Search results for: pathway

Authors: Panadda Rojpibulstit, Suthathip Kittisenachai, Songchan Puthong, Sirikul Manochantr, Pornpen Gamnarai, Sasichai Kangsadalampai, Sittiruk Roytrakul

Abstract:

Hepatocellular carcinoma (HCC) is the most primary hepatic cancer worldwide. Nowadays a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is continually developed in HCC treatment. In this regard, after establishing and consequently exploring Hep88 mAb’s tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb’s specific Ag from both membrane and cytoplasmic fractions of HepG2 cell line was identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E.coli BL21 (DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) andalpha-enolase. In addition, gradual appearance of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Taken together, paraptosis-like programmed cell death (PCD) of HepG2 is induced by binding of mortalin (HSPA9) and alpha-enolase to Hep88 mAb. Mortalin depletion by formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independent of p53-mediated apoptosis. Additionally, Hep88 mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. These results imply that Hep88 mAb might be a promising tool for development of an effective treatment of HCC in the next decade.

Keywords: Hepatocellular carcinoma, Monoclonal antibody, Paraptosis-like program cell death, Transmission electron microscopy, mortalin (HSPA9), alpha-enolase

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Authors: Katarína Koňariková, Georgios A. Perdikaris, Lucia Andrezálová, Zdeňka Ďuračková, Lucia Laubertová, Helena Gbelcová, Ingrid Žitňanová

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Introduction: The continuous demand for new anti-cancer drugs has stimulated chemotherapeutic research based on the use of essential metalloelements with the aim to develop potential drugs with lower toxicity and higher antiproliferative activity against tumors. Copper(II) and its complexes play an important role as suitable species for antiproliferative tests. Objectives: The central objective of the current study was to investigate the potential in vitro anti-proliferative effects of N-salicylidene-L-glutamato copper (II) complexes and molecular mechanism of apoptosis induced by tested complexes. In our project we tested N-salicylidene-L-glutamato copper (II) complexes ZK1 - [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK0 - ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O); MK1 - [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol); MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] at concentration range 0.001-100 µmol/L against human colon carcinoma cell line HT-29 and human breast carcinoma cell line MCF-7. Methods: Viability was assessed by direct counting of 0.4% trypan blue dye-excluding cells after 24, 48 and 72 hour cultivations with or without copper complex and by MTT assay. To analyze the type of cell death and its mechanism induced by our copper complex we used different methods. To distinguish apoptosis from necrosis we used electrophoretic analysis, to study the activity of caspases 8 and 9 – luminometric analysis and caspase activity 3 colorimetric assay. Results: The observed anti-proliferative effect of the copper complexes appeared to be dose-, time- and cell line- dependent. Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) than to ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)). Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex than human breast carcinoma cells MCF-7. IC50 decreased with time of incubation (24, 48 and 72h) for HT-29, but increased for MCF-7. By electrophoresis we found apoptotic cell death induced by our copper complexes in HT-29 at concentrations 1, 10, 50 and 100 µmol/L after 48h (ZK1) and 72h (MK0, MK1) and in MCF-7 we did not find apoptosis. We also studied molecular mechanism of apoptosis in HT-29 induced by copper complexes. We found active caspase 9 in HT-29 after ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)) influence and active caspase 8 after MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) influence. Conclusion: Our copper complexes showed cytotoxic activities against human colon carcinoma cells HT-29 and breast cancer cell line MCF-7 in vitro. Apoptosis was activated by mitochondrial pathway (intrinsic pathway) in case of ZK1 [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK1 [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol) and MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] copper complexes and by death receptors (extrinsic pathway) in case of MK0 [Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O copper complex in HT-29.

Keywords: apoptosis, copper complex, cancer, carcinoma cell line

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Authors: Hany M. Fayed, Rehab F. Abdel-Rahman, Alyaa F. Hessin, Hanan A. Ogaly, Gihan F. Asaad, Abeer A. A. Salama, Sahar Abdelrahman, Mahmoud S. Arbid, Marwan Abd Elbaset Mohamed

Abstract:

Liver fibrosis is a serious global health problem that occurs as a result of a variety of chronic liver disorders. Apigenin, a flavonoid found in many plants, has several pharmacological properties. The aim of this study was to evaluate the antifibrotic efficacy of apigenin (APG) against experimentally induced hepatic fibrosis in rats via using thioacetamide (TAA) and to explore the possible underlying mechanisms. TAA (100 mg/kg, i.p.) was given three times each week for two weeks to induce liver fibrosis. After TAA injections, APG was given orally (5 and 10 mg/kg) daily for two weeks. Biochemical, molecular, histological and immunohistochemical analyses were performed on blood and liver tissue samples. The functioning of the liver, oxidative stress, inflammation, and liver fibrosis indicators were all evaluated. The findings showed that TAA markedly increased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as the levels of malondialdehyde (MDA), focal adhesion kinase (FAK), hypoxia-inducible factor-1 (HIF-1), nuclear factor-κB (NF-κB), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) with a reduction in albumin, total protein, A/G ratio, GSH content and interleukin-10 (IL-10). Moreover, TAA elevated the content of collagen I, α -smooth muscle actin (α-SMA), and hydroxyproline in the liver. The treatment with APG in a dose-dependent manner has obviously prevented these alterations and amended the harmful effects induced by TAA. The histopathological and immunohistochemical observations supported this biochemical evidence. The higher dose of APG produced the most significant antifibrotic effect. As a result of these data, APG appears to be a promising antifibrotic drug and could be used as a new herbal medication or dietary supplement in the future for the treatment of liver fibrosis. This effect might be related to the inhibition of the HIF-1/FAK signaling pathway.

Keywords: apigenin, FAK, HIF-1, liver fibrosis, rat, thioacetamide

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Authors: Boitumelo Setlhare, Mduduzi P. Mokoena, Ademola O. Olaniran

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Agricultural and industrial activities have led to increasing production of xenobiotics such as 2,4-dichlorophenol (2,4-DCP), a derivative of 2,4-dichlorophenoxyacetic acid (2,4-D), which is a widely used herbicide. Bioremediation offers an efficient, cost-effective and environmentally friendly method for degradation of the compound through the activities of the various microbial enzymes involved in the catabolic pathway. The aim of this study was to isolate and characterize bacterial isolate indigenous to contaminated sites in Durban, South Africa for 2,4-DCP degradation. One bacterium capable of utilizing 2,4-DCP as sole carbon source was isolated using culture enrichment technique and identified as Pseudomonas chlororaphis strain UFB2 via PCR amplification and analysis of 16S rRNA gene sequence. This isolate was able to degrade up to 75.11% of 2,4-DCP in batch cultures within 10 days, with the degradation rate constant of 0.14 mg/l/d. Phylogenetic analysis revealed the relatedness of this bacterial isolate to other Pseudomonas sp. previously characterized for chlorophenol degradation. PCR amplification of the catabolic genes involved in 2,4-DCP degradation revealed the presence of the correct amplicons for phenol hydroxylase (600 bp), catechol 1,2-dioxygenase (214 bp), muconate isomerase (851 bp), cis-dienelactone hydrolase (577 bp), and trans-dienelactone hydrolase (491 bp) genes. Enzyme assays revealed activity as high as 21840 mU/mg, 15630 mU/mg, 2340 mU/mg and 1490 mU/mg obtained for phenol hydroxylase, catechol 1,2-dioxygenase, cis-dienelactone hydroxylase and trans-dienelactone hydroxylase, respectively. The absence of catechol 2,3-dioxygenase gene and the corresponding enzyme in this isolate suggests that the organism followed ortho-pathway for 2,4-DCP degradation. Furthermore, the absence of malaycetate reductase genes showed that the bacterium may not be able to completely mineralize 2,4-DCP. Further studies are required to optimize 2,4-DCP degradation by this isolate as well as to elucidate the mechanism of 2,4-DCP degradation.

Keywords: biodegradation, catechol 1, 2-dioxygenase, 2, 4-dichlorophenol, phenol hydroxylase, Pseudomonas chlororaphis

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Authors: M. Cardenas-Garcia, P. P. Gonzalez-Perez

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Intercellular communication is a necessary condition for cellular functions and it allows a group of cells to survive as a population. Throughout this interaction, the cells work in a coordinated and collaborative way which facilitates their survival. In the case of cancerous cells, these take advantage of intercellular communication to preserve their malignancy, since through these physical unions they can send signs of malignancy. The Wnt/β-catenin signaling pathway plays an important role in the formation of intercellular communications, being also involved in a large number of cellular processes such as proliferation, differentiation, adhesion, cell survival, and cell death. The modeling and simulation of cellular signaling systems have found valuable support in a wide range of modeling approaches, which cover a wide spectrum ranging from mathematical models; e.g., ordinary differential equations, statistical methods, and numerical methods– to computational models; e.g., process algebra for modeling behavior and variation in molecular systems. Based on these models, different simulation tools have been developed from mathematical ones to computational ones. Regarding cellular and molecular processes in cancer, its study has also found a valuable support in different simulation tools that, covering a spectrum as mentioned above, have allowed the in silico experimentation of this phenomenon at the cellular and molecular level. In this work, we simulate and explore the complex interaction patterns of intercellular communication in cancer cells using the Cellulat bioinformatics tool, a computational simulation tool developed by us and motivated by two key elements: 1) a biochemically inspired model of self-organizing coordination in tuple spaces, and 2) the Gillespie’s algorithm, a stochastic simulation algorithm typically used to mimic systems of chemical/biochemical reactions in an efficient and accurate way. The main idea behind the Cellulat simulation tool is to provide an in silico experimentation environment that complements and guides in vitro experimentation in intra and intercellular signaling networks. Unlike most of the cell signaling simulation tools, such as E-Cell, BetaWB and Cell Illustrator which provides abstractions to model only intracellular behavior, Cellulat is appropriate for modeling both intracellular signaling and intercellular communication, providing the abstractions required to model –and as a result, simulate– the interaction mechanisms that involve two or more cells, that is essential in the scenario discussed in this work. During the development of this work we made evident the application of our computational simulation tool (Cellulat) for the modeling and simulation of intercellular communication between normal and cancerous cells, and in this way, propose key molecules that may prevent the arrival of malignant signals to the cells that surround the tumor cells. In this manner, we could identify the significant role that has the Wnt/β-catenin signaling pathway in cellular communication, and therefore, in the dissemination of cancer cells. We verified, using in silico experiments, how the inhibition of this signaling pathway prevents that the cells that surround a cancerous cell are transformed.

Keywords: cancer cells, in silico approach, intercellular communication, key molecules, modeling and simulation

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Authors: M. Sasajima, M. Watanabe, T. Yamaguchi Y. Kurosawa, Y. Koike

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In very narrow pathways, the speed of sound propagation and the phase of sound waves change due to the air viscosity. We have developed a new Finite Element Method (FEM) that includes the effects of air viscosity for modeling a narrow sound pathway. This method is developed as an extension of the existing FEM for porous sound-absorbing materials. The numerical calculation results for several three-dimensional slit models using the proposed FEM are validated against existing calculation methods.

Keywords: simulation, FEM, air viscosity, slit

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Authors: Yusuf E. Mustapha, Inioluwa A Akindoyeni, Oluwatoyin G. Ezekiel, Ifeoluwa O. Awogbindin, Ebenezer O. Farombi

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Background: Parkinson's disease (PD) is the most prevalent motor disorder. Available therapies are palliative with no effect on disease progression. Kolaviron (KV), a natural anti-inflammatory and antioxidant agent, has been reported to possess neuroprotective effects in Parkinsonian flies and rats. Objective: The present study investigates the neuroprotective effect of KV, focusing on the DJ1/Nrf2 signaling pathway. Methodology: All-trans retinoic acid (ATRA, 10 mg/kg, i.p.) was used to inhibit Nrf2. Murine model of PD was established with four doses of MPTP (20 mg/kg i.p.) at 2 hours interval. MPTP mice were pre-treated with either KV (200 mg/kg/day p.o), ATRA, or both conditions for seven days before PD induction. Motor behaviour was evaluated, and markers of oxidative stress/damage and its regulators were assessed with immunofluorescence and ELISA techniques. Results: MPTP-treated mice covered less distance with reduced numbers of anticlockwise rotations, heightened freezing, and prolonged immobility when compared to control. However, KV significantly attenuated these deficits. Pretreatment of MPTP mice with KV upregulated Nrf2 expression beyond MPTP level with a remarkable reduction in Keap1 expression and marked elevation of DJ-1 level, whereas co-administration with ATRA abrogated these effects. KV treatment restored MPTP-mediated depletion of endogenous antioxidant, striatal oxidative stress, oxidative damage, and inhibition of acetylcholinesterase activity. However, ATRA treatment potentiated acetylcholinesterase inhibition and attenuated the protective effect of KV on the level of nitric oxide and activities of catalase and superoxide dismutase. Conclusion: Kolaviron protects Parkinsonian mice by stabilizing and activating the Nrf2 signaling pathway. Thus, kolaviron can be explored as a pharmacological lead in PD management.

Keywords: Garcinia kola, Kolaviron, Parkinson Disease, Nrf2, behavioral incompetence, neurodegeneration

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Authors: Qi LI, Xu Lin, Nengming Lin

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Objective: The aim of this study was to investigate the role of desmocollin 2 (DSC2) protein in the proliferation, migration and drug resistance of lung cancer cells. Method: CCK-8 assays and colony formation assays were used to evaluate the effect of dsc2 regulation on cancer cell viability and colony formation. Transwell assays and wound healing assays were also performed. Cell flow double staining was used to detect the apoptosis rate of cells with DSC2, which was added cisplatin. Western blot assay was used to detect cell cycle, PI3k/Akt and apoptosis-related proteins. Results: Our data showed that dsc2 is upregulated in clinical lung cancer tissues compared with pericarcinomatous tissues, and it is differentially expressed in lung cancer cell lines. The down-regulation of dsc2 in A549 and H358 lung cancer cells significantly suppressed the cell proliferation, metastasis, and motility. In contrast, the opposite effects were observed in overexpression of dsc2 both in H23 and PC9 cell lines. In addition to lung adenocarcinoma cell lines, we also examined its expression in lung squamous cell lines, such as H226. Western blotting showed that dsc2 could reduce the level of phosphorylated Akt (Ser 473) and p-mTOR. Thus, it is speculated that dsc2 up-regulation promotes proliferation and invasiveness through activation of the PI3K/AKT pathway. Also, knockdown of dsc2 in A549 and H226 could significantly decreased in the levels of cyclinB and wee1 protein. Additionally, flow cytometry showed that dsc2 knockdown combined with cisplatin could significantly enhance cell apoptosis rate. Conclusion: These data suggest that dsc2 promotes the proliferation and migration of lung cancer cells in vitro. Also, the results suggested that dsc2 could affect the cell cycle and apoptosis of lung cells. Furthermore, knockdown of dsc2 could sensitize cisplatin in both lung adenocarcinoma and lung squamous cell lines. Thus we suggested that dsc2 can be used as a therapeutic target for lung cancer.

Keywords: desmocollin 2, cisplatin, lung cancer, PI3K/AKT, lung squamous cell

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Authors: Andrés Quiroz, Emilio Hormazabal, Ana Mutis, Fernando Ortega, Manuel Chacón-Fuentes, Leonardo Parra

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Red clover (Trifolium pratense L.) is an important forage species in temperate regions of the world. The main limitation of this species worldwide is a lack of persistence related to the high mortality of plants due to a complex of biotic and abiotic factors, determining a life span of two or three seasons. Because of the importance of red clover in Chile, a red clover breeding program was started at INIA Carillanca Research Center in 1989, with the main objective of improving the survival of plants, forage yield, and persistence. The main selection criteria for selecting new varieties have been based on agronomical parameters and biotic factors. The main biotic factor associated with red clover mortality in Chile is Hylastinus obscurus (Coleoptera: Curculionidae). Both larval and adults feed on the roots, causing weakening and subsequent death of clover plants. Pesticides have not been successful for controlling infestations of this root borer. Therefore, alternative strategies for controlling this pest are a high priority for red clover producers. Currently, the role of semiochemical in the interaction between H. obscurus and red clover plants has been widely studied for our group. Specifically, from the red clover foliage has been identified limonene is eliciting repellency from the root borer. Limonene is generated in the plant from two independent biosynthetic pathways, the mevalonic acid, and deoxyxylulose pathway. Mevalonate pathway enzymes are localized in the cytosol, whereas the deoxyxylulose phosphate pathway enzymes are found in plastids. In summary, limonene can be determinated by enzymatic bioassay using GPP as substrate and by limonene synthase expression. Therefore, the main objective of this work was to study genetic variation of limonene in material provided by INIA´s Red Clover breeding program. Protein extraction was carried out homogenizing 250 mg of leave tissue and suspended in 6 mL of extraction buffer (PEG 1500, PVP-30, 20 mM MgCl2 and antioxidants) and stirred on ice for 20 min. After centrifugation, aliquots of 2.5 mL were desalted on PD-10 columns, resulting in a final volume of 3.5 mL. Protein determination was performed according to Bradford with BSA as a standard. Monoterpene synthase assays were performed with 50 µL of protein extracts transferred into gas-tight 2 mL crimp seal vials after addition of 4 µL MgCl₂ and 41 µL assay buffer. The assay was started by adding 5 µL of a GPP solution. The mixture was incubated for 30 min at 40 °C. Biosynthesized limonene was quantified in a GC equipped with a chiral column and using synthetic R and S-limonene standards. The enzymatic the production of R and S-limonene from different Superqueli-Carillanca genotypes is shown in this work. Preliminary results showed significant differences in limonene content among the genotypes analyzed. These results constitute an important base for selecting genotypes with a high content of this repellent monoterpene towards H. obscurus.

Keywords: head space, limonene enzymatic determination, red clover, Hylastinus obscurus

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Authors: Hala Raslan, Noha Eltaweel, Hanaa Rasmi, Solaf Kamel, May Magdy, Sherif Ismail, Khalda Amr

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Introduction: Rheumatoid arthritis (RA) is an inflammatory, autoimmune disorder with progressive joint damage. Osteoarthritis (OA) is a degenerative disease of the articular cartilage that shows multiple clinical manifestations or symptoms resembling those of RA. Genetic predisposition is believed to be a principal etiological factor for RA and OA. In this study, we aimed to measure the expression of the top deregulated miRNAs that might be the cause of pathogenesis in both diseases, according to our latest NGS analysis. Six of the deregulated miRNAs were selected as they had multiple target genes in the RA pathway, so they are more likely to affect the RA pathogenesis.Methods: Eighty cases were recruited in this study; 45 rheumatoid arthiritis (RA), 30 osteoarthiritis (OA) patients, as well as 20 healthy controls. The selection of the miRNAs from our latest NGS study was done using miRwalk according to the number of their target genes that are members in the KEGG RA pathway. Total RNA was isolated from plasma of all recruited cases. The cDNA was generated by the miRcury RT Kit then used as a template for real-time PCR with miRcury Primer Assays and the miRcury SYBR Green PCR Kit. Fold changes were calculated from CT values using the ΔΔCT method of relative quantification. Results were compared RA vs Controls and OA vs Controls. Target gene prediction and functional annotation of the deregulated miRNAs was done using Mienturnet. Results: Six miRNAs were selected. They were miR-15b-3p, -128-3p, -194-3p, -328-3p, -542-3p and -3180-5p. In RA samples, three of the measured miRNAs were upregulated (miR-194, -542, and -3180; mean Rq= 2.6, 3.8 and 8.05; P-value= 0.07, 0.05 and 0.01; respectively) while the remaining 3 were downregulated (miR-15b, -128 and -328; mean Rq= 0.21, 0.39 and 0.6; P-value= <0.0001, <0.0001 and 0.02; respectively) all with high statistical significance except miR-194. While in OA samples, two of the measured miRNAs were upregulated (miR-194 and -3180; mean Rq= 2.6 and 7.7; P-value= 0.1 and 0.03; respectively) while the remaining 4 were downregulated (miR-15b, -128, -328 and -542; mean Rq= 0.5, 0.03, 0.08 and 0.5; P-value= 0.0008, 0.003, 0.006 and 0.4; respectively) with statistical significance compared to controls except miR-194 and miR-542. The functional enrichment of the selected top deregulated miRNAs revealed the highly enriched KEGG pathways and GO terms. Conclusion: Five of the studied miRNAs were greatly deregulated in RA and OA, they might be highly involved in the disease pathogenesis and so might be future therapeutic targets. Further functional studies are crucial to assess their roles and actual target genes.

Keywords: MiRNAs, expression, rheumatoid arthritis, osteoarthritis

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Authors: Shalini Arora, Sri Sivakumar

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The deep removal of high molecular weight sulphur compounds (e.g., 4,6, dimethyl dibenzothiophene) is challenging due to their steric hindrance. Hydrogenation desulfurization (HYD) pathway is the main pathway to remove these sulfur compounds, and it is mainly governed by the number of type 2 sites. The formation of type 2 sites can be enhanced by modulating the pore structure and the interaction between the active metal and support. To this end, we report the enhanced HDS catalytic activity of ultrasmall NiMo supported on amorphous alumina (A-Al₂O₃) catalysts by one pot colloidal synthesis method followed by calcination and sulfidation. The amorphous alumina (A-Al₂O₃) was chosen as the support due to its lower surface energy, better physicochemical properties, and enhanced acidic sites (due to the dominance of tetra and penta coordinated [Al] sites) than crystalline alumina phase. At 20% metal oxide composition, NiMo supported on A-Al₂O₃ catalyst showed 1.4 and 1.2 times more reaction rate constant and turn over frequency (TOF) respectively than the conventional catalyst (wet impregnated NiMo catalysts) for HDS reaction of dibenzothiophene reactant molecule. A-Al₂O₃ supported catalysts represented enhanced type 2 sites formation (because this catalystpossesses higher sulfidation degree (80%) and NiMoS sites (19.3 x 10¹⁷ sites/mg) with desired optimum stacking degree (2.5) than wet impregnated catalyst at same metal oxide composition 20%) along with higher active metal dispersion, Mo edge site fraction. The experimental observations were also supported by DFT simulations. Lower heat of adsorption (< 4.2 ev for MoS2 interaction and < 3.15 ev for Ni doped MoS2 interaction) values for A-Al₂O₃ confirmed the presence of weaker metal-support interaction in A-Al₂O₃ in contrast to crystalline ℽ-Al₂O3. The weak metal-support interaction for prepared catalysts clearly suggests the higher formation of type 2 sites which leads to higher catalytic activity for HDS reaction.

Keywords: amorphous alumina, colloidal, desulfurization, metal-support interaction

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Authors: Salma A. El-Marasy, Rehab F. Abdel-Rahman, Reham M. Abd-Elsalam

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The burden of stroke is intensely increasing worldwide. Brain injury following transient or permanent focal cerebral ischemia develops ischemic stroke as a consequence of a complex series of pathophysiological events. The aim of this study is to evaluate the possible neuroprotective effect of a dipeptidyl peptidase-4 inhibitor, vildagliptin, independent on its insulinotropic properties in non-diabetic rats subjected to cerebral ischemia. Anaesthetized Wistar rats were subjected to either left middle cerebral artery occlusion (MCAO) or sham operation followed by reperfusion after 30 min of MCAO. The other three groups were orally administered vildagliptin at 3 dose levels (2.5, 5, 10 mg/kg) for 3 successive weeks before subjected to left focal cerebral ischemia/reperfusion and till the end of the study. Neurological deficit scores and motor activity were assessed 24h following reperfusion. 48h following reperfusion, rats were euthanized and their left brain hemispheres were harvested and used in the biochemical, histopathological, and immunohistochemical investigations. Vildagliptin pretreatment improved neurological score deficit, locomotor activity and motor coordination in MCAO rats. Moreover, vildagliptin reduced malondialdehyde (MDA), elevated reduced glutathione (GSH), phosphotylinosital 3 kinase (PI3K), phosphorylated of protein kinase B (p-AKT), and mechanistic target of rapamycin (mTOR) brain contents in addition to reducing protein expression of caspase-3. Also, vildagliptin showed a dose-dependent attenuation in neuronal cell loss and histopathological alterations in MCAO rats. This study proves that vildagliptin exerted the neuroprotective effect in a dose-dependent manner as shown in amelioration of neuronal cell loss and histopathological damage in MCAO rats, which may be mediated by attenuating neuronal and motor deficits, it’s anti-oxidant property, activation of PI3K/AKT/mTOR pathway and its anti-apoptotic effect.

Keywords: caspase-3, cerebral ischemia, dipeptidyl peptidase-4 inhibitor, oxidative stress, PI3K/AKT/mTOR pathway, rats, vildagliptin

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Authors: Fadia Gafri, Kathryn Mckintosh, Louise Young, Alan Harvey, Simon Mackay, Andrew Paul, Robin Plevin

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Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor found originally to play a key role in regulating inflammation. However considerable evidence links this pathway to the suppression of apoptosis, cellular transformation, proliferation and invasion (Aggarwal et al., 2006). Moreover, recent studies have also linked inflammation to cancer progression making NF-κB overall a promising therapeutic target for drug discovery (Dobrovolskaia & Kozlov, 2005). In this study we examined the effect of the natural product canthin-6-one (SU182) as part of a CRUK small molecule drug discovery programme for effects upon the NF-κB pathway. Initial studies demonstrated that SU182 was found to have good potency against the inhibitory kappa B kinases (IKKs) at 30M in vitro. However, at concentrations up to 30M, SU182 had no effect upon TNFα stimulated loss in cellular IκBα or p65 phosphorylation in the keratinocyte cell line NCTC2544. Nevertheless, 30M SU182 reduced TNF-α / PMA-induced NF-κB-linked luciferase reporter activity to (22.9 ± 5%) and (34.6± 3 %, P<0.001) respectively, suggesting an action downstream of IKK signalling. Indeed, SU182 neither decreased NF-κB-DNA binding as assayed by EMSA nor prevented the translocation of p65 (NF-κB) to the nucleus assessed by immunofluorescence and subcellular fractionation. In addition to the inhibition of transcriptional activity of TNFα-induced NF-κB reporter activity SU182 significantly reduced PMA-induced AP-1-linked luciferase reporter activity to about (48± 9% at 30M, P<0.001) . This mode of inhibition was not sufficient to prevent the activation of NF-κB dependent induction of other proteins such as COX-2 and iNOS, or activated MAP kinases (p38, JNK and ERK1/2) in LPS stimulated RAW 264.7 macrophages. Taken together these data indicate the potential for SU182 to interfere with the transcription factors NF-κB and AP-1 at transcriptional level. However, no potential anti-inflammatory effect was indicated, further investigation for other NF-κB dependent proteins linked to survival are also required to identify the exact mechanism of action.

Keywords: Canthin-6-one, NF-κB, AP-1, phosphorylation, Nuclear translocation, DNA-binding activity, inflammatory proteins.

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Authors: Harika Atmaca, Emir Bozkurt, Latife Merve Oktay, Selim Uzunoglu, Ruchan Uslu, Burçak Karaca

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Microarrays have been developed for highly parallel enzyme-linked immunosorbent assay (ELISA) applications. The most common protein arrays are produced by using multiple monoclonal antibodies, since they are robust molecules which can be easily handled and immobilized by standard procedures without loss of activity. Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate, Ecteinascidia turbinata, has been shown to have antitumor effects. Here, we used a novel proteomic approach to explore the mechanism of action of trabectedin in prostate cancer cell line PC-3 by apoptosis antibody microarray. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. Trabectedin induced cytotoxicity and apoptosis in prostate cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL R2/DR5, TNF R1/TNFRSF1A, FADD were significantly increased by 4.0-, 21.0-, 4.20- and 11.5-fold by trabectedin treatment in PC-3 cells. Moreover, mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, and Cleaved Caspase-3 expressions were induced by 2.68-, 2.07-, 2.8-, and 4.5-fold and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 3.5- and 5.2-fold in PC-3 cells. Proteomic (antibody microarray) analysis suggests that the mechanism of action of trabectedin may be exerted via the induction of both intrinsic and extrinsic apoptotic pathways. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

Keywords: trabectedin, prostate cancer, omic protein expression profile, apoptosis

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Authors: Shreya Sara Ittycheria, K. C. Sivakumar, Shijulal Nelson Sathi, Priya Srinivas

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hCG, a heterodimer composed of α and β subunits, is a peptide hormone having numerous biological functions. Although hCG is expressed by placenta during pregnancy, ectopic β-hCG secretion is observed in many non-trophoblastic tumors including that of breast. In-vitro and in-vivo studies done in the lab, have proved that BRCA1 defective cancers express β-hCG and when β-hCG is expressed or supplemented, it promotes tumor progression and exhibits resistance to carboplatin and ABT888, in such cancers but not in BRCA1 wild type cancers. In cancer cells, instead of binding to its regular receptor, LH-CGR, β-hCG binds with Transforming Growth Factor Receptor 2 (TGFβRII) and phosphorylates it resulting in faster tumor progression through the Smad signaling pathway. Targeting β-hCG could be a potential therapeutic strategy for managing BRCA1 defective cancers. Here, molecular docking and dynamic simulation studies were done to identify potential small molecule inhibitors against β-hCG as there are currently no such inhibitors reported. The binding sites of TGFβRII on β-hCG were identified from the top 10 predicted complexes from Z Dock. Virtual screening of selected commercially available small molecules from various libraries such as ZINC, NCI and Life Chemicals amounting to a total of 50,025 molecules were done. Four potential small molecule inhibitors were identified, RgcbPs-1, RgcbPs-2, RgcbPs-3 and RgcbPs-4 with binding affinities -60.778 kcal/mol, -45.447 kcal/mol, -65.2268 kcal/mol and -82.040 kcal/mol respectively. Further, 100ns Molecular Dynamics (MD) simulation showed that these molecules form stable complexes with β-hCG. RgcbPs-1 maintains hydrogen bonds with Q54, L52, Q46, C100, G36, C57, C38 residues, RgcbPs-2 maintains hydrogen bonds with A83 residue, RgcbPs-3 maintains hydrogen bonds with C57, Y58, R94, G101 residues and RgcbPs-4 maintains hydrogen bonds with G36, C38, T40, C57, D99, C100, G101 and L104 residues of β-hCG all of which coincide with the TGFβRII binding site on β-hCG. These results show that these two inhibitors could be used either singly or in combination for inhibiting β-hCG from binding to TGFβRII and thereby directly inhibiting the tumorigenesis pathway.

Keywords: β-hCG, breast cancer, dynamic simulations, molecular docking, small molecule inhibitors, virtual screening.

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Authors: Razieh Zarei, Hassan zm, Abolghasem Ajami, Darush Moslemi, Narges Afsary, Amrollah Mostafa-zade

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Introduction: IL-23 is responsible for the differentiation and expansion of Th17/ThIL-17 cells from naive CD4+ T cells. Therefore, may be IL-23/IL17 axis involve in a variety of allergic and autoimmune diseases, such as RA, MS, inflammatory bowel disease (IBD), and asthma. TGF-β is also share for the differentiation Th17 producing IL-17 and CD4+CD25+Foxp3hiT regulatory cells from naïve CD4+ T cells which are involved in the regulation of immune response, maintaining immunological self-tolerance and immune homeostasis ,and the control of autoimmunity and cancer surveillance. Therefore, T regulatory cells play a key role in autoimmunity, allergy, cancer, infectious disease, and the induction of transplantation tolerance. Vitamin A and it's derivatives (retinoids) inhibit or reverse the carcinogenic process in some types of cancers in oral cavity,head and neck, breast, skin, liver, and blood cells. Shark is a murine organism and its cartilage has antitumor peptides to prevent angiogenesis, in vitro. Our purpose is whether simultaneous oral treatment vitamin A and shark cartilage can modulate IL-23/IL-17 and CD4CD25Foxp3 T regulatory cell/TGF-β pathways and Th1/Th2 immunity in patients with gastric cancer. Materials and Methods: First investigated an imbalanced supernatant of cytokines exist in patients with gastric cancer by ELISA. Associated with cytokines measuring such as IL-23,IL-17,TGF-β,IL-4 and γ-IFN, then flow cytometry was employed to determine whether the peripheral blood mononuclear cells such as CD4+CD25+Foxp3highT regulatory cells in patients with gastric cancer were changed correspondingly. Results: An imbalance between IL-17 secretion and TGF-β/Foxp3 t regulatory cell pathway and so, Th1 immunity (γ-IFN production) and TH2 immunity (IL-4 secretion) was not seen in patients with gastric cancer treated by vitamin A and shark cartilage. But, the simultaneously presented down-regulation of IL-23 indicated, at least cytokine level. Conclusion: Il-23, as a pro-angiogenesis cytokine, probably, help to tumor growth. Hence, suggested that down-regulation of IL-23, at least cytokine level, is useful for anti-tumor immune responses in patients with gastric cancer.

Keywords: IL-23/IL17 axis, TGF-β/CD4CD25Foxp3 T regulatory pathway, γ-IFN, IL-4, shark cartilage and gastric cancer

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Authors: A. O. Ogunkeyede, D. Amuchi, A. A. Adebayo

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Heavy metal contaminations in soil have received great attention. Anthropogenic activities such as vehicular emission, industrial activities and constructions have resulted in elevated concentration of heavy metals in the surface soils. The metal particles can be free from the surface soil when they are disturbed and re-entrained in air, which necessitated the need to investigate surface soil at market environment where adults and children are present on daily basis. This study assesses concentration of heavy metal pollution, ecological and health risk factors in surface soil at Effurun market. 8 samples were collected at household material (EMH), fish (EMFs), fish and commodities (EMF-C), Abattoir (EMA 1 & 2), fruit sections (EMF 1 & 2) and lastly main road (EMMR). The samples were digested and analyzed in triplicate for contents of Lead (Pb), Nickel (Ni), Cadmium (Cd) and Copper (Cu). The mean concentration of the Pb mg/kg (112.27 ± 1.12) and Cu mg/kg (156.14 ± 1.10) were highest in the abattoir section (EMA 1). The mean concentrations of the heavy metal were then used to calculate the ecological and health risk for people within the market. Pb contamination at EMMR, EMF 2, EMFs were moderately while Pb shows considerable contamination at EMH, EMA 1, EMA 2 and EMF-C sections of the Effurun market. The ecological risk factor varies between low to moderate pollution for Pb and EMA 1 has the highest potential ecological risk that falls within moderate pollution. The hazard quotient results show that dermal exposure pathway is the possible means of heavy metal exposure to the traders while ingestion is the least sources of exposure to adult. The ingestion suggested that children around the EMA 1 have the highest possible exposure to children due to hand-to-mouth and object-to-mouth behaviour. The results further show that adults at the EMA1 will have the highest exposure to Pb due to inhalation during burning of cow with tyre that contained Pb and Cu. The carcinogenic risk values of most sections were higher than acceptable values, while Ni at EMMR, EMF 1 & 2, EMFs and EMF-C sections that were below the acceptable values. The cancer risk for inhalation exposure pathway for Pb (1.01E+17) shows a significant level of contamination than all the other sections of the market. It suggested that the people working at the Abattoir were very prone to cancer risk.

Keywords: carcinogenic, ecological, heavy metal, risk

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Authors: Fitria D. Ayuningtyas, Anggraini Barlian

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Green turtle (Chelonia mydas) is one of TSD (Temperature-dependent Sex Determination, TSD) animals which sex is determined by the egg’s incubation temperature. GSD (Genotypic Sex Determination) homologous genes such as Wilms’ Tumor (Wt1) and Forkhead Box L2 (FoxL2) play a role in TSD animal sex determination process. Wt1 plays a role in both male pathway, as a transcription factor for Sf1 gene and in female pathway, as a transcription factor for Dax1. FoxL2 plays a role specifically in female sex determination, and known as transcriptional factor for Aromatase gene. Until now, research on the pattern of Wt1 and FoxL2 genes expression in C.mydas has not been conducted yet. The aim of this research is to know the pattern of Wt1 and FoxL2 genes expression in Mesonephros-Gonad (MG) complexes of Chelonia mydas embryos incubated in masculinizing temperature (MT) and feminizing temperature (FT). Eggs of C.mydas incubated in 3 different stage of TSP (Thermosensitive Period) at masculinizing temperature (26±10C, MT) and feminizing temperature (31±10C FT). Mesonefros-gonad complexes were isolated at Pre-TSP stage (FT at days 14th, MT at days 24th), TSP stage (FT at days 24th, MT at days 36th) and differentiated stage (FT at days 40th, MT at days 58th). RNA from mesonephros-gonad (MG) complexes were converted into cDNA by RT-PCR process, and the pattern of Wt1 and FoxL2 genes expression is analyzed by quantitative Real Time PCR (qPCR) method, β-actin gene is used as an internal control. The pattern of Wt1 gene expression in Pre-TSP stage was almost the same between MG complexes incubated at MT or FT, while TSP and differentiation stage, the pattern of Wt1 gene expression in MG complexes incubated at MT or FT was increased. Wt1 gene expression of MG complexes that incubated at FT was higher than at MT. There was a difference pattern between Wt1 gene expression in this research compared to the previous research in protein level. It could be assumed that the difference caused by post-transcriptional regulation mechanisms before mRNA of Wt1 gene translated into protein structure. The pattern of FoxL2 gene expression in Pre-TSP stage was almost the same between MG complexes that incubated at MT and FT, and increased in both TSP and differentiated stage. The FoxL2 gene expression in MG complexes that incubated in FT is higher than MT on TSP and differentiated stage. Based on the results of this research, it can be assumed that Wt1 and FoxL2 gene were expressed in MG complexes that incubated both at MT and FT since Pre-TSP stage. The pattern of Wt1 gene expression was increased in every stage of gonadal development, and so do the pattern of FoxL2 gene expression. Wt1 and FoxL2 gene expressions were higher in MG complexes incubated at FT than MT.

Keywords: chelonia mydas, FoxL2, gene expression, TSD, Wt1

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Authors: T. C. C. Soo, S. Bhassu

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Unlike the common presence of both innate and adaptive immunity in vertebrates, crustaceans, in particular, shrimps, have been discovered to possess only innate immunity. This further emphasizes the importance of innate immunity within shrimps in pathogenic resistance. Under the study of pathogenic immune challenge, different shrimp species actually exhibit varying degrees of immune resistance towards the same pathogen. Furthermore, even within the same shrimp species, different batches of challenged shrimps can have different strengths of immune defence. Several important pathways are activated within shrimps during pathogenic infection. One of them is JAK-STAT pathway that is activated during bacterial, viral and fungal infections by which STAT(Signal Transducer and Activator of Transcription) gene is the core element of the pathway. Based on theory of Central Dogma, the genomic information is transmitted in the order of DNA, RNA and protein. This study is focused in uncovering the important evolutionary patterns present within the DNA (non-coding region) and RNA (coding region). The three shrimp species involved are Macrobrachium rosenbergii, Penaeus monodon and Litopenaeus vannamei which all possess commercial significance. The shrimp species were challenged with a famous penaeid shrimp virus called white spot syndrome virus (WSSV) which can cause serious lethality. Tissue samples were collected during time intervals of 0h, 3h, 6h, 12h, 24h, 36h and 48h. The DNA and RNA samples were then extracted using conventional kits from the hepatopancreas tissue samples. PCR technique together with designed STAT gene conserved primers were utilized for identification of the STAT coding sequences using RNA-converted cDNA samples and subsequent characterization using various bioinformatics approaches including Ramachandran plot, ProtParam and SWISS-MODEL. The varying levels of immune STAT gene activation for the three shrimp species during WSSV infection were confirmed using qRT-PCR technique. For one sample, three biological replicates with three technical replicates each were used for qRT-PCR. On the other hand, DNA samples were important for uncovering the structural variations within the genomic region of STAT gene which would greatly assist in understanding the STAT protein functional variations. The partially-overlapping primers technique was used for the genomic region sequencing. The evolutionary inferences and event predictions were then conducted through the Bayesian Inference method using all the acquired coding and non-coding sequences. This was supplemented by the construction of conventional phylogenetic trees using Maximum likelihood method. The results showed that adaptive evolution caused STAT gene sequence mutations between different shrimp species which led to evolutionary divergence event. Subsequently, the divergent sites were correlated to the differing expressions of STAT gene. Ultimately, this study assists in knowing the shrimp species innate immune variability and selection of disease resistant shrimps for breeding purpose. The deeper understanding of STAT gene evolution from the perspective of both purifying and adaptive approaches not only can provide better immunological insight among shrimp species, but also can be used as a good reference for immunological studies in humans or other model organisms.

Keywords: gene evolution, JAK-STAT pathway, immunology, STAT gene

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Authors: Michael P. Mannino, Gerald W. Hart

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The majority of glucose metabolism proceeds through glycolytic pathways such as glycolysis or pentose phosphate pathway, however, about 5% is shunted through the hexosamine biosynthetic pathway, producing uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). This precursor can then be incorporated into complex oligosaccharides decorating the cell surface or remain as an intracellular post-translational-modification (PTM) of serine/threonine residues (O-GlcNAcylation, OGN), which has been identified on over 4,000 cytosolic or nuclear proteins. Intracellular OGN has major implications on cellularprocesses, typically by modulating protein localization, protein-protein interactions, protein degradation, and gene expression. Additionally, OGN is known to have an extensive cross-talk with phosphorylation, be in a competitive or cooperative manner. Unlike other PTMs there are only two cycling enzymes that are capable of adding or removing the GlcNAc moiety, O-linked N-aceytl glucosamine Transferase (OGT) and O-linked N-acetyl glucoamidase (OGA), respectively. The activity of OGT has been shown to be sensitive to cellular UDP-GlcNAc levels, even changing substrate affinity. Owing to this and that the concentration of UDP-GlcNAc is related to the metabolisms of glucose, amino acid, fatty acid, and nucleotides, O-GlcNAc is often referred to as a nutrient sensing rheostat. Indeed OGN is known to regulate several signaling pathways as a result of nutrient levels, such as insulin signaling. Dysregulation of OGN is associated with several disease states such as cancer, diabetes, and neurodegeneration. Improvements in glycomics over the past 10-15 years has significantly increased the OGT substrate pool, suggesting O-GlcNAc’s involvement in a wide variety of signaling pathways. However, O-GlcNAc’s role at the receptor level has only been identified in a case-by-case basis of known pathways. Examining the OGN of the plasma membrane (PM) may better focus our understanding of O-GlcNAc-effected signaling pathways. In this current study, PM fractions were isolated from several cell types via ultracentrifugation, followed by purification and MS/MS analysis in several cell lines. This process was repeated with or without OGT/OGA inhibitors or with increased/decreased glucose levels in media to ascertain the importance of OGN. Various pathways are followed up on in more detailed studies employing methods to localize OGN at the PM specifically.

Keywords: GlcNAc, nutrient sensitive, post-translational-modification, receptor

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Authors: Nima Samie, Batoul Sadat Haerian, Sekaran Muniandy, M. S. Kanthimathi

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Colon and breast cancers are categorized as the most prevalent types of cancer worldwide. Recently, the broad clinical application of metal complex compounds has led to the discovery of potential therapeutic drugs. The aim of this study was to evaluate the cytotoxic action of a selected nickel complex compound (NCC) against human colon and breast cancer cells. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, MCF-7 and Hs 190.T cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content , measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results showed that our nickel complex compound displayed a potent suppressive effect on HT-29, WiDr, MCF-7 and Hs 190.T after 24 h of treatment with IC50 value of 2.02±0.54, 2.13±0.65, 3.76±015 and 3.14±0.45 µM respectively. This cytotoxic effect on normal cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the nickel complex compound. Activation of this pathway was further evidenced by significant activation of caspase 9 and 3/7.The nickel complex compound (NCC) was also shown activate the extrinsic pathways of apoptosis by activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. The results of this study suggest that the nickel complex compound is a potent anti-cancer agent inducing both intrinsic and extrinsic pathways as well as cell cycle arrest in colon and breast cancer cells.

Keywords: nickel complex, apoptosis, cytoskeletal rearrangement, colon cancer, breast cancer

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Authors: Chengcao Sun, Cuili Yang, Shujun Li, Ruilin Xue, Liang Wang, Yongyong Xi, Dejia Li

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Backgrounds: Sulforaphane, one of the most important isothiocyanates in the human diet, is known to have chemopreventive and antioxidant activities in different tissues via activation of NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). However, its effects on muscular dystrophy remain unknown. This work was undertaken to evaluate the effects of Sulforaphane on Duchenne muscular dystrophy (DMD). Methods: 4-week-old mdx mice were treated with SFN by gavage (2 mg/kg body weight per day) for 8 weeks. Blood was collected from eye socket every week, and tibial anterior, extensor digitorum longus, gastrocnemius, soleus, triceps brachii muscles and heart samples were collected after 8-week gavage. Force measurements and mice exercise capacity assays were detected. GSH/GSSG ratio, TBARS, CK and LDH levels were analyzed by spectrophotometric methods. H&E staining was used to analyze histological and morphometric of skeletal muscles of mdx mice, and Evas blue dye staining was made to detect sarcolemmal integrity of mdx mice. Further, the role of Sulforaphane on Nrf2/ARE signaling pathway was analyzed by ELISA, western blot and qRT-PCR. Results: Our results demonstrated that SFN treatment increased the expression and activity of muscle phase II enzymes NQO1 and HO-1 with Nrf2 dependent manner. SFN significantly increased skeletal muscle mass, muscle force (~30%), running distance (~20%) and GSH/GSSG ratio (~3.2 folds) of mdx mice, and decreased the activities of plasma creatine phosphokinase (CK) (~45%) and lactate dehydrogenase (LDH) (~40%), gastrocnemius hypertrophy (~25%), myocardial hypertrophy (~20%) and MDA levels (~60%). Further, SFN treatment also reduced the central nucleation (~40%), fiber size variability, inflammation and improved the sarcolemmal integrity of mdx mice. Conclusions: Collectively, these results show that SFN can improve muscle function, pathology and protect dystrophic muscle from oxidative damage in mdx mice through Nrf2 signaling pathway, which indicate Nrf2 may have clinical implications for the treatment of patients with muscular dystrophy.

Keywords: sulforaphane, duchenne muscular dystrophy, Nrf2, oxidative stress

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Authors: Andleeb Zahra, Itrat Rubab, Sumaira Malik, Amina Khan, Muhammad Jawad Khan, M. Qaiser Fatmi

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Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. Recent studies have highlighted the role of miRNA in disease pathology, indicating its potential use in an early diagnostic tool. miRNAs are small, double stranded, non-coding RNAs that regulate gene expression by deregulating mRNAs. miRNAs play important roles in modifying various cellular processes such as cell growth, differentiation, apoptosis, and immune response. Dis-regulated expression of miRNAs is known to affect the cell growth, and this may function as tumor suppressors or oncogenes in various cancers. Objectives: The main objectives of this study were to characterize the extracellular miRNAs involved in oral cancer (OC) to assist early detection of cancer as well as to propose a list of genes that can potentially be used as biomarkers of OC. We used gene expression data by microarrays already available in literature. Materials and Methods: In the first step, a total of 318 miRNAs involved in oral carcinoma were shortlisted followed by the prediction of their target genes. Simultaneously, the differentially expressed genes (DEGs) of oral carcinoma from all experiments were identified. The common genes between lists of DEGs of OC based on experimentally proven data and target genes of each miRNA were identified. These common genes are the targets of specific miRNA, which is involved in OC. Finally, a list of genes was generated which may be used as biomarker of OC. Results and Conclusion: In results, we included some of pathways in cancer to show the change in gene expression under the control of specific miRNA. Ingenuity pathway analysis (IPA) provided a list of major biomarkers like CDH2, CDK7 and functional enrichment analysis identified the role of miRNA in major pathways like cell adhesion molecules pathway affected by cancer. We observed that at least 25 genes are regulated by maximum number of miRNAs, and thereby, they can be used as biomarkers of OC. To better understand the role of miRNA with respect to their target genes further experiments are required, and our study provides a platform to better understand the miRNA-OC relationship at genomics level.

Keywords: biomarkers, gene expression, miRNA, oral carcinoma

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Authors: Manabu Sasajima, Takao Yamaguchi, Yoshio Koike, Mitsuharu Watanabe

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This paper discusses the propagation of sound waves in air, specifically in narrow rectangular pathways of an occluded-ear simulator for acoustic measurements. In narrow pathways, both the speed of sound and the phase of the sound waves are affected by the damping of the air viscosity. Herein, we propose a new finite-element method (FEM) that considers the effects of the air viscosity. The method was developed as an extension of existing FEMs for porous, sound-absorbing materials. The results of a numerical calculation for a three-dimensional ear-simulator model using the proposed FEM were validated by comparing with theoretical lumped-parameter modeling analysis and standard values.

Keywords: ear simulator, FEM, simulation, viscosity

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Authors: Seyedahmad Hosseini, Mohammadjavad Sotoudeheian

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Introduction: Allergic asthma is a heterogeneous immuno-inflammatory disease based on Th-2-mediated inflammation. Histopathologic abnormalities of the airways characteristic of asthma include epithelial damage and subepithelial collagen deposition. Objectives: Human bronchial epithelial cell genome expression of TNF‑α, IL‑6, ICAM‑1, VCAM‑1, nuclear factor (NF)‑κB signaling pathways up-regulate during inflammatory cascades. Moreover, immunofluorescence assays confirmed the nuclear translocation of NF‑κB p65 during inflammatory responses. An absolute LDH leakage assays suggestedLPS-inducedcells injury, and the associated mechanisms are co-incident events. LPS-induced phosphorylation of ERKand JNK causes inflammation in epithelial cells through inhibition of ERK and JNK activation and NF-κB signaling pathway. Furthermore, the inhibition of NF-κB mRNA expression and the nuclear translocation of NF-κB lead to anti-inflammatory events. Likewise, activation of SUMF2 which inhibits IL-13 and reduces Th2-cytokines, NF-κB, and IgE levels to ameliorate asthma. On the other hand, TNFα-induced mucus production reduced NF-κB activation through inhibition of the activation status of Rac1 and IκBα phosphorylation. In addition, bradykinin B2 receptor (B2R), which mediates airway remodeling, regulates through NF-κB. Bronchial B2R expression is constitutively elevated in allergic asthma. In addition, certain NF-κB -dependent chemokines function to recruit eosinophils in the airway. Besides, bromodomain containing 4 (BRD4) plays a significant role in mediating innate immune response in human small airway epithelial cells as well as transglutaminase 2 (TG2), which is detectable in saliva. So, the guanine nucleotide-binding regulatory protein α-subunit, Gα16, expresses a κB-driven luciferase reporter. This response was accompanied by phosphorylation of IκBα. Furthermore, expression of Gα16 in saliva markedly enhanced TNF-α-induced κB reporter activity. Methods: The applied method to form NF-κB activation is the electromobility shift assay (EMSA). Also, B2R-BRD4-TG2 complex detection by immunoassay method within saliva with EMSA of NF-κB activation may be a novel biomarker for asthma diagnosis and follow up. Conclusion: This concept introduces NF-κB signaling pathway as potential asthma biomarkers and promising targets for the development of new therapeutic strategies against asthma.

Keywords: NF-κB, asthma, saliva, T-helper

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Authors: Priyanka Mokashi, Aparna Khanna, Nancy Pandita

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Diabetes mellitus is a chronic metabolic disorder which will be the 7th leading cause of death by 2030. The current line of treatment for the diabetes mellitus is oral antidiabetic drugs (biguanides, sulfonylureas, meglitinides, thiazolidinediones and alpha-glycosidase inhibitors) and insulin therapy depending upon the type 1 or type 2 diabetes mellitus. But, these treatments have their disadvantages, ranging from the developing of resistance to the drugs and adverse effects caused by them. Alternative to these synthetic agents, natural products provides a new insight for the development of more efficient and safe drugs due to their therapeutic values. Enicostema littorale blume (A. Raynal) is a traditional Indian plant belongs to the Gentianaceae family. It is widely distributed in Asia, Africa, and South America. There are few reports on Swrtiamarin, major component of this plant for its antidiabetic activity. However, the antidiabetic activity of flavonoids from E. littorale and their mechanism of action have not yet been elucidated. Flavonoids have a positive relationship with disease prevention and can act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, adipocytes, hepatocytes and skeletal myofibers. They may exert beneficial effects in diabetes by (i) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (ii) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (iii) increasing glucose uptake in hepatocytes, skeletal muscle and white adipose tissue (iv) reducing insulin resistance, inflammation and oxidative stress. Therefore, we have isolated four flavonoid rich fractions, Fraction A (FA), Fraction B (FB), Fraction C (FC), Fraction D (FD) from crude alcoholic hot (AH) extract from E. littorale, identified by LC/MS. Total eight flavonoids were identified on the basis of fragmentation pattern. Flavonoid FA showed the presence of swertisin, isovitexin, and saponarin; FB showed genkwanin, quercetin, isovitexin, FC showed apigenin, swertisin, quercetin, 5-O-glucosylswertisin and 5-O-glucosylisoswertisin whereas FD showed the presence of swertisin. Further, these fractions were assessed for their antidiabetic activity on stimulating glucose uptake in insulin-resistant HepG2 cell line model (IR/HepG2). The results showed that FD containing C-glycoside Swertisin has significantly increased the glucose uptake rate of IR/HepG2 cells at the concentration of 10 µg/ml as compared to positive control Metformin (0.5mM) which was determined by glucose oxidase- peroxidase method. It has been reported that enhancement of glucose uptake of cells occurs due the translocation of Glut4 vesicles to cell membrane through IR/IRS1/AKT pathway. Therefore, we have studied expressions of three genes IRS1, AKT and Glut4 by real-time PCR to evaluate whether they follow the same pathway or not. It was seen that the glucose uptake rate has increased in FD treated IR/HepG2 cells due to the activation of insulin receptor substrate-1 (IRS1) followed by protein kinase B (AKT) through phosphoinositide 3-kinase (PI3K) leading to translocation of Glut 4 vesicles to cell membrane, thereby enhancing glucose uptake and insulin sensitivity of insulin resistant HepG2 cells. Hence, the up-regulation indicated the mechanism of action through which FD (Swertisin) acts as antidiabetic candidate in the treatment of type 2 diabetes mellitus.

Keywords: E. littorale, glucose transporter, glucose uptake rate, insulin resistance

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Authors: Khiem M. Nguyen, Ming C. Yang

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Chloroplast pigments are extremely important during photosynthesis since they play essential roles in light absorption and energy transfer. Therefore, understanding the efficiency of chlorophyll (Chl) biosynthesis could facilitate enhancement in photo-assimilates accumulation, and ultimately, in crop yield. The Chl-deficient mutants have been used extensively to study the Chl biosynthetic pathways and the biogenesis of the photosynthetic apparatus. Rice (Oryza sativa L.) is one of the most leading food crops, serving as staple food for many parts of the world. To author’s best knowledge, Chl b–lacking rice has been found; however the molecular mechanism of Chl biosynthesis still remains unclear compared to wild-type rice. In this study, the ultrastructure analysis, photosynthetic properties, and transcriptome profile of wild-type rice (Norin No.8, N8) and its Chl b-lacking mutant (Chlorina 1, C1) were examined. The finding concluded that total Chl content and Chl b content in the C1 leaves were strongly reduced compared to N8 leaves, suggesting that reduction in the total Chl content contributes to leaf color variation at the physiological level. Plastid ultrastructure of C1 possessed abnormal thylakoid membranes with loss of starch granule, large number of vesicles, and numerous plastoglobuli. The C1 rice also exhibited thinner stacked grana, which was caused by a reduction in the number of thylakoid membranes per granum. Thus, the different Chl a/b ratio of C1 may reflect the abnormal plastid development and function. Transcriptional analysis identified 23 differentially expressed genes (DEGs) and 671 transcription factors (TFs) that were involved in Chl metabolism, chloroplast development, cell division, and photosynthesis. The transcriptome profile and DEGs revealed that the gene encoding PsbR (PSII core protein) was down-regulated, therefore suggesting that the lower in light-harvesting complex proteins are responsible for the lower photosynthetic capacity in C1. In addition, expression level of cell division protein (FtsZ) genes were significantly reduced in C1, causing chloroplast division defect. A total of 19 DEGs were identified based on KEGG pathway assignment involving Chl biosynthesis pathway. Among these DEGs, the GluTR gene was down-regulated, whereas the UROD, CPOX, and MgCH genes were up-regulated. Observation through qPCR suggested that later stages of Chl biosynthesis were enhanced in C1, whereas the early stages were inhibited. Plastid structure analysis together with transcriptomic analysis suggested that the Chl a/b ratio was amplified both by the reduction in Chl contents accumulation, owning to abnormal chloroplast development, and by the enhanced conversion of Chl b to Chl a. Moreover, the results indicated the same Chl-cycle pattern in the wild-type and C1 rice, indicating another Chl b degradation pathway. Furthermore, the results demonstrated that normal grana stacking, along with the absence of Chl b and greatly reduced levels of Chl a in C1, provide evidence to support the conclusion that other factors along with LHCII proteins are involved in grana stacking. The findings of this study provide insight into the molecular mechanisms that underlie different Chl a/b ratios in rice.

Keywords: Chl-deficient mutant, grana stacked, photosynthesis, RNA-Seq, transcriptomic analysis

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Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

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The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

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Authors: K. Thiel, P. Patrikainen, C. Nagy, D. Fitzpatrick, E.-M. Aro, P. Kallio

Abstract:

Photosynthetic cyanobacteria have been recognized as potential future biotechnological hosts for the direct conversion of CO₂ into chemicals of interest using sunlight as the solar energy source. However, in order to develop commercially viable systems, the flux of electrons from the photosynthetic light reactions towards specified target chemicals must be significantly improved. The objective of the study was to investigate whether the autotrophic production efficiency of specified end-metabolites can be improved in engineered cyanobacterial cells by rescuing excited electrons that are normally lost to molecular oxygen due to the cyanobacterial flavodiiron protein Flv1/3. Natively Flv1/3 dissipates excess electrons in the photosynthetic electron transfer chain by directing them to molecular oxygen in Mehler-like reaction to protect photosystem I. To evaluate the effect of flavodiiron inactivation on autotrophic production efficiency in the cyanobacterial host Synechocystis sp. PCC 6803 (Synechocystis), sucrose was selected as the quantitative reporter and a representative of a potential end-product of interest. The concept is based on the native property of Synechocystis to produce sucrose as an intracellular osmoprotectant when exposed to high external ion concentrations, in combination with the introduction of a heterologous sucrose permease (CscB from Escherichia coli), which transports the sucrose out from the cell. In addition, cell growth, photosynthetic gas fluxes using membrane inlet mass spectrometry and endogenous storage compounds were analysed to illustrate the consequent effects of flv deletion on pathway flux distributions. The results indicate that a significant proportion of the electrons can be lost to molecular oxygen via Flv1/3 even when the cells are grown under high CO₂ and that the inactivation of flavodiiron activity can enhance the photosynthetic electron flux towards optionally available sinks. The flux distribution is dependent on the light conditions and the genetic context of the Δflv mutants, and favors the production of either sucrose or one of the two storage compounds, glycogen or polyhydroxybutyrate. As a conclusion, elimination of the native Flv1/3 reaction and concomitant introduction of an engineered product pathway as an alternative sink for excited electrons could enhance the photosynthetic electron flux towards the target endproduct without compromising the fitness of the host.

Keywords: cyanobacterial engineering, flavodiiron proteins, redirecting electron flux, sucrose

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Authors: Dhruvit S. Berawala, Jann R. Ursin, Obrad Slijepcevic

Abstract:

Shale gas is one of the most rapidly growing forms of natural gas. Unconventional natural gas deposits are difficult to characterize overall, but in general are often lower in resource concentration and dispersed over large areas. Moreover, gas is densely packed into the matrix through adsorption which accounts for large volume of gas reserves. Gas production from tight shale deposits are made possible by extensive and deep well fracturing which contacts large fractions of the formation. The conventional reservoir modelling and production forecasting methods, which rely on fluid-flow processes dominated by viscous forces, have proved to be very pessimistic and inaccurate. This paper presents a new approach to forecast shale gas production by detailed modeling of gas desorption, diffusion and non-linear flow mechanisms in combination with statistical representation of these processes. The representation of the model involves a cube as a porous media where free gas is present and a sphere (SiC: Sphere in Cube model) inside it where gas is adsorbed on to the kerogen or organic matter. Further, the sphere is considered consisting of many layers of adsorbed gas in an onion-like structure. With pressure decline, the gas desorbs first from the outer most layer of sphere causing decrease in its molecular concentration. The new available surface area and change in concentration triggers the diffusion of gas from kerogen. The process continues until all the gas present internally diffuses out of the kerogen, gets adsorbs onto available surface area and then desorbs into the nanopores and micro-fractures in the cube. Each SiC idealizes a gas pathway and is characterized by sphere diameter and length of the cube. The diameter allows to model gas storage, diffusion and desorption; the cube length takes into account the pathway for flow in nanopores and micro-fractures. Many of these representative but general cells of the reservoir are put together and linked to a well or hydraulic fracture. The paper quantitatively describes these processes as well as clarifies the geological conditions under which a successful shale gas production could be expected. A numerical model has been derived which is then compiled on FORTRAN to develop a simulator for the production of shale gas by considering the spheres as a source term in each of the grid blocks. By applying SiC to field data, we demonstrate that the model provides an effective way to quickly access gas production rates from shale formations. We also examine the effect of model input properties on gas production.

Keywords: adsorption, diffusion, non-linear flow, shale gas production

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