantly, transfection with CDK5 cDNA into CSP neurons significantly increased the susceptibility to ventricular arrhythmias (arrhythmia score was 1.7±0.9 in sham+CDK5 group vs. 0 in sham group) in anesthetized sham rats. Based on above data, we conclude that CDK5 induces cardiac sympathetic overactivation via phosphorylation of Cav 2.2‐α subunits in CSP neurons, and further increases the susceptibility of ventricular arrhythmias in sham rats.Support or Funding InformationThis study was supported by National Heart, Lung, and Blood Institute Grant (R01HL137832‐01 to Y.‐L. Li)
Proteomic and Phosphoproteomic Analysis of Skeletal Muscle During Hindlimb Immobilization
Authors: Lin Kuan-Hung , Blanco Rocky , Wilson Gary M. , Steinert Nate D. , Coon Joshua J. , Hornberger Troy A. 
Published: April 2020

The maintenance of skeletal muscle mass is required for health and issues associated with the quality of life. It is known that immobilization leads to the loss of muscle mass, and a decrease in protein synthesis is one of the primary mechanisms that drive this response. Moreover, previous studies have shown that protein synthesis is largely regulated by changes in the phosphorylation state of the various factors that can control protein synthesis (e.g., initiation and elongation factors). In this study, we took advantage of the powerful capabilities of mass spectrometry (MS) to identify the proteomic and phosphoproteomic alterations that occur in response to immobilization. In total, we were able to identify and quantify alterations in 15,020 unique phosphorylation sites and 4,646 proteins. To our surprise, the rate of protein synthesis decreased within 6 hours of immobilization, but no phosphorylation sites were significantly altered at this time. At later time points (i.e., 24 and 72 hours), the rate of protein synthesis further decreased, but again this was not associated with major changes in protein phosphorylation. To verify the proteomic and phosphoproteomic results, we performed western blot analysis to assess the phosphorylation state of various proteins that have been implicated in the regulation of protein synthesis. The results from the western blot analysis were highly congruent with the MS results. Hence, when taken together, our results strongly suggest that alterations in protein phosphorylation do not play a major role in immobilization‐induced decrease in protein synthesis and muscle mass.Support or Funding InformationNational Institutes for Health grant 1R01AR057347 to T.A.H.
Beta‐Arrestin‐1 Alters Glucocorticoid Signaling and the Glucocorticoid Receptor Transcriptome
Authors: Petrillo Maria G. , Diaz-Jimenez David , Oakley Robert H. , Cidlowski John A. 
Published: April 2020

The glucocorticoid receptor (GR) is a transcription factor essential for life which regulates gene expression following glucocorticoid (GC) binding. Ligand‐activated GR regulates transcription of genes involved in development, metabolism, and immune system function. Interestingly, GC‐mediated transcriptional profile is cell‐type specific, but the mechanism regulating selective gene transcription in a cell‐specific manner is still unclear. Selection of target genes, directionality of regulation, and magnitude of the response are largely thought to be dictated by the interplay between the GR and co‐regulatory elements as well as chromatin remodelers. We have discovered that β‐arrestin‐1, a known regulator of G‐protein‐coupled receptor signaling, is a protein binding partner of the GR. Upon glucocorticoid exposure, GR‐β‐arrestin‐1 complexes were detected in the nucleus of cells where GR is transcriptionally active. Moreover, depletion of β‐arrestin‐1 expression altered the GR‐mediated transcription profile by reducing gene responsiveness to glucocorticoids. This protein partnership raised the possibility that β‐arrestin‐1 may be a regulator of the GR transcriptional activity. To investigate if β‐arrestin‐1 contributes to the glucocorticoid‐dependent gene regulation, we performed RNA‐seq and Assay for Transposase‐Accessible Chromatin (ATAC)‐seq from control and β‐arrestin‐1 knockdown A549 cells treated with or without the synthetic glucocorticoid dexamethasone (Dex). RNA‐seq analysis identified three patterns of gene regulation: I) glucocorticoid‐responsive genes whose regulation depends on β‐arrestin‐1 expression (group 1; 720 genes), II) glucocorticoid‐responsive genes that are independent of β‐arrestin‐1 expression (group 2; 1886 genes), and III) genes whose transcription is blocked in control cells but become de‐novo glucocorticoid‐responsive genes upon β‐arrestin‐1 depletion (group 3; 986 genes). For group 1 genes, integration of RNA‐seq with ATAC‐seq analyses revealed that glucocorticoid responsive elements (GREs) near glucocorticoid‐induced genes became inaccessible when β‐arrestin‐1 expression was depleted. In contrast, evaluation of the chromatin state at the levels of the GRE regions of group 3 genes showed that β‐arrestin‐1 maintained the chromatin in a closed state, thus impeding GR from binding. Consistent with this finding, β‐arrestin‐1 depletion facilitated chromatin opening at those regions leading to GR‐dependent transcription. In conclusion, our study demonstrates that β‐arrestin‐1 is a novel coregulator of the GR that contributes to defining the cell‐type specificity of glucocorticoids by modulating the chromatin architecture.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIEHS, National Institutes of Health.