Search results for: purified protein

Authors: Yamina Boukari, Nashiru Billa, Andrew Morris, Stephen Doughty, Kevin Shakesheff

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Poly (lactic-co-glycolic) acid (PLGA) is a synthetic polymer that can be used in bone tissue engineering with the aim of creating a scaffold in order to support the growth of cells. The formation of microspheres from this polymer is an attractive strategy that would allow for the development of an injectable system, hence avoiding invasive surgical procedures. The aim of this study was to develop a microsphere delivery system for use as an injectable scaffold in bone tissue engineering and evaluate various formulation parameters on its properties. Porous and lysozyme-containing PLGA microspheres were prepared using the double emulsion solvent evaporation method from various molecular weights (MW). Scaffolds were formed by sintering to contain 1 -3mg of lysozyme per gram of scaffold. The mechanical and physical properties of the scaffolds were assessed along with the release of lysozyme, which was used as a model protein. The MW of PLGA was found to have an influence on microsphere size during fabrication, with increased MW leading to an increased microsphere diameter. An inversely proportional relationship was displayed between PLGA MW and mechanical strength of formed scaffolds across loadings for low, intermediate and high MW respectively. Lysozyme release from both microspheres and formed scaffolds showed an initial burst release phase, with both microspheres and scaffolds fabricated using high MW PLGA showing the lowest protein release. Following the initial burst phase, the profiles for each MW followed a similar slow release over 30 days. Overall, the results of this study demonstrate that lysozyme can be successfully incorporated into porous PLGA scaffolds and released over 30 days in vitro, and that varying the MW of the PLGA can be used as a method of altering the physical properties of the resulting scaffolds.

Keywords: bone, microspheres, PLGA, tissue engineering

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Authors: Faheema Khan

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To investigate the ability of sensitive and tolerant genotype of Glycine max to adapt to a saline environment in a field, we examined the growth performance, water relation and activities of antioxidant enzymes in relation to photosynthetic rate, chlorophyll a fluorescence, photosynthetic pigment concentration, protein and proline in plants exposed to salt stress. Ten soybean genotypes (Pusa-20, Pusa-40, Pusa-37, Pusa-16, Pusa-24, Pusa-22, BRAGG, PK-416, PK-1042, and DS-9712) were selected and grown hydroponically. After 3 days of proper germination, the seedlings were transferred to Hoagland’s solution (Hoagland and Arnon 1950). The growth chamber was maintained at a photosynthetic photon flux density of 430 μmol m−2 s−1, 14 h of light, 10 h of dark and a relative humidity of 60%. The nutrient solution was bubbled with sterile air and changed on alternate days. Ten-day-old seedlings were given seven levels of salt in the form of NaCl viz., T1 = 0 mM NaCl, T2=25 mM NaCl, T3=50 mM NaCl, T4=75 mM NaCl, T5=100 mM NaCl, T6=125 mM NaCl, T7=150 mM NaCl. The investigation showed that genotype Pusa-24, PK-416 and Pusa-20 appeared to be the most salt-sensitive. genotypes as inferred from their significantly reduced length, fresh weight and dry weight in response to the NaCl exposure. Pusa-37 appeared to be the most tolerant genotype since no significant effect of NaCl treatment on growth was found. We observed a greater decline in the photosynthetic variables like photosynthetic rate, chlorophyll fluorescence and chlorophyll content, in salt-sensitive (Pusa-24) genotype than in salt-tolerant Pusa-37 under high salinity. Numerous primers were verified on ten soybean genotypes obtained from Operon technologies among which 30 RAPD primers shown high polymorphism and genetic variation. The Jaccard’s similarity coefficient values for each pairwise comparison between cultivars were calculated and similarity coefficient matrix was constructed. The closer varieties in the cluster behaved similar in their response to salinity tolerance. Intra-clustering within the two clusters precisely grouped the 10 genotypes in sub-cluster as expected from their physiological findings.Salt tolerant genotype Pusa-37, was further analysed by 2-Dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the Present study, 173 protein spots were identified. Of these, 40 proteins responsive to salinity were either up- or down-regulated in Pusa-37. Proteomic analysis in salt-tolerant genotype (Pusa-37) led to the detection of proteins involved in a variety of biological processes, such as protein synthesis (12 %), redox regulation (19 %), primary and secondary metabolism (25 %), or disease- and defence-related processes (32 %). In conclusion, the soybean plants in our study responded to salt stress by changing their protein expression pattern. The photosynthetic, biochemical and molecular study showed that there is variability in salt tolerance behaviour in soybean genotypes. Pusa-24 is the salt-sensitive and Pusa-37 is the salt-tolerant genotype. Moreover this study gives new insights into the salt-stress response in soybean and demonstrates the power of genomic and proteomic approach in plant biology studies which finally could help us in identifying the possible regulatory switches (gene/s) controlling the salt tolerant genotype of the crop plants and their possible role in defence mechanism.

Keywords: glycine max, salt stress, RAPD, genomic and proteomic variability

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Authors: Yasmin M. Attia, Hanan S. El-Abhar, Mahmoud M. Al Marzabani, Samia A. Shouman

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Background: Tamoxifen (TAM) is the most commonly used hormone therapy for the treatment of early and metastatic breast cancer. Although it significantly decreases the tumor recurrence rate and provides an overall benefit, as much as 20–30% of women still relapse during or after long-term therapy. 3-Bromopyruvate (3-BP) is a promising agent with impressive antitumor effects in several models of animal tumors and cell lines. Aim: This study was designed to investigate the combined effect of (TAM) and (3-BP) in breast cancer cells and to explore their molecular interaction via assessment of apoptotic, angiogenic, and metastatic markers. Methods: In vitro cytotoxicity study was carried out for both compounds to determine the combination regimen producing a synergistic effect and mechanistic pathways were studied using RT-PCR and western techniques. Moreover, the anti-oncolytic and anti-angiogenic potentials were assessed in mice bearing solid Ehrlich carcinoma (SEC). Results: The combined treatment significantly increased the expressions and protein levels of caspase 7, 9, and 3 and decreased of angiogenic markers VEGF, HIF-1α, and HK2 compared to cells treated with either drug individually. However, there were no significant changes in MMP-2 and MMP-9 protein levels. Interestingly, the in vivo results supported the in vitro findings; there was a decrease in the tumor volume and VEFG using immunohistochemistry in the combination-treated groups compared to either TAM or 3-BP treated one. Conclusion: 3-BP synergizes the cytotoxic effect of TAM by increasing apoptosis and decreasing angiogenesis which makes this combination a promising regimen to be applied clinically.

Keywords: tamoxifen, 3-bromopyruvate, breast cancer, cytotoxicity, angiogenesis

Procedia PDF Downloads 205

Authors: Javier Enciso-Benavides, Fredy Fabian, Carlos Castaneda, Luis Alfaro, Alex Choque, Aparicio Aguilar, Javier Enciso

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Background: The use of biomarkers in breast cancer diagnosis, therapy, and prognosis has gained increasing interest. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and may cause relapse. Therefore, due to the importance of diagnosis, therapy, and prognosis, several biomarkers that characterize CSCs have been identified; however, in treatment-naïve triple-negative breast tumors, there is an urgent need to identify new biomarkers and therapeutic targets. According to this, the aim of this study was to identify serum proteins associated with cancer stem cells and pluripotency in women with triple-negative breast tumors in order to subsequently identify a biomarker for this type of breast tumor. Material and Methods: Whole blood samples from 12 women with histopathologically diagnosed triple-negative breast tumors were used after obtaining informed consent from the patient. Blood serum was obtained by conventional procedure and frozen at -80ºC. Identification of cancer stem cell-associated proteins was performed by matrix-assisted laser desorption/ionisation-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS), protein analysis was obtained using the AB Sciex TOF/TOF™ 5800 system (AB Sciex, USA). Sequences not aligned by ProteinPilot™ software were analyzed by Protein BLAST. Results: The following proteins related to pluripotency and cancer stem cells were identified by MALDI TOF/TOF mass spectrometry: A-chain, Serpin A12 [Homo sapiens], AIEBP [Homo sapiens], Alpha-one antitrypsin, AT {internal fragment} [human, partial peptide, 20 aa] [Homo sapiens], collagen alpha 1 chain precursor variant [Homo sapiens], retinoblastoma-associated protein variant [Homo sapiens], insulin receptor, CRA_c isoform [Homo sapiens], Hydroxyisourate hydrolase [Streptomyces scopuliridis], MUCIN-6 [Macaca mulatta], Alpha-actinin-3 [Chrysochloris asiatica], Polyprotein M, CRA_d isoform, partial [Homo sapiens], Transcription factor SOX-12 [Homo sapiens]. Recommendations: The serum proteins identified in this study should be investigated in the exosome of triple-negative breast cancer stem cells and in the blood serum of women without breast cancer. Subsequently, proteins found only in the blood serum of women with triple-negative breast cancer should be identified in situ in triple-negative breast cancer tissue in order to identify a biomarker to study the evolution of this type of cancer, or that could be a therapeutic target. Conclusions: Eleven cancer stem cell-related serum proteins were identified in 12 women with triple-negative breast cancer, of which MUCIN-6, retinoblastoma-associated protein variant, transcription factor SOX-12, and collagen alpha 1 chain are the most representative and have not been studied so far in this type of breast tumor. Acknowledgement: This work was supported by Proyecto CONCYTEC–Banco Mundial “Mejoramiento y Ampliacion de los Servicios del Sistema Nacional de Ciencia Tecnología e Innovacion Tecnologica” 8682-PE (104-2018-FONDECYT-BM-IADT-AV).

Keywords: triple-negative breast cancer, MALDI TOF/TOF MS, serum proteins, cancer stem cells

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Authors: Bhawna Chandravanshi, Ramesh Bhonde

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In the present study, we focused on the improvisation of islet survival in hypoxia. The Islet-like cell aggregates (ICAs) derived from Wharton's jelly mesenchymal stem cells (WJ-MSC) were cultured with and without WJ-MSC for 48h in hypoxia and normoxia and tested for their direct trophic effect on β cell survival. The WJ MSCs themselves secreted insulin upon glucose challenge and expressed the pancreatic markers at both transcription and translational level (C-peptide, Insulin, Glucagon and Glut 2). Direct contact of MSCs with ICAs facilitate the highest viability under hypoxia as evidenced by fluorescein diacetate/propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytokine analysis of the co-cultured ICAs revealed amplification of anti-inflammatory cytokine-like TGFβ and TNFα accompanied by depletion of pro-inflammatory cytokines. The increment in VEGF and PDGFa was also seen showing their ability to vascularize upon transplantation. This was further accompanied by reduction in total reactive oxygen species, nitric oxide, and super oxide ions and down-regulation of Caspase3, Caspase8, p53 and up regulation of Bcl2 confirming prevention of apoptosis in ICAs. There was a significant reduction in the expression of p38 protein in the presence of MSCs making the ICAs responsive to glucose. Taken together our data demonstrate for the first time that the WJ-MSC expressed pancreatic markers and their supplementation protected engineered islets against hypoxia, oxidative stress, and inflammatory cytokines by inhibiting p38 MAPK protein.

Keywords: hypoxia, islet-like cell aggregates, inflammatory cytokines, oxidative stress

Procedia PDF Downloads 235

Authors: M. Cabral, A. Verdin, G. Garçon, A. Touré, C. Diop, M. Fall, S. Bouhsina, D. Dewaele, F.Cazier, A. Tall Dia, P. Shirali, A. Diouf

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This case-control study dealt with the health adverse effects within the population neighboring the Mbeubeuss waste dump, which is located near the district of Malika (Diamalaye II) in Dakar (Senegal). All the household and industrial waste arising from Dakar are stored in this open landfill without being covered and are therefore possible sources of Pb and Cd contaminated air emissions and lixiviates. The objective of this study is part of improving the health of the population neighboring Mbeubeuss by determining Pb and Cd concentrations both in environment and humans, and studying possible renal function alterations within the adults. Soil and air samples were collected in the control site (Darou Salam) and the waste dump neighboring site (Diamalaye II). Control and exposed adults were recruited as living in Darou Salam (n = 52) and in Diamalaye II (n = 77). Pb and Cd concentrations in soil, air and biological samples were determined. Moreover, we were interested in analyzing some impregnation (zinc protoporphyrin, d-aminolevulinic acid dehydratase) and oxidative stress biomarkers (malonedialdehyde, gluthatione status), in addition to several nephrotoxicity parameters (creatinuria, proteinuria, lactate dehydrogenase, CC16 protein, glutathione S-transferase-alpha and retinol binding protein) in blood and/or urine. The results showed the significant Pb and Cd contamination of the soil and air samples derived from the landfill, and therefore of the neighboring population of adults. This critical exposure to environmental Pb and Cd had some harmful consequences for their health, as shown by the reported oxidative stress and nephrotoxicity signs.

Keywords: Pb and Cd environmental exposure, impregnation markers, landfill, nephrotoxicity markers

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Authors: Yew Mun Yip, Dawei Zhang

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Proteins are the biological machinery that executes specific vital functions in every cell of the human body by folding into their 3D structures. When a protein misfolds from its native structure, the machinery will malfunction and lead to misfolding diseases. Although in vitro experiments are able to conclude that the mutations of the amino acid sequence lead to incorrectly folded protein structures, these experiments are unable to decipher the folding process. Therefore, molecular dynamic (MD) simulations are employed to simulate the folding process so that our improved understanding of the folding process will enable us to contemplate better treatments for misfolding diseases. MD simulations make use of force fields to simulate the folding process of peptides. Secondary structures are formed via the hydrogen bonds formed between the backbone atoms (C, O, N, H). It is important that the hydrogen bond energy computed during the MD simulation is accurate in order to direct the folding process to the native structure. Since the atoms involved in a hydrogen bond possess very dissimilar electronegativities, the more electronegative atom will attract greater electron density from the less electronegative atom towards itself. This is known as the polarization effect. Since the polarization effect changes the electron density of the two atoms in close proximity, the atomic charges of the two atoms should also vary based on the strength of the polarization effect. However, the fixed atomic charge scheme in force fields does not account for the polarization effect. In this study, we introduce the polarized structure-specific backbone charge (PSBC) model. The PSBC model accounts for the polarization effect in MD simulation by updating the atomic charges of the backbone hydrogen bond atoms according to equations derived between the amount of charge transferred to the atom and the length of the hydrogen bond, which are calculated from quantum-mechanical calculations. Compared to other polarizable models, the PSBC model does not require quantum-mechanical calculations of the peptide simulated at every time-step of the simulation and maintains the dynamic update of atomic charges, thereby reducing the computational cost and time while accounting for the polarization effect dynamically at the same time. The PSBC model is applied to two different β-peptides, namely the Beta3s/GS peptide, a de novo designed three-stranded β-sheet whose structure is folded in vitro and studied by NMR, and the trpzip peptides, a double-stranded β-sheet where a correlation is found between the type of amino acids that constitute the β-turn and the β-propensity.

Keywords: hydrogen bond, polarization effect, protein folding, PSBC

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Authors: Mahzad Yousefi, Azin Tavakoli

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Periodontal disease is an inflammatory reaction; therefore, it is predicted that changes may occur in some inflammatory parameters that can be detected in routine blood tests. The objective of this study was to evaluate the hematological and biochemistry changes that occur in dogs affected with advanced stages of periodontal disease. 87 dogs were diagnosed with periodontal disease (PD group), and 76 healthy dogs entered the study. The PD dogs had been affected with periodontitis stage 3 or 4 and were candidates for any dental extractions. The healthy dogs were either referred for annual checkups or for issuing health travel certificates that their owners asked for complete lab tests. Neither the diseased nor healthy subjects had a history of infectious, or other general health problems or surgery in the past 3 months. Age, as well as all hematologic including PCV, WBC and RBC count, Hb, MCV, MCH, MCHC, PLT, CBC, NLR, and biochemistry data, including total protein, albumin, glucose, BUN, Creatinine, ALT, AST, and ALP, were recorded and analyzed statistically. Results confirmed that aging has a significant direct relationship with the advanced stages of periodontal disease. Mild leukocytosis occurred in the diseased group; however, it was not significant. Also, the mean total protein of the PD group was lower than that of the healthy dogs, and serum levels of albumin were found to be lower significantly in the diseased group (P<0.05). Mean ±SD amount of Platelet, MCH, and ALT were significantly higher in the diseased group in comparison to the healthy dogs (P<0.05). No significant differences were reported in other evaluated parameters. It is concluded that CBC indices of PD dogs are not systemic inflammatory; however, only a decrease in albumin showed inflammatory responses. Some indices in routine laboratory tests can be changed significantly during advanced stages of the periodontal disease dogs.

Keywords: periodontal disease, dogs, hematological factors, advanced stages, blood tests

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Authors: M. Y. Abubakar, M. Yunisa, A. N. Muhammad

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Constraining the production Clarias gariepinus and Oreochromis niloticus culture is the prohibitive cost of feed. We assess the performance of the species fingerlings on diets substituted with composite. Four dietary treatments (0%, 25%, 45%, and 75%) for C. gariepinus and five (0%, 25%, 50%, 75%, and whole food composite) for O. niloticus were formulated and each fed to 15 fingerlings for C. gariepinus and 10 fingerlings for O. niloticus stocked in 75ltrs plastic bowls, replicated trice in a completely randomized design. The experiment lasted 56 days. Percent survival rate was significantly (p < 0.05) higher (57.78 ± 9.69) in C. gariepinus fed diet III. The growth and nutrient utilization indices were least in the fish fed diet IV, which was significantly (p < 0.05) lower than in other treatments. Fish fed dietary treatment III, recorded the best in growth and nutrient utilization indices and was significantly higher (p < 0.05) than those fed dietary treatments I & II which were non-significant (p > 0.05) and higher than those fed 75% substitution. Better profit index was in the fish fed diet with 50% substitution level. For O. niloticus, the survival (172.62 ± 39.03) was significantly higher (p < 0.05) in those fed 25% substituted diet. For growth indices, the least performed were those fed whole composite while other treatments were non-significant (p > 0.05) different from each other. In terms of nutrient utilization, fish fed diet substituted at 0%, 25%, 50% and 75% food composite had similar food conversion ratio and protein efficiency ratio. However, there was no significant difference in the profit index among the whole treatment. It can be concluded that food composite from Sokoto house-holds can optimally replace groundnut cake up to 50% level as a protein source in the diets of Clarias gariepinus and O. niloticus fingerlings without adverse effects on survival, growth, and nutrient utilization.

Keywords: food composite, nutrient utilization, C. gariepinus, O. niloticus household, substitution levels

Procedia PDF Downloads 143

Authors: F. Yılmaz, Y. Saylan, A. Derazshamshir, S. Atay, A. Denizli

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Quartz crystal microbalance (QCM), high-resolution mass-sensing technique, measures changes in mass on oscillating quartz crystal surface by measuring changes in oscillation frequency of crystal in real time. Protein adsorption techniques via hydrophobic interaction between protein and solid support, called hydrophobic interaction chromatography (HIC), can be favorable in many cases. Some nanoparticles can be effectively applied for HIC. HIC takes advantage of the hydrophobicity of proteins by promoting its separation on the basis of hydrophobic interactions between immobilized hydrophobic ligands and nonpolar regions on the surface of the proteins. Lysozyme is found in a variety of vertebrate cells and secretions, such as spleen, milk, tears, and egg white. Its common applications are as a cell-disrupting agent for extraction of bacterial intracellular products, as an antibacterial agent in ophthalmologic preparations, as a food additive in milk products and as a drug for treatment of ulcers and infections. Lysozyme has also been used in cancer chemotherapy. The aim of this study is the synthesis of hydrophobic nanoparticles for Lysozyme detection. For this purpose, methacryoyl-L-phenylalanine was chosen as a hydrophobic matrix. The hydrophobic nanoparticles were synthesized by micro-emulsion polymerization method. Then, hydrophobic QCM nanosensor was characterized by Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM) and zeta size analysis. Hydrophobic QCM nanosensor was tested for real-time detection of Lysozyme from aqueous solution. The kinetic and affinity studies were determined by using Lysozyme solutions with different concentrations. The responses related to a mass (Δm) and frequency (Δf) shifts were used to evaluate adsorption properties.

Keywords: nanosensor, HIC, lysozyme, QCM

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

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Authors: Shreya Ghosh, Akansha Garg, Chayanika Kala, Ashwani Kumar Thakur

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Background: Granuloma formation is one of the characteristic features of tuberculosis. Besides, chronic inflammation underlying tuberculosis is often indicated by an increase in the concentration of serum amyloid A (SAA) protein. The connection between tuberculosis and SAA-driven secondary amyloidosis is well documented. However, SAA-derived amyloid deposition start sites are not well understood in tuberculosis and other chronic inflammatory conditions. It was hypothesized that granuloma could be a potential site for an amyloid deposition because both SAA protein and proteases that cleave SAA into aggregation-prone fragments are reported to be present in the granuloma. Here the authors have shown the presence of SAA-derived amyloid deposits in the granuloma of tuberculosis patients. Methodology: Over a period of two years, tuberculosis patients were screened, and biopsies were collected from the affected organs of the patients. The gold standard, Congo red dye staining, was used to identify amyloid deposits in the tissue sections of tuberculosis patients containing granulomatous structure. Results: 11 out of 150 FFPE biopsy specimens of tuberculosis patients showed eosinophilic hyaline-rich deposits surrounding granuloma. Upon Congo red staining, these deposits exhibited characteristic apple-green birefringence under polarized light, confirming amyloid deposits. Further, upon immunohistochemical staining with anti-SAA, the amyloid enriched areas showed positive immunoreactivity. Conclusion: In this pilot study, we have shown that granuloma can be a potential site for serum amyloid A-derived amyloid formation in tuberculosis patients. Moreover, the presence of amyloid gave significant cues that granuloma might be a probable amyloid deposition start in tuberculosis patients. This study will set a stage to expand the clinical and fundamental research in the understanding of amyloid formation in granuloma underlying tuberculosis and chronic inflammatory conditions.

Keywords: amyloid, granuloma, periphery, serum amyloid A, tuberculosis

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

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For chemotherapy, a drug delivery system should be able to specifically target cancer cells and deliver the therapeutic dose without affecting normal cells. Folate receptors (FR) can be considered key targets since they are commonly over-expressed in cancer cells and they are the molecular marker used in this study. Here, cyclodextrin (CD) has being studied as a vehicle for delivering the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within the CD cavity. In this study, β-CD has been modified using folic acid so as to specifically target the FR molecular marker. Thus, the system studied here for drug delivery consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 9.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 10.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 8.5 for A549 and 132.6 µM ± 12.1 and 288.1 µM ± 16.3 for BEAS-2B. These results demonstrate that MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug.

Keywords: cyclodextrins, cancer treatment, drug delivery, folate receptors, reduced folate carriers

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Authors: Shu-Pin Huang, Pai-Chi Teng, Hao-Han Chang, Chia-Hsin Liu, Yung-Lun Lin, Shu-Chi Wang, Hsin-Chih Yeh, Chih-Pin Chuu, Jiun-Hung Geng, Li-Hsin Chang, Wei-Chung Cheng, Chia-Yang Li

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The emergence of immune checkpoint inhibitors (ICIs) targeting proteins like PD-1 and PD-L1 has changed the treatment paradigm of bladder cancer. However, not all patients benefit from ICIs, with some experiencing early death. There's a significant need for biomarkers associated with the PD-1 pathway in bladder cancer. Current biomarkers focus on tumor PD-L1 expression, but a more comprehensive understanding of PD-1-related biology is needed. Our study has developed a seven-gene risk score panel, employing a comprehensive bioinformatics strategy, which could serve as a potential prognostic and predictive biomarker for bladder cancer. This panel incorporates the FYN, GRAP2, TRIB3, MAP3K8, AKT3, CD274, and CD80 genes. Additionally, we examined the relationship between this panel and immune cell function, utilizing validated tools such as ESTIMATE, TIDE, and CIBERSORT. Our seven-genes panel has been found to be significantly associated with bladder cancer survival in two independent cohorts. The panel was also significantly correlated with tumor infiltration lymphocytes, immune scores, and tumor purity. These factors have been previously reported to have clinical implications on ICIs. The findings suggest the potential of a PD-1 pathway-based transcriptomic panel as a prognostic and predictive biomarker in bladder cancer, which could help optimize treatment strategies and improve patient outcomes.

Keywords: bladder cancer, programmed cell death protein 1, prognostic biomarker, immune checkpoint inhibitors, predictive biomarker

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Authors: Uchegbu Nneka Nkechi, Okoye Ogochukwu Peace

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The evaluation of the visco-elastic properties and microbial quality of a formulated oat-based dietetic food were investigated. Oat flour, pumpkin seed flour, carrot flour and skimmed milk powder were blended in varying proportions to formulate a product with codes OCF, which contains 70% oat flour, 10 % carrot flour, 10 % pumpkin seed flour and 10% skimmed milk powder, OCF which contains 65 % oat flour, 10 % carrot flour, 10 % pumpkin seed flour and 15 % skimmed milk powder, OCF which contains 60 % oat flour, 10 % carrot flour, 10 % pumpkin seed flour and 20 % skimmed milk powder, OCF which contains 55 % oat flour, 10 % carrot flour, 10 % pumpkin seed flour and 25 % skimmed milk powder and OF with 95 % oat as the commercial control. All the samples were assessed for their proximate composition, microbial quality and visco-elastic properties. The moisture content was highest at sample OF (10.73%) and lowest at OCF (7.10%) (P<0.05). Crude protein ranged from 13.38%-22.86%, with OCF having the highest (P<0.05) protein content and OF having the lowest. Crude fat was 3.74% for OCF and 6.31% for OF. Crude fiber ranged from 3.58% - 17.39% with OF having the lowest (P<0.05) fiber content and OCF having the highest. Ash content was 1.30% for OCF and 2.75% for OCF. There was no mold growth in the samples. The total viable ml/wl count ranged from 1.5×10³ cfu/g - 2.6×10³ cfu/g, with OCF having the lowest and OF having the highest (P<0.05) total viable count. The peak viscosity of the sample ranged from 75.00 cP-2895.00 cP, with OCF having the lowest and OF having the highest value. The final viscosity was 130.50 cP in OCF and 3572.50 cP in OF. The setback viscosity was 58.00 cP in OCF and 1680.50 cP in OF. The peak time was 6.93 mins in OCF to 5.57 mins in OF. There was no pasting temperature for all samples except the OF, which had 86.43. Sample OF was the highest in terms of overall acceptability. This study showed that the oat-based composite flour produced had a nutritional profile that would be acceptable for the aged population.

Keywords: dietetic, pumpkin, visco-elastic, microbial

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Authors: Arfan Ali, Idrees Ahmad Nasir

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The critical evaluation of tolerance in tomato plants against the induced saline soil were assessed by transcript analysis of genes coding for products potentially involved in stress tolerance. A reverse transcriptase PCR experiment was performed with Hsp90-1, MT2, and GR1like protein genes using RNA isolated from different tissues of tomato plants. Four strains of Bacillus magisterium were inoculated with 100 Mm & 200 Mm concentrations of salt. Eleven treatments each ten replica pots were installed in green house experiment and the parameters taken into account were morphological (length, weight, number of leaves, leaf surface area), chemical (anthocyanin, chlorophyll-a, chlorophyll-b, carotenoids) and biological (gene expression). Results bare a response i.e. highest response of MT2 like gene was at 24 hpi and the highest levels of GR1 like protein transcript accumulation were detected at 36 hpi. The chemical and morphological parameters at diverse salt concentrations bequeath superlative response amongst strains which candidly flank on Zm7 and Zm4. Therefore, Bacillus magisterium Zm7 strains and somehow Zm4 strain can be used in saline condition to make plants tolerant. The overall performance of strains Zm7, Zm6, and Zm4 was found better for all studied traits under salt stress conditions. Significant correlations among traits root length, shoot length, number of leaves, leaf surface area, carotenoids, anthocyanin, chlorophyll-a and chlorophyll-b were found and suggested that the salt tolerance in tomato may be improved through the use of PGPR strains.

Keywords: Bacillus magisterium, gene expression glutathione reductase, metallothionein, PGPR, Rhizobacteria, saline

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Authors: Sachin K. Samuchiwal, Barbara Balestrieri, Amanda Paskavitz, Hannah Raff, Joshua A. Boyce

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IL-33, a recently discovered member of the IL-1 cytokine family, binds to the TLR/IL1R super family receptor ST2 and induces type 2 immune responses. IL-33 is constitutively expressed in structural cells at barrier sites such as skin, lung, and intestine, and also inducibly expressed by hematopoietic cells including macrophages. Stimulation of macrophages by Lipopolysaccharide (LPS) can induce de novo IL-33 expression, and also causes the production of prostaglandin-E2 (PGE2) via cyclooxygenase (COX)-2 and microsomal PGE2 synthase-1 (mPGES-1). Because PGE2 can regulate macrophage functions through both autocrine and paracrine mechanisms, the potential interplay of endogenous PGE2 on IL-33 production was explored. Bone-marrow derived murine macrophages (bmMF) that lack either mPGES-1 or EP2 receptor expression were stimulated with LPS in the absence or presence of exogenous PGE2 along with pharmacological agonists and antagonists. The study results demonstrate that endogenous PGE2 markedly enhances LPS-induced IL-33 production by bmMFs via EP2 receptors. Moreover, exogenous PGE2 can amplify LPS-induced IL-33 expression dominantly by EP2 and partly by EP4 receptors by a pathway involving cAMP and exchange protein activated by cAMP (EPAC), but not protein kinase A (PKA). Though both IL-33 production and PGE2 generation in response to LPS require activation of both p38 MAPK and NF-κB, PGE2 did not influence this activation. In conclusion, it is demonstrated that endogenous PGE2 signaling through EP2 and EP4 receptors is a prerequisite for LPS-induced IL-33 production in bmMFs and the underlying cAMP mediated pathway involves EPAC. Since IL-33 is a critical pro-inflammatory cytokine in various pathological disorders, this PGE2-EP2/EP4-cAMP mediated pathway can be exploited to intervene in IL-33 driven pathologies.

Keywords: bone marrow macrophages, EPAC, IL-33, PGE2

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Authors: Olfat Shaker, Miriam Wadie, Reham Ali, Ayman Yosry

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Colorectal cancer (CRC) is a major cause of mortality and morbidity and accounts for over 9% of cancer incidence worldwide. Silent information regulator 2 homolog 1 (SIRT1) gene is located in the nucleus and exert its effects via modulation of histone and non-histone targets. They function in the cell via histone deacetylase (HDAC) and/or adenosine diphosphate ribosyl transferase (ADPRT) enzymatic activity. The aim of this work was to study the relationship between SIRT1 polymorphism and its protein level in colorectal cancer patients in comparison to control cases. This study includes 2 groups: thirty healthy subjects (control group) & one hundred CRC patients. All subjects were subjected to: SIRT-1 serum level was measured by ELISA and gene polymorphisms of rs12778366, rs375891 and rs3740051 were detected by real time PCR. For CRC patients clinical data were collected (size, site of tumor as well as its grading, obesity) CRC patients showed high significant increase in the mean level of serum SIRT-1 compared to control group (P<0.001). Mean serum level of SIRT-1 showed high significant increase in patients with tumor size ≥5 compared to the size < 5 cm (P<0.05). In CRC patients, percentage of T allele of rs12778366 was significantly lower than controls, CC genotype and C allele C of rs 375891 were significantly higher than control group. In CRC patients, the CC genotype of rs12778366, was 75% in rectosigmoid and 25% in cecum & ascending colon. According to tumor size, the percentage of CC genotype was 87.5% in tumor size ≥5 cm. Conclusion: serum level of SIRT-1 and T allele, C allele of rs12778366 and rs 375891 respectively can be used as diagnostic markers for CRC patients.

Keywords: CRC, SIRT1, polymorphisms, ELISA

Procedia PDF Downloads 197

Authors: Przemyslaw Nogly, Tobias Weinert, Daniel James, Sergio Carbajo, Dmitry Ozerov, Antonia Furrer, Dardan Gashi, Veniamin Borin, Petr Skopintsev, Kathrin Jaeger, Karol Nass, Petra Bath, Robert Bosman, Jason Koglin, Matthew Seaberg, Thomas Lane, Demet Kekilli, Steffen Brünle, Tomoyuki Tanaka, Wenting Wu, Christopher Milne, Thomas A. White, Anton Barty, Uwe Weierstall, Valerie Panneels, Eriko Nango, So Iwata, Mark Hunter, Igor Schapiro, Gebhard Schertler, Richard Neutze, Jörg Standfuss

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Ultrafast isomerization of retinal is the primary step in a range of photoresponsive biological functions including vision in humans and ion-transport across bacterial membranes. We studied the sub-picosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin using an X-ray laser. Twenty snapshots with near-atomic spatial and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket prior to passing through a highly-twisted geometry and emerging in the 13-cis conformation. The aspartic acid residues and functional water molecules in proximity of the retinal Schiff base respond collectively to formation and decay of the initial excited state and retinal isomerization. These observations reveal how the protein scaffold guides this remarkably efficient photochemical reaction.

Keywords: bacteriorhodopsin, free-electron laser, retinal isomerization mechanism, time-resolved crystallography

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Authors: Mohammad A. M. Kewisha

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Xerophilic fungi , which are responsible for many cases of biodeterioration monuments, have been known as an interesting source of antimicrobial compounds. Sixty nine fungal strains, isolated from different localities and species inside Egyptian museums, were screened for antimicrobial activity against some bacterial species and unicellular fungi. The most potent antimicrobial activity was obtained by Asperigillus fumigatus which was identified by ITS4 ……. and showed activity against Staphylococcus aureus with 20 mm and C. albicans with18 mm of inhibition zone. Different parameters were optimized to enhance this activity. The culture grown under stationary conditions for 8 days at 30°C and pH 8 gave the best antimicrobial activity. Moreover, both starch and yeast extract showed the most suitable carbon and nitrogen sources, respectively. The antimicrobial compound was purified and subjected to spectroscopic characterization, which revealed that the antimicrobial compound might be 5,7 ethoxy, 4\,5\ methoxy isorhamnetin -3- O- galactoside. This study suggests that Aspergillus fumagates as a potential candidate offering a better scope for the production, purification and isolation of broad-spectrum antimicrobial compounds. These findings will facilitate the scale-up and further purification to ascertain the compounds responsible for antimicrobial activity, which can be exploited for the treatment of biodeterioration monuments and pharmaceutical applications.

Keywords: antimicrobial activity, asperigillus fumigatus, Identification by ITS4, Staphylococcus aureus, C.albicans

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Authors: Ahmad Abdi, M. Golzadeh Gangeraj, Alireza Barari, S. Shirali, S. Amini

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Aims: Diabetes is one of the common metabolic diseases in the world that has the dire adverse effects such as nephropathy, retinopathy and cardiovascular problems. Pharmaceutical and non-pharmaceutical strategies for control and treatment of diabetes are provided. Exercise and nutrition as non-drug strategies for the prevention and control of diabetes are considered. Exercises may increase oxidative stress and myocardium injury, thus it is necessary to take nutrition strategies to help diabetic athletes. Methods: This study was a semi-experimental research. Therefore, 24 men with type 2 diabetes were selected and randomly divided in four groups (1. control, 2. saffron extract, 3. aerobic exercises, 4. compound aerobic exercises and saffron extract). Saffron extract with 100 mg/day was used. Aerobic exercises, three days a week, for eight weeks, with 55-70% of maximum heart rate were performed. At the end, levels of Heart-type fatty acid-binding protein (HFABP) and Troponin T were measured. Data were analyzed by Paired t, One-way ANOVA and Tukey tests. Results: The serum Troponin T increased significantly in saffron extract, aerobic exercises and compound saffron extract -aerobic exercises in type 2 diabetic men(P=0.024, P =0.013, P=0.005 respectively). Saffron extract consumption (100 mg/day) and aerobic exercises did not significantly influence the serum HFABP (P =0.365, P =0.188 respectively). But serum HFABP decreased significantly in compound saffron extract -aerobic exercises group (P =0.003). Conclusions: Raised cardiac Troponin T and HFABP concentration accepted as the standard biochemical markers for the diagnosis of cardiac injury. Saffron intake may beneficially protect the myocardium from injuries. Compound saffron extract -aerobic exercises can decrease levels of Troponin T and HFABP in men with type 2 diabetes.

Keywords: Saffron, aerobic exercises, type 2 diabetes, HFABP, troponin T

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Authors: Tamar Grdzelishvili, Zaza Sinauridze

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Introduction: Preeclampsia is a complication of the second trimester of pregnancy, which is characterized by high morbidity and multiorgan damage. Many complex pathogenic mechanisms are now implicated to be responsible for this disease (1). Preeclampsia is one of the leading causes of maternal mortality worldwide. Statistics are enough to convince you of the seriousness of this pathology: about 100,000 women die of preeclampsia every year. It occurs in 3-14% (varies significantly depending on racial origin or ethnicity and geographical region) of pregnant women, in 75% of cases - in a mild form, and in 25% - in a severe form. During severe pre-eclampsia-eclampsia, perinatal mortality increases by 5 times and stillbirth by 9.6 times. Considering that the only way to treat the disease is to end the pregnancy, the main thing is timely diagnosis and prevention of the disease. Identification of high-risk pregnant women for PE and giving prophylaxis would reduce the incidence of preterm PE. First-trimester screening model developed by the Fetal Medicine Foundation (FMF), which uses the Bayes-theorem to combine maternal characteristics and medical history together with measurements of mean arterial pressure, uterine artery pulsatility index, and serum placental growth factor, has been proven to be effective and have superior screening performance to that of traditional risk factor-based approach for the prediction of PE (2) Methods: Retrospective single center screening study. The study population consisted of women from the Tbilisi maternity hospital “Pineo medical ecosystem” who met the following criteria: they spoke Georgian, English, or Russian and agreed to participate in the study after discussing informed consent and answering questions. Prior to the study, the informed consent forms approved by the Institutional Review Board were obtained from the study subjects. Early assessment of preeclampsia was performed between 11-13 weeks of pregnancy. The following were evaluated: anamnesis, dopplerography of the uterine artery, mean arterial blood pressure, and biochemical parameter: Pregnancy-associated plasma protein A (PAPP-A). Individual risk assessment was performed with performed by Fast Screen 3.0 software ThermoFisher scientific. Results: A total of 513 women were recruited and through the study, 51 women were diagnosed with preeclampsia (34.5% in the pregnant women with high risk, 6.5% in the pregnant women with low risk; P<0.000 1). Conclusions: First-trimester screening combining maternal factors with uterine artery Doppler, blood pressure, and pregnancy-associated plasma protein-A is useful to predict PE in a routine care setting. More patient studies are needed for final conclusions. The research is still ongoing.

Keywords: first-trimester, preeclampsia, screening, pregnancy-associated plasma protein

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Authors: Muhammad S. Sajid, Asim Shamim, Muhammad Nisar Khan, Ashfaq A. Chatta, Muhammad Saqib

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Goats (Capra hircus) are a valued asset for resource poor farmers globally. But the parasitic infection especially Haemonchus contortus (Trichostrongylid), impact the health and production of goats globally. The present study intended to evaluate resilient and resistance to Haemonchus contortus in indigenous goat breeds (Teddy and Beetal) of Punjab, Pakistan. Out of 60, 30 goats of each breed were divided into 6 groups and each group contain five goats. Two group of each breed received challenged infection with 12000 and 18000 L3 (third stage) larvae of Haemonchus contortus under two infection protocol that is early and trickle and remaining two group of each breed was kept as control. Resilient and resistance of each breed was then measured on the basis of their phenotypic markers like: faecal egg counts, packed cell volume, FAMACHA score system, body weight, total protein, albumin and worm count on 2nd, 4th, 6th, and 8th week of post infection. Variation in response of each goat breeds to Haemonchus contortus was observed. Teddy breed showed significant (P < 0.05)resistance as compared to Beetal. It is probably first attempt to report an evaluation of goat breed response towards Haemonchus contortus in Pakistan. It was concluded that Teddy goats have a greater genetic tendency to resist against to the Haemonchus contortus infection and this breed could be kept and bred from the economic point of view. Evaluation of genetic markers are like: gene, protein expression, Immunoglobulin, Histamines and interleukins determination are recommended for future studies which can be helpful to be fined resistant breed of goats.

Keywords: goat, beetal, teddy, haemonchus contortus, resistance, resilience, phenotypic markers

Procedia PDF Downloads 337

Authors: K. Karapetyan, F. Tkhruni, A. Aghajanyan, T. S. Balabekyan, L. Arstamyan

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Introduction: Globalization of the food supply has created the conditions favorable for the emergence and spread of food-borne and especially dangerous pathogens (EDP) in developing countries. The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the processed foods product. Antimicrobial compounds of lactic acid bacteria (LAB) possess bactericidal or bacteriostatic activity against intestinal pathogens, spoilage organisms and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella. Endemic strains of LAB were isolated. The strains, showing broad spectrum of antimicrobial activity against food spoiling microorganisms, were selected. The genotyping by 16S rRNA sequencing, GS-PCR, RAPD PCR methods showed that they were presented by Lactobacillus rhamnosus109, L.plantarum 65, L.plantarum 66 and Enterococcus faecium 64 species. LAB are deposited in "Microbial Depository Center" (MDC) SPC "Armbiotechnology". Methods: LAB strains were isolated from different dairy products from rural households from the highland regions of Armenia. Serially diluted samples were spread on MRS (Merck, Germany) and hydrolyzed milk agar (1,2 % w/v). Single colonies from each LAB were individually inoculated in liquid MRS medium and incubated at 37oC for 24 hours. Culture broth with biomass was centrifuged at 10,000 g during 20 min for obtaining of cell free culture broth (CFC). The antimicrobial substances from CFC broth were purified by the combination of adsorption-desorption and ion-exchange chromatography methods. Separation of bacteriocins was performed using a HPLC method on "Avex ODS" C18 column. Mass analysis of peptides recorded on the device API 4000 in the electron ionization mode. The spot-on-lawn method on the test culture plated in the solid medium was applied. The antimicrobial activity is expressed in arbitrary units (AU/ml). Results. Purification of CFC broth of LAB allowed to obtain partially purified antimicrobial preparations which contains bacteriocins with broad spectrum of antimicrobial activity. Investigation of their main biochemical properties shown, that inhibitory activity of preparations is partially reduced after treatment with proteinase K, trypsin, pepsin, suggesting a proteinaceous nature of bacteriocin-like substances containing in CFC broth. Preparations preserved their activity after heat treatment (50-121 oC, 20 min) and were stable in the pH range 3–8. The results of SDS PAAG electrophoresis show that L.plantarum 66 and Ent.faecium 64 strains have one bacteriocin (BCN) with maximal antimicrobial activity with approximate molecular weight 2.0-3.0 kDa. From L.rhamnosus 109 two BCNs were obtained. Mass spectral analysis indicates that these bacteriocins have peptide bonds and molecular weight of BCN 1 and BCN 2 are approximately 1.5 kDa and 700 Da. Discussion: Thus, our experimental data shown, that isolated endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms of different taxonomic group, such as Salmonella sp., Esherichia coli, Bacillus sp., L.monocytogenes, Proteus mirabilis, Staph. aureus, Ps. aeruginosa. Obtained results proved the perspectives for use of endemic strains in the preservation of foodstuffs. Acknowledgments: This work was realized with financial support of the Project Global Initiatives for Preliferation Prevention (GIPP) T2- 298, ISTC A-1866.

Keywords: antimicrobial activity, bacteriocins, endemic strains, food safety

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Authors: Rashmi Shukla, Yamini Bhusan Tripathi

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Diabetic nephropathy (DN) is characterized as diabetic kidney disease which involves many pathways e.g. hyperactivated protein kinase c (PKC), polyol pathway, excess production of advanced glycation end product (AGEs) & free radical accumulation etc. All of them results to hypoxia followed by apoptosis of podocytes, glomerulosclerosis, extracellular matrix (ECM) accumulation and fibrosis resulting to irreversible changes in kidney. This is continuously rising worldwide and there are not enough specific drugs, to retard its progress. Due to increasing side effects of allopathic drugs, interest in herbal remedies is growing. Earlier, we have reported that PTY-2 (a phytomedicine, derived from Pueraria tuberosa Linn.) inhibits the accumulation of extracellular matrix (ECM) through activation of MMP-9. Present study exhibited the therapeutic potential of Pueraria tuberosa in the prevention of podocytes apoptosis and modulation of nephrin expression in streptozotocin (STZ) induced DN rats. DN rats were produced by maintaining persistent hyperglycemia for 8 weeks by intra-peritoneal injection of 55 mg/kg streptozotocin (STZ). These rats were randomly divided in 2 groups, i.e. DN control, and DN+ water extract of Pueraria tuberosa (PTW). One group of age-matched normal rats served as non-diabetic control (group-1), The STZ induced DN rats (group-2) and DN+PTW treated rats (group-3). The PTW was orally administered (0.3g/kg) daily to group-2 rats and drug vector (1 ml of 10% tween 20) in control rats. The treatments were continued for 20 days and blood and urine samples were collected. Rats were then sacrificed to investigate the expression Bcl2, Bax and nephroprotective protein i.e. nephrin in kidney glomerulus. The effect of PTW was evaluated, we have found that the PTW significantly(p < .001) reversed the raised serum urea, serum creatinine, urine protein and improved the creatinine clearance in STZ induce diabetic nephropathy in rats and also significantly(p < .001) prevented the rise in urine albumin excretion. The Western blot analysis of kidney tissue homogenate showed increased expression of Bcl2 in PTW treated rats. The RT-PCR showed the increased expression and accumulation of nephrin mRNA. The confocal photomicrographs also supported the reduction of Bax and a simultaneous increase in Bcl2 and nephrin in glomerular podocytes. Hence, our finding suggests that the nephroprotective role of PTW is mediated via restoration of nephrin thus prevents the podocytes apoptosis and ameliorates diabetic nephropathy. The clinical trial of PTW would prove to be a potential food supplement/ drug of alternative medicine for patients with diabetic nephropathy in early stage.

Keywords: Pueraria tuberosa, diabetic nephropathy, anti-apoptosis, nephrin

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Authors: Maria Del Carmen Millan-Linares, Ana Lemus Conejo, Rocio Toscano, Alvaro Villanueva, Francisco Millan, Justo Pedroche, Sergio Montserrat-De La Paz

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GPETAFLR (Glycine-Proline-Glutamine-Threonine-Alanine-Phenylalanine-Leucine-Arginine) is a peptide isolated from Lupinus angustifolius L. protein hydrolysate (LPH). Herein, the effect of this peptide was investigated in two different models of neuroinflammation: in the immortalized murine microglia cell line BV-2 and in a high-fat-diet-induced obesity mouse model. Methods and Results: Effects of GPETAFLR on neuroinflammation were evaluated by RT-qPCR, flow cytometry, and ELISA techniques. In BV-2 microglial cells, Lipopolysaccharides (LPS) enhanced the release of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) whereas GPETAFLR decreased pro-inflammatory cytokine levels and increased the release of the anti-inflammatory cytokine IL-10 in BV2 microglial cells. M1 (CCR7 and iNOS) and M2 (Arg-1 and Ym-1) polarization markers results showed how the GPETAFLR octapeptide was able to decrease M1 polarization marker expression and increase the M2 polarization marker expression compared to LPS. Animal model results indicate that GPETAFLR has an immunomodulatory capacity, both decreasing pro-inflammatory cytokine IL-6 and increasing the anti-inflammatory cytokine IL-10 in brain tissue. Polarization markers in the brain tissue were also modulated by GPETAFLR that decreased the pro-inflammatory expression (M1) and increased the anti-inflammatory expression (M2). Conclusion: Our results suggest that GPETAFLR isolated from LPH has significant potential for management of neuroinflammatory conditions and offer benefits derived from the consumption of Lupinus angustifolius L. in the prevention of neuroinflammatory-related diseases.

Keywords: GPETAFLR peptide, BV-2 cell line, neuroinflammation, cytokines, high-fat-diet

Procedia PDF Downloads 114

Authors: Roshni Raha, Karthikeyan S.

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The world's largest livestock population resides in India. Existing strategies must be modified to increase the production of livestock and their by-products in order to meet the demands of the growing human population. Even though India leads the world in both milk production and the number of cows, average production is not very healthy and productive. This may be due to the animals' poor nutrition caused by a chronic under-availability of high-quality fodder and feed. This article explores Azolla pinnata to be a promising source to produce high-quality unconventional feed and fodder for effective livestock production and good quality breeding in India. This article is an exploratory study using a literature survey and experimentation analysis. In the realm of agri-biotechnology, azolla sp gained attention for helping farmers achieve sustainability, having minimal land requirements, and serving as a feed element that doesn't compete with human food sources. It has high methionine content, which is a good source of protein. It can be easily digested as the lignin content is low. It has high antioxidants and vitamins like beta carotene, vitamin A, and vitamin B12. Using this concept, the paper aims to investigate and develop a model of using azolla plants as a novel, high-potential feed source to combat the problems of low production and poor quality of animals in India. A representative sample of animal feed is collected where azolla is added. The sample is ground into a fine powder using mortar. PITC (phenylisothiocyanate) is added to derivatize the amino acids. The sample is analyzed using HPLC (High-Performance Liquid Chromatography) to measure the amino acids and monitor the protein content of the sample feed. The amino acid measurements from HPLC are converted to milligrams per gram of protein using the method of amino acid profiling via a set of calculations. The amino acid profile data is then obtained to validate the proximate results of nutrient enhancement of the composition of azolla in the sample. Based on the proximate composition of azolla meal, the enhancement results shown were higher compared to the standard values of normal fodder supplements indicating the feed to be much richer and denser in nutrient supply. Thus azolla fed sample proved to be a promising source for animal fodder. This would in turn lead to higher production and a good breed of animals that would help to meet the economic demands of the growing Indian population. Azolla plants have no side effects and can be considered as safe and effective to be immersed in the animal feed. One area of future research could begin with the upstream scaling strategy of azolla plants in India. This could involve introducing several bioreactor types for its commercial production. Since azolla sp has been proved in this paper as a promising source for high quality animal feed and fodder, large scale production of azolla plants will help to make the process much quicker, more efficient and easily accessible. Labor expenses will also be reduced by employing bioreactors for large-scale manufacturing.

Keywords: azolla, fodder, nutrient, protein

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Authors: Giap Pham Ngoc Tram, Tran Hong Quan, Tran Tieu Yen, Nguyen Phung Tien

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The use of natural ingredients to enhance the nutritional and sensory properties of food products has gained significant interest in recent years. This study focuses on the effect of Golden oyster mushroom powder (GOMP) on the physiochemical, antioxidative, and sensory properties of noodles. The aim of this study is to investigate the influence of GOMP on the nutritional, antioxidant, and sensory properties of noodles. The study determined the color, moisture, total ash, protein, total phenolic, flavonoid contents, water activity, and antioxidant activity of GOMP and noodles. The incorporation of GOMP at levels of 5-15% increased the ash, protein, flavonoid, and total phenolic contents of the noodles. It also enhanced their antioxidant activities, as evidenced by improved DPPH radical scavenging activity and metal chelating activity. However, the incorporation of GOMP resulted in a decrease in the L* and b* values of the noodles. Furthermore, the GOMP-enriched noodles exhibited a lower cutting force compared to the control. This study highlights the potential of GOMP as a nutritional and antioxidant ingredient in noodle preparation. It adds to the existing literature by providing evidence of the positive effects of GOMP on the nutritional and functional properties of noodles. The researchers collected data on the physiochemical properties, nutritional contents, and antioxidant activities of GOMP and noodles. Statistical analysis was then performed to assess the differences between the control and GOMP-enriched noodles. The results of this study demonstrate that the inclusion of GOMP at the amount of 5-15% can increase the nutritional and antioxidant properties of noodles without significantly impacting sensory attributes.

Keywords: oyster mushroom, noodles, antioxidant activity, phytochemical, sensory property

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Authors: Soma Banerjee, Aamen Talukdar, Argha Mandal, Dipankar Chaudhuri

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Japanese Encephalitis is a major infectious disease with nearly half the world’s population living in areas where it is prevalent. Currently, treatment for it involves only supportive care and symptom management through vaccination. Due to the lack of antiviral drugs against Japanese Encephalitis Virus (JEV), the quest for such agents remains a priority. For these reasons, simulation studies of drug targets against JEV are important. Towards this purpose, docking experiments of the kinase inhibitors were done against the chosen target NS3 helicase as it is a nucleoside binding protein. Previous efforts regarding computational drug design against JEV revealed some lead molecules by virtual screening using public domain software. To be more specific and accurate regarding finding leads, in this study a proprietary software Schrödinger-GLIDE has been used. Druggability of the pockets in the NS3 helicase crystal structure was first calculated by SITEMAP. Then the sites were screened according to compatibility with ATP. The site which is most compatible with ATP was selected as target. Virtual screening was performed by acquiring ligands from databases: KinaseSARfari, KinaseKnowledgebase and Published inhibitor Set using GLIDE. The 25 ligands with best docking scores from each database were re-docked in XP mode. Protein structure alignment of NS3 was performed using VAST against MMDB, and similar human proteins were docked to all the best scoring ligands. The low scoring ligands were chosen for further studies and the high scoring ligands were screened. Seventy-three ligands were listed as the best scoring ones after performing HTVS. Protein structure alignment of NS3 revealed 3 human proteins with RMSD values lesser than 2Å. Docking results with these three proteins revealed the inhibitors that can interfere and inhibit human proteins. Those inhibitors were screened. Among the ones left, those with docking scores worse than a threshold value were also removed to get the final hits. Analysis of the docked complexes through 2D interaction diagrams revealed the amino acid residues that are essential for ligand binding within the active site. Interaction analysis will help to find a strongly interacting scaffold among the hits. This experiment yielded 21 hits with the best docking scores which could be investigated further for their drug like properties. Aside from getting suitable leads, specific NS3 helicase-inhibitor interactions were identified. Selection of Target modification strategies complementing docking methodologies which can result in choosing better lead compounds are in progress. Those enhanced leads can lead to better in vitro testing.

Keywords: antivirals, docking, glide, high-throughput virtual screening, Japanese encephalitis, ns3 helicase

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Authors: H. M. Takematsu, B. R. De Camargo, E. F. Noronha

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The use of hydrolyzing enzymes for degradation of lignocellulosic biomass is of great concern for the production of second generation ethanol. Although many hollocelulases have already been described in the literature, much more has to be discovered. Therefore, the aim of this study to evaluate hollocelulase production of P. polonicum grown in liquid media containing sugarcane bagasse as the carbon source. From a collection of twenty fungi isolated from Cerrado biome soil, P. polonicum was molecular identified by sequencing of ITS4, βtubulin and Calmodulin genes, and has been chosen to be further investigated regarding its potential production of hydrolyzing enzymes. Spore suspension (1x10-6 ml-1) solution was inoculated in sterilized minimal liquid medium containing 0,5%(w/v) of non-pretreated sugarcane bagasse as the carbon source, and incubated in shaker incubator at 28°C and 120 rpm. The supernatant obtained, was subjected to enzymatic assays to analyze xylanase, mannanase, pectinase and endoglucanase activities. Xylanase activity showed better results (67,36 UI/mg). Xylanases bands were indicated by zymogram and SDS-PAGE, and one of them was partially purified and characterized. It showed maximum activity at 50 °C, was thermostable for 72h at 40°C, and pH5.0 was the optimum observed. This study presents P. polonicum as an interesting source of hollocelulases, especially xylanase, for lignocellulose bio-conversion processes with commercial use.

Keywords: sugarcane bagasse, Cerrado biome , hollocelulase, lignocellulosic biomass

Procedia PDF Downloads 264