Rigosertib and Cholangiocarcinoma: A Cell Cycle Affair

Rigosertib is multi-kinase inhibitor that could represent an interesting therapeutic option for non-resectable patients with cholangiocarcinoma, a very aggressive hepatic cancer with limited effective treatments. The Western blotting technique was used to evaluate alterations in the expression of proteins involved in the regulation of the cell cycle of cholangiocarcinoma EGI-1 cells. Our results show an increase in EMI1 and Cyclin B protein levels after Rigosertib treatment. Moreover, the phosphorylation of CDK1 is significantly reduced by Rigosertib, while PLK1 expression increased after 24 h of treatment and decreased after 48 h. Finally, we evaluated the role of p53. Its levels increase after Rig treatment, and, as shown in the cell viability experiment with the p53 inhibitor Pifithrin, its activity is necessary for the effects of Rigosertib against the cell viability of EGI-1 cells. In conclusion, we hypothesized the mechanism of the action of Rigosertib against cholangiocarcinoma EGI-1 cells, highlighting the importance of proteins involved in the regulation of cell cycles. The CDK1-Cyclin B complex and p53 play an important role, explaining the Block in the G2/M phase of the cell cycle and the effect on cell viability

Serological responses to a soluble recombinant circumsporozoite protein-VK210 of Plasmodium vivax (rPvCSP-VK210) among Iranian malaria patients

Background Circumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay). Method Genomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E.coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni–NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test. Results The recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively. Conclusion The sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.

Nanoencapsulation of Hirudo medicinalis proteins in liposomes as a nanocarrier for inhibiting angiogenesis through targeting VEGFA in the Breast cancer cell line (MCF-7)

Introduction: Breast cancer is the most serious cause of women’s death throughout the world. Using nanocarrier vehicles to the exact site of cancer upgrades the therapeutic efficiency of the drugs. Capsulation of active proteins in the vesicular liposomes’ hydrophilic core is essential to develop a therapeutic protein carrier system. We aimed to encapsulate the medicinal leech saliva extract (LSE) and assess the inhibition of angiogenesis of breast cancer cells by targeting vascular endothelial growth factor A (VEGFA). Methods: In this research, enhanced formulation of liposomal protein was determined by zeta potential analysis, droplet size, drug release assay, and transmission electron microscopy (TEM). Furthermore, a cytotoxicity assay of liposomal LSE was performed to determine the cytotoxic activity of components. For assessing the expression of VEGFA, P53, and hypoxia-inducible factor subunit alpha (HIF1a) genes, Real-Time PCR was applied. Results: Nano liposome was chosen as an enhanced formulation due to its much smaller size (46.23 nm). Liposomal LSE had more practical actions on the MCF-7 cells. As noticed by DAPI staining, apoptosis was extensively greater in treated MCF-7 cells. Wound healing assay demonstrated that MCF-7 cells could not sustain growth at the presence of liposomal LSE and expression of the VEGFA gene was declined in treated cells. Downregulation of VEGFA was evaluated with western blotting technique. Conclusion: It can be concluded that our investigation of the tests confirmed the fact that nano liposomal LSE is a novel promising formulation for anticancer drugs and can significantly improve the penetration of protein drugs to cancer cells.

Primary Anastomosis versus Colostomy in Patients with Penetrating Colonic Injuries; A Comparative Study

Background: Penetrating colon injuries treatment has been a controversial topic of discussion in medical literature. The literature shows that randomized trials comparing primary repair versus bypass showed no significant difference in complication rates between the two groups. While other trials comparing complication rates between the two groups showed that the primary repair has lower complication rates as compared to the colostomy. Methodology: Randomized control trail was conducted in six months of duration from 1stJanuary 2020 to 31stDecember 2020in surgical unit of DHQ Teaching Hospital / SMC (UOS), Sargodha. After taking Ethical approval from the hospital, the study was conducted on 300 patients sample size calculated through open Epi sample size calculator with margin of error 5% and confidence interval 95%. Randomized blotting technique was used for randomization to overcome biasness. Results: Group 01 of primary anastomosis had 97 (64.67%) males and 53 (35.33%) females while group 02 of colostomy had 92 (66.34%) males and 58 (33.66%) females. complications intra-abdominal abscess was analyzed between both groups. In group 01 primary anastomosis, only 4 (2.66%) patients develop intra-abdominal abscess while in rest of patients i.e. 146(97.34%) showed efficacy to primary anastomosis. In group 02 of colostomy, 27 (18%) patients develop intra-abdominal abscess while rest of patients i.e. 123(82%) patients showed efficacy towards colostomy. Conclusion: In penetrating colon injuries, anastomosis showed more efficacy, safe to use and excellent results on the basis of post-operative complications as compared to colostomy. Keywords: Primary anastomosis, defunctioning in colostomy, penetrating colonic injuries, effectiveness

The effectiveness of Rutin for prevention of surgical induced endometriosis development in a rat model

Apoptosis and antioxidant mechanisms are pathways for the treatment of endometriosis (Endo). Rutin (Rtn) is an antioxidant flavonol that induces apoptosis. This study, for first time, was conducted to evaluate the effects of rutin on Endo through apoptosis and antioxidant mechanisms. The experimental Endo was induced in 24 rats and then the animals were subdivided into Endo-sole, 3000 and 6000 µg/kg rutin (Rtn-3000 and Rtn-6000) and vitamin C groups. After 4 weeks, the expression of Bcl2, Bax, anti Pro Caspase-9, cleaved Caspase-9, pro PARP, pro Cleaved PARP, Pro PARP, pro mTOR and mTOR were assessed by western blotting technique. The protein concentrations of malondialdehyde (MDA), total antioxidant capacity, and super oxide dismutase and gutathione peroxidase were also evaluated. TUNEL staining was also used for the detection of apoptosis. Caspase-9 and concentration of antioxidants were higher in the treated groups compared to Endo-sole group (P < 0.05). The results also showed that rutin decreased the expression of Bcl2 and MDA concentration (P < 0.05). The results for TUNEL staining showed that the animals treated with Rtn-6000 and vitamin C showed higher apoptosis. Rutin induces apoptosis by the expression of Bcl-2, Bax and caspase and also antioxidant activity by increasing antioxidants concentrations.

Regenerative role of Genistein treatment on Fibrotic and inflammatory Biomarkers Alteration in the Lung of Estrogen deficient Rats

Background Phytoestrogens are suggested to have estrogenic effects in the pulmonary system and have been revealed with a few adverse side effects. In this study, we tried to investigate the effect of genistein treatment on estrogen deficiency-induced lung injury and demonstrating whether genistein supplementation could replace estrogen hormone in postmenopausal women. Methods Forty adult female rats were divided into four groups; sham: rats that underwent surgery without ovariectomy, OVX: rats that underwent ovariectomies, OVX.E: ovariectomized rats with eight weeks period of estrogen treatment (20µg/kg/day), OVX.Gen: ovariectomized rats with eight week period of genistein treatment (1mg/kg/day). At the end of the experiment, lung tissue was removed and inflammatory and fibrotic biomarkers were evaluated with western blotting technique. Hematoxylin-eosin and immunohistochemical staining were used to evaluate histomorphological changes in the lung tissue. Results Genistein treatment restored ERK1/2, TGFβ1, MMP2, and IL1β, Bcl-2, and caspase3 expression levels, implying the effectiveness of genistein supplementation in targeting estrogen deficiency symptoms. Conclusions Genistein supplementation exerted protective effects against ovariectomy-induced lung injury with reducing inflammation and fibrosis, moreover, it can be recommended as a natural alternative to postmenopausal hormone therapy.

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

BackgroundDiagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) forToxoplasmain blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment.Methology/principle findingsHere we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopropylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml ofT.gondiiantigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1)T.gondiiserology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected,T.gondiiseropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence ofT.gondiiantigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic.Conclusion/significancesOur results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

Effect of PI3K/AKT/mTOR Signaling Pathway on Regulating and Controlling the Anti-Invasion and Metastasis of Hepatoma Cells by Bufalin

Background: Autophagy plays a "double-edged sword" in the process of tumorigenesis, development and metastasis. Objective: In this study, we explored the effect of PI3K/AKT/mTOR autophagy related signaling pathway on regulating and controlling the invasion and metastasis of liver cancer cells by Bufalin. Methods: The cell counting , migration , adhesion and invasion assay were used to evaluate the effect of Bufalin on the cell proliferation, invasion and metastasis. The protein expression of PI3K/AKT/mTOR signaling pathway were detected by Western Blotting technique. Results: After inhibiting autophagy of HCC-LM3 cells, the inhibitory effect of Bufalin on adhesion, migration and invasion of HCC-LM3 cells was significantly enhanced. The synergistic inhibition was strongest when different autophagy inhibitors were combined with 3MA and CQ. After inhibiting autophagy, Bufalin significantly inhibited the protein expression of P-AKT, Cyclin D1, MMP-2, MMP-9 and VEGF in HCC-LM3 cells. The protein expression of PTEN and E-Cadherin in HCC-LM3 cells was significantly increased. Conclusion: The present study shows that the anti-tumor effect of Bufalin mainly inhibit the proliferation, extracellular matrix degradation and angiogenesis of HCC by influencing autophagy. These findings confirm the capability of Bufalin in inhibiting metastasis of HCC and in parallel to current patents could be applied as a novel therapeutic strategy in the prevention of metastasis of HCC.

The mushroom, Cordyceps cicadae, ameliorates renal interstitial fibrosis via TLR2-mediated pathways

Purpose: To evaluate the mushroom, Cordyceps cicadae, for its ability to suppress tissue fibrosis and Toll-like receptors 2 (TLR 2) pathway activation in a mouse model of renal interstitial fibrosis (RIF).Methods: Cordyceps cicadae powder was obtained from BioAsia Group (Shanghai, China). RIF was induced via unilateral ureteral obstruction (UUO) in male C57Bl/6 mice. Animals were treated via the intragastric administration of Cordyceps cicadae powder (0.1g, 0.3 g/ml/100 g/day), beginning 24 h prior to UUO, and the treatment was continued for the following 14 days. Changes in tissue histology were then assessed via hematoxylin and eosin, and Sirius red stainings. Tissue macrophages were characterized based upon their expression of inducible nitric oxide synthase (iNOS) and interleukin-10 (IL-10), while Western blotting technique was used to measure the levels of TLR2, Myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB)/p-NF-κB in samples from these animals.Results: Treatment with Cordyceps cicadae powder is associated with a shift in macrophage phenotype that in turn decreased the production of extracellular matrix and alleviated RIF occurrence in mice model.Conclusion: This mechanistic study highlights the novel potential approach for treating and preventing RIF using Cordyceps cicadae powder. Keywords: Renal interstitial fibrosis, TLR2-mediated pathway, Cordyceps cicadae

Network Pharmacology Analysis and Molecular Characterization of the Herbal Medicine Formulation Qi-Fu-Yin for the Inhibition of the Neuroinflammatory Biomarker iNOS in Microglial BV-2 Cells: Implication for the Treatment of Alzheimer’s Disease

Aberrant microglial activation drives neuroinflammation and neurodegeneration in Alzheimer’s disease (AD). The present study is aimed at investigating whether the herbal formula Qi-Fu-Yin (QFY) could inhibit the inflammatory activation of cultured BV-2 microglia. A network pharmacology approach was employed to predict the active compounds of QFY, protein targets, and affected pathways. The representative pathways and molecular functions of the targets were analyzed by Gene Ontology (GO) and pathway enrichment. A total of 145 active compounds were selected from seven herbal ingredients of QFY. Targets (e.g., MAPT, APP, ACHE, iNOS, and COX-2) were predicted for the selected active compounds based on the relevance to AD and inflammation. As a validation, fractions were sequentially prepared by aqueous extraction, ethanolic precipitation, and HPLC separation, and assayed for downregulating two key proinflammatory biomarkers iNOS and COX-2 in lipopolysaccharide- (LPS-) challenged BV-2 cells by the Western blotting technique. Moreover, the compounds of QFY in 90% ethanol downregulated iNOS in BV-2 cells but showed no activity against COX-2 induction. Among the herbal ingredients of QFY, Angelicae Sinensis Radix and Ginseng Radix et Rhizoma contributed to the selective inhibition of iNOS induction. Furthermore, chemical analysis identified ginsenosides, especially Rg3, as antineuroinflammatory compounds. The herbal formula QFY may ameliorate neuroinflammation via downregulating iNOS in microglia.