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Hilsa species are broadly described as largely anadromous fish with a large valuable population size. It is well-known as one of the most critical commercial and occurs in marine, estuarine and riverine environments Hilsa shad, Tenualosa ilisha locally known as Sbour, migrates to the freshwater environment of the Shatt Al-Arab River systems for breeding. It was determined the genetic variation in 70 specimens of T. ilisha from four sites at the Shatt Al-Arab River, the mtDNA cytochrome-b gene was selected as a molecular marker for detecting genetic diversity, origin, and divergence of the population through comparing them with 6 samples from different locations at Indo-Pacific. The AMOVA analysis showed that the variation between groups is 60.97% and 39.02% within population indicating statistically significant P value (P

Antagonistic interactions among different functional guilds of nematodes have been recognized for quite some time, but the underlying explanatory mechanisms are unclear. We investigated responses of tomato (Solanum lycopersicum) to two functional guilds of nematodes - plant parasite (Meloidogyne javanica) and entomopathogens (Heterorhabditis bacteriophora, Steinernema feltiae belowground, and S. carpocapsae) - as well as a leaf mining insect (Tuta absoluta) aboveground. Our results indicate that entomopathogenic nematodes (EPNs): 1) reduced root knot nematode (RKN) infestation belowground, 2) reduced herbivore (T. absoluta) host preference and performance aboveground, and 3) induced overlapping plant defense responses by rapidly activating polyphenol oxidase (PPO) and guaiacol peroxidase (GP) activity in roots, but simultaneously suppressing this activity in aboveground tissues. Concurrently, we investigated potential plant signaling mechanisms underlying these interactions using transcriptome analyses. We found that both entomopathogens and plant parasites triggered immune responses in plant roots with shared gene expression. Secondary metabolite transcripts induced in response to the two nematode functional guilds were generally overlapping and showed an analogous profile of regulation. Likewise, we show that EPNs modulate plant defense against RKN invasion, in part, by suppressing active expression of antioxidant enzymes. Inoculations of roots with EPN triggered an immune response in tomato via up-regulated phenylpropanoid metabolism and synthesis of protease inhibitors (PIs) in plant tissues, which may explain decreased egg laying and developmental performance exhibited by herbivores on EPN-inoculated plants. Furthermore, changes induced in the volatile organic compound (VOC)-related transcriptome indicated that M. javanica and/or S. carpocapsae inoculation of plants triggered both direct and indirect defenses. Our results support the hypothesis that plants 'mistake' subterranean EPNs for parasites, and these otherwise beneficial worms activate a battery of plant defenses associated with systemic acquired resistance (SAR) and/or induced systemic resistance (ISR) with concomitant antagonistic effects on temporally co-occurring subterranean plant pathogenic nematodes and terrestrial herbivores.

In this study, the antimicrobial effect of exopolysaccharide (EPS) extracted from Enterococcus strains [E. durans K48 (MT437,248), E. faecium R114 (MT437,249) and E. faecium T52 (MT437,250)] isolated from Kishk was applied against some foodborne pathogenic bacteria using well diffusion and microdilution methods. The antioxidant activity of EPS was also evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and ferric reducing antioxidant power (FRAP) method. The cytotoxicity effect of EPS on human Gingival Fibroblast (HGF) cell line was also assessed. The results obtained by antimicrobial test showed that the most resistant bacteria to the examined EPS was Listeria monocytogenes, and the most susceptible were Staphylococcus aureus and E. faecalis. The results showed that the DPPH inhibitory percentage of EPS (25 mg/mL) from E. durans K48, E. faecium R114, and E. faecium T52 was 53%, 58% and 64%, respectively. EPS from E. faecium T52 displayed the highest reducing power, but statistically, there was no significant difference between the reducing power of EPS T52 and EPS R114 (P ≥ 0.05). The lowest toxicity percentage of EPS k48, EPS T52, and EPS R114 on normal human cell line at a concentration of 0.2 mg/mL was 10%, 15%, and 13%, respectively, which was statistically significant (P < 0.05). The obtained results in the present study indicate that EPS from the examined LAB strains with no in vitro cytotoxicity can be a potential source of natural antioxidant and antibacterial agent to be used in food and pharmaceutical industries., This work was financially supported by a Grant (No. 47333) from Ferdowsi University of Mashhad (Research affairs), Iran.

Trabajo presentado en el XIII International Scientific Agriculture Symposium AGROSYM 2022, celebrado en Sarajevo (Bosnia y Herzegovina), del 6 al 9 de octubre de 2022, Exopolysaccharides are high molecular weight polymers composed of sugar subunits. Produced exopolysaccharides by lactic acid bacteria (LAB) play a significant role in improvement of organoleptic properties of fermented dairy products such as yogurt. Diversely, the probiotic function of these bacteria and the prebiotic properties of their produced biopolymers promote consumer¿s health. For this purpose, a traditional dairy product known as ¿Kishk¿ was selected. 143 strains of lactic acid bacteria were isolated from Iranian Kishk in Khorasan Province and cultured in formulated MRS mediums with different sugars such as glucose, fructose, sucrose and, lactose (40 g/L) and incubated in anaerobic conditions at 30 and 37°C for 48 hours. The microscopic features of the isolates were assessed and the production of exopolysaccharide in the culture medium was evaluated by disk and ruthenium red methods. The phenol-sulfuric and weight method were used to quantify exopolysaccharide production. Results showed pH of Kishk samples ranged from 3.60 to 4.08 and the average of total mesophilic count and LAB count of samples were 6.50 and 5.89 log CFU/g, respectively. Analysis of data exhibited 79 out of 143 lactic acid bacteria isolates were exopolysaccharide producer and 70% of them were cocci. The average of maximum and minimum production by weight method were 2.61 g/L and 0.08 g/L, respectively. The average of highest and the lowest amount of exopolysaccharide by phenol sulfuric method were measured 1.87 g/L and 0.06 g/L, respectively. This study indicates the potential of exopolysaccharide production by Iranian native species from dairy products.

One of the most promising ways to diagnose cancer especially colorectal cancer (CRC) is to trace its epigenetic events. In this article, a discovery step for detection of methylated DNA markers (MDMs) was performed using SureSelectXT Methyl-Seq in CRC case and control groups in addition to several methylation profiling datasets (GSE48684, GSE53051, GSE77718, GSE101764, and GSE42752). In silico validation of MDMs in colorectal and other cancers was conducted by Lnc2met. MethyLight assay was run on 40 and 47 case and control formalin-fixed paraffin-embedded tissues, respectively and the performance of selected genes were classified by support vector machine (SVM). As a result, 180 regions were identified among all common genes. In addition to SEPT9 and SFRP2, the best three MDM regions were selected from SLC30A10, AKR1B1 and GALNT14. Based on all assays, the best performance was accomplished by SEPT9/AKR1B1 with 98% sensitivity, 99% specificity, 125 positive likelihood ratio, 0.02 negative likelihood ratio and 5074 diagnostic odds ratio. Our results indicate that the AKR1B1/SEPT9 methylation panel detects CRC with a higher performance than SEPT9 methylation, which is a commercial diagnostic test for CRC. However, the creation of a clinically valuable test derived from this study requires performance evaluation in liquid biopsies.

Sheep satellite cells more than satellite cells of the rat and mouse are similar to human satellite cells. These cells are widely used in the modeling and treatment of diseases like heart insufficiency, neurological diseases, muscular dystrophy, cerebral cell transplantation for the treatment of migraines, screening, and the production of new drugs. This study was aimed to isolate and culture primary satellite cells (PSCs) obtained from sheep fetus, and perform clonal expansion of transfected PSCs. Skeletal muscle tissues of hind limbs were collected from sheep fetuses obtained from a local abattoir. After enzymatic digestion, flasks were replaced after 3 hours to isolate non-myogenic cells, such as fibroblasts. After six days, the cells were differentiated to myoblasts. Using a differentiation medium containing the horse serum, myotube cells were observed in the flask, indicating that the cultured cells were satellite cells. The mRNA expression of the PAX7 gene was used to confirm the presence of satellite cells. In addition, the results showed that satellite cells grow in a culture medium containing 5% FBS without differentiation, while 10% FBS initiates their differentiation.

One of the most effective methods for increasing the antimicrobial activity of a substance is to combine it with one or more other antimicrobial agents. The aim of the present study was to evaluate the antimicrobial effect of buforin I and nisin alone and investigate the synergistic action of these compounds against the most important food spoilage microorganisms in clouding B. subtilis, S. epidermidis, L. innocua, E. coli, S. Enteritidis, A. oryzae, R. glutinis and G. candidum. The results of MIC and MBC/MFC examinations showed that buforin I had higher antimicrobial activity than nisin on all the microbial strains used in this study (p≤0.5). E.coli was the most resistant to both antimicrobial agents, while Listeria innocua and Staphylococcus epidermidis were the most sensitive to nisin and buforin I, respectively. The results of synergistic interaction between buforin I and nisin indicated that the combination of buforin I and nisin on B. subtilis, S. epidermidis and A. oryzae showed synergistic effect, while it had no effect on S. Enteritidis and Geotrichum candidum. The combination of buforin I and nisin showed partial synergistic effect on Listeria innocua, Escherichia coli, Rhodotorula glutinis. Assessment of viability of the microorganisms under the antimicrobial agents alone and in combination with each other at MICs and FICs indicated that use of these antimicrobial agents in combination enhances antimicrobial activity at lower concentrations of both agents. The present study investigated the antimicrobial properties of buforin I against food spoilage microorganisms for the first time and suggests that its use alone or in combination with nisin may provide a clear horizon for the application of antimicrobial peptides as natural preservatives. Thus, the combination of antimicrobial peptides and traditional antimicrobial food preservative could be a promising option for the prevention of contamination, spoilage, and infestation of food and beverage products.

Objective Salmonella is a Gram-negative, anaerobic, flagellated bacterium of Salmonella type in Enterobacteriaceae family, which is known as a zoonosis infectious agent. In recent years, different types of antibiotics have been used to overcome the Salmonella infection; however, the increasing number of antibiotic-resistant bacteria, the provision of alternative methods seems be necessary. Specific immunoglobulin Y (IgY) originated from egg yolk causes passive immunity in the fetus, and extracting these antibodies from egg yolk is cheaper and more feasible than other antibodies. Numerous studies have shown that the egg yolk IgY has immunogenic function against a wide range of bacterial and viral infections in mammals. The purpose of this study was to produce specific immunoglobulin Y against Salmonella typhimurium and Salmonella enteritidis bacteria in laying hens. Materials and Methods In the current study, six pullets were inoculated and boosted with one milliliter of killed Salmonella typhimurium and Salmonella enteritidis. Injections were done fortnightly until one week before egg production. After breast muscle injection of chickens, the eggs were collected, and the IgY was extracted from yolk by the Polson method with polyethylene glycol 6000. The produced IgY was approved by SDS-PAGE method. The specific indirect ELISA done by specificity test for purified IgY also approved the specific IgY against both Salmonella typhimurium and Salmonella enteritidis. Results The results of SDS - PAGE demonstrated the presence of two protein bands of 27 and 67 kDa representing light and heavy chains of IgY, respectively. The specific indirect ELISA method proved that the specific IgY against Salmonella typhimurium and Salmonella enteritidis was produced in the egg yolk. Conclusions It might be possible to administer the egg yolk containing specific IgY against both Salmonella typhimurium and Salmonella enteritidis, to induce passive immunity for preventing some pathogenic diseases in calves such as diarrhea.

Background: Due to the heating of amino acids with edible oils to high temperatures, different amounts of acrylamide are produced. Objective: The purpose of this study was to compare the level of acrylamide in the tahdig of bread and tahdig of potato prepared with the common liquid and solid edible oils, including sunflower, corn, canola, frying oil and solid oils. Methods: The tahdig of bread and potato was prepared under the same temperature and time with different oils. Acrylamide isolation was performed on a solid-phase extraction (SPE) cartridge and acrylamide was determined using High-performance liquid chromatography (HPLC). Results: The highest amount of acrylamide was obtained with sunflower oil in the tahdig of potato (194.091 mg/Kg) and the lowest amount of acrylamide was obtained with solid oil in the tahdig of bread (48.54 mg/Kg). For all the oils, the acrylamide content of the tahdig of potato was higher than bread. Conclusion: This study clearly demonstrated the involvement of the kind of oils in the formation of acrylamide in the tahdig of bread.

The novel coronavirus (SARS-CoV-2) has without a doubt escalated to become a global crisis. Taking into consideration our limited knowledge regarding the virus, all the efforts to provide better understanding or explore the solutions are highly welcomed. In this article, 88 conventional drugs, 16 vitamins, and 63 natural (plant) compounds were chosen to perform a binding simulation with the reported COVID-19 main protease (Mpro) in search for probable inhibitors. Based on docking results, various vitamins (B9, A, K, and E vitamins) exhibited a significantly strong interaction with the studied receptor which might refer to the importance of these supplements in daily diets. Additionally, the strong ligand-protein interactions of some conventional drugs such as Pleconaril, Adefovir dipivoxil, and Stavudine in addition to plant-based compounds such as Curcumin (Curcuma longa), Anolignan A (Anogeissus acuminata), and Phyllamyricin B (Phyllanthus myrtifolius) render these compounds promising and recommended for further studies.

Recently, ‘solid tumor biopsies’ have been challenged by the emergence of ‘liquid biopsies’, which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC). Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR. The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76. We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.

Lactoferrin is one of the most important components in milk. Lactoferrin is an 80 kDa iron-binding glycoprotein that involves a diverse range of biological activities. However, the antimicrobial properties are its most widely studied function. The aim of this study was to assay the antimicrobial and antibiofilm activities of bovine lactoferrin against five of the most important foodborne pathogenic bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Listeria innocua, in planktonic and biofilm forms. Bovine lactoferrin was purchased, MIC and MBC were determined for the mentioned bacterial strains and the effect of lactoferrin concentrations on biofilm formation was evaluated using microtiter plate method. Based on the results, lactoferrin had a greater effect on gram-positive bacteria, L. monocytogenes and L. innocua were the most sensitive strains. MIC for L. monocytogenes, L. innocua, S. aureus and P. aeruginosa was 6, 6, 8 and 14 mg/mL, respectively, Nevertheless, MIC for gram negative E. coli was not observed, even at 16 mg/mL. The result demonstrated that MBC for L. monocytogenes, L. innocua and S. aureus were 10, 12 and 16 mg/mL, respectively. However, MBC for P. aeruginosa and E. coli were not observed for the lactoferrin concentrations evaluated. The highest biofilm formation inhibitory effects of bovine lactoferrin were against L. monocytogenes and L. innocua. To our knowledge, this is the first report on the antibiofilm activity of bovine lactoferrin against foodborne pathogenic bacteria. Our findings suggest lactoferrin to possibly use as an anti-biofilm agent in both food and pharmaceutical industries.

Sheep has been an essential source of food to the inhabitants of the Iranian plateau for centuries. Furthermore, this geographic area is considered the original place of sheep domestication. Phylogenetic studies are highly important in understanding the evolutionary relationships among species. This understanding assists in decision making and planning for genetic resources conservation programs. Analyzing sequences of mitochondrial genome regions provides more reliable evidence regarding the genetic diversity and evolutionary origin of the species, due to the high rate of mitochondrial genome evolution compared to the nucleus. The aim of this study was to use the sequence of mitochondrial control region to investigate the phylogenetic relationship and genetic distances between some domestic sheep breeds of Iran and other Middle Eastern countries. For this purpose, blood samples were collected from Zel, Dalagh, and Mehrabani sheep breeds. After DNA extraction, polymerase chain reaction (PCR) was carried out using specific primers for the targeted mitochondrial genome. Then, PCR products were purified and a standard sequencing was performed. Sequences obtained from this study were compared with other (NCBI) registered sequences of Middle Eastern breeds. Analysis of phylogenetic tree of the main haplotypes of sheep revealed that Zel breed was grouped in the haplotype B with other thin-tailed breeds, such as Karayaka and Sakiz. On the other hand, Mehrabani sheep breed was placed in the haplotype A along with other Middle Eastern fat-tailed breeds such as Naeimi and Saidi. This study provided additional proof for the use of control region sequence as a precise method of genetic distance estimation among sheep breeds.

Computational prediction of signal peptides is one of the most important steps in genetic engineering experiments. The periplasmic expression cause the reducing in the inherent destructive behavior of Bofurin I against its host and also reducing its susceptibility to proteolytic degradation. In order to predict the best signal peptides for expression of Buforin I in E. coli, 103 signal sequences were retired from signal peptide databases. Since the purpose of this study was to introduce the optimal signal peptides for periplasmic expression, first, sub-cellular localization site of signal peptides was analyzed. Then, n, h, and c regions of signal peptide, signal peptide probability and physico-chemical features were investigated. Base on the results, MalE, hofQ, papK, ugpB, zraP, and sfmC were introduced as the best signal peptides. For increasing the half-life of mRNA and the increasing the stability of the mRNA against exonuclease activity, secondary structures of mRNA including Shine-Dalgarno, untranslated region of ompA, start codon, signal peptide and sequences of Buforin I were analyzed. Based on the total free energy pilot evaluated and mRNA conformations, papK seemed more appropriate than the rest of the signal peptides. The obtained result of this study can be used for design the periplasmic expression constructs.

Clostridium perfringens-induced necrotic enteritis (NE) is an economically important disease of broiler chickens. The present study evaluated the effect of C. perfringens on the intestinal histomorphometry, enteric microbial colonization, and host immune responses using 3 experimental NE reproduction methods. The experimental groups consisted of 1) unchallenged Control diet (corn-soybean meal), 2) Control diet + Eimera inoculation at d 11 followed by C. perfringens challenge at d 15 (ECp), 3) Wheat-based diet + C. perfringens challenge (WCp), and 4) Wheat-based diet + Eimeria inoculation followed by C. perfringens challenge (WECp). The results showed that chickens receiving ECp and WECp had reduced (P < 0.05) bird performance coupled with enteric gross lesions and epithelial damage at d 17 and 24 of age compared to unchallenged control birds. These ECp and WECp administered birds also had increased (P < 0.05) ileal colonization by clostridia and E. coli at d 17 and 24, while the resident Lactobacillus counts were reduced (P < 0.05) at d 24 of age. Furthermore, at d 24, jejunal transcription of IL-6, IL-10, annexin-A1 and IL-2 genes was upregulated (P < 0.05) in the ECp group, whereas the transcription of TNF receptor associated factor (TRAF)-3 gene was increased (P < 0.05) in WECp treated birds when compared to unchallenged control group. Additionally, stimulation of chicken splenocytes and cecal tonsilocytes with virulent C. perfringens bacilli or their secretory proteins resulted in a higher (P < 0.05) frequency of T cells and their upregulation of MHC-II molecule, as determined by flow cytometry. These findings suggest that C. perfringens, while inducing epithelial damage and changes in microbiota, can also trigger host immune responses. Furthermore, NE reproduction methods using coccidia with or without the wheat-based dietary predisposition seem to facilitate an optimal NE reproduction in broiler chickens and thus, may provide better avenues for future C. perfringens research.

This study aimed to monitor the effect of including rumen-protected L-carnitine (Carneon 20 Rumin-Pro, Kaesler Nutrition GmbH, Cuxhaven, Germany) in the transition diet on the productive and metabolic responses of multiparous high-producing Holstein dairy cows. Thirty-two multiparous cows were allocated in a completely randomized design to receive the same diet plus 60 g fat prill containing 85% palmitic acid (control, n = 16) or 100 g rumen-protected L-carnitine (RLC, n = 16); at 28 days before expected calving until 28 days in milk (DIM). Fat prill was included in the control diet to balance the palmitic acid content of both experimental diets. Milk production over the 28 DIM for the control and RLC groups was 46.5 and 47.7 kg, respectively. Milk fat content tended to increase upon rumen-protected L-carnitine inclusion (p = 0.1). Cows fed rumen-protected L-carnitine had higher fat- and energy-corrected milk compared with the control group. Pre- and post-partum administration of L-carnitine decreased both high- and low-density lipoprotein concentrations in peripheral blood of post-partum cows. The results of this study indicated that the concentration of triglycerides and beta-hydroxybutyrate was not significantly different between the groups, whereas the blood non-esterified fatty acid concentration was markedly decreased in cows supplemented with L-carnitine. Animals in the RLC group had a significant (p < 0.05) lower blood haptoglobin concentration at 7 and 14 DIM than the control. Animals in the RLC group had a lower concentration of blood enzymes than those of the control group. The mRNA abundance of Toll-like receptors 4, cluster of differentiation 14, and myeloid differential protein 2 did not significantly change upon the supplementation of L-carnitine in the transition diet. In summary, the dietary inclusion of RLC improved dairy cow's performance during the early lactation period. Greater production, at least in part, is driven by improved energy utilization efficiency and enhanced metabolic status in animals during the periparturient period.

DNA methylation is a fundamental epigenetic process and have a critical role in many biological processes. The study of DNA methylation at a large scale of genomic levels is widely conducted by several techniques that are next-generation sequencing (NGS)-based methods. Methylome data revealed by DNA methylation next-generation sequencing (mNGS), should be always verified by another technique which they usually have a high cost. In this study, we offered a low-cost approach to corroborate the mNGS data. In this regard, mNGS was performed on 6 colorectal cancer (case group) and 6 healthy individual colon tissue (control group) samples. An R-script detected differentially methylated regions (DMRs), was further validated by high resolution melting (MS-HRM) analysis. After analyzing the data, the algorithm found 194 DMRs. Two locations with the highest level of methylation difference were verified by MS-HRM, which their results were in accordance with the mNGS. Therefore, in the present study, we suggested MS-HRM as a simple, accurate and low-cost method, useful for confirming methylation sequencing results.

Background:Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens.Objective:n this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated.Method:Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay.Results:The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH.Conclusion:Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

Gamma amino butyric acid (GABA) is used as drugs, food ingredients, and dietary supplements. l-glutamate is converted to GABA by the decarboxylation reaction, which is catalyzed by the glutamate decarboxylase (GAD). Escherichia coli is widely being used to express proteins. However, without appropriate signal peptide, it cannot be applied for secretory proteins. Selecting a suitable signal peptide (SP) is a critical step in the secretory production of different proteins. In silico identification of suitable SP is a reliable and cost-effective alternative to experimental approaches. In previous studies, the localization of proteins was not considered and the SPs of periplasmic, membranes and extracellular were compared. Therefore, this study aimed to predict the best SP for the expression of recombinant GAD in the outer membrane of E. coli only. Also, we compared twelve servers to evaluate protein localization, solubility, and secretory pathway. In the present study, 127 SPs were taken from the Signal Peptide database. The localization site, physico-chemical properties, location of cleavage sites, regions and D-score of them were determined by ProtComp, ProtParam, and SignalP 3.0 and 4.1 servers, respectively. To rank SPs based on the secretion properties, PRED-TAT and SignalP 5.0 webservers were used. Based on the results, the localization site of 13 SPs was in the outer membrane of E. coli. Among them, the most suitable candidates seemed to be torT with a reasonably high D-score, aliphatic index, and GRAVY, followed by ccmH and then pspE. TorT accelerates GAD scale-up production and might be useful in future experimental research.