Search results for: recombinant proteins

Authors: Kalyan S. Ghosh, B. L. Dhananjaya

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Snake bite causes fatal injuries in multi-organs and even many deaths due to several adverse physiological effects of various phospholipases (PLA2s) present in snake venom. Though these PLA2s bear highly homologues sequences and also structure but exhibit a different extent of those pharmacological effects. In this study, we have explored the difference in the neurotoxicity of two PLA2 namely PLA2-V, PLA2-VIIIa present in the venom from Vipera russellii. Bioinformatics studies on sequences of these two proteins along with detailed structural comparison enable us to explore the differences unambiguously. The identification of the residues involved in neurotoxicity will further lead towards proper designing of inhibitors against such killing effects of the venom.

Keywords: electrostatic potential, homology modeling, hydrophobicity, neurotoxicity, Phospholipase A2

Procedia PDF Downloads 407

Authors: Alexander A. Osmolovskiy, Anastasia V. Orekhova, Daria M. Bednenko, Yelyzaveta Boiko

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Micromycetes from Aspergillus genus can produce proteases capable of promoting proteolysis of hemostasis proteins or, along with hydrolytic activity, to show the ability to convert proenzymes of this system activating them into an active form. At the same time, practical medicine needs specific activators for quantitation of the level of some plasma enzymes, especially protein C and factor X, the lack of which leads to the development of thromboembolic diseases. Thus, some micromycetes of the genus Aspergillus were screened for the ability to synthesize extracellular proteases with promising activity for designing anti-thrombotic and diagnostic preparations. Such standard methods like salting out, electrophoresis, isoelectrofocusing were used for isolation, purification and study of physicochemical properties of proteases. Enzyme activity was measured spectrophotometrically fibrin as a substrate of the reaction and chromogenic peptide substrates of different proteases of the human hemostasis system. As a result of the screening, four active producers were selected: Aspergillus janus 301, A. flavus 1, A. terreus 2, and A. ochraceus L-1. The enzyme of A. janus 301 showed the greatest fibrinolytic activity (around 329.2 μmol Tyr/(ml × min)). The protease produced by A. terreus 2 had the highest plasmin-like activity (54.1 nmol pNA/(ml × min)), but fibrinolytic activity was lower than A. janus 301 demonstrated (25.2 μmol Tyr/(ml × min)). For extracellular protease of micromycete A. flavus a high plasmin-like activity was also shown (39.8 nmol pNA / (ml × min)). Moreover, according to our results proteases one of the fungi - A. terreus 2 were able to activate protein C of human plasma - the key factor of the human anticoagulant hemostasis system. This type of activity was 39.8 nmol pNA/(ml × min)). It was also shown that A. ochraceus L-1 could produce extracellular proteases with protein C and factor X activator activities (65.9 nmol pNA/(ml × min) and 34.6 nmol pNA/(ml × min) respectively). The maximum accumulation of the proteases falls on the 4th day of cultivation. Using isoelectrofocusing was demonstrated that the activation of both proenzymes might proceed via limited proteolysis induced by proteases of A. ochraceus L-1. The activatory activity of A. ochraceus L-1 proteases toward essential hemostatic proenzymes, protein C and X factor may be useful for practical needs. It is well known that similar enzymes, activators of protein C and X factor isolated from snake venom, South American copperhead Agkistrodon contortrix contortrix and Russell’s viper Daboia russelli russeli, respectively, are used for the in vitro diagnostics of the functional state of these proteins in blood plasma. Thus, the proteases of Aspergillus genus can be used as cheap components for enzyme thrombolytic preparations.

Keywords: anti-trombotic drugs, fibrinolysis, diagnostics, proteases, micromycetes

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Authors: Kuate Tueguem William Norbert, Ngoh Dooh Jules Patrice, Kone Sangou Abdou Nourou, Mboussi Serge Bertrand, Chewachang Godwill Mih, Essome Sale Charles, Djuissi Tohoto Doriane, Ambang Zachee

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The objectives of this study were to evaluate the effects of foliar application of 24-epibrassinolide (EBR) on the development of rice helminthosporiosis caused by Bipolaris oryzae and its influence on the improvement of growth parameters and induction of the synthesis of defense substances in the rice plants. The experimental asset up involved a multifactorial split-plot with two varieties (NERICA 3 and local variety KAMKOU) and five treatments (T0: control, T1: EBR, T2: BANKO PLUS (fungicide), T3: NPK (chemical fertilizer), T4: mixture: NPK + BANKO PLUS + EBR) with three repetitions. Agro-morphological and epidemiological parameters, as well as substances for plant resistance, were evaluated over two growing seasons. The application of the EBR induced significant growth of the rice plants for the 2015 and 2016 growing seasons on the two varieties tested compared to the T0 treatment. At 74 days after sowing (DAS), NERICA 3 showed plant heights of 58.9 ± 5.4; 83.1 ± 10.4; 86.01 ± 9.4; 69.4 ± 11.1 and 87.12 ± 7.4 cm at T0; T1; T2; T3, and T4, respectively. Plant height for the variety KAMKOU varied from 87,12 ± 8,1; 88.1 ± 8.1 and 92.02 ± 6.3 cm in T1, T2, and T3 to 74.1 ± 8.6 and 74.21 ± 11.4 cm in T0 and T3. In accordance with the low rate of expansion of helminthosporiosis in experimental plots, EBR (T1) significantly reduced the development of the disease with severities of 0.0; 1.29, and 2.04%, respectively at 78; 92, and 111 DAS on the variety NERICA 3 compared with1; 3.15 and 3.79% in the control T0. The reduction of disease development/severity as a result of the application of EBR is due to the induction of acquired resistance of rice varieties through increased phenol (13.73 eqAG/mg/PMF) and total protein (117.89 eqBSA/mg/PMF) in the T1 treatment against 5.37 eqAG/mg/PMF and 104.97 eqBSA/mg/PMF in T0 for the NERICA 3 variety. Similarly, on the KAMKOU variety, 148.53 eqBSA/mg/PMF were protein and 6.10 eqAG/mg/PMF of phenol in T1. In summary, the results show the significant effect of EBR on plant growth, yield, synthesis of secondary metabolites and defense proteins, and disease resistance. The EBR significantly reduced losses of rice grains by causing an average gain of about 1.55 t/ha compared to the control and 1.00 t/ha compared to the NPK-based treatment for the two varieties studied. Further, the enzymatic activities of PPOs, POXs, and PR2s were higher in leaves from treated EBR-based plants. These results show that 24-epibrassinolide can be used in the control of helminthosporiosis of rice to reduce disease and increase yields.

Keywords: Oryza sativa, 24-epibrassinolide, helminthosporiosis, secondary metabolites, PR proteins, acquired resistance

Procedia PDF Downloads 164

Authors: Elizalde D. Bana

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Healing restores continuity and form through cell replication; hence, conserving structural integrity. In response to the worldwide pressing problem of chronic wounds in the healthcare delivery system, the researcher aims to provide effective intervention to preserve the structural integrity of the person. The wound healing effects of cryopreserved and lyophilized amniotic membrane (AM) of a term fetus embedded into two (2) concentrations (1.5 % and 1.0 %) of absorption-based ointment has been evaluated in vivo using the excision wound healing model 1x1 cm size. The total protein concentration in full term fetus was determined by the Biuret and Bradford methods, which are based on UV-visible spectroscopy. The percentages of protein presence in 9.5 mg (Mass total sample) of Amniotic membrane ranges between 14.77 – 14.46 % in Bradford method, while slightly lower to 13.78 – 13.80 % concentration in Biuret method, respectively. Bradford method evidently showed higher sensitivity for proteins than Biuret test. Overall, the amniotic membrane is composed principally of proteins in which a copious amount of literature substantially proved its healing abilities. After which, an area of 1 cm by 1 cm skin tissue was excised to its full thickness from the dorsolateral aspect of the isogenic mice and was applied twice a day with the ointment formulation having two (2) concentrations for the diabetic group and non-diabetic group. The wounds of each animal were left undressed and its area was measured every other day by a standard measurement formula from day 2,4,6,8,10,12 and 14. By the 14th day, the ointment containing 1.5 % of AM in absorption-based ointment applied to non-diabetic and diabetic group showed 100 % healing. The wound areas in the animals treated with the standard antibiotic, Mupirocin Ointment (Brand X) showed a 100% healing by the 14th day but with traces of scars, indicating that AM prepared from cryopreservation and lyophilization, at that given concentration, had a better wound healing property than the standard antibiotic. Four (4) multivariate tests were used which showed a significant interaction between days and treatments, meaning that the ointments prepared in two differing concentrations and induced in different groups of the mice had a significant effect on the percent of contraction over time. Furthermore, the evaluations of its effectiveness to wound healing were all significant although in differing degrees. It is observed that the higher the concentrations of amniotic membrane, the more effective are the results.

Keywords: wounds, healing, amniotic membrane ointments, biomedical, stem cell

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Authors: R. Prinsloo

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Amniotic fluid stem cells (AFSC) have been shown to contribute towards the amelioration of Acute Renal Failure (ARF), but the mechanisms underlying the renoprotective effect are largely unknown. Therefore, the main goal of the current study was to evaluate the therapeutic efficacy of AFSC in a cisplatin-induced rat model of ARF and to investigate the underlying mechanisms responsible for its renoprotective effect. To study the therapeutic efficacy of AFSC, ARF was induced in Wistar rats by an intra-peritoneal injection of cisplatin, and five days after administration, the rats were randomized into two groups and injected with either AFSC or normal saline intravenously. On day 8 and 12 after cisplatin injection, i.e., day 3 and day7 post-therapy respectively, the blood biochemical parameters, histopathological changes, apoptosis, and expression of pro-apoptotic, anti-apoptotic and autophagy-related proteins in renal tissues were studied in both groups of rats. Administration of AFSC in ARF rats resulted in improvement of renal function and attenuation of renal damage as reflected by significant decrease in blood urea nitrogen, serum creatinine levels, tubular cell apoptosis as assessed by Bax/Bcl2 ratio, and expression of the pro-apoptotic proteins viz. PUMA, Bax, cleaved caspase-3 and cleaved caspase-9 as compared to saline-treated group. Furthermore, in the AFSC-treated group as compared to saline-treated group, there was a significant increase in the activation of autophagy as evident by increased expression of LC3-II, ATG5, ATG7, Beclin1 and phospho-AMPK levels with a concomitant decrease in phospho-p70S6K and p62 expression levels. To further confirm whether the protective effects of AFSC on cisplatin-induced apoptosis were dependent on autophagy, chloroquine, an autophagy inhibitor was administered by the intra-peritoneal route. Chloroquine administration led to significant reduction in the anti-apoptotic effects of the AFSC therapy and further deterioration in the renal structure and function caused by cisplatin. Collectively, our results put forth that AFSC ameliorates cisplatin-induced ARF through induction of autophagy and inhibition of apoptosis. Furthermore, the protective effects of AFSC were blunted by chloroquine, highlighting that activation of autophagy is an important mechanism of action for the protective role of AFSC in cisplatin-induced renal injury.

Keywords: best interest of the child, children's rights, parent and child relationship, parental responsibilities and rights

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Authors: Debbarma Asis, Guha Chandan

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Tumor Protein-53 (P53) is one of the tumor suppressor proteins. P53 regulates the cell cycle that conserves stability by preventing genome mutation. It is named so as it runs as 53-kilodalton (kDa) protein on Polyacrylamide gel electrophoresis although the actual mass is 43.7 kDa. Experimental evidence has indicated that P53 cancer mutants loses tumor suppression activity and subsequently gain oncogenic activities to promote tumourigenesis. Tumor-specific DNA has recently been detected in the plasma of breast cancer patients. Detection of tumor-specific genetic materials in cancer patients may provide a unique and valuable tumor marker for diagnosis and prognosis. Commercially available MDA-468 breast cancer cell line was used for the proposed study.

Keywords: tumor protein (P53), cancer mutants, MDA-468, tumor suppressor gene

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Authors: Noura Shehab-Eldeen, Mohamed Elsherbeeny, Hossam Elmetwally, Mohamed Salama, Ahmed Lotfy, Mohamed Elgamal, Hussein Sheashaa, Mohamed Sobh

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Mitochondrial toxins including papaverine may be implicated in the etiology and pathogenesis of Parkinson's disease. The aim was to detect the effect of papaverine on the proliferation and viability of neural stem cells. Rat neural progenitor cells were isolated from embryos (E14) brains. The dispersed tissues were allowed to settle, then, The supernatant was centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s solution cultured as free-floating neurospheres Dulbecco’s modified Eagle medium (DMEM) and Hams F12 (3:1) supplemented with B27 (Invitrogen GmBH, Karlsruhe, Germany), 20 ng/mL epidermal growth factor (EGF; Biosource, Karlsruhe, Germany), 20 ng/mL recombinant human fibroblast growth factor (rhFGF; R&D Systems, Wiesbaden-Nordenstadt, Germany), and penicillin and streptomycin (1:100; Invitrogen) at 37°C with 7.5% CO2 . Differentiation was initiated by growth factor withdrawal and plating onto a poly-d-lysine/ laminin matrix. The neurospheres were fed every 2-3 days by replacing 50% of the culture media with fresh media. The culture suspension was transferred to a dish containing 16 wells. The wells were divided as follows: 4 wells received no papaverine (control), 4 wells 1 u, 4 wells 5 u and 4 wells 10 u of papaverine solution. In the next 2 weeks, photography (0,4,5,11days) and viability test were done. The photographs were analysed. Results : papaverine didn't affect proliferation of neurospheres, while it affected viability compared to control , this was dose related. Conclusion: This indicates the harmful effect of papaverine suggesting it to be a candidate neurotoxin causing Parkinsonism.

Keywords: neurospheres, neural stem cells, papaverine, Parkinsonism

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Authors: Jeongyeob Hong, Myeonghoon Park, Taeson Yoon

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After the advent of Achaeans which utilize different metabolism pathway and contain conspicuously different cellular structure, they have been recognized as possible materials for developing quality of human beings. Among diverse Achaeans, in this paper, we compared 16s RNA Sequences of four different species of Thermococcus: Achaeans genus specialized in sulfur-dealing metabolism. Four Species, Barophilus, Kodakarensis, Hydrothermalis, and Onnurineus, live near the hydrothermal vent that emits extreme amount of sulfur and heat. By comparing ribosomal sequences of aforementioned four species, we found similarities in their sequences and expressed protein, enabling us to expect that certain ribosomal sequence or proteins are vital for their survival. Apriori algorithms and Decision Tree were used. for comparison.

Keywords: Achaeans, Thermococcus, apriori algorithm, decision tree

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Authors: Jayanwita Sarkar, Usha Chakraborty, Bishwanath Chakraborty

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Plants are constantly interacting with various abiotic and biotic stresses. In changing climate scenario plants are continuously modifying physiological processes to adapt to changing environmental conditions which profoundly affect plant-pathogen interactions. Spot blotch in wheat is a fast-rising disease in the warmer plains of South Asia where the rise in minimum average temperature over most of the year already affecting wheat production. Hence, the study was undertaken to explore the role of elevated temperature in spot blotch disease development and modulation of antioxidative responses by plant growth promoting rhizobacteria (PGPR) for biocontrol of spot blotch at high temperature. Elevated temperature significantly increases the susceptibility of wheat plants to spot blotch causing pathogen Bipolaris sorokiniana. Two PGPR Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) isolated from wheat (Triticum aestivum L.) and blady grass (Imperata cylindrical L.) rhizophere respectively, showing in vitro antagonistic activity against Bipolaris sorokiniana were tested for growth promotion and induction of resistance against spot blotch in wheat. GC-MS analysis showed that Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) produced antifungal and antimicrobial compounds in culture. Seed priming with these two bacteria significantly increase growth, modulate antioxidative signaling and induce resistance and eventually reduce disease incidence in wheat plants at optimum as well as elevated temperature which was further confirmed by indirect immunofluorescence assay using polyclonal antibody raised against Bipolaris sorokiniana. Application of the PGPR led to enhancement in activities of plant defense enzymes- phenylalanine ammonia lyase, peroxidase, chitinase and β-1,3 glucanase in infected leaves. Immunolocalization of chitinase and β-1,3 glucanase in PGPR primed and pathogen inoculated leaf tissue was further confirmed by transmission electron microscopy using PAb of chitinase, β-1,3 glucanase and gold labelled conjugates. Activity of ascorbate-glutathione redox cycle related enzymes such as ascorbate peroxidase, superoxide dismutase and glutathione reductase along with antioxidants such as carotenoids, glutathione and ascorbate and osmolytes like proline and glycine betain accumulation were also increased during disease development in PGPR primed plant in comparison to unprimed plants at high temperature. Real-time PCR analysis revealed enhanced expression of defense genes- chalcone synthase and phenyl alanineammonia lyase. Over expression of heat shock proteins like HSP 70, small HSP 26.3 and heat shock factor HsfA3 in PGPR primed plants effectively protect plants against spot blotch infection at elevated temperature as compared with control plants. Our results revealed dynamic biochemical cross talk between elevated temperature and spot blotch disease development and furthermore highlight PGPR mediated array of antioxidative and molecular alterations responsible for induction of resistance against spot blotch disease at elevated temperature which seems to be associated with up-regulation of defense genes, heat shock proteins and heat shock factors, less ROS production, membrane damage, increased expression of redox enzymes and accumulation of osmolytes and antioxidants.

Keywords: antioxidative enzymes, defense enzymes, elevated temperature, heat shock proteins, PGPR, Real-Time PCR, spot blotch, wheat

Procedia PDF Downloads 143

Authors: Selvaprakash Ramalingam, Rabi N. Sahoo, Dharmendra Saraswat, A. Kumar, Rajeev Ranjan, Joydeep Mukerjee, Viswanathan Chinnasamy, K. K. Chaturvedi, Sanjeev Kumar

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Rice is one of the most important staple food crops in the world. Among the various diseases that affect rice crops, rice blast is particularly significant, causing crop yield and economic losses. While the plant has defense mechanisms in place, such as chemical indicators (proteins, salicylic acid, jasmonic acid, ethylene, and azelaic acid) and resistance genes in certain varieties that can protect against diseases, susceptible varieties remain vulnerable to these fungal diseases. Early prediction of rice blast (RB) disease is crucial, but conventional techniques for early prediction are time-consuming and labor-intensive. Hyperspectral remote sensing techniques hold the potential to predict RB disease at its asymptomatic stage. In this study, we aimed to demonstrate the prediction of RB disease at the asymptomatic stage using non-imaging hyperspectral ASD spectroradiometer under controlled laboratory conditions. We applied statistical spectral discrimination theory to identify unknown spectra of M. Oryzae, the fungus responsible for rice blast disease. The infrared (IR) region was found to be significantly affected by RB disease. These changes may result in alterations in the absorption, reflection, or emission of infrared radiation by the affected plant tissues. Our research revealed that the protein spectrum in the IR region is impacted by RB disease. In our study, we identified strong correlations in the region (Amide group - I) around X 1064 nm and Y 1300 nm with the Lambda / Lambda derived spectra methods for protein detection. During the stages when the disease is developing, typically from day 3 to day 5, the plant's defense mechanisms are not as effective. This is especially true for the PB-1 variety of rice, which is highly susceptible to rice blast disease. Consequently, the proteins in the plant are adversely affected during this critical time. The spectral contour plot reveals the highly correlated spectral regions 1064 nm and Y 1300 nm associated with RB disease infection. Based on these spectral sensitivities, we developed new spectral disease indices for predicting different stages of disease emergence. The goal of this research is to lay the foundation for future UAV and satellite-based studies aimed at long-term monitoring of RB disease.

Keywords: rice blast, asymptomatic stage, spectral sensitivity, IR

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Authors: Michio Kurosu

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Alterations in glycosylation not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Identification of cell type-specific glycoconjugates (tumor markers) has led to the discovery of new assay systems for certain cancers via immunodetection reagents. N- and O-linked glycans are the most abundant forms of glycoproteins. Recent studies of cancer immunotherapy are based on the immunogenicity of truncated O-glycan chains (e.g., Tn, sTn, T, and sLea/x). The prevalence of N-linked glycan changes in the development of tumor cells is known; however, therapeutic antibodies against N-glycans have not yet been developed. This is due to the lack of specificity of N-linked glycans between normal/healthy and cancer cells. Abnormal branching of N-linked glycans has been observed, particularly in solid cancer cells. While the discovery of drug-like glycosyltransferase inhibitors that block the biosynthesis of specific branching has a very low likelihood of success, altered glycosylation levels can be exploited by suppressing N-glycan biosynthesis through the inhibition of dolichyl-phosphate N-acetylglucosaminephosphotransferase1 (DPAGT1) activity. Inhibition of DPAGT1 function leads to changes of O-glycosylation on proteins associated with mitochondria and zinc finger binding proteins (indirect effects). On the basis of dynamic crosstalk between DPAGT1 and Snail/Slung/ZEB1 (a family of transcription factors that promote the repression of the adhesion molecules), we have developed pharmacologically acceptable selective DPAGT1 inhibitors. Tunicamycin kills a wide range of cancer and healthy cells in a non-selective manner. In sharp contrast, our DPAGT1 inhibitors display strong cytostatic effects against 16 solid cancers, which require the overexpression of DPAGT1 in their progression but do not affect the cell viability of healthy cells. The identified DPAGT1 inhibitors possess impressive anti-metastatic ability in various solid cancer cell lines and induce their mitochondrial structural changes, resulting in apoptosis. A prototype DPAGT1 inhibitor, APPB has already been proven to shrink solid tumors (e.g., pancreatic cancers, triple-negative breast cancers) in vivo while suppressing metastases and has strong synergistic effects when combined with current cytotoxic drugs (e.g., paclitaxel). At this conference, our discovery of selective DPAGT1 inhibitors with drug-like properties and proof-of-pharmaceutical concept studies of a novel DPAGT1 inhibitor are presented.

Keywords: DPAGT1 inhibitors, anti-metastatic drugs, natural product based drug designs, cytostatic effects

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Authors: Somayeh Fani, Mahmood Ameen Abdulla

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Costus speciosus is an important Malaysian medicinal plant commonly used traditionally in the treatment of many aliments. The present investigation is designed to elucidate preventive effects of ethanolic extracts of C. speciosus rhizome against absolute ethanol-induced gastric mucosal injury in Sprague-Dawley rats. Five groups of rats were orally pre-treated with vehicle, carboxymethylcellulose (CMC) as normal control group (Group 1), ethanol as ulcer control group (Group 2), omeprazole 20 mg/kg (reference group) (Group 3), and 250 and 500 mg/kg of C. speciosus extract (experimental groups) (Group 4 and 5), respectively. An hour later, CMC was given orally to Group 1 rats and absolute ethanol was given orally to Group 2-5 rats to generate gastric mucosal injury. After an additional hour, the rats were sacrificed. Grossly, ulcer control group exhibited severe of gastric mucosal hemorrhagic injury and increased in ulcer area, whereas groups pre-treated with omeprazole or plant’s rhizomes exhibited the significant reduction of gastric mucosal injury. Significant increase in the pH and mucous of gastric content was observed in rats re-treated with C. speciosus rhizome. Histology, ulcer control rats, demonstrated remarkable disruption of gastric mucosa, increased in edema and inflammatory cells infiltration of submucosal layer compared to rats pre-treated with rhizomes extract. Periodic acid Schiff staining for glycoprotein, rats pre-fed with C. speciosus C. displayed remarkably intense uptake of magenta color by glandular gastric mucosa compared with ulcer control rats. Immunostaining of gastric epithelium, rats pre-treatment with rhizome extract provide evidence of up-regulation of HSP70 and down-regulation of Bax proteins compared to ulcer control animals. Gastric tissue homogenate, C. speciosus significantly increased the activity of superoxide dismutase (SOD), and catalase (CAT), increased the level of non-protein sulfhydryl (NP-SH) and decreased the level of lipid peroxidation after ethanol administration. Acute toxicity test did not show any signs of toxicity. The mechanisms implicated the gasrtoprotective property of C. speciosus depend upon the antisecretory activity, increased in gastric mucus glycoprotein, up-regulation of HSP70 protein and down-regulation of Bax proteins, reduction in the lipid peroxidation and increase in the level of NP-SH and antioxidant enzymes activity in gastic homogenate.

Keywords: antioxidant, Costus speciosus, gastric ulcer, histology, omeprazole

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Authors: Marcin Stasiulewicz, Sebastian Filipkowski, Aneta Panuszko

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Background: The hallmark of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, is an aggregation of the abnormal forms of peptides and proteins. Water is essential to functioning biomolecules, and it is one of the key factors influencing protein folding and misfolding. However, the hydration studies of proteins are complicated due to the complexity of protein systems. The use of model compounds can facilitate the interpretation of results involving larger systems. Objectives: The goal of the research was to characterize the properties of the hydration water surrounding the two three-residue K peptide fragments INS (Isoleucine - Asparagine - Serine) and NSR (Asparagine - Serine - Arginine). Methods: Fourier-transform infrared spectra of aqueous solutions of the tripeptides were recorded on Nicolet 8700 spectrometer (Thermo Electron Co.) Measurements were carried out at 25°C for varying molality of solute. To remove oscillation couplings from water spectra and, consequently, obtain narrow O-D semi-heavy water bands (HDO), the isotopic dilution method of HDO in H₂O was used. The difference spectra method allowed us to isolate the tripeptide-affected HDO spectrum. Results: The structural and energetic properties of water affected by the tripeptides were compared to the properties of pure water. The shift of the values of the gravity center of bands (related to the mean energy of water hydrogen bonds) towards lower values with respect to the ones corresponding to pure water suggests that the energy of hydrogen bonds between water molecules surrounding tripeptides is higher than in pure water. A comparison of the values of the mean oxygen-oxygen distances in water affected by tripeptides and pure water indicates that water-water hydrogen bonds are shorter in the presence of these tripeptides. The analysis of differences in oxygen-oxygen distance distributions between the tripeptide-affected water and pure water indicates that around the tripeptides, the contribution of water molecules with the mean energy of hydrogen bonds decreases, and simultaneously the contribution of strong hydrogen bonds increases. Conclusions: It was found that hydrogen bonds between water molecules in the hydration sphere of tripeptides are shorter and stronger than in pure water. It means that in the presence of the tested tripeptides, the structure of water is strengthened compared to pure water. Moreover, it has been shown that in the vicinity of the Asparagine - Serine - Arginine, water forms stronger and shorter hydrogen bonds. Acknowledgments: This work was funded by the National Science Centre, Poland (grant 2017/26/D/NZ1/00497).

Keywords: amyloids, K-peptide, hydration, FTIR spectroscopy

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Authors: Keivan Jamshidi, Fateme Behbahani, Sara Omidi, Nadia Shahi, Alireza Farkhonde

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Amyloidosis represents a heterogenous group of diseases that have in common the deposition of fibrils composed of proteins of beta-pleated sheet structure, which can be specifically identified by histochemistry using the Congo red or similar stains. Between October 2013 to April 2014 (6 months) different patterns of renal amyloidosis was diagnosed on histopathological examination of kidneys belong to 196 out of 7065 slaughtered sheep subjected to postmortem examination. Microscopic examination of renal tissue sections stained with H&E and CR staining techniques revealed 3 patterns of renal amyloid deposition; including glomerular (22.72%), medullary (68.18%), and vascular (9.09%) were recognized. Renal medullary amyloidosis (RMA) was detected as the most prevalence pattern of renal amyloidosis in domestic sheep.

Keywords: sheep, amyloidosis, kidney, slaughterhouse

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Authors: Ewa Mojs, Katarzyna Wiechec, Maia Kubiak, Wlodzimierz Samborski

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Interactions between mother’s psychological condition and child’s health status are complex and derive from the nature of the mother-child relationship. The aim of the study was to analyze the issue of anxiety amongst mothers of short children in the aspect of growth hormone therapy. The study was based on a group of 101 mothers of originally short-statured children – 70 with growth hormone deficiency (GHD) treated with recombinant human growth hormone (rhGH) and 31 undergoing the diagnostic process, without any treatment. Collected medical data included child's gender, height and weight, chronological age, bone age delay, and rhGH therapy duration. For all children, the height SDS and BMI SDS were calculated. To evaluate anxiety in mothers, the Spielberger State-Trait Anxiety Inventory (STAI) was used. Obtained results revealed low trait anxiety levels, with no statistically significant differences between the groups. State anxiety levels were average when mothers of all children were analyzed together, but when divided into groups, statistical differences appeared. Mothers of children without diagnosis and treatment had significantly higher levels of state anxiety than mothers of children with GHD receiving appropriate therapy. These results show, that the occurrence of growth failure in children is not related to high maternal trait anxiety, but the lack of diagnosis and lack of appropriate treatment generates higher levels of maternal state anxiety than the process of rh GH therapy in the offspring. Commencement of growth hormone therapy induce a substantial reduction of the state anxiety in mothers, and the duration of treatment causes its further decrease.

Keywords: anxiety, development, growth hormone deficiency, motherhood

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Authors: Kerekoppa Puttaiah Bhatta Ramesha, Uday Kannegundla, Praseeda Mol, Lathika Gopalakrishnan, Jagish Kour Reen, Gourav Dey, Manish Kumar, Sakthivel Jeyakumar, Arumugam Kumaresan, Kiran Kumar M., Thottethodi Subrahmanya Keshava Prasad

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Spermatozoa are the highly specialized transcriptionally and translationally inactive haploid male gamete. The understanding of proteome of sperm is indispensable to explore the mechanism of sperm motility and fertility. Though there is a large number of human sperm proteomic studies, in-depth proteomic information on Bos indicus spermatozoa is not well established yet. Therefore, we illustrated the profile of sperm proteome in indigenous cattle, Malnad gidda (Bos Indicus), using high-resolution mass spectrometry. In the current study, two semen ejaculates from 3 breeding bulls were collected employing the artificial vaginal method. Using 45% percoll purification, spermatozoa cells were isolated. Protein was extracted using lysis buffer containing 2% Sodium Dodecyl Sulphate (SDS) and protein concentration was estimated. Fifty micrograms of protein from each individual were pooled for further downstream processing. Pooled sample was fractionated using SDS-Poly Acrylamide Gel Electrophoresis, which is followed by in-gel digestion. The peptides were subjected to C18 Stage Tip clean-up and analyzed in Orbitrap Fusion Tribrid mass spectrometer interfaced with Proxeon Easy-nano LC II system (Thermo Scientific, Bremen, Germany). We identified a total of 6773 peptides with 28426 peptide spectral matches, which belonged to 1081 proteins. Gene ontology analysis has been carried out to determine the biological processes, molecular functions and cellular components associated with sperm protein. The biological process chiefly represented our data is an oxidation-reduction process (5%), spermatogenesis (2.5%) and spermatid development (1.4%). The highlighted molecular functions are ATP, and GTP binding (14%) and the prominent cellular components most observed in our data were nuclear membrane (1.5%), acrosomal vesicle (1.4%), and motile cilium (1.3%). Seventeen percent of sperm proteins identified in this study were involved in metabolic pathways. To the best of our knowledge, this data represents the first total sperm proteome from indigenous cattle, Malnad Gidda. We believe that our preliminary findings could provide a strong base for the future understanding of bovine sperm proteomics.

Keywords: Bos indicus, Malnad Gidda, mass spectrometry, spermatozoa

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Authors: Soniya Nityanand, Ekta Minocha, Manali Jain, Rohit Anthony Sinha, Chandra Prakash Chaturvedi

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Amniotic fluid stem cells (AFSC) have been shown to contribute towards the amelioration of Acute Renal Failure (ARF), but the mechanisms underlying the renoprotective effect are largely unknown. Therefore, the main goal of the current study was to evaluate the therapeutic efficacy of AFSC in a cisplatin-induced rat model of ARF and to investigate the underlying mechanisms responsible for its renoprotective effect. To study the therapeutic efficacy of AFSC, ARF was induced in Wistar rats by an intra-peritoneal injection of cisplatin, and five days after administration, the rats were randomized into two groups and injected with either AFSC or normal saline intravenously. On day 8 and 12 after cisplatin injection, i.e., day 3 and day7 post-therapy respectively, the blood biochemical parameters, histopathological changes, apoptosis and expression of pro-apoptotic, anti-apoptotic and autophagy-related proteins in renal tissues were studied in both groups of rats. Administration of AFSC in ARF rats resulted in improvement of renal function and attenuation of renal damage as reflected by significant decrease in blood urea nitrogen, serum creatinine levels, tubular cell apoptosis as assessed by Bax/Bcl2 ratio, and expression of the pro-apoptotic proteins viz. PUMA, Bax, cleaved caspase-3 and cleaved caspase-9 as compared to saline-treated group. Furthermore, in the AFSC-treated group as compared to saline-treated group, there was a significant increase in the activation of autophagy as evident by increased expression of LC3-II, ATG5, ATG7, Beclin1 and phospho-AMPK levels with a concomitant decrease in phospho-p70S6K and p62 expression levels. To further confirm whether the protective effects of AFSC on cisplatin-induced apoptosis were dependent on autophagy, chloroquine, an autophagy inhibitor was administered by the intra-peritoneal route. Chloroquine administration led to significant reduction in the anti-apoptotic effects of the AFSC therapy and further deterioration in the renal structure and function caused by cisplatin. Collectively, our results put forth that AFSC ameliorates cisplatin-induced ARF through induction of autophagy and inhibition of apoptosis. Furthermore, the protective effects of AFSC were blunted by chloroquine, highlighting that activation of autophagy is an important mechanism of action for the protective role of AFSC in cisplatin-induced renal injury.

Keywords: amniotic fluid stem cells, acute renal failure, autophagy, cisplatin

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Authors: S. Mowafi, A. Abou El-Kheir, M. Abou Taleb, H. El-Sayed

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Two proteinic biopolymers; namely keratin and sericin, were extracted from their respective natural resources by simple appropriate methods. The said proteins were dissolved in the appropriate solvents followed by regeneration in a form of film polyvinyl alcohol. Protein-starch-potassium iodide (PSPI) composite was prepared by anchoring starch and potassium iodide mixture onto the film surface using appropriate polymeric material. The possibility of using PSPI composite for determination of the concentration of chlorine ions in domestic as well as industrial water was examined. The concentration of chlorine in water was determined spectrophotometrically by measuring the intensity of blue colour of formed between starch and the released iodine obtained by interaction of potassium iodide chlorine in the tested water sample.

Keywords: chlorine, protein, potassium iodide, water

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Authors: Sara Przetocka, Krzysztof Żak, Grzegorz Dubin, Tadeusz Holak

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Toll-like receptors (TLRs) play central role in the innate immune response and inflammation by recognizing pathogen-associated molecular patterns (PAMPs). A fundamental basis of TLR signalling is dependent upon the recruitment and association of adaptor molecules that contain the structurally conserved Toll/interleukin-1 receptor (TIR) domain. MyD88 (myeloid differentiation primary response gene 88) is the universal adaptor for TLRs and cooperates with Mal (MyD88 adapter-like protein, also known as TIRAP) in TLR4 response which is predominantly used in inflammation, host defence and carcinogenesis. Up to date two possible models of MyD88, Mal and TLR4 interactions have been proposed. The aim of our studies is to confirm or abolish presented models and accomplish the full structural characterisation of TIR domains interaction. Using molecular cloning methods we obtained several construct of MyD88 and Mal TIR domain with GST or 6xHis tag. Gel filtration method as well as pull-down analysis confirmed that recombinant TIR domains from MyD88 and Mal are binding in complexes. To examine whether obtained complexes are homo- or heterodimers we carried out cross-linking reaction of TIR domains with BS3 compound combined with mass spectrometry. To investigate which amino acid residues are involved in this interaction the NMR titration experiments were performed. 15N MyD88-TIR solution was complemented with non-labelled Mal-TIR. The results undoubtedly indicate that MyD88-TIR interact with Mal-TIR. Moreover 2D spectra demonstrated that simultaneously Mal-TIR self-dimerization occurs which is necessary to create proper scaffold for Mal-TIR and MyD88-TIR interaction. Final step of this study will be crystallization of MyD88 and Mal TIR domains complex. This crystal structure and characterisation of its interface will have an impact in understanding the TLR signalling pathway and possibly will be used in development of new anti-cancer treatment.

Keywords: cancer, MyD88, TIR domains, Toll-like receptors

Procedia PDF Downloads 262

Authors: M. Irfan Fareed, Muhammad Tahir, Alvina Gul Kazi

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Castor bean (Ricinus communis; family Euphorbiaceae) is cultivated for the production of oil and as an ornamental plant throughout tropical regions. Leaf samples from castor bean plants with leaf curl and vein thickening were collected from areas around Okara (Pakistan) in 2011. PCR amplification using diagnostic primers showed the presence of a begomovirus and subsequently the specific pair (BurNF 5’- CCATGGTTGTGGCAGTTGATTGACAGATAC-3’, BurNR 5’- CCATGGATTCACGCACAGGGGAACCC-3’) was used to amplify and clone the whole genome of the virus. The complete nucleotide sequence was determined to be 2,759 nt (accession No. HE985227). Alignments showed the highest levels of nucleotide sequence identity (98.8%) with Cotton leaf curl Burewala virus (CLCuBuV; accession No. JF416947) No. JF416947). The virus in castor beans lacks on intact C2 gene, as is typical of CLCuBuV in cotton. An amplification product of ca. 1.4 kb was obtained in PCR with primers for betasatellites and the complete nucleotide sequence of a clone was determined to be 1373 nt (HE985228). The sequence showed 96.3% nucleotide sequence identity to the recombinant Cotton leaf curl Multan betasatellite (CLCuMB; JF502389). This is the first report of CLCuBuV and its betasatellite infecting castor bean, showing this plant species as an alternate host of the virus. Already many alternate host have been reported from different alternate host like tobacco, tomato, hibiscus, okra, ageratum, Digera arvensis, habiscus, Papaya and now in Ricinus communis. So, it is suggested that these alternate hosts should be avoided to grow near cotton growing regions.

Keywords: Ricinus communis, begomovirus, betasatellite, agriculture

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Authors: Shiva Shakori Poshteh

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Lung cancer is a highly prevalent and devastating disease, representing a significant global health concern with profound implications for healthcare systems and society. Its high incidence, mortality rates, and late-stage diagnosis contribute to its formidable nature. To address these challenges, nanoparticle-based drug delivery has emerged as a promising therapeutic strategy. Curcumin (CUR), a natural compound derived from turmeric, has garnered attention as a potential nanomedicine for lung cancer treatment. Nanoparticle formulations of CUR offer several advantages, including improved drug delivery efficiency, enhanced stability, controlled release kinetics, and targeted delivery to lung cancer cells. CUR exhibits a diverse array of effects on cancer cells. It induces apoptosis by upregulating pro-apoptotic proteins, such as Bax and Bak, and downregulating anti-apoptotic proteins, such as Bcl-2. Additionally, CUR inhibits cell proliferation by modulating key signaling pathways involved in cancer progression. It suppresses the PI3K/Akt pathway, crucial for cell survival and growth, and attenuates the mTOR pathway, which regulates protein synthesis and cell proliferation. CUR also interferes with the MAPK pathway, which controls cell proliferation and survival, and modulates the Wnt/β-catenin pathway, which plays a role in cell proliferation and tumor development. Moreover, CUR exhibits potent antioxidant activity, reducing oxidative stress and protecting cells from DNA damage. Utilizing CUR as a standalone treatment is limited by poor bioavailability, lack of targeting, and degradation susceptibility. Nanoparticle-based delivery systems can overcome these challenges. They enhance CUR’s bioavailability, protect it from degradation, and improve absorption. Further, Nanoparticles enable targeted delivery to lung cancer cells through surface modifications or ligand-based targeting, ensuring sustained release of CUR to prolong therapeutic effects, reduce administration frequency, and facilitate penetration through the tumor microenvironment, thereby enhancing CUR’s access to cancer cells. Thus, nanoparticle-based CUR delivery systems promise to improve lung cancer treatment outcomes. This article provides an overview of lung cancer, explores CUR nanoparticles as a treatment approach, discusses the benefits and challenges of nanoparticle-based drug delivery, and highlights prospects for CUR nanoparticles in lung cancer treatment. Future research aims to optimize these delivery systems for improved efficacy and patient prognosis in lung cancer.

Keywords: lung cancer, curcumin, nanomedicine, nanoparticle-based drug delivery

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Authors: Fatima Bouazza, Khadija Khedid, Lamiae Amallah, Aziz Mouhaddach, Basma Boukour, Jihane Ennadir, Rachida Hassikou

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In order to combine milk and its derived products conservation and flavoring, Moroccans often used aromatic and medicinal plants. These plant extracts are endowed with several nutritive and therapeutic properties. This study constitutes a first national assessment of physico-chemical quality of sheep’s milk from moroccan Sardi breed and the evaluation of the antibacterial effect of three medicinal plants extracts: Aloe barbadensis Miller, Thymus satureioides and Mentha pulegium on flora isolated from this sheep's milk. 100 milk samples were collected in four regions of Morocco. The bacteria isolated were identified by classical and molecular methods (16S rRNA sequencing) and tested, according to the disk method, for their sensitivity to several antibiotics. The physico-chemical analyzes of sheep’s milk concerned the pH, titratable acidity, density, dry extract, freezing point and contents of: fat, proteins, lactose and calcium. The essential oils (EOs) of T. satureioides and M .pulegium were extracted by hydrodistillation and analyzed by GC / MS, while the Aloe vera leaf pulp was analyzed by the methods of Harborne and HPLC. A total number of 125 bacteria have been identified. Significant resistance to chemical antibiotics has been noted in LABs. The average temperature value of milk is around 57.15 °C, the pH is 6.56, the titratable acidity is around 3.4 ° D, the density is 1.035g / cm³ , the total dry extract is around 169.5g / l, the ash (9.8g / l), the freezing point (- 0.556 °C) while the average fat content is 67.85g / l . The samples richest in fat belong to the region of Settat, cradle of the Sardi breed, with a maximum average value of 74.4g / l. The average protein is 56g / l, lactose (39.92g / l), and calcium (1.855g / l). Analysis of the major components of EOs revealed the dominance of borneol in the case of T. satureioides and of pulegone in M. pulegium. Aloe vera gel contains alkaloids, flavonoids, catechic tannins, saponins and 1.60 µg / ml of aloin. The plant extracts have a bactericidal effect on E. coli, Klebsiellaoxytoca and Staphylococci and bacteriostatic effect on LABs of technological interest (Lactobacillus). As a result of this study, it is believed that the consumption of sardi sheep’s milk would be of nutritional benefit. Its richness in fat and proteins predisposes it for biotechnological development in the manufacture of cheese and yogurt. Also, the use of aromatic and medicinal plants, as natural additives would be of great benefit to flavor and maintain its quality.

Keywords: sheep’s milk, lactic flora, antimicrobial power, aloe barbadensis miller, thymus satureioides, mentha pulegium

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Authors: José Maria, Vânia Laranjo, Luís Abrunhosa, António Inês

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The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Keywords: carboxypeptidase, lactic acid bacteria, mycotoxins, ochratoxin a.

Procedia PDF Downloads 437

Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun

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Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.

Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation

Procedia PDF Downloads 358

Authors: A. R. Diniz, P. Borrego, I. Bártolo, N. Taveira

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Cell-to-cell transmission plays a critical role in the spread of HIV-1 infection in vitro and in vivo. Inhibition of HIV-1 cell-associated infection by antiretroviral drugs and neutralizing antibodies (NAbs) is more difficult compared to cell-free infection. Limited data exists on cell-associated infection by HIV-2 and its inhibition. In this work, we determined the ability of entry inhibitors to inhibit HIV-1 and HIV-2 cell-to cell fusion as a proxy to cell-associated infection. We developed a method in which Hela-CD4-cells are first transfected with a Tat expressing plasmid (pcDNA3.1+/Tat101) and infected with recombinant vaccinia viruses expressing either the HIV-1 (vPE16: from isolate HTLV-IIIB, clone BH8, X4 tropism) or HIV-2 (vSC50: from HIV-2SBL/ISY, R5 and X4 tropism) envelope glycoproteins (M.O.I.=1 PFU/cell).These cells are added to TZM-bl cells. When cell-to-cell fusion (syncytia) occurs the Tat protein diffuses to the TZM-bl cells activating the expression of a reporter gene (luciferase). We tested several entry inhibitors including the fusion inhibitors T1249, T20 and P3, the CCR5 antagonists MVC and TAK-779, the CXCR4 antagonist AMD3100 and several HIV-2 neutralizing antibodies (Nabs). All compounds inhibited HIV-1 and HIV-2 cell fusion albeit to different levels. Maximum percentage of HIV-2 inhibition (MPI) was higher for fusion inhibitors (T1249- 99.8%; P3- 95%, T20-90%) followed by co-receptor antagonists (MVC- 63%; TAK-779- 55%; AMD3100- 45%). NAbs from HIV-2 infected patients did not prevent cell fusion up to the tested concentration of 4μg/ml. As for HIV-1, MPI reached 100% with TAK-779 and T1249. For the other antivirals, MPIs were: P3-79%; T20-75%; AMD3100-61%; MVC-65%.These results are consistent with published data. Maraviroc had the lowest IC50 both for HIV-2 and HIV-1 (IC50 HIV-2= 0.06 μM; HIV-1=0.0076μM). Highest IC50 were observed with T20 for HIV-2 (3.86μM) and with TAK-779 for HIV-1 (12.64μM). Overall, our results show that entry inhibitors in clinical use are less effective at preventing Env mediated cell-to-cell-fusion in HIV-2 than in HIV-1 which suggests that cell-associated HIV-2 infection will be more difficult to inhibit compared to HIV-1. The method described here will be useful to screen for new HIV entry inhibitors.

Keywords: cell-to-cell fusion, entry inhibitors, HIV, NAbs, vaccinia virus

Procedia PDF Downloads 286

Authors: Guo Wenyu, Qu Youli

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A practical and simple self-indexing data structure, Partitioned Elias-Fano (PEF) - Compressed Suffix Arrays (CSA), is built in linear time for the CSA based on PEF indexes. Moreover, the PEF-CSA is compared with two classical compressed indexing methods, Ferragina and Manzini implementation (FMI) and Sad-CSA on different type and size files in Pizza & Chili. The PEF-CSA performs better on the existing data in terms of the compression ratio, count, and locates time except for the evenly distributed data such as proteins data. The observations of the experiments are that the distribution of the φ is more important than the alphabet size on the compression ratio. Unevenly distributed data φ makes better compression effect, and the larger the size of the hit counts, the longer the count and locate time.

Keywords: compressed suffix array, self-indexing, partitioned Elias-Fano, PEF-CSA

Procedia PDF Downloads 229

Authors: Mohammadhosein Rahimi, Mohammad Raouf Hosseini, Mehran Bakhshi, Alireza Baghbanan

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Silver ions (Ag⁺) and their compounds are consequentially toxic to microorganisms, showing biocidal effects on many species of bacteria. Silver-phosphate (or silver orthophosphate) is one of these compounds, which is famous for its antimicrobial effect and catalysis application. In the present study, a green method was presented to synthesis silver-phosphate nanoparticles using Sporosarcina pasteurii. The composition of the biosynthesized nanoparticles was identified as Ag₃PO₄ using X-ray Diffraction (XRD) and Energy Dispersive Spectroscopy (EDS). Also, Fourier Transform Infrared (FTIR) spectroscopy showed that Ag₃PO₄ nanoparticles was synthesized in the presence of biosurfactants, enzymes, and proteins. In addition, UV-Vis adsorption of the produced colloidal suspension approved the results of XRD and FTIR analyses. Finally, Transmission Electron Microscope (TEM) images indicated that the size of the nanoparticles was about 20 nm.

Keywords: bacteria, biosynthesis, silver-phosphate, Sporosarcina pasteurii, nanoparticle

Procedia PDF Downloads 427

Authors: Walter Vera-Ortega, Idoia Busnadiego, Sam J. Wilson

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Introduction: Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the acquired immunodeficiency syndrome (AIDS), a leading cause of death worldwide infecting millions of people each year. Despite intensive research in vaccine development, therapies against HIV-1 infection are not curative, and the huge genetic variability of HIV-1 challenges to drug development. Current animal models for HIV-1 research present important limitations, impairing the progress of in vivo approaches. Macaques require a CD8+ depletion to progress to AIDS, and the maintenance cost is high. Mice are a cheaper alternative but need to be 'humanized,' and breeding is not possible. The development of an HIV-1 clone able to replicate in mice is a challenging proposal. The lack of human co-factors in mice impedes the function of the HIV-1 accessory proteins, Tat and Rev, hampering HIV-1 replication. However, Tat and Rev function can be replaced by constitutive/chimeric promoters, codon-optimized proteins and the constitutive transport element (CTE), generating a novel HIV-1 clone able to replicate in mice without disrupting the amino acid sequence of the virus. By minimally manipulating the genomic 'identity' of the virus, we propose the generation of an HIV-1 clone able to replicate in mice to assist in antiviral drug development. Methods: i) Plasmid construction: The chimeric promoters and CTE copies were cloned by PCR using lentiviral vectors as templates (pCGSW and pSIV-MPCG). Tat mutants were generated from replication competent HIV-1 plasmids (NHG and NL4-3). ii) Infectivity assays: Retroviral vectors were generated by transfection of human 293T cells and murine NIH 3T3 cells. Virus titre was determined by flow cytometry measuring GFP expression. Human B-cells (AA-2) and Hela cells (TZMbl) were used for infectivity assays. iii) Protein analysis: Tat protein expression was determined by TZMbl assay and HIV-1 capsid by western blot. Results: We have determined that NIH 3T3 cells are able to generate HIV-1 particles. However, they are not infectious, and further analysis needs to be performed. Codon-optimized HIV-1 constructs are efficiently made in 293T cells in a Tat and Rev independent manner and capable of packaging a competent genome in trans. CSGW is capable of generating infectious particles in the absence of Tat and Rev in human cells when 4 copies of the CTE are placed preceding the 3’LTR. HIV-1 Tat mutant clones encoding different promoters are functional during the first cycle of replication when Tat is added in trans. Conclusion: Our findings suggest that the development of an HIV-1 Tat-Rev independent clone is challenging but achievable aim. However, further investigations need to be developed prior presenting our HIV-1 clone as a candidate model for research.

Keywords: codon-optimized, constitutive transport element, HIV-1, long terminal repeats, research model

Procedia PDF Downloads 282

Authors: Fabienne Vozy, Anick Lepage

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Insects are displaying high nutritional value, low greenhouse gas emissions, low land use requirements and high food conversion efficiency. They can contribute to the food chain and be one of many solutions to protein shortages. Currently, in North America, nutritional entomology is under-developed and the needs to better understand its benefits remain to convince large-scale producers and consumers (both for human and agricultural needs). As such, large-scale production of mealworms offers a promising alternative to replacing traditional sources of protein and fatty acids. To proceed orderly, it is required to collect more data on the nutritional values of insects such as, a) Evaluate the diets of insects to improve their dietary value; b) Test the breeding conditions to optimize yields; c) Evaluate the use of by-products and organic residues as sources of food. Among the featured technical parameters, relative humidity (RH) percentage and temperature, optimal substrates and hydration sources are critical elements, thus establishing potential benchmarks for to optimize conversion rates of protein and fatty acids. This research is to establish the combination of the most influential rearing parameters with local food residues, to correlate the findings with the nutritional value of the larvae harvested. 125 same-monthly old adults/replica are randomly selected in the mealworm breeding pool then placed to oviposit in growth chambers preset at 26°C and 65% RH. Adults are removed after 7 days. Larvae are harvested upon the apparition of the first nymphosis signs and batches, are analyzed for their nutritional values using wet chemistry analysis. The first samples analyses include total weight of both fresh and dried larvae, residual humidity, crude proteins (CP%), and crude fats (CF%). Further analyses are scheduled to include soluble proteins and fatty acids. Although they are consistent with previous published data, the preliminary results show no significant differences between treatments for any type of analysis. Nutritional properties of each substrate combination have yet allowed to discriminate the most effective residue recipe. Technical issues such as the particles’ size of the various substrate combinations and larvae screen compatibility are to be investigated since it induced a variable percentage of lost larvae upon harvesting. To address those methodological issues are key to develop a standardized efficient procedure. The aim is to provide producers with easily reproducible conditions, without incurring additional excessive expenditure on their part in terms of equipment and workforce.

Keywords: entomophagy, nutritional value, rearing parameters optimization, Tenebrio molitor

Procedia PDF Downloads 87

Authors: Leonard Lipovich, Pattaraporn Thepsuwan, Anton-Scott Goustin, Juan Cai, Donghong Ju, James B. Brown

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Two-thirds of human genes do not encode any known proteins. Aside from long non-coding RNA (lncRNA) genes with recently-discovered functions, the ~40,000 non-protein-coding human genes remain poorly understood, and a role for their transcripts as de-facto unconventional messenger RNAs has not been formally excluded. Ribosome profiling (Riboseq) predicts translational potential, but without independent evidence of proteins from lncRNA open reading frames (ORFs), ribosome binding of lncRNAs does not prove translation. Previously, we mass-spectrometrically documented translation of specific lncRNAs in human K562 and GM12878 cells. We now examined lncRNA translation in human MCF7 cells, integrating strand-specific Illumina RNAseq, Riboseq, and deep mass spectrometry in biological quadruplicates performed at two core facilities (BGI, China; City of Hope, USA). We excluded known-protein matches. UCSC Genome Browser-assisted manual annotation of imperfect (tryptic-digest-peptides)-to-(lncRNA-three-frame-translations) alignments revealed three peptides hypothetically explicable by 'stop-to-nonstop' in-frame replacement of stop codons by amino acids in two ORFs of the lncRNA MMP24-AS1. To search for this phenomenon genomewide, we designed and implemented a novel pipeline, matching tryptic-digest spectra to wildcard-instead-of-stop versions of repeat-masked, six-frame, whole-genome translations. Along with singleton putative stop-to-nonstop events affecting four other lncRNAs, we identified 24 additional peptides with stop-to-nonstop in-frame substitutions from multiple positive-strand MMP24-AS1 ORFs. Only UAG and UGA, never UAA, stop codons were impacted. All MMP24-AS1-matching spectra met the same significance thresholds as high-confidence known-protein signatures. Targeted resequencing of MMP24-AS1 genomic DNA and cDNA from the same samples did not reveal any mutations, polymorphisms, or sequencing-detectable RNA editing. This unprecedented apparent gene-specific violation of the genetic code highlights the importance of matching peptides to whole-genome, not known-genes-only, ORFs in mass-spectrometry workflows, and suggests a new mechanism enhancing the combinatorial complexity of the proteome. Funding: NIH Director’s New Innovator Award 1DP2-CA196375 to LL.

Keywords: genetic code, lncRNA, long non-coding RNA, mass spectrometry, proteogenomics, ribo-seq, ribosome, RNAseq

Procedia PDF Downloads 206