Search results for: tumor protein (P53)

Authors: Muhammad A. Khan, Waseem Shahzad

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Analysis of protein sequences is carried out for the purpose to discover their structural and ancestry relationship. Sequence similarity determines similar protein structures, similar function, and homology detection. Biological sequences composed of amino acid residues or nucleotides provide significant information through sequence alignment. In this paper, we present a new similarity/dissimilarity measure to sequence alignment based on the primary structure of a protein. The approach finds the distance between the two given sequences using the novel sequence alignment algorithm and a mathematical model. The algorithm runs at a time complexity of O(n²). A distance matrix is generated to construct a phylogenetic tree of different species. The new similarity/dissimilarity measure outperforms other existing methods.

Keywords: alignment, distance, homology, mathematical model, phylogenetic tree

Procedia PDF Downloads 157

Authors: Dicky Eka Putra, S. P. Nadia Chairunissa, Lidia Paramita, Roza Hartati, Ice Yolanda Puri

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Background: Soy bean and the products such as tofu, tempeh and soy milk are famous in the community. Moreover, another product is tofu flour which is not familiar in Indonesia yet and it is well known as Okara. There are massive differences of energy, protein and carbohydrate between them which is know as good for protein sources as well. Unfortunately, it is seldom used as food variety. Basically, it can be benefit in order to create many products for example cakes, snacks and some desserts. Aim: the study was in order to promote the benefit of tofu flour as school feeding of elementary school and baby porridge and also to compare the nutrient. Method: Soy pulp was filtered and steamed approximately 30 minutes. Then, it was put at a plate under sunrise or barked on the oven for 10 hours at 800C. When it have dried and milling and tofu flour is ready to be used. Result: Tofu flour could be used as substitute of flour and rice flour when people want to cook some foods. In addition, some references said that soy bean is good for a specific remedy for the proper functioning of the heart, liver, kidneys, stomach, and bowels, constipation, as a stimulant for the lungs, for eradication of poison from the system, improving the complexion by cleaning the skin of impurities, and stimulating the growth and appearance of the hair. Discussion: Comparing between soy bean, tofu and tofu flour which has difference amount of nutrients. For example energy 382 kcal, 79 kcal and 393 kcal respectively and also protein 30.2 kcal, 7.8 kcal, and 17.4 kcal. In addition, carbohydrate of soy pulp was high than soy bean and tofu (30.1 kcal). Finally, local should replace flour, rice and gelatin rice flour with tofu flour.

Keywords: tofu flour, protein, soy bean, school feeding

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Authors: Meziani Samira, Menadi Noreddine, Labga Lahouaria, Chenni Fatima Zohra, Toumi Asma

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A comparative study between two varieties of soya beans was carried out in this work. The method consists of studying and proceeding to prepare a by-product (Flour) from two varieties of soybeans, a Chinese variety imported and marketed in Algeria. The chemical composition of ash, protein and fat was determined in this study. The minerals, namely potassium and sodium, were measured by flame spectrophotometer. In addition, the estimation of the polyphenol content and evaluation of the antioxidant activity Ferric Reducing Antioxidant Power assay (FRAP) f the methanol extracts of the flours were also carried out. The result revealed that soy flour from two cultivars, on average, contained 8% moisture, more than 50% protein, 1.58-1.87g fat, and 0.28-0.30g of ash. A slight difference was found for contents of 489 mg/ml of K + and 20 mg/ml of NA +. In addition, the phenolic content of the methanolic extracts gives a value of almost 37 mg EAG / g for both cultivars of soy flour. The estimated Reductive Antioxidant Iron (FRAP) potency of soy flour might be related to its polyphenol richness, which is similar to the variety of China. The flour Soya varieties tested contained a significant amount of protein and phenolic compounds with good antioxidant properties.

Keywords: soye beans, soya flour, protein, total polyphenols

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Authors: Alyaa Abdlwahab

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Cutaneous leishmaniasis represents a serious health problem in Syria; this problem has become noticeably aggravated after the civil war in the country. Leishmania tropica parasite is the main cause of cutaneous leishmaniasis in Syria. In order to control the disease, we need an effective vaccine against leishmania parasite. DNA vaccination remains one of the favorable approaches that have been used to face cutaneous leishmaniasis. Ribosomal protein S4 is responsible for important roles in Leishmania parasite life. DNA vaccine based on S4 gene has been used against infections by many species of Leishmania parasite but leishmania tropica parasite, so this gene represents a good candidate for DNA vaccine construction. After proving the existence of ribosomal protein S4 gene in a Syrian strain of Leishmania tropica (LCED Syrian 01), sequencing it and cloning it into pCI plasmid, BALB/C mice were inoculated with pCI-S4 DNA vaccine. The immune response was determined by monitoring the lesion progression in inoculated BALB/C mice for six weeks after challenging mice with Leishmania tropica (LCED Syrian 01) parasites. IL-12, IFN-γ, and IL-4 were quantified in draining lymph nodes (DLNa) of the immunized BALB/C mice by using the RT-qPCR technique. The parasite burden was calculated in the final week for the footpad lesion and the DLNs of the mice. This study proved the existence and the expression of the ribosomal protein S4 gene in Leishmania tropica (LCED Syrian 01) promastigotes. The sequence of ribosomal protein cDNA S4 gene was determined and published in Genbank; the gene size was 822 bp. Expression was also demonstrated at the level of cDNA. Also, this study revealed that pCI-S4 DNA vaccine induces TH1\TH2 response in immunized mice; this response prevents partially developing a dermal lesion of Leishmania.

Keywords: ribosomal protein S4, DNA vaccine, Leishmania tropica, BALB\c

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Authors: Merry Meryam Martgrita, Marselina Irasonia Tan

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One of several aggresive cancer is cancer that overexpress c-erbB2 receptor along with the expression of estrogen receptor. Components of extracellular matrix play an important role to increase cancer cells proliferation, migration and invasion. Both components can affect cancer development by regulating the signal transduction pathways in cancer cells. In recent research, SKOV-3 ovarian cancer cell line, that overexpress c-erbB2 receptor was cultured on type IV collagen and treated with estradiol-17β, to reveal the role of both components on RNA and protein level of c-erbB2 receptor. In this research we found a modulation phenomena of increasing and decreasing of c-erbB2 RNA level and a stabilisation phenomena of c-erbB2 protein expression due to estradiol-17β and type IV collagen. It seemed that estradiol-17β has an important role to increase c-erbB2 transcription and the stability of c-erbB2 protein expression. Type IV collagen has an opposite role. It blocked c-erbB2 transcription when it bound to integrin receptor in SKOV-3 cells.

Keywords: c-erbB2, estradiol-17β, SKOV-3, type IV collagen

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Authors: Aarti Agarwal, Ajmal Khan

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Background and Objective: Breathing difficulty is most distressing symptom for the patient and their caregivers providing palliative care to individuals with advanced malignancy. It needs to be tackled effectively and sometimes preemptively to provide relief from respiratory obstruction. Interventional procedures like tracheal stenting are becoming increasingly popular as a part of palliation for respiratory symptoms. We present a case of esophageal tumor earlier stented by Gastroenterologist to maintain esophageal patency, but the tumor outgrew to produce tracheal infiltration and thereby causing airway obstruction. Method and Result: 62-year-old man presented with unresectable Carcinoma oesophagus with inability to swallow. A metallic stent was placed by the gastroenterologist, to maintain esophageal patency and enable patient to swallow. Two months later, the patient returned to hospital in emergency with respiratory distress. CT neck and thorax revealed tumor infiltration through posterior tracheal wall. Lower extent of the tumor was till 1 cm above the carina. Airway stenting with Tracheo bronchial stent with Y configuration was planned under general anaesthesia with airway blocks. Superior Laryngeal Nerve Block, Glossopharyngeal block and Trans tracheal infiltration of local anaesthetics were performed. The patient was sedated with Fentanyl, Midazolam and propofol infusion but was breathing spontaneously. Once the rigid bronchoscope was placed inside trachea, breathing was supported with oxygen and sevoflurane. Initially, the trachea was cleared of tumor by coring. After creating space, tracheal stent was positioned and deployed. After stent placement patient was awakened, suctioned and nebulized. His respiratory stridor relieved instantaneously and was shifted to recovery. Conclusion: Airway blocks help in decreasing the incidence and severity of coughing during airway instrumentation thereby help in proper stent placement. They also reduce the requirement of general anaesthetics and hasten the post stenting recovery. Airway stent provided immediate relief to patient from symptoms of respiratory difficulty. Decision for early tracheal stenting may be taken for a select group of patients with high propensity for local spread, thereby avoiding respiratory complications and providing better quality of life in patients with inoperable malignancy.

Keywords: tracheal stent, respiratory difficulty, esophageal tumor, anaesthetic management

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Authors: Tahra ElObeid, Omnya Ahmed, Reem Al-Sharshani, Doaa Dalloul, Jannat Alnattei

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Background: Camelus dormedarius camels are also called ‘the Arabian camels’ and are present in the desert area of North Africa and the Middle East. Recently, camel’s milk has a great attention globally because of their proteins and peptides that have been reported to be beneficial for the health and in the management of many diseases. Objectives: This study was designed to investigate the antioxidant, antimicrobial activity and to evaluate the total phenolic content of camel’s milk proteins in Qatar. Methods: Fresh two camel’s milk samples from Omani breed and called Muhajer (camel’s milk A and B) were collected on the 1st of the December. Both samples were from the same location Al- Shahaniyah, Doha, Qatar, but from different local private farms and feeding system. Camel’s milk A and B were defatted by centrifugation and their proteins were extracted by acid and thermal precipitation. The antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Total phenolic compound (TPC) was evaluated by Folin-Ciocalteu reagent (FCR). On the other hand, the antimicrobial activity against eight different type of pathogenic bacteria was evaluated by disc diffusion method and the zone of inhibition was measured. Results: The of the total phenolic content of whole milk in both camel’s milk A and B were significantly the highest among the protein extracts. The % of the DPPH radical inhibition of casein protein in both camel’s milk A and B were significantly the highest among the protein extracts. In this study, there were marked changes in the antibacterial activity in the different camel milk protein extracts. All extracts showed bacterial overgrowth. Conclusion: The antioxidant activity of the camel milk protein extracts correlated to their unique phenolic compounds and bioactive protein peptides. The antimicrobial activity was not detected perhaps due to the technique, the quality, or the extraction method. Overall, camel's milk exhibits a high antioxidant activity, which is responsible for many health benefits besides the nutritional values.

Keywords: camels milk, antioxidant content, antimicrobial activity, proteins, Qatar

Procedia PDF Downloads 192

Authors: J. F. Pollo Gaspary, F. Peron Gaspary, E. M. Simão, R. Concatto Beltrame, G. Orengo de Oliveira, M. S. Ristow Ferreira, F. Sartori Thies, I. F. Minello, F. dos Santos de Oliveira

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Background: The immune system has evolved several mechanisms to protect the host against cancer, and it has now been suggested that the expansion of its functions may prevent tumor growth and control the symptoms of cancer patients. Two techniques, ozone therapy and pulsed electromagnetic fields (PEMF), are independently associated with an increase in the immune system functions and they maybe help palliative care of patients in these conditions. Case Report: A patient with rectal adenocarcinoma with metastases decides to interrupt the clinical chemotherapy protocol due to refractoriness and side effects. As a palliative care alternative treatment it is suggested to the patient the use of ozone therapy associated with PEMF techniques. Results: The patient reports an improvement in well-being, in autonomy and in pain control. Imaging tests confirm a pause in tumor growth despite more than 60 days without using classic treatment. These results associated with palliative care alternative treatment stimulate the return to the chemotherapy protocol. Discussion: This case illustrates that these two techniques can contribute to the control of tumor growth and refractory symptoms, such as pain, probably by enhancing the immune system. Conclusions: The potential use of the combination of these two therapies, ozone therapy and PEMF therapy, can contribute to palliation of cancer patients, alone or in combination with pharmacological therapies. The conduct of future investigations on this paradigm can elucidate how much these techniques contribute to the survival and well-being of these patients.

Keywords: cancer, complementary and alternative medicine , ozone therapy, palliative care, PEMF therapy

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Authors: Ravinder Singh, Ankita Gurao, Saroj Bandhan, Sudhir Kumar Kashyap

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The Bos taurus Beta defensin 3 is a defensin peptide secreted by neutrophils and epithelial that exhibits anti-microbial activity. It is one of the crucial components forming an innate defense against intra mammary infections in livestock. The beta defensin 3 by virtue of its anti-microbial activity inhibits major mastitis pathogens including Staphylococcus aureus and Pseudomonas aeruginosa etc, which are also responsible for biofilm formation leading to antibiotic resistance phenomenon. Therefore, the defensin may prove as a non-conventional option to treat mastitis. In this study, computational analysis has been performed including sequence comparison among species and homology modeling of Bos taurus beta defensin 3 protein. The assessments of protein structure were done using the protein structure and model assessment tools integrated in Swiss Model server, which employs various local and global quality evaluation parameters. Further, molecular docking was also carried out between the defensin peptide and the components of biofilm to gain insight into various interactions and structural differences crucial for functionality of this protein.

Keywords: beta defensin 3, bos taurus, docking, homology modeling

Procedia PDF Downloads 271

Authors: Achraf Al Faraj, Asma Sultana Shaik, Baraa Al Sayed

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Specific and effective targeting of drug delivery systems (DDS) to cancerous sites remains a major challenge for a better diagnostic and therapy. Recently, SWCNTs with their unique physicochemical properties and the ability to cross the cell membrane show promising in the biomedical field. The purpose of this study was first to develop a biocompatible iron oxide tagged SWCNTs as diagnostic nanoprobes to allow their noninvasive detection using MRI and their preferential targeting in a breast cancer murine model by placing an optimized flexible magnet over the tumor site. Magnetic targeting was associated to specific antibody-conjugated SWCNTs active targeting. The therapeutic efficacy of doxorubicin-conjugated SWCNTs was assessed, and the superiority of diffusion-weighted (DW-) MRI as sensitive imaging biomarker was investigated. Short Polyvinylpyrrolidone (PVP) stabilized water soluble SWCNTs were first developed, tagged with iron oxide nanoparticles and conjugated with Endoglin/CD105 monoclonal antibodies. They were then conjugated with doxorubicin drugs. SWCNTs conjugates were extensively characterized using TEM, UV-Vis spectrophotometer, dynamic light scattering (DLS) zeta potential analysis and electron spin resonance (ESR) spectroscopy. Their MR relaxivities (i.e. r1 and r2*) were measured at 4.7T and their iron content and metal impurities quantified using ICP-MS. SWCNTs biocompatibility and drug efficacy were then evaluated both in vitro and in vivo using a set of immunological assays. Luciferase enhanced bioluminescence 4T1 mouse mammary tumor cells (4T1-Luc2) were injected into the right inguinal mammary fat pad of Balb/c mice. Tumor bearing mice received either free doxorubicin (DOX) drug or SWCNTs with or without either DOX or iron oxide nanoparticles. A multi-pole 10x10mm high-energy flexible magnet was maintained over the tumor site during 2 hours post-injections and their properties and polarity were optimized to allow enhanced magnetic targeting of SWCNTs toward the primary tumor site. Tumor volume was quantified during the follow-up investigation study using a fast spin echo MRI sequence. In order to detect the homing of SWCNTs to the main tumor site, susceptibility-weighted multi-gradient echo (MGE) sequence was used to generate T2* maps. Apparent diffusion coefficient (ADC) measurements were also performed as a sensitive imaging biomarker providing early and better assessment of disease treatment. At several times post-SWCNT injection, histological analysis were performed on tumor extracts and iron-loaded SWCNT were quantified using ICP-MS in tumor sites, liver, spleen, kidneys, and lung. The optimized multi-poles magnet revealed an enhanced targeting of magnetic SWCNTs to the primary tumor site, which was found to be much higher than the active targeting achieved using antibody-conjugated SWCNTs. Iron-loading allowed their sensitive noninvasive tracking after intravenous administration using MRI. The active targeting of doxorubicin through magnetic antibody-conjugated SWCNTs nanoprobes was found to considerably decrease the primary tumor site and may have inhibited the development of metastasis in the tumor-bearing mice lung. ADC measurements in DW-MRI were found to significantly increase in a time-dependent manner after the injection of DOX-conjugated SWCNTs complexes.

Keywords: single-walled carbon nanotubes, nanomedicine, magnetic resonance imaging, cancer diagnosis and therapy

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Authors: Xuechun Kan, Dongdong Li, Fan Li, Peidang Liu

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Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with a poor prognosis, and radiotherapy is one of the main treatment methods. However, due to the obvious resistance of tumor cells to radiotherapy, high dose of ionizing radiation is required during radiotherapy, which causes serious damage to normal tissues near the tumor. Therefore, how to improve radiotherapy resistance and enhance the specific killing of tumor cells by radiation is a hot issue that needs to be solved in clinic. Recent studies have shown that silver-based nanoparticles have strong radiosensitization, and silver nanoclusters (AgNCs) also provide a broad prospect for tumor targeted radiosensitization therapy due to their ultra-small size, low toxicity or non-toxicity, self-fluorescence and strong photostability. Aptamer 5TR1 is a 25-base oligonucleotide aptamer that can specifically bind to mucin-1 highly expressed on the membrane surface of TNBC 4T1 cells, and can be used as a highly efficient tumor targeting molecule. In this study, AgNCs were synthesized by DNA template based on 5TR1 aptamer (NC-T5-5TR1), and its role as a targeted radiosensitizer in TNBC radiotherapy was investigated. The optimal DNA template was first screened by fluorescence emission spectroscopy, and NC-T5-5TR1 was prepared. NC-T5-5TR1 was characterized by transmission electron microscopy, ultraviolet-visible spectroscopy and dynamic light scattering. The inhibitory effect of NC-T5-5TR1 on cell activity was evaluated using the MTT method. Laser confocal microscopy was employed to observe NC-T5-5TR1 targeting 4T1 cells and verify its self-fluorescence characteristics. The uptake of NC-T5-5TR1 by 4T1 cells was observed by dark-field imaging, and the uptake peak was evaluated by inductively coupled plasma mass spectrometry. The radiation sensitization effect of NC-T5-5TR1 was evaluated through cell cloning and in vivo anti-tumor experiments. Annexin V-FITC/PI double staining flow cytometry was utilized to detect the impact of nanomaterials combined with radiotherapy on apoptosis. The results demonstrated that the particle size of NC-T5-5TR1 is about 2 nm, and the UV-visible absorption spectrum detection verifies the successful construction of NC-T5-5TR1, and it shows good dispersion. NC-T5-5TR1 significantly inhibited the activity of 4T1 cells and effectively targeted and fluoresced within 4T1 cells. The uptake of NC-T5-5TR1 reached its peak at 3 h in the tumor area. Compared with AgNCs without aptamer modification, NC-T5-5TR1 exhibited superior radiation sensitization, and combined radiotherapy significantly inhibited the activity of 4T1 cells and tumor growth in 4T1-bearing mice. The apoptosis level of NC-T5-5TR1 combined with radiation was significantly increased. These findings provide important theoretical and experimental support for NC-T5-5TR1 as a radiation sensitizer for TNBC.

Keywords: 5TR1 aptamer, silver nanoclusters, radio sensitization, triple-negative breast cancer

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Authors: Ahreum Choi, Otgontuya Tsogbadrakh, Kwang-Hwan Jung

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Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied. These retinal-binding proteins have divided into two types. The type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. For type I rhodopsin, there are two main functions that are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is one of the microbial rhodopsin with main function as photo-sensory transduction. Although ASR is expressed fairly well in Escherichia coli, the expression level is relatively less compare to Proteorhodopsin. In this study, full length of ASR was used to test for the expression influence by codon usage in E. coli. Eight amino acids of codon at N-terminal part of ASR were changed randomly with designed primers, which allow 8,192 nucleotide different cases. The codon changes were screened for the preferable codons of each residue, which have given higher expression yield. Among those 57 selected mutations, there are 24 color-enhanced E. coli colonies that contain ASR proteins, and it showed better expression level than the wild type ASR codon usage. This strongly suggests that high codon usage of only partial N-terminal of protein can increase the expression level of whole protein.

Keywords: 7-transmembrane, all-trans retinal, rhodopsin, codon-usage, protein expression

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Authors: Swati Srivastava, Mattia Lauriola, Yuval Gilad, Adi Kimchi, Yosef Yarden

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The glucocorticoid receptor (GR) has been proposed to play important, but incompletely understood roles in cancer. Glucocorticoids (GCs) are widely used as co-medication of various carcinomas, due to their ability to reduce the toxicity of chemotherapy. Furthermore, GR antagonism has proven to be a strategy to treat triple negative breast cancer and castration-resistant prostate cancer. These observations suggest differential GR involvement in cancer subtypes. The goal of our study has been to elaborate the current understanding of GR signaling in tumor progression and metastasis. Our study involves two cellular models, non-tumorigenic breast epithelial cells (MCF10A) and Ewing sarcoma cells (CHLA9). In our breast cell model, the results indicated that the GR agonist dexamethasone inhibits EGF-induced mammary cell migration, and this effect was blocked when cells were stimulated with a GR antagonist, namely RU486. Microarray analysis for gene expression revealed that the mechanism underlying inhibition involves dexamenthasone-mediated repression of well-known activators of EGFR signaling, alongside with enhancement of several EGFR’s negative feedback loops. Because GR mainly acts primarily through composite response elements (GREs), or via a tethering mechanism, our next aim has been to find the transcription factors (TFs) which can interact with GR in MCF10A cells.The TF-binding motif overrepresented at the promoter of dexamethasone-regulated genes was predicted by using bioinformatics. To validate the prediction, we performed high-throughput Protein Complementation Assays (PCA). For this, we utilized the Gaussia Luciferase PCA strategy, which enabled analysis of protein-protein interactions between GR and predicted TFs of mammary cells. A library comprising both nuclear receptors (estrogen receptor, mineralocorticoid receptor, GR) and TFs was fused to fragments of GLuc, namely GLuc(1)-X, X-GLuc(1), and X-GLuc(2), where GLuc(1) and GLuc(2) correspond to the N-terminal and C-terminal fragments of the luciferase gene.The resulting library was screened, in human embryonic kidney 293T (HEK293T) cells, for all possible interactions between nuclear receptors and TFs. By screening all of the combinations between TFs and nuclear receptors, we identified several positive interactions, which were strengthened in response to dexamethasone and abolished in response to RU486. Furthermore, the interactions between GR and the candidate TFs were validated by co-immunoprecipitation in MCF10A and in CHLA9 cells. Currently, the roles played by the uncovered interactions are being evaluated in various cellular processes, such as cellular proliferation, migration, and invasion. In conclusion, our assay provides an unbiased network analysis between nuclear receptors and other TFs, which can lead to important insights into transcriptional regulation by nuclear receptors in various diseases, in this case of cancer.

Keywords: epidermal growth factor, glucocorticoid receptor, protein complementation assay, transcription factor

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Authors: Vijay Kumar Kutala, Addepalli Pavani, M. Amresh Rao, Naushad Sm

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In this study, we evaluated the impact of CYP2C9*2 and CYP2C9*3 variants on binding and hydroxylation of warfarin. In silico data revealed that warfarin forms two hydrogen bonds with protein backbone i.e. I205 and S209, one hydrogen bond with protein side chain i.e. T301 and stacking interaction with F100 in CYP2C9*1. In CYP2C9*2 and CYP2C9*3 variants, two hydrogen bonds with protein backbone are disrupted. In double variant, all the hydrogen bonds are disrupted. The distances between C7 of S-warfarin and Fe-O in CYP2C9*1, CYP2C9*2, CYP2C9*3 and CYP2C9*2/*3 were 5.81A°, 7.02A°, 7.43° and 10.07°, respectively. The glide scores (Kcal/mol) were -7.698, -7.380, -6.821 and -6.986, respectively. Increase in warfarin/7-hydroxy warfarin ratio was observed with increase in variant alleles. To conclude, CYP2C9*2 and CYP2C9*3 variants result in disruption of hydrogen bonding interactions with warfarin and longer distance between C7 and Fe-O thus impairing warfarin 7-hydroxylation due to lower binding affinity of warfarin.

Keywords: warfarin, CYP2C9 polymorphism, personalized medicine, in Silico

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Authors: Eleni Poulou-Sidiropoulou, Charalampos N. Bompas, Martina Samiotaki, Alexios Vlamis-Gardikas

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The glutaredoxin (Grx) and thioredoxin (Trx) systems keep the intracellular environment reduced in almost all organisms. In Escherichia coli (E. coli), the Grx system relies on NADPH+ to reduce GSH reductase (GR), the latter reducing oxidized diglutathione to glutathione (GSH) which in turn reduces cytosolic Grxs, the electron donors for different intracellular substrates. In the Trx system, GR and GSH are replaced by Trx reductase (TrxR). Three of the Grxs of E. coli (Grx1, 2, 3) are reduced by GSH, while Grx4 is likely reduced by TrxR. Trx1 and Grx1 from E. coli may reduce ribonucleotide reductase Ia to ensure a constant supply of deoxyribonucleotides for the synthesis of DNA. The role of the other three Grxs is relatively unknown, especially for Grx2 that may amount up to 1 % of total cellular protein in the stationary phase of growth. The protein is known as a potent antioxidant, but no specific functions have been attributed to it. Herein, affinity chromatography of cellular extracts on immobilized Grx2, followed by MS analysis of the resulting eluates, was employed to identify protein ligands that could provide insights into the biological role of Grx2. Ionic, strong non-covalent, and covalent (disulfide) interactions with relevant proteins were detected. As a means of verification, the identified ligands were subjected to in silico docking with monothiol Grx2. In other experiments, protein extracts from E. coli cells lacking the gene for Grx2 (grxB) were compared to those of wild type. Taken together, the two approaches suggest that Grx2 is involved in protein synthesis, nucleotide metabolism, DNA damage repair, stress responses, and various metabolic processes. Grx2 appears as a versatile protein that may participate in a wide range of biological pathways beyond its known general antioxidant function.

Keywords: Escherichia coli, glutaredoxin 2, interactome, thiol-disulfide oxidoreductase

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Authors: Shiva Emami, Jenny Persson, Bengt Johansson Lindbom

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Group A streptococcus (GAS) is a prevalent human pathogen, causing a wide range of infections and diseases. One of the most well-known virulence factors in GAS is M protein, a surface protein that facilitates bacterial invasion. In this study, we used a recombinant GAS strain (GAS-2W) expressing M protein containing a hyper immunogenic peptide (2W). Mice were immunized three times with heat-killed-GAS subcutaneously at three weeks intervals. Three weeks post last immunization, mice were challenged intraperitoneally with a lethal dose of live GAS. In order to investigate the impact of IFN-ƴ and antibodies in protection against GAS infection, we used a mouse model knock-out for IFN-ƴ (IFN-ƴ KO). We observed immunization with GAS-2W strain can increase protection against GAS infection in mice compared with the original GAS strain. Higher levels of antibodies against M1 protein were measured in GAS-2W-immunized mice. There was also a significant increase in IgG2c response in mice immunized with GAS2W. By using IFN-ƴ KO mice, we showed that not a high level of total IgG, but IgG2c was correlated with protection through the i.p challenge. It also emphasizes the importance of IFN-ƴ cytokine to combat GAS by isotype switching to IgG2c (which is opsonic for phagocytosis). Our data indicate the crucial role of IFN-ƴ in the protective immune response that, together with IgG2c, can induce protection against GAS.

Keywords: Group A streptococcus, IgG2c, IFN-γ, protection

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Authors: Ravinder Singh, C Rajesh, Saroj Badhan, Shailendra Mishra, Ranjit Singh Kataria

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Heat shock protein 60 and 70 are crucial chaperones that guide appropriate folding of denatured proteins under heat stress conditions. HSP60 and HSP70 provide assistance in correct folding of a multitude of denatured proteins. The heat shock factors are the family of some transcription factors which controls the regulation of gene expression of proteins involved in folding of damaged or improper folded proteins during stress conditions. Under normal condition heat shock proteins bind with HSF-1 and act as its repressor as well as aids in maintaining the HSF-1’s nonactive and monomeric confirmation. The experimental protein structure for all these proteins in Bubalus bubalis is not known till date. Therefore computational approach was explored to identify three-dimensional structure analysis of all these proteins. In this study, an extensive in silico analysis has been performed including sequence comparison among species to comparative modeling of Bubalus bubalis HSP60, HSP70 and HSF-1 protein. The stereochemical properties of proteins were assessed by utilizing several scrutiny bioinformatics tools to ensure model accuracy. Further docking approach was used to study interactions between Heat shock proteins and HSF-1.

Keywords: Bubalus bubalis, comparative modelling, docking, heat shock protein

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Authors: Farnaz Firuzian, Farhad Choobdar, Ali Mazouri

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As Very Low Birth Weight (VLBW) neonates cannot tolerate enteral feeding, parenteral nutrition (PN) must be administered shortly after birth. To find an optimal combination of nutrition, in this study, we compare administering high glucose versus high protein intake as a component of total parenteral nutrition (TPN) to test their effect on birth weight (BW) regain in VLBW. This study employs a multi-arm randomized trial: 145 newborns with BW < 1500 g were randomized to control (C) or experimental groups: high glucose (G) or high protein (P). All samples in each group received the same TPN regimens except glucose and protein intake: Glocuse was provided by dextrose water (DW) serum: 7-15 g/kg/d (10% DW) in groups C and P versus 8.75-18.75 g/kg/d (12.5% DW) in group G. Protein provided by amino acids 3 g/kg/d for groups C and G versus 4 g/kg/d for group P. Outcomes (weight, height, and head circumference) was monitored on a daily basis until the BW was regained. Data has been gathered recently and is being processed. We hypothesize that neonates with higher amino acid intake will result in sooner BW regain than other groups. The result will be presented at the conference. The findings of this study not only can help optimize nutrition, cost reduction, and shorter NICU admission of VLBW neonates at the hospital level but eventually contribute to reduced healthcare-associated infections (HAIs) and an improved health economy.

Keywords: very low birth weight neonates, weight gain, parenteral nutrition, glucose, amino acids

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Authors: Lukas Stiburek, Jana Cesnekova, Josef Houstek, Jiri Zeman

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Cellular function depends on mitochondrial function and integrity that is therefore maintained by several classes of proteins possessing chaperone and/or proteolytic activities. In this work, we focused on characterization of LACE1 (lactation elevated 1) function in mitochondrial protein homeostasis maintenance. LACE1 is the human homologue of yeast mitochondrial Afg1 ATPase, a member of SEC18-NSF, PAS1, CDC48-VCP, TBP family. Yeast Afg1 was shown to be involved in mitochondrial complex IV biogenesis, and based on its similarity with CDC48 (p97/VCP) it was suggested to facilitate extraction of polytopic membrane proteins. Here we show that LACE1, which is a mitochondrial integral membrane protein, exists as part of three complexes of approx. 140, 400 and 500 kDa and is essential for maintenance of fused mitochondrial reticulum and lamellar cristae morphology. Using affinity purification of LACE1-FLAG expressed in LACE1 knockdown background we show that the protein physically interacts with mitochondrial inner membrane protease YME1L. We further show that human LACE1 exhibits significant pro-apoptotic activity and that the protein is required for normal function of the mitochondrial respiratory chain. Thus, our work establishes LACE1 as a novel factor with the crucial role in mitochondrial homeostasis maintenance.

Keywords: LACE1, mitochondria, apoptosis, protease

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Authors: Asif Bilal, Fouzia Tanvir, Sibtain Ahmad

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Estrogen receptor alpha, also known as estrogen receptor-1, is highly involved in risk of mammary carcinoma. The objectives of this study were to identify non-synonymous SNPs of estrogen receptor and their association with breast cancer and to identify the chemotherapeutic responses of phytochemicals against it via in-silico study design. For this purpose, different online tools. to identify pathogenic SNPs the tools were SIFT, Polyphen, Polyphen-2, fuNTRp, SNAP2, for finding disease associated SNPs the tools SNP&GO, PhD-SNP, PredictSNP, MAPP, SNAP, MetaSNP, PANTHER, and to check protein stability Mu-Pro, I-Mutant, and CONSURF were used. Post-translational modifications (PTMs) were detected by Musitedeep, Protein secondary structure by SOPMA, protein to protein interaction by STRING, molecular docking by PyRx. Seven SNPs having rsIDs (rs760766066, rs779180038, rs956399300, rs773683317, rs397509428, rs755020320, and rs1131692059) showing mutations on I229T, R243C, Y246H, P336R, Q375H, R394S, and R394H, respectively found to be completely deleterious. The PTMs found were 96 times Glycosylation; 30 times Ubiquitination, a single time Acetylation; and no Hydroxylation and Phosphorylation were found. The protein secondary structure consisted of Alpha helix (Hh) is (28%), Extended strand (Ee) is (21%), Beta turn (Tt) is 7.89% and Random coil (Cc) is (44.11%). Protein-protein interaction analysis revealed that it has strong interaction with Myeloperoxidase, Xanthine dehydrogenase, carboxylesterase 1, Glutathione S-transferase Mu 1, and with estrogen receptors. For molecular docking we used Asiaticoside, Ilekudinuside, Robustoflavone, Irinoticane, Withanolides, and 9-amin0-5 as ligands that extract from phytochemicals and docked with this protein. We found that there was great interaction (from -8.6 to -9.7) of these ligands of phytochemicals at ESR1 wild and two mutants (I229T and R394S). It is concluded that these SNPs found in ESR1 are involved in breast cancer and given phytochemicals are highly helpful against breast cancer as chemotherapeutic agents. Further in vitro and in vivo analysis should be performed to conduct these interactions.

Keywords: breast cancer, ESR1, phytochemicals, molecular docking

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Authors: Hamza Javar Magnier, Robin Curtis

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There have been rapid advances in the upstream processing of protein therapeutics, which has shifted the bottleneck to downstream purification and formulation. Finding liquid formulations with shelf lives of up to two years is increasingly difficult for some of the newer therapeutics, which have been engineered for activity, but their formulations are often viscous, can phase separate, and have a high propensity for irreversible aggregation1. We explore means to develop improved predictive ability from a better understanding of how protein-protein interactions on formulation conditions (pH, ionic strength, buffer type, presence of excipients) and how these impact upon the initial steps in protein self-association and aggregation. In this work, we study the initial steps in the aggregation pathways using a minimal protein model based on square-well potentials and discontinuous molecular dynamics. The effect of model parameters, including range of interaction, stiffness, chain length, and chain sequence, implies that protein models fold according to various pathways. By reducing the range of interactions, the folding- and collapse- transition come together, and follow a single-step folding pathway from the denatured to the native state2. After parameterizing the model interaction-parameters, we developed an understanding of low-temperature conformational properties and fluctuations, and the correlation to the folding transition of proteins in isolation. The model fluctuations increase with temperature. We observe a low-temperature point, below which large fluctuations are frozen out. This implies that fluctuations at low-temperature can be correlated to the folding transition at the melting temperature. Because proteins “breath” at low temperatures, defining a native-state as a single structure with conserved contacts and a fixed three-dimensional structure is misleading. Rather, we introduce a new definition of a native-state ensemble based on our understanding of the core conservation, which takes into account the native fluctuations at low temperatures. This approach permits the study of a large range of length and time scales needed to link the molecular interactions to the macroscopically observed behaviour. In addition, these models studied are parameterized by fitting to experimentally observed protein-protein interactions characterized in terms of osmotic second virial coefficients.

Keywords: protein folding, native-ensemble, conformational fluctuation, aggregation

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Authors: Oqba Basal, Andras Szabo

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Using mineral fertilization is increasing worldwide, as it is claimed to be majorly responsible for achieving high yields; however, the negative impacts of mineral fertilization on soil and environment are becoming more obvious, with alternative methods being more necessary and applicable, especially with the current climatic changes which have imposed serious abiotic stresses, such as drought. An experiment was made during 2017 growing season in Debrecen, Hungary to investigate the effects of inoculation and N fertilization on the seed yield and protein concentration of the soybean (Glycine max (L.) Merr.) cultivar (Panonia Kincse) under three different irrigation regimes: severe drought stress (SD), moderate drought stress (MD) and control with no drought stress (ND). Three N fertilizer rates were applied: no N fertilizer (0 N), 35 kg ha⁻¹ of N fertilizer (35 N) and 105 kg ha⁻¹ of N fertilizer (105 N). Half of the seeds in each treatment was inoculated with Bradyrhizobium japonicum inoculant, and the other half was not inoculated. The results showed significant differences in the seed yield associated with inoculation, irrigation and the interaction between them, whereas there were no significant differences in the seed yield associated with fertilization alone or in interaction with inoculation or irrigation or both. When seeds were inoculated, yield was increased when (35 N) was applied compared to (0 N) but not significantly; however, the high rate of N fertilizer (105 N) reduced the yield to a level even less than (0 N). When seeds were not inoculated, the highest rate of N increased the yield the most compared to the other two N fertilizer rates whenever the drought was present (moderate or severe). Under severe drought stress, inoculation was positively and significantly correlated with yield; however, adding N fertilizer increased the yield of uninoculated plants compared to the inoculated ones, regardless of the rate of N fertilizer. Protein concentration in the seeds was significantly affected by irrigation and by fertilization, but not by inoculation. Protein concentration increased as the N fertilization rate increased, regardless of the inoculation or irrigation treatments; moreover, increasing the N rate reduced the correlation coefficient of protein concentration with the irrigation. It was concluded that adding N fertilizer is not always recommended, especially when seeds are inoculated before being sown; however, it is very important under severe drought stress to sustain yield. Enhanced protein concentrations could be achieved by applying N fertilization, whether the seeds were pre-inoculated or not.

Keywords: drought stress, N fertilization, protein concentration, soybean

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Authors: Nusaiba Al-Nemrawi, Belal Al-Husein

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Methotrexate (MTX) is a stoichiometric inhibitor of dihydrofolate reductase, which is essential for DNA synthesis. MTX is a chemotherapeutic agent used for treating many types of cancer cells. However, cells’ resistant to MTX is very common and its pharmacokinetic behavior is highly problematic. of MTX within tumor cells, we propose encapsulation of antitumor drugs in nanoparticulated systems. Chitosan (CS) is a naturally occurring polymer that is biocompatibe, biodegradable, non-toxic, cationic and bioadhesive. CS nanoparticles (CS-NPs) have been used as drug carrier for targeted delivery. Titanium dioxide (TiO2), a natural mineral oxide, which is used in biomaterials due to its high stability and antimicrobial and anticorrosive properties. TiO2 showed a potential as a tumor suppressor. In this study a new formulation of MTX loaded in CS NPs (CS-MTX NPs) and coated with Titanium oxide (TiO2) was prepared. The mean particle size, zeta potential, polydispersity index were measured. The interaction between CS NPs and TiO2 NPs was confirmed using FTIR and XRD. CS-MTX NPs was studied in vitro using the tumor cell line MCF-7 (human breast cancer). The results showed that CS-MTX has a size around 169 nm and as they were coated with TiO2, the size ranged between and depending on the ratio of CS-MTX to TiO2 ratio used in the preparation. All NPs (uncoated and coated carried positive charges and were monodispersed. The entrapment efficacy was around 65%. Both FTIR and XRD proved that TiO2 interacted with CS-MTX NPs. The drug invitro release was controlled and sustained over days. Finally, the studied in vitro using the tumor cell line MCF-7 suggested that combining nanomaterials with anticancer drugs CS-MTX NPs may be more effective than free MTX for cancer treatment. In conclusion, the combination of CS-MTX NPs and TiO2 NPs showed excellent time-dependent in vitro antitumor behavior, therefore, can be employed as a promising anticancer agent to attain efficient results towards MCF-7 cells.

Keywords: Methotrexate, Titanium dioxide, Chitosan nanoparticles, cancer

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Authors: Sarita Puri, Tapan K. Chaudhuri

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Chaperonin GroEL is a tetradecameric Escherichia coli protein having identical subunits of 57 kDa. The elucidation of thermodynamic parameters related to stability for the native GroEL is not feasible as it undergoes irreversible unfolding because of its large size (800kDa) and multimeric nature. Nevertheless, it is important to determine the thermodynamic stability parameters for the highly stable GroEL protein as it helps in folding and holding of many substrate proteins during many cellular stresses. Properly folded monomers work as building-block for the formation of native tetradecameric GroEL. Spontaneous refolding behavior of monomeric GroEL makes it suitable for protein-denaturant interactions and thermodynamic stability based studies. The urea mediated unfolding is a three state process which means there is the formation of one intermediate state along with native and unfolded states. The heat mediated denaturation is a two-state process. The unfolding process is reversible as observed by the spontaneous refolding of denatured protein in both urea and head mediated refolding processes. Analysis of folding/unfolding data provides a measure of various thermodynamic stability parameters for the monomeric GroEL. The proposed mechanism of unfolding of monomeric GroEL is a three state process which involves formation of one stable intermediate having folded apical domain and unfolded equatorial, intermediate domains. Research in progress is to demonstrate the importance of specific residues in stability and oligomerization of GroEL protein. Several mutant versions of GroEL are under investigation to resolve the above mentioned issue.

Keywords: equilibrium unfolding, monomeric GroEl, spontaneous refolding, thermodynamic stability

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Authors: Saba Belgheisi

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Food packaging, is an important discipline in the area of food technology, concerns preservation and protection of foods. The objective of this research was to determine of the effect of whey protein based edible coating on the moisture loss and sensory attributes of fresh mutton after 0, 1, 3 and 5 days at 5° C. The moisture content, moisture loss and sensory attributes (juiciness, color and odor) of the coated and uncoated samples were analyzed. The results showed that, moisture content, moisture loss, juiciness and color of the coated and uncoated samples have significant differences (p < 0.05) at the intervals of 0 to 1 and 1 to 3 days of storage. But no significant difference was observed at interval time 3 to 5 days of storage (p > 0.05). Also, there was no significant differences in the odor values of the coated and uncoated samples (p > 0.05). Therefore, the coated samples had consistently more moisture, juiciness and colored values than uncoated samples after 3 days at 5° C. So, whey protein edible coating could enhance product presentation and eliminate the need for placing absorbent pads at the bottom of the trays.

Keywords: coating, whey protein, mutton, moisture, sensory

Procedia PDF Downloads 443

Authors: Sodamade A., Bolaji K. A.

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Problems associated with protein malnutrition are still prevalent in third-world countries, leading to the constant search for plants that could serve as nutrients and medicinal purposes. Huckleberry is one of the plants that has been proven useful locally in the treatment of numerous ailments and diseases. A fresh sample of the plant (Vaccinium pavifolium) was collected from a vegetable garden situated near the Erelu dam of the Emmanuel Alayande College of Education Campus, Oyo. The sample was authenticated at the Forestry Research Institute of Nigeria (FRIN) Ibadan. The leaves of the plant were plucked and processed for leaf protein concentrates before proximate composition, mineral analysis phytochemical and antimicrobial properties were determined using a standard method of analysis. The results of proximate constituents showed; moisture content; 9.89±0.051g/100g, Ash; 3.23±0.12g/100g, crude fat; 3.96±0.11g/100g and 61.27±0.56g/100g of Nitrogen free extractive. The mineral analysis of the sample showed; Mg; 0.081±0.00mg/100g, Ca; 42.30±0.05mg/100g, Na; 27.57±0.09mg/100g, K; 6.81±0.01mg/100g, P; 8.90±0.03mg/100g, Fe; 0.51±0.00mg/100g, Zn; 0.021±0.00mg/100g, Cd; 0.04±0.04mg/100g, Pb; 0.002±0.00mg/100g, Cr; 0.041±0.00mg/100g Cadmium and Mercury were not detected in the sample. The result of phytochemical analysis of leaf protein concentrates of the Huckleberry showed the presence of Alkaloid, Saponin, Flavonoid, Tanin, Coumarin, Steroids, Terpenoids, Cardiac glycosides, Glycosides, Quinones, Anthocyanin, phytosterols, and phenols. Ethanolic extracts of the Vaccinium parvifolium L. leaf protein concentrates showed that it contains bioactive compounds that are capable of combating the following microorganisms; Staphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis, Pseudomonas aeruginosa, Klebisialae pneumonia and Proteus mirabilis. The results of the analysis of Vaccinium parvifolium L. leaf protein concentrates showed that the sample contains valuable nutrient and mineral constituents, and phytochemical compounds that could make the sample useful for medicinal activities.

Keywords: leaf protein concentrates, vaccinium parvifolium, nutritional characteristics, mineral composition, antimicrobial activity

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Authors: Hemalatha Ganapathyswamy, Thirukkumar Subramani

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Celiac disease is an autoimmune response to dietary wheat gluten. Gluten is the main structure forming protein in bread and hence developing gluten-free bread is a technological challenge. The study aims at using nonwheat flours like barnyard millet and lentil flour to replace wheat in bread formulations. Other characteristics of these grains, such as high protein, soluble fiber, mineral content and bioactive components make them attractive alternatives to traditional gluten-free ingredients in the production of high protein, gluten-free bread. The composite flour formulations for the development of gluten-free bread were optimized using lentil flour (50 to 70 g), barnyard millet flour (0 to 30 g) and corn flour (0 to 30 g) by means of response surface methodology with various independent variables for physical, sensorial and nutritional characteristics. The optimized composite flour which had a desirability value of 0.517, included lentil flour –62.94 g, barnyard millet flour– 24.34 g and corn flour– 12.72 g with overall acceptability score 8.00/9.00. The optimized gluten-free bread formulation had high protein (14.99g/100g) and fiber (1.95g/100g) content. The glycemic index of the gluten-free bread was 54.58 rendering it as low glycemic which enhances the functional benefit of the gluten-free bread. Since the standardised gluten-free bread from barnyard millet and lentil flour are high protein, and gluten-free with low glycemic index, the product would serve as an ideal therapeutic food in the management of both celiac disease and diabetes mellitus with better nutritional value.

Keywords: gluten free bread, lentil, low glycemic index, response surface methodology

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae causes a wide range of infections, including urinary tract infections, sepsis, bacteremia, pneumonia, and liver abscesses. The emergence of multi-drug resistance in this bacterium led to a major setback for clinical management. WHO also endorsed a need for finding alternative therapy to antibiotics for the treatment of these infections. Development of vaccines and passive antibody therapy has been proven as a potent alternative to antibiotics in the case of MDR, XDR, and PDR Klebsiella infections. Siderophore receptors have been demonstrated to be overexpressed for the internalization of iron siderophore complexes during infections in most Gram-negative bacteria. For the present study, immune response to siderophore receptors to establish this protein as a potential immunogen for the development of therapeutic leads was explored. Clinical strains of Klebsiella pneumoniae were grown in iron-deficient conditions, and the iron-regulated outer membrane proteins were extracted and characterized through mass spectrometry for specific identification. The gene for identified protein was cloned in pET- 28a vector and expressed in E. coli. The native protein and the recombinant protein were isolated and purified and used as antigens for the generation of immune response in BALB/c mice. The native protein of Klebsiella pneumoniae grown in iron-deficient conditions was identified as FepA (Ferrienterobactin receptor) and other siderophore receptors. This 80 kDa protein generated an immune response in BALB/c mice. The antiserum from mice after subsequent booster doses was collected and showed binding with FepA protein in western blot and phagocytic uptake of the K. pneumoniae in the presence antiserum from immunized mice also observed from the animal studies after bacterial challenge post immunisation in mice have shown bacterial clearance. The antiserum from mice showed binding and clearance of the Klebsiella pneumoniae bacteria in vitro and in vivo. These antigens used for generating an active immune response in mice can further be used for therapeutic monoclonal antibody development against Klebsiella pneumoniae infections.

Keywords: antiserum, FepA, Klebsiella pneumoniae, multi drug resistance, siderophore receptor

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Authors: Y. Ruchi, A. Prerna, S. Deepshikha

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Amyotrophic lateral sclerosis (ALS), also known as “Lou Gehrig’s disease”. It is a neurodegenerative disease associated with degeneration of motor neurons in the cerebral cortex, brain stem, and spinal cord characterized by distal muscle weakness, atrophy, normal sensation, pyramidal signs and progressive muscular paralysis reflecting. ALS2 is a juvenile autosomal recessive disorder, slowly progressive, that maps to chromosome 2q33 and is associated with mutations in the alsin gene, a putative GTPase regulator. In this paper we have done homology modeling of alsin2 protein using multiple templates (3KCI_A, 4LIM_A, 402W_A, 4D9S_A, and 4DNV_A) designed using the Prime program in Schrödinger software. Further modeled structure is used to identify effective binding sites on the basis of structural and physical properties using sitemap program in Schrödinger software, structural and function analysis is done by using Prosite and ExPASy server that gives insight into conserved domains and motifs that can be used for protein classification. This paper summarizes the structural, functional and binding site property of alsin2 protein. These binding sites can be potential drug target sites and can be used for docking studies.

Keywords: ALS, binding site, homology modeling, neuronal degeneration

Procedia PDF Downloads 371

Authors: Hamzeh Alipour, Masoumeh Bagheri, Abbasali Raz, Javad Dadgar Pakdel, Kourosh Azizi, Aboozar Soltani, Mohammad Djaefar Moemenbellah-Fard

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Background: Maggot debridement therapy is an appropriate, effective, and controlled method using sterilized larvae of Luciliasericata (L.sericata) to treat wounds. Netrin-A is an enzyme in the Laminins family which secreted from salivary gland of L.sericata with a central role in neural regeneration and angiogenesis. This study aimed to production of new recombinant Netrin-A protein of Luciliasericata larvae by baculovirus expression vector system (BEVS) in SF9. Material and methods: In the first step, gene structure was subjected to the in silico studies, which were include determination of Antibacterial activity, Prion formation risk, homology modeling, Molecular docking analysis, and Optimization of recombinant protein. In the second step, the Netrin-A gene was cloned and amplified in pTG19 vector. After digestion with BamH1 and EcoR1 restriction enzymes, it was cloned in pFastBac HTA vector. It was then transformed into DH10Bac competent cells, and the recombinant Bacmid was subsequently transfected into insect Sf9 cells. The expressed recombinant Netrin-A was thus purified in the Ni-NTA agarose. This protein evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay method. Results: The Bacmid vector structure with Netrin-A was successfully constructed and then expressed as Netrin-A protein in the Sf9 cell lane. The molecular weight of this protein was 52 kDa with 404 amino acids. In the in silico studies, fortunately, we predicted that recombinant LSNetrin-A have Antibacterial activity and without any prion formation risk.This molecule hasa high binding affinity to the Neogenin and a lower affinity to the DCC-specific receptors. Signal peptide located between amino acids 24 and 25. The concentration of Netrin-A recombinant protein was calculated to be 48.8 μg/ml. it was confirmed that the characterized gene in our previous study codes L. sericata Netrin-A enzyme. Conclusions: Successful generation of the recombinant Netrin-A, a secreted protein in L.sericata salivary glands, and because Luciliasericata larvae are used in larval therapy. Therefore, the findings of the present study could be useful to researchers in future studies on wound healing.

Keywords: blowfly, BEVS, gene, immature insect, recombinant protein, Sf9

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