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Hepatic stellate cells (HSCs) play a key role in liver fibrosis. Succinate and succinate receptor (GPR91) signaling pathway are involved in the activation, proliferation, and migration of HSCs. We investigated whether succinate may induce hepatic fibrosis. The mice were randomly divided into 2 groups —the control group (chow diet-fed mice, n = 26) and sodium succinate group (2% sodium succinate + chow diet, n = 38). Each diet was provided for 16 weeks. Mice administered an oral diet of 2% sodium succinate for sixteen weeks lost body weight and had increased serum alanine transaminase and hepatic triglyceride contents compared to those in the control mice. Moreover, mice fed with sodium succinate showed increased expression of the alpha smooth muscle actin protein and gene in the liver at 8 weeks of feeding and increased fibrosis in their histology at 16 weeks of feeding. However, the expression of the GPR91 protein and mRNA increased at 4 weeks of feeding, but decreased at 8 and 16 weeks of feeding. These results suggest that an oral succinate diet could induce liver damage and liver fibrosis in mice and that GPR91 signaling might be an early marker or sensor of hepatic fibrosis development.

Gamma-aminobutyric acid (GABA) transaminase—also called GABA aminotransferase (GABA-AT)—deficiency is a rare autosomal recessive disorder characterized by a severe neonatal-infantile epileptic encephalopathy with symptoms such as seizures, hypotonia, hyperreflexia, developmental delay, and growth acceleration. GABA transaminase deficiency is caused by mutations in GABA-AT, the enzyme responsible for the catabolism of GABA. Mutations in multiple locations on GABA-AT have been reported and their locations have been shown to influence the onset of the disease and the severity of symptoms. We examined how GABA-AT mutations influence the structural stability of the enzyme and GABA-binding affinity using computational methodologies such as molecular dynamics simulation and binding free energy calculation to understand the underlying mechanism through which GABA-AT mutations cause GABA-AT deficiency. GABA-AT 3D model depiction was carried out together with seven individual mutated models of GABA-AT. The structural stability of all the predicted models was analyzed using several tools and web servers. All models were evaluated based on their phytochemical values. Additionally, 100 ns MD simulation was carried out and the mutated models were evaluated using RMSD, RMSF, Rg, and SASA. gmxMMPBSA free energy calculation was carried out. Moreover, RMSD and free energy calculations were also compared with those obtained using online web servers. Our study demonstrates that P152S, Q296H, and R92Q play a more critical role in the structural instability of GABA-AT compared with the other mutated models: G465R, L211F, L478P, and R220K.

We investigated the effect of the acetylcholine muscarinic receptor inhibitor benztropine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Benztropine inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 6.11 ± 0.80 μM and Hill coefficient of 0.62 ± 0.03. Benztropine shifted the steady-state activation curves toward a more positive potential, and the steady-state inactivation curves toward a more negative potential, suggesting that benztropine inhibited Kv channels by affecting the channel voltage sensor. Train pulse (1 or 2 Hz)-induced Kv currents were effectively reduced by the benztropine treatment. Furthermore, recovery time constants of Kv current inactivation increased significantly in response to benztropine. These results suggest that benztropine inhibited vascular Kv channels in a use (state)-dependent manner. The inhibitory effect of benztropine was canceled by pretreatment with the Kv 1.5 inhibitor, but there was no obvious change after pretreatment with Kv 2.1 or Kv7 inhibitors. In conclusion, benztropine inhibited the Kv current in a concentration- and use (state)-dependent manner. Inhibition of the Kv channels by benztropine primarily involved the Kv1.5 subtype. Restrictions are required when using benztropine to patients with vascular disease.

Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1) is an orphan nuclear receptor encoded by the NR0B1 gene. The functional study showed that DAX1 is a physiologically significant target for EWS/FLI1-mediated oncogenesis, particularly Ewing Sarcoma (ES). In this study, a three-dimensional DAX1 structure was modeled by employing a homology modeling approach. Furthermore, the network analysis of genes involved in Ewing Sarcoma was also carried out to evaluate the association of DAX1 and other genes with ES. Moreover, a molecular docking study was carried out to check the binding profile of screened flavonoid compounds against DAX1. Therefore, 132 flavonoids were docked in the predicted active binding pocket of DAX1. Moreover, the pharmacogenomics analysis was performed for the top ten docked compounds to evaluate the ES-related gene clusters. As a result, the five best flavonoid-docked complexes were selected and further evaluated by Molecular Dynamics (MD) simulation studies at 100 ns. The MD simulation trajectories were evaluated by generating RMSD, hydrogen bond plot analysis, and interaction energy graphs. Our results demonstrate that flavonoids showed interactive profiles in the active region of DAX1 and can be used as potential therapeutic agents against DAX1-mediated augmentation of ES after in-vitro and in-vivo evaluations.

Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism Mycobacterium tuberculosis includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs in vitro and in vivo. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated naïve T cells, polarized CD4+ and CD8+ T cells to secrete IFN-γ, and induced T-cell–mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA251–264-pulsed ex vivo greatly strengthened immune priming in vivo, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy. Cancer Res; 71(8); 2858–70. ©2011 AACR.

Supplementary Methods, Figures 1-6 from Enhanced Efficacy of Therapeutic Cancer Vaccines Produced by Co-Treatment with Mycobacterium tuberculosis Heparin-Binding Hemagglutinin, a Novel TLR4 Agonist

Proinsulin C-peptide has a protective effect against diabetic complications; however, its role in hyperglycemia-induced pulmonary fibrosis is unknown. In this study, we investigated the inhibitory effect of C-peptide on hyperglycemia-induced pulmonary fibrosis and the molecular mechanism of C-peptide action in the lungs of diabetic mice and in human pulmonary microvascular endothelial cells (HPMVECs). We found that, in the lungs of diabetic mice, C-peptide supplementation using osmotic pumps attenuated hyperglycemia-induced pulmonary fibrosis and expression of fibrosis-related proteins. In HPMVECs, C-peptide inhibited vascular endothelial growth factor-induced adherens junction disruption and endothelial cell permeability by inhibiting reactive oxygen species generation and transglutaminase (TGase) activation. In the lungs, C-peptide supplementation suppressed hyperglycemia-induced reactive oxygen species generation, TGase activation, and microvascular leakage. C-peptide inhibited hyperglycemia-induced inflammation and apoptosis, which are involved in the pathological process of pulmonary fibrosis. We also demonstrated the role of TGase2 in hyperglycemia-induced vascular leakage, inflammation, apoptosis, and pulmonary fibrosis in the lungs of diabetic TGase2-null (Tgm2−/−) mice. Furthermore, we demonstrated a long-term inhibitory effect of systemic delivery of C-peptide using K9-C-peptide hydrogels on hyperglycemia-induced fibrosis in diabetic lungs. Overall, our findings suggest that C-peptide alleviates hyperglycemia-induced pulmonary fibrosis by inhibiting TGase2-mediated microvascular leakage, inflammation, and apoptosis in diabetes.

Ewing sarcoma (ES) is a highly malignant carcinoma prevalent in children and most frequent in the second decade of life. It mostly occurs due to t(11;22) (q24;q12) translocation. This translocation encodes the oncogenic fusion protein EWS/FLI (Friend leukemia integration 1 transcription factor), which acts as an aberrant transcription factor to deregulate target genes essential for cancer. Traditionally, flavonoids from plants have been investigated against viral and cancerous diseases and have shown some promising results to combat these disorders. In the current study, representative flavonoid compounds from various subclasses are selected and used to disrupt the RNA-binding motif of EWS, which is required for EWS/FLI fusion. By blocking the RNA-binding motif of EWS, it might be possible to combat ES. Therefore, molecular docking experiments validated the binding interaction patterns and structural behaviors of screened flavonoid compounds within the active region of the Ewing sarcoma protein (EWS). Furthermore, pharmacogenomics analysis was used to investigate potential drug interactions with Ewing sarcoma-associated genes. Finally, molecular dynamics simulations were used to investigate the stability of the best selected docked complexes. Taken together, daidzein, kaempferol, and genistein exhibited a result comparable to ifosfamide in the proposed in silico study and can be further analyzed as possible candidate compounds in biological in vitro studies against ES.

Plasmodium vivax (P. vivax) is one of the highly prevalent human malaria parasites. Due to the presence of extravascular reservoirs and recurrent infections from latent liver stages, P. vivax is extremely challenging to manage and eradicate. Traditionally, herbs have been widely used to combat various diseases. Flavonoids from plants are investigated against viral and infectious diseases and showed some promising results to combat these disorders. In the present study, in silico approaches are utilized to study the effect of flavonoids against P. vivax to inhibit malarial invasion of human red blood cells (RBC). Duffy binding protein (DBP) is a malarial protein responsible for binding the Duffy antigen receptor for chemokines (DARC) on human red blood cells and causes malarial invasion. The main focus is to block the DBP binding site to restrict the formation of the DBP-DARC complex. A molecular docking study was performed to analyze the interaction of flavonoid molecules with the DARC-binding site of DBP of P. vivax. Furthermore, molecular dynamic simulation studies are carried out to study the stability of top-docked complexes. The results showed the good effectiveness of flavonoids such as daidzein, genistein, kaempferol, and quercetin in the DBP binding site. These flavonoids bind with DBP and blocked its active site. Furthermore, the binding of these four ligands was maintained throughout the 50 ns simulation maintaining stable hydrogen bond formation with the active site residues of DBP. The present study suggests that flavonoids might be good candidates and novel agents against DBP-mediated RBC invasion of P. vivax.

Diabetes mellitus (DM) is a metabolic disease closely related to cardiovascular disease. The dipeptidyl peptidase-4 inhibitor teneligliptin is used to treat DM and has recently been shown to have a cardiovascular protective effect against diseases such as hypertension and heart failure. The present study demonstrates the vasodilatory effect of teneligliptin using aortic rings pre-contracted with phenylephrine. Teneligliptin induced a vasodilatory effect in a dose-dependent manner, with and without endothelium. In addition, pretreatment with the nitric oxide synthase inhibitor L-NAME and small-conductance Ca

Background Babesia is an intraerythrocytic parasite often misdiagnosed as a malaria parasite, leading to inappropriate treatment of the disease especially in co-endemic areas. In recent years, optical diffraction tomography (ODT) has shown great potential in the field of pathogen detection by quantification of three-dimensional (3D) imaging tomograms. The 3D imaging of biological cells is crucial to investigate and provide valuable information about the mechanisms behind the pathophysiology of cells and tissues. Methods The early ring stage of P. falciparum were obtained from stored stock of infected RBCs and of B. microti were obtained from infected patients during diagnosis. The ODT technique was applied to analyze and characterize detailed differences between P. falciparum and B. microti ring stage at the single cell level. Based on 3D quantitative information, accurate measurement was performed of morphological, biochemical, and biophysical parameters. Results Accurate measurements of morphological parameters indicated that the host cell surface area at the ring stage in B. microti was significantly smaller (140.2 ± 17.1 µm2) than that in P. falciparum (159.0 ± 15.2 µm2), and sphericities showed higher levels in B. microti-parasitized cells (0.66 ± 0.05) than in P. falciparum (0.60 ± 0.04). Based on biochemical parameters, host cell hemoglobin level was significantly higher and membrane fluctuations were respectively more active in P. falciparum-infected cells (30.25 ± 2.96 pg; 141.3 ± 24.68 nm) than in B. microti (27.28 ± 3.52 pg; 110.1 ± 38.83 nm). The result indicates that P. falciparum more actively altered host RBCs than B. microti. Conclusion Although P. falciparum and B. microti often show confusable characteristics under the microscope, and the actual three-dimensional properties are different. These differences could be used in differential clinical diagnosis of erythrocytes infected with B. microti and P. falciparum. Graphical Abstract

Plasmodium vivax (P. vivax) is one of the human’s most common malaria parasites. P. vivax is exceedingly difficult to control and eliminate due to the existence of extravascular reservoirs and recurring infections from latent liver stages. Traditionally, licorice compounds have been widely investigated against viral and infectious diseases and exhibit some promising results to combat these diseases. In the present study, computational approaches are utilized to study the effect of licorice compounds against P. vivax Duffy binding protein (DBP) to inhibit the malarial invasion to human red blood cells (RBCs). The main focus is to block the DBP binding site to Duffy antigen receptor chemokines (DARC) of RBC to restrict the formation of the DBP–DARC complex. A molecular docking study was performed to analyze the interaction of licorice compounds with the DARC binding site of DBP. Furthermore, the triplicates of molecular dynamic simulation studies for 100 ns were carried out to study the stability of representative docked complexes. The leading compounds such as licochalcone A, echinatin, and licochalcone B manifest competitive results against DBP. The blockage of the active region of DBP resulting from these compounds was maintained throughout the triplicates of 100 ns molecular dynamic (MD) simulation, maintaining stable hydrogen bond formation with the active site residues of DBP. Therefore, the present study suggests that licorice compounds might be good candidates for novel agents against DBP-mediated RBC invasion of P. vivax.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium. Understanding the biological features of various parasite forms is important for the optical diagnosis and defining pathological states, which are often constrained by the lack of ambient visualization approaches. Here, we employ a label-free tomographic technique to visualize the host red blood cell (RBC) remodeling process and quantify changes in biochemical properties arising from parasitization. Through this, we provide a quantitative body of information pertaining to the influence of host cell environment on growth, survival, and replication of P. falciparum and P. vivax in their respective host cells: mature erythrocytes and young reticulocytes. These exquisite three-dimensional measurements of infected red cells demonstrats the potential of evolving 3D imaging to advance our understanding of Plasmodium biology and host-parasite interactions.

Inflammasomes are a group of intracellular multiprotein platforms that play important roles in immune systems. Benzyl isothiocyanate (BITC) is a constituent of cruciferous plants and has been confirmed to exhibit various biological activities. The modulatory effects of BITC on inflammasome-mediated interleukin (IL)-1β expression and its regulatory mechanisms in

The Plasmodium ovale curtisi (Poc) prevalence has increased substantially in sub-Saharan African countries as well as regions of Southeast Asia. Poc parasite biology has not been explored much to date; in particular, the invasion mechanism of this malaria parasite remains unclear. In this study, the binding domain of the Duffy binding protein of P. ovale curtisi (PocDBP) was characterized as an important ligand for reticulocyte invasion. The homologous region of the P. vivax Duffy binding protein in PocDBP, named PocDBP-RII herein, was selected, and the recombinant PocDBP-RII protein was expressed in an Escherichia coli system. This was used to analyze reticulocyte binding activity using fluorescence-activated cell sorting and immune serum production in rabbits. The binding specificity was proven by treating reticulocytes with trypsin, chymotrypsin and neuraminidase. The amino acid sequence homology in the N-terminal Cys-rich region was found to be ~ 44% between PvDBP and PocDBP. The reticulocyte binding activity of PocDBP-RII was significantly higher than the erythrocyte binding activity and was concentration dependent. Erythrocyte binding was reduced significantly by chymotrypsin treatment and inhibited by an anti-PocDBP-RII antibody. This finding suggests that PocDBP may be an important ligand in the reticulocyte invasion process of P. ovale curtisi.

We investigated the effect of asenapine, a commonly used atypical antipsychotic, on voltage-dependent K

Coagulation is a potential defense mechanism that involves activating a series of zymogens to convert soluble fibrinogen to insoluble fibrin clots to prevent bleeding and hemorrhagic complications. To prevent the extra formation and diffusion of clots, the counterbalance inhibitory mechanism is activated at levels of the coagulation pathway. Contrariwise, this system can evade normal control due to either inherited or acquired defects or aging which leads to unusual clots formation. The abnormal formations and deposition of excess fibrin trigger serious arterial and cardiovascular diseases. Although heparin and heparin-based anticoagulants are a widely prescribed class of anticoagulants, the clinical use of heparin has limitations due to the unpredictable anticoagulation, risk of bleeding, and other complications. Hence, significant interest has been established over the years to investigate alternative therapeutic anticoagulants from natural sources, especially from marine sources with good safety and potency due to their unique chemical structure and biological activity. This review summarizes the coagulation cascade and potential macromolecular anticoagulants derived from marine flora and fauna.

Inflammasomes are a group of intracellular multiprotein platforms that play important roles in immune systems. Benzyl isothiocyanate (BITC) is a constituent of cruciferous plants and has been confirmed to exhibit various biological activities. The modulatory effects of BITC on inflammasome-mediated interleukin (IL)-1β expression and its regulatory mechanisms in Pseudomonas aeruginosa (P. aeruginosa) LPS/ATP-stimulated THP-1 cells was investigated. Monocytic THP-1 cells were treated with phorbol myristate acetate (PMA) to induce differentiation into macrophages. Enzyme-linked immunosorbent assays (ELISA) were performed to measure the levels of IL-1β produced in P. aeruginosa LPS/ATP-exposed THP-1 cells. Western blotting was performed to examine the BITC modulatory mechanisms in inflammasome-mediated signaling pathways. BITC inhibited IL-1β production in P. aeruginosa LPS/ATP-induced THP-1 cells. BITC also inhibited activation of leucine-rich repeat protein-3 (NLRP3) and caspase-1 in P. aeruginosa LPS/ATP-induced THP-1 cells. Furthermore, we show that mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation in P. aeruginosa LPS was attenuated by BITC. These BITC-mediated modulatory effects on IL-1β production may have therapeutic potential for inflammasome-mediated disorders such as a nasal polyp.

Aims We investigated the changes in large-conductance Ca2+-activated K+ (BKCa) channels from human umbilical arterial smooth muscle cells experiencing gestational diabetes mellitus (GDM). Main methods Whole-cell patch-clamp technique, arterial tone measurement, RT-PCR, Quantitative real-time PCR, western blot were performed in human umbilical arterial smooth muscle cells. Key findings Whole-cell BKCa current density was decreased in the GDM group compared with the normal group. The vasorelaxant effects of the synthetic BKCa channel activator NS-1619 (10 μM) were impaired in the GDM group compared with the normal group. Reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analyses suggested that the mRNA, total RNA, and protein expression levels of the BKCa channel were decreased in the GDM group relative to the normal group. In addition, the expression levels of protein kinase A and protein kinase G, which regulate BKCa channel activity, remained unchanged between the groups. Applying the BKCa channel inhibitor paxilline (10 μM) induced vasoconstriction and membrane depolarization of isolated umbilical arteries in the normal group but showed less of an effect on umbilical arteries in the GDM group. Significance Our results demonstrate for the first time impaired BKCa current and BKCa channel-induced vasorelaxation activities that were not caused by impaired BKCa channel-regulated protein kinases, but by decreased expression of the BKCa channels, in the umbilical arteries of GDM patients.

Aims Canagliflozin is an anti-diabetic agent and sodium glucose co-transporter-2 inhibitor. Despite numerous clinical trials demonstrating its beneficial effects on blood pressure, the cellular mechanisms underlying the effects of canagliflozin on vascular reactivity have yet to be clarified. We investigated the vasodilatory effect of canagliflozin on aortic rings isolated from rabbits. Main methods We used rabbit thoracic aortic rings and its arterial tone was tested by using wire myography system. Key findings Canagliflozin caused concentration-dependent vasodilation in aortic rings pre-constricted with phenylephrine or high K+. However, the degree of canagliflozin-induced vasodilation of the aortic rings pre-constricted with high K+ was less than that of rings pre-constricted with phenylephrine. Application of 4-aminopyridine, a voltage-dependent K+ (Kv) channel inhibitor, reduced canagliflozin-induced vasodilation. However, pre-incubation of an inwardly rectifying K+ channel inhibitor, a large-conductance Ca2+-activated K+ channel inhibitor, and an ATP-sensitive K+ inhibitor did not modulate the vasodilatory effects of canagliflozin. Indeed, canagliflozin increased Kv currents in aortic smooth muscle cells. Pre-treatment with thapsigargin or cyclopiazonic acid, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors, reduced the vasodilatory effects of canagliflozin. Conversely, pre-treatment with a Ca2+ channel inhibitor, adenylyl cyclase/PKA inhibitors, and guanylyl cyclase/PKG inhibitors did not modulate the vasodilatory effects of canagliflozin. Endothelium removal, and pre-treatment with the nitric oxide synthase inhibitor L-NAME, and small- and intermediate-conductance Ca2+-activated K+ channel inhibitor apamin and TRAM-34, did not diminish the vasodilatory effects of canagliflozin. Significance Our results indicate that canagliflozin induces vasodilation, which is dependent on the robust SERCA activity and Kv channel activation.

Abalone viscera (AV) is one of the byproducts of the seafood processing industry. The low molecular weight (&lt, 5 kDa) peptides (LMW-AV) obtained from gastrointestinal digestion of AV could suppress allergenic responses on activated HMC-1 human mast cells in our previous study. Regarding the allergenic response of LMW-AV, in the present study, we further investigated the potential of oral administration of LMW-AV against atopic dermatitis (AD) in a dermatitis-induced model stimulated with Dermatophagoides farinae. The results demonstrated that the LMW-AV reduced a number of clinical symptoms, such as the severity of the dermatitis and serum immunoglobulin E levels. Moreover, LMW-AV could inhibit the expression of chemokines and cytokines. The histological analysis indicated that the LMW-AV has suppressed the eosinophil count and the mast cell infiltration into the upper dermis. The results suggest that LMW-AV can be considered as a promising candidate for AD treatment.