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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. [ABSTRACT FROM AUTHOR]

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 below‐ground, and S. carpocapsae)—as well as a leaf mining insect (Tuta absoluta) above‐ground. Our results indicate that entomopathogenic nematodes (EPNs): (1) reduced root knot nematode (RKN) infestation below‐ground, (2) reduced herbivore (T. absoluta) host preference and performance above‐ground, and (3) induced overlapping plant defence responses by rapidly activating polyphenol oxidase and guaiacol peroxidase activity in roots, but simultaneously suppressing this activity in above‐ground tissues. Concurrently, we investigated potential plant signalling 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 defence against RKN invasion, in part, by suppressing active expression of antioxidant enzymes. Inoculations of roots with EPN triggered an immune response in tomato via upregulated phenylpropanoid metabolism and synthesis of protease inhibitors 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‐related transcriptome indicated that M. javanica and/or S. carpocapsae inoculation of plants triggered both direct and indirect defences. Our results support the hypothesis that plants "mistake" subterranean EPNs for parasites, and these otherwise beneficial worms activate a battery of plant defences associated with systemic acquired resistance and/or induced systemic resistance with concomitant antagonistic effects on temporally co‐occurring subterranean plant pathogenic nematodes and terrestrial herbivores. [ABSTRACT FROM AUTHOR]

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<0.05). Furthermore, the maximum likelihood phylogenetic tree showed two main clusters of all T. ilisha groups representing two stocks of separated breeding grounds have a common ancestor. [ABSTRACT FROM AUTHOR]

There is a continuing need to prevent the increasing use of common antibiotic and find the replacement to combat the drug/antibiotic resistant bacteria such as antimicrobial peptides (AMPs) such as thanatin peptide. In this study, recombinant thanatin peptide was expressed in the HEK293 cell line. Then the antimicrobial properties of this peptide on some poultry and farm animal's pathogen strains were assessed. The thermal-stability of thanatin was predicted in various temperatures through in silico analysis. Afterwards, according to Minimum Inhibitory Concentration (MIC) results, Escherichia coli and Pseudomonas aeruginosa were chosen to test the hypothesis of LptA/LptD–thanatin interaction, computationally. Relative amino acid sequences and crystallography structures were retrieved and missed tertiary structures were predicted. The interaction of thanatin with LptA and LptD of Escherichia coli and Pseudomonas aeruginosa were analyzed subsequently. The antibacterial activity of thanatin peptide was evaluated between 6.25 and 100 μg/mL using minimum inhibitory concentration. Also, the amounts of minimum bactericidal concentrations (MBC) were between 12.5 and 200 μg/mL. The bioinformatics analysis followed by the in vitro assessment, demonstrated that thanatin would be thermally stable in the body temperature of poultry and farm animals. Thanatin could penetrate to the outer membrane domain of LptD in Escherichia coli and it could block the transition path of this protein while the entrance of LptD in Pseudomonas aeruginosa was blocked for thanatin by extra residues in comparison with Escherichia coli LptD. In addition, the quality of interaction, with regard to the number and distance of interactions which leads to higher binding energy for thanatin and LptD of Escherichia coli was much better than Pseudomonas aeruginosa. But the site and quality of interaction for thanatin and LptA was almost the same for Escherichia coli and Pseudomonas aeruginosa. Accordingly, thanatin can prevent the assembly of LptA periplasmic bridge in both pathogens. The antibacterial and thermal stability of the thanatin peptide suggested that thanatin peptide might serve as a natural alternative instead of common antibiotics in the veterinary medicine. The outcome of this in silico study supports the MIC results. Therefore, a probable reason for different level of activity of thanatin against Escherichia coli and Pseudomonas aeruginosa might be the quality of LptA/LptD–thanatin interaction. [ABSTRACT FROM AUTHOR]

Myostatin is a known negative regulator of muscle tissue growth. Thus, an inhibitor of myostatin may be therapeutically useful as an anabolic agent for the muscle tissue. A promising gene-silencing approach for gene therapy is DNA interference (DNAi), a sequence that is complementary to the promoter region of a target gene. To confer resistance to nuclease digestion, several modifications such as methylphosphonate or phosphorothioate have been proposed, wherein a nonbridging oxygen atom in the oligonucleotide phosphate backbone is replaced by sulphur. The aim of the present study was to assess the effectiveness of the DNAi molecule with phosphorothioate (PS) and without phosphorothioate (WPS) modification for inhibition of myostatin gene expression in mice. Eighteen four-week-old male BALB/c mice were randomly divided into three groups: DNAi-PS (n = 6), DNAi-WPS (n = 6) and control (n = 6). Intraperitoneal injections of DNAi (10 mg/kg) were given once a week, and mice body weights were measured weekly and sacrificed after three weeks. The expression of myostatin was assessed using real-time quantitative polymerace chain reaction. For histological evaluation, the skeletal muscle tissue was dissected from the biceps. The results were analysed by a t-test. Results demonstrated that administration of DNAi intraperitoneally with modification could suppress myostatin expression by up to 70%. Leg weight and histological analysis proved that chemically modified DNAi significantly suppressed the myostatin gene in mice. Overall, the results on DNA-induced gene silencing by antisense DNA oligonucleotides in animals can provide insight into the treatment of inherited diseases. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor (IGF) axis is fundamental for mammalian growth and development. However, no comprehensive reference data on gene expression across tissues and pre- and postnatal developmental stages are available for any given species. Here we provide systematic promoter- and splice variant specific information on expression of IGF system components in embryonic (Day 48), fetal (Day 153), term (Day 277, placenta) and juvenile (Day 365–396) tissues of domestic cow, a major agricultural species and biomedical model. Analysis of spatiotemporal changes in expression of IGF1, IGF2, IGF1R, IGF2R, IGFBP1-8 and IR genes, as well as lncRNAs H19 and AIRN, by qPCR, indicated an overall increase in expression from embryo to fetal stage, and decrease in expression from fetal to juvenile stage. The stronger decrease in expression of lncRNAs (average ―16-fold) and ligands (average ―12.1-fold) compared to receptors (average ―5.7-fold) and binding proteins (average ―4.3-fold) is consistent with known functions of IGF peptides and supports important roles of lncRNAs in prenatal development. Pronounced overall reduction in postnatal expression of IGF system components in lung (―12.9-fold) and kidney (―13.2-fold) are signatures of major changes in organ function while more similar hepatic expression levels (―2.2-fold) are evidence of the endocrine rather than autocrine/paracrine role of IGFs in postnatal growth regulation. Despite its rapid growth, placenta displayed a more stable expression pattern than other organs during prenatal development. Quantitative analyses of contributions of promoters P0-P4 to global IGF2 transcript in fetal tissues revealed that P4 accounted for the bulk of transcript in all tissues but skeletal muscle. Demonstration of IGF2 expression in fetal muscle and postnatal liver from a promoter orthologous to mouse and human promoter P0 provides further evidence for an evolutionary and developmental shift from placenta-specific P0-expression in rodents and suggests that some aspects of bovine IGF expression may be closer to human than mouse. [ABSTRACT FROM AUTHOR]

Current study hypothesized that dietary l‐carnitine (LC) inclusion during the mating period ameliorates both metabolic status and reproductive performance of ewes. Seventy Baluchi ewes (52 ± 4.2 kg of bodyweight and 18 ± 6 months old of age) were enrolled in this study. Animals were randomly allocated into two dietary treatments, control (only basal diet) or basal diet plus supplementation with a rumen‐protected LC (Carneon 20 Rumin‐pro; 20% LC; Kaesler Nutrition GmbH) at the rate of 10 g/head/day from 21 days before until 35 days after introducing rams to the ewes (MP). Feed intake was monitored by subtracting the ort from feed offered. Blood sample collection was conducted on Days −10, +10 and +20 relative to MP. Pregnancy was confirmed on Day 30 post‐MP. Feed intake of the ewes in the LC group was higher than the control (p < 0.05). LC supplementation increased the cholesterol concentration in the ewes (p < 0.05). Blood urea concentration of animals in the LC group was significantly lower than the control (p < 0.05). The mRNA expression of toll‐like receptor 4 was evidently lower in animals supplemented with LC than the control (p < 0.05). Both lambing and fecundity rates in the LC group tended to be higher compared with the control. LC supplementation showed potential to alter certain metabolites in the ewes. A tendency for higher lambing rate may partly be driven by dams efficient energy partitioning to support foetal growth and maintaining pregnancy. [ABSTRACT FROM AUTHOR]

The productivity of beef cows depends on early reproduction traits such as puberty and has an economic impact on the efficiency of production system. Imprinted genes modulate many important endocrine processes such as growth, the onset of puberty and maternal reproductive and behavior. The role of imprinted genes in puberty is a challenging subject since they show the reciprocal role of maternal and paternal genomes in progeny. Although, there are evidences of the involvement of imprint genes in puberty in human, the role of this type of genes in the onset of puberty in cattle has not been studied yet. Here we examined the expression of 27 imprinted genes in pre and post puberty in a bovine model to find differentially expressed imprinted genes in maternal-paternal purebreds and reciprocal crosses across eight tissues and discussed the task of these genes in this crucial process of development and in onset of puberty. DLK1 and MKRN3 that previously described as cause of the central precocious puberty (CPP) in human were differentially expressed in this study. Functional annotation analysis of differentially imprinted genes in different tissues showed significant biological processes of cellular response to growth factor stimulus, response to growth factor, response to parathyroid hormone, developmental growth and the importance of alternative splicing. The results of this study have implications in understanding the role of imprinted genes in the onset of puberty in cattle. [ABSTRACT FROM AUTHOR]

Background: Lactoferrin is a versatile protein with important modulatory functions in inflammation and immune response. This glycoprotein can bind and sequester iron and LPS, thereby intervening in certain signaling pathways and biological processes. In the present meta‐analysis, we aimed to pool experimental data regarding the immunomodulatory effects of lactoferrin and its derived peptides on the NF‐κB signaling pathway. Materials: We searched PubMed, Google Scholar, and Web of Science databases and obtained all related articles published before April 2022. Finally, 25 eligible studies were selected, and their reports were analyzed. Methods: We used Review Manager Version 5.2 to compute the standardized mean difference (SMD) and its 95% confidence interval. In addition, the source of heterogeneity was explored using meta‐regression and sensitivity analysis. The symmetry of the funnel plot and Egger's test were also used to evaluate publication bias utilizing Comprehensive Meta‐Analysis Version 2. Results: Comparing the group of cells and animals exposed to lipopolysaccharide alone with the group that received pretreatment with lactoferrin and its derivatives, we observed significant reductions in TNF‐α, IL‐1 beta, and IL‐6 levels by 8.73 pg/mL, 2.21 pg/mL, and 3.24 pg/mL, respectively, in the second group. Additionally, IKK‐β, p‐IκB, and NF‐κB (p65) levels were significantly lower by 7.37‐fold, 15.02‐fold, and 3.88‐fold, respectively, in various cells and tissues. Conclusion: Based on the results of this meta‐analysis, lactoferrin and its derived peptides can be considered potent prophylactic and therapeutic candidates against inflammation‐associated diseases by targeting the NF‐kB pathway. [ABSTRACT FROM AUTHOR]

DNA-based approaches can now be utilized as low-risk methods to change gene expression. It appears that this approach has the ability to partially replace RNA-based approaches for altering gene expression, which in the majority of cases leads to immunological responses in patients. When utilized as a technique to silence target gene expression, DNA interference (DNAi) is a single-stranded DNA created to complement the upstream region of a gene. This DNAi molecule is stabilized using a variety of chemical changes, including phosphorothioates, methylphosphonate setC, etc. Several studies of the efficient application of DNA-based methods both in eukaryotic cell lines and the therapy of various disorders, such as Duchenne muscular dystrophy, cancer, etc., have been mentioned. Understanding the DNAi process, its transfer carriers, stabilization techniques, and their limitations is crucial for advancing these applications and predicting the future of DNAi both in basic science and the treatment of disorders brought on by abnormal gene expression. The main purpose of this review is introducing benefits of using DNAi in gene silencing. this review has discussed about different applications of DNAi in drug discovery and treatment, criteria of designing DNAi, possible modifications, introducing different types of carriers and limitations of DNAi administration. [ABSTRACT FROM AUTHOR]

Today, ostrich breeding has been widely developed in Iran and other countries due to the ability of this animal to produce quality meat, leather, and oil. However, one of the main problems in breeding them is sex determination using aggressive techniques with low accuracy. This study aimed to determine the sex of immature ostriches using specific primers in a multiplex PCR reaction. This study considered 20 specimens of unspecified immature and six specimens (three adult males and females) of known-sex African ostriches as controls. SS and OSFES primers were used to amplify part of the female-specific sequence and 18S primer was used as a control in a PCR reaction. The presence of SS and OSFES bands in gel electrophoresis indicated the amplification of the desired parts related to the female sex and the absence of these bands indicates the male sex of the species. In total, out of 20 African ostriches studied, 50% of them belonged to females and 50% of them belonged to males. Later, with the growth of immature individuals, the results of this experiment were confirmed. In this study, it was found that the use of feather samples for DNA extraction and multiplex PCR is a suitable, accurate, and cost-effective method in identifying and determining the sex of young ostrich and leads to more real and reliable results, avoiding stress in birds. [ABSTRACT FROM AUTHOR]

Rapid degradation of barley grain (BG, Hordeum vulgare) starch in the rumen can seriously impair rumen fermentation efficiency. Some strategies to curb the negative effects of grain feeding and hamper dysfermentation rely on the usage of phytogenic substances or organic acids. In order to process BGs, they were steeped in 5% lactic acid (BGLA), oregano (BGORE) or thyme (BGTHY) extracts for 48 h. Therefore, an in situ study was conducted to assess the effect of either processed BG or unprocessed BG (control; BGCTRL) on ruminal degradation kinetics (a; soluble fraction, b; potential degradable fraction, c; fractional degradation rate) and effective rumen degradability (ERD) of dry matter (DM), crude protein (CP) and starch. In vitro trials with a 2 × 2 × 4 factorial design were also used to assess the effect of diets which contained intact or processed BGs with different CP [160 (P16) vs. 170 (P17)] g/kg DM,) and ruminal protein degradability percentages [90 (low degradability; LD) vs. 115 (high degradability; HD) g/kg DM] on rumen gas production characteristics (b; asymptotic gas volume, c; the constant rate of gas production), yield of microbial crude protein (MCP) and effective utilizable crude protein in the duodenum (EuCP). In situ data demonstrated that BGLA compared with BGCTRL had significantly lower fractions of “a” (0.22 vs. 0.26, P=0.03) and “c” (0.10 vs. 0.17, P<0.01) and ERD of starch (0.53 vs. 0.64, P=0.01). The treatment of BGs with the plant extracts, however, was not able to change the in situ parameters relatively to BGCTRL. Results of the in vitro trials indicated that diets containing processed BG had higher MCP when compared with BGCTRL (19.74 vs. 15.85 mg/250 mg DM, P<0.01). Lactic acid and ORE-treated barley decreased the gas production constant rate (c; mL/h) and gas volume after 2 h compared with BGCTRL (P≤0.05). Our study revealed that processed BG can alter the rumen starch degradation pattern, and rumen gas production parameters and increase MCP and EuCP. [ABSTRACT FROM AUTHOR]

KRAS mutation is responsible for 40–50% of colorectal cancers (CRCs). RNA-seq data and bioinformatics methods were used to analyze the transcriptional profiles of KRAS mutant (mtKRAS) in comparison with the wild-type (wtKRAS) cell lines, followed by in-silico and quantitative real-time PCR (qPCR) validations. Gene set enrichment analysis showed overrepresentation of KRAS signaling as an oncogenic signature in mtKRAS. Gene ontology and pathway analyses on 600 differentially-expressed genes (DEGs) indicated their major involvement in the cancer-associated signal transduction pathways. Significant hub genes were identified through analyzing PPI network, with the highest node degree for PTPRC. The evaluation of the interaction between co-expressed DEGs and lncRNAs revealed 12 differentially-expressed lncRNAs which potentially regulate the genes majorly enriched in Rap1 and RAS signaling pathways. The results of the qPCR showed the overexpression of PPARG and PTGS2, and downregulation of PTPRC in mtKRAS cells compared to the wtKRAS one, which confirming the outputs of RNA-seq analysis. Further, significant upregualtion of miR-23b was observed in wtKRAS cells. The comparison between the expression level of hub genes and TFs with expression data of CRC tissue samples deposited in TCGA databank confirmed them as distinct biomarkers for the discrimination of normal and tumor patient samples. Survival analysis revealed the significant prognostic value for some of the hub genes, TFs, and lncRNAs. The results of the present study can extend the vision on the molecular mechanisms involved in KRAS-driven CRC pathogenesis. [ABSTRACT FROM AUTHOR]

Myostatin (MSTN) is primarily expressed in skeletal muscle tissue and acts as a negative regulator of skeletal muscle growth by inhibiting differentiation and proliferation of myoblasts. Inhibition of MSTN expression could result in muscular hypertrophy. An effective therapeutic approach based on specific silencing of a target gene is provided by RNA interference. The distribution of biologically active small interfering RNAs (siRNAs) inside the target cells/tissue, is a significant problem due to the limited stability and delivery of siRNAs. Strategies depending on vector delivery have also a limited clinical utility due to safety concerns. Thus direct application of active siRNAs in vivo is the preferred strategy. We described the efficiency of intramuscular and intraperitoneal injections of MSTN-siRNA conjugated with cholesterol into the skeletal muscle of mice. The designed siRNA molecule was complementary to the exon II of the mouse MSTN gene. Mice were injected with a weekly dose of 10 μg/kg conjucated siRNA-cholestrol intraperitoneally or intramuscularly. Our findings suggested that within a few weeks of application, siRNA-treated mice showed a significant increase in muscle mass and suppressed MSTN gene expression. Even though both types of injections increased muscle weight, intramuscular siRNA injections suppressed the MSTN gene more effectively, whereas intraperitoneal RNA injections had a more significant impact on total body weight. The cholesterol-conjugated siRNA platform discussed here may hold promise for treating several skeletal muscle-related diseases, such as atrophic muscle disease, muscular dystrophy, and type II diabetes. [ABSTRACT FROM AUTHOR]

KRAS mutation is responsible for 40–50% of colorectal cancers (CRCs). RNA-seq data and bioinformatics methods were used to analyze the transcriptional profiles of KRAS mutant (mtKRAS) in comparison with the wild-type (wtKRAS) cell lines, followed by in-silico and quantitative real-time PCR (qPCR) validations. Gene set enrichment analysis showed overrepresentation of KRAS signaling as an oncogenic signature in mtKRAS. Gene ontology and pathway analyses on 600 differentially-expressed genes (DEGs) indicated their major involvement in the cancer-associated signal transduction pathways. Significant hub genes were identified through analyzing PPI network, with the highest node degree for PTPRC. The evaluation of the interaction between co-expressed DEGs and lncRNAs revealed 12 differentially-expressed lncRNAs which potentially regulate the genes majorly enriched in Rap1 and RAS signaling pathways. The results of the qPCR showed the overexpression of PPARG and PTGS2, and downregulation of PTPRC in mtKRAS cells compared to the wtKRAS one, which confirming the outputs of RNA-seq analysis. Further, significant upregualtion of miR-23b was observed in wtKRAS cells. The comparison between the expression level of hub genes and TFs with expression data of CRC tissue samples deposited in TCGA databank confirmed them as distinct biomarkers for the discrimination of normal and tumor patient samples. Survival analysis revealed the significant prognostic value for some of the hub genes, TFs, and lncRNAs. The results of the present study can extend the vision on the molecular mechanisms involved in KRAS-driven CRC pathogenesis. [ABSTRACT FROM AUTHOR]

• To date, a few methylated single gene blood biomarkers have been developed for cancer detection including colorectal cancer (CRC) such as methylated SEPT9 gene. • 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. • The creation of a clinically valuable diagnostic test for CRC derived from these studies requires performance evaluation in liquid biopsies. 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. [ABSTRACT FROM AUTHOR]

Purpose: 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). Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

In the present study, a novel chimeric peptide was derived from camel lactoferrin designed with a considerable anti-HCV activity and its neutralization mechanism was predicted by molecular modelling tools. A novel anti-HCV peptide derived from camel lactoferrin (cLF36) was designed and expressed it recombinantly in HEK-293-T cells. Anti-viral activity of this peptide was evaluated against hepatitis C virus by Real-time PCR assay in vitro. Finally, to have a better insight into the mode of action of peptide on HCV entry inhibition, we examined the interaction of cLF36 with envelope glycoprotein E2 by molecular dynamic simulation. This chimeric peptide had significant inhibitory effects on both HCV entry (44 µg/mL) and viral replication (88 µg/mL) under in vitro (p > 0.01). Moreover, cLF36 peptide was not toxic to HEK cells as a normal cell at twofold of its anti-viral concentrations for HCV entry and even at concentrations as high as 250 µg/mL exhibited minimal hemolysis (2.5%) against human RBCs (red blood cells). The results of in silico analysis showed that cLF36 interacted with β-sandwich and front layer of E2 protein as two potential CD81 binding sites. We generated and characterized a new camel lactoferrin derived HCV inhibitors. This peptide blocked HCV entry and also intracellular HCV replication in cell culture experiment. [ABSTRACT FROM AUTHOR]

High temperature is one of the main environmental factors causing economic losses to the poultry industry, as it reduces growth and production performance of chickens. The heat shock proteins (HSPs) play a key role in cellular defense mechanisms during exposure in a hot environment. The aim of this study was to evaluate the expression level of the candidate genes in the liver of Khorasan native chickens under acute heat stress. Sixteen 42 days old chickens were divided into two groups; the control (25 °C and 50% humidity) and heat-treated (42 °C and 50% humidity), and then the liver was sampled. The level of gene expression of HSPB1, HSPB9, SERPINH1, HSPA2 and HSP110 were evaluated using the reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) method. The results of the analysis of variance revealed that the expression of HSPA2 and HSP110 was significantly increased. In the biological processes of gene ontology, three processes had FDR < 0.01. HSPA2 and HSPB1 involved in the processes that stimulated cells against increasing temperature. The results indicated that Khorasan native chickens have suitable resistance to acute heat stress. Furthermore, HSPA2 has the ability to express under high ambient temperature in order to protect the structure of cellular proteins. [ABSTRACT FROM AUTHOR]

1. The objective of the study was to investigate the influence of maternal and parent of origin effects (POE) on genetic variation of Iranian native fowl on economic traits. 2. Studied traits were body weights at birth (BW0), at eight (BW8) and 12 weeks of age (BW12), age (ASM) and weight at sexual maturity (WSM), egg number (EN) and average egg weight (AEW). 3. Several models, including additive, maternal additive genetics, permanent environmental effects and POE were compared using Wombat software. Bayesian Information Criterion (BIC) was used to identify the best model for each trait. The chance of reranking of birds between models was investigated using Spearman correlation and Wilcoxon rank test. 4. Based on the best model, direct heritability estimates for BW0, BW8, BW12, ASM, WSM, EN and AEW traits were 0.05, 0.21, 0.23, 0.30, 0.39, 0.22 and 0.38, respectively. Proportion of variance due to paternal POE for BW8 was 4% and proportion of variance due to maternal POE for BW12 was 5%. 5. Estimated maternal heritability for BW0 was 0.30 and for BW8 and BW12 were 0.00 and 0.01, respectively, which shows that maternal heritability was reduced by age. 6. Based on the results, considering POE for BW8 and BW12 and maternal genetic effects for BW0 improved the accuracy of estimations and avoid reranking of birds for these traits. [ABSTRACT FROM AUTHOR]

Genomic imprinting results in monoallelic expression of genes in mammals and flowering plants. Understanding the function of imprinted genes improves our knowledge of the regulatory processes in the genome. In this study, we have employed classification and clustering algorithms with attribute weighting to specify the unique attributes of both imprinted (monoallelic) and biallelic expressed genes. We have obtained characteristics of 22 known monoallelically expressed (imprinted) and 8 biallelic expressed genes that have been experimentally validated alongside 208 randomly selected genes in bovine (Bos taurus). Attribute weighting methods and various supervised and unsupervised algorithms in machine learning were applied. Unique characteristics were discovered and used to distinguish mono and biallelic expressed genes from each other in bovine. To obtain the accuracy of classification, 10-fold cross-validation with concerning each combination of attribute weighting (feature selection) and machine learning algorithms, was used. Our approach was able to accurately predict mono and biallelic genes using the genomics and proteomics attributes. [ABSTRACT FROM AUTHOR]

Imprinted genes display biased expression of paternal and maternal alleles in mammals. They are marked through epigenetic process during gametogenesis. Characterization of imprinted genes has expanded our understanding of the regulation and function of genes. In the current study, 22 experimentally validated imprinted genes in bovine (Bos Taurus) were analysed. Several supervised machine learning algorithms and attribute weighting methods were used to find characteristics of different types of imprinted genes and suggest a classification method for finding maternally and paternally expressed genes in bovine. For assessing the best model and comparing attributes in other organisms, we have also conducted a comparative analysis for human and sheep imprinted genes. According to the results of the present study, GC contents 10 and 100 kb upstream, Gly and Gln amino acids, Ile/ATC codon usage, LINE and SINE in 100kbup and length of first intron were significantly different between the maternal and paternal genes in cattle. Considering all species together, we found that GC content 100 kb up, LINE 100 kb up and the frequency of amino acids like Gly, Gln and Met were the most important attributes for identifying the paternal and maternal imprinted genes. These findings could imply conservation pattern in the attributes among these species. [ABSTRACT FROM AUTHOR]

The lack of cost-effective methods for producing antimicrobial peptides has made it impossible to use their high potential as a new and powerful class of antimicrobial agents. In recent years, extensive research has been conducted to decrease the cost of recombinant proteins production through microorganisms, transgenic animals, and plants. Well-known genetic and physiological characteristics, short-term proliferation, and ease of manipulation make E. coli expression system a valuable host for recombinant proteins production. Expression in periplasmic space is recommended to reduce the inherently destructive behavior of antimicrobial peptides against the expressing microorganism and to decline susceptibility to proteolytic degradation. In this study, a pET-based expression system was used to express buforin I at E. coli periplasmic space, and its antimicrobial, hemolytic, and cell toxicity activities as well as structural stability were evaluated. The hemolysis activity and cytotoxicity of His-tagged buforin I were negligible and its antimicrobial activity did not show a significant difference compared to synthetic buforin I. In addition, in silico investigating of stability of native and His-tagged buforin I showed that RMSF, RMSD and Rg curves had followed a similar trend during 150 ns simulation. Furthermore, evaluating the modelled structures, FTIR and X-ray methods of both peptides indicated an insignificant structural difference. It was concluded that the recombinant buforin I could be a viable alternative to some currently used antibiotics by successfully expressing it in the pET-based expression system. [ABSTRACT FROM AUTHOR]

Identification of selection signatures may provide a better understanding of domestication process and candidate genes contributing to this process. In this study, two populations of domestic and wild goats from Iran were analyzed to identify selection signatures. RSB, iHS, and XP‐EHH statistics were used in order to identify robust selection signatures in the goat genome. Genotype data of domestic and wild goats from the NextGen project was used. The data was related to 18 Capra aegagrus (wild goat) and 20 Capra hircus (domestic goat) from Iran. The iHS method indicated 675 and 441 selection signatures in C. aegagrus and C. hircus, respectively. RSB and XP‐EHH methods showed about 370 and 447 selection signatures in C. aegagrus and C. hircus, respectively. These selection signatures were mainly associated with milk production, fleece trait, mammary epithelial cells, reproduction, and immune system. [ABSTRACT FROM AUTHOR]

Objective(s): Early, specific, and sensitive detection methods of COVID-19 are essential for force stopping its worldwide infection. Although CT images of the lung and/or viral RNA extraction followed by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) are widely used; they have some limitations. Here, we developed a highly sensitive magnetic bead-based viral RNA extraction assay followed by rRT-PCR. Materials and Methods: Case group included oropharyngeal/nasopharyngeal and blood samples from 30 patients diagnosed positive by PCR test for COVID-19 and control group included 30 same samples from COVID-19 negative PCR test individuals. RNA was extracted, using viral RNA extraction kit as well as using our hand-made capture bead-based technique. A one-step cDNA synthesis and Real Time PCR was conducted. A two-step comparison of the different viral RNA extraction methods for oropharyngeal/nasopharyngeal and blood samples was performed. Student t-test was applied with a P<0.05 considered statistically significant. Results: In the case group, all 30 mucosal samples extracted either with viral RNA extraction kit or with beads-based assay were COVID-19 positive although in the latter category, Cqs were much lower. Although 43% of plasma samples extracted by bead-based method were found to be positive but no plasma samples extracted with column-based kit were detected positive by Real Time PCR. Conclusion: Bead-based RNA extraction method can reduce RNA loss by its single-tube performance and enhance the test sensitivity. It is also more sensitive to lower viral loads as shown in the detection of blood samples and the lower Cqs of mucosal samples. [ABSTRACT FROM AUTHOR]

• SNP markers were used to study genetic diversity of domestic and wild Iranian sheep and goats. • Inbreeding coefficients (FGRM, FHOM and FROH), linkage disequilibrium (LD), and effective population size (Ne) were used. • Wild species had lower genetic diversity than domestic species. • Lower genetic diversity for wild sheep and goat species was due to decrease in Ne in recent generations. • Wild sheep population is in a more critical conditions from genetic diversity point of view. In present study, genetic diversity of domestic and wild Iranian sheep and goats were evaluated using inbreeding coefficients (F GRM , F HOM and F ROH), linkage disequilibrium (LD), and effective population size (Ne). Genomic data of Iranian sheep and goat species were obtained from the NEXTGEN project. Animals consisted of 20 domestic sheep (Ovis aries), 14 wild sheep (Ovis orientalis), 20 domestic goats (Capra hircus) and 18 wild goats (Capra aegagrus). Mutations equivalent to the IlluminaSNPchip50 K were extracted to study mutations for each animal. Based on F GRM , F HOM and F ROH , higher rates of inbreeding were observed in wild species than domestic species. Wild sheep had the highest rate of inbreeding among studied populations. Our results showed a decreasing in LD with increasing in distance between pairs of SNPs so that the average LD in short distance was in the range of 0.11−0.12, and in long distance was in the range of 0.091−0.093. The analysis of LD also demonstrated higher genetic diversity in domestic sheep and goat than wild species and wild sheep had the lowest genetic diversity among studied populations. The results indicated that Ne in 13 generations ago was higher in domestic species compared to wild species. In general, present results revealed that wild species had lower genetic diversity compared to the domestic species in Iran. Meanwhile, wild sheep population is in critical conditions from genetic diversity point of view and required more attention. Designing appropriate breeding programs to improve genetic diversity in wild species, especially in wild sheep in Iran is necessary to protect them from extinction. [ABSTRACT FROM AUTHOR]

• This is the first study for evaluating the meta-analysis of GWAS and gene network analysis across countries for milk production traits in Holstein cows. • 770 significant SNPs associated with milk production traits were detected across countries. • The most significant lead SNPs for milk production traits were identified on BTA14. These SNPs were rs109421300 SNP near the DGAT1 gene, rs109146371 SNP near the PPP1R16A gene, rs134432442 SNP near the CPSF1 gene, rs109968515 SNP near the CYHR1 gene, and rs111018678 SNP near the TRAPPC9 gene. • Lead SNPs with pleiotropic effects for milk production traits (e.g., rs109421300 and rs109350371 SNPs) were detected in multi-trait meta-analysis. • Regulation of cation channel activity, ion channel complex, and phosphoric diester hydrolase activity were the most significant enriched GO terms for milk production traits. A large number of genome-wide association studies (GWAS) in livestock, especially in dairy cows, provide favorable conditions to integrate multiple independent studies. Methods such as meta-analysis provide the identification of effective QTLs with higher precision and power. A meta-analysis for milk production traits between different countries was conducted using the GWAS summary statistics (i.e., P-value, sample size, allele effects, and etc.) in Holstein cows. In the present study, METAL software was used for the weighted Z-score model. Gene network analysis was used as a complementary method to improve our knowledge of the genome structure of milk production traits and was implemented through the STRING plug-in in Cytoscape software. The Cytoscape ClueGO plug-in was also used for GO enrichment in order to identify biological process, cellular component, and molecular function associated with genomic regions. The aim of this study was to improve the power of QTLs detection and identify the biological mechanisms associated with milk production traits. Data were obtained from 26 published studies from 2010 to 2019. A total of 2,072 SNPs were identified for milk production traits, of which 1,583 SNPs were significant (P < 0.05). Meta-analysis identified 9 QTLs for milk yield, 36 QTLs for fat percentage, and 10 QTLs for protein percentage. Some QTLs were confirmed on BTA14, e.g., BTA14:1801116 close to the DGAT1 gene (milk yield, P = 2.6 × 10 − 131 ; fat percentage, P = 4.8 × 10 − 347 ; protein percentage, P = 7.6 × 10 − 24 ) and BTA14:1651311 close to the PPP1R16A gene (milk yield, P = 2.3 × 10 − 162 ; fat percentage, P = 3.5 × 10 − 153 ). We identified pleiotropic effects of lead SNPs for milk production traits, e.g., one SNP (rs109421300) at BTA14 had pleiotropic effects on milk yield, fat percentage, and protein percentage traits. The most important SNPs for studied traits across countries implicated to network scoring and visualization were including: rs109421300 (DGAT1 gene) for milk yield, fat percentage, and protein percentage; rs109146371 (PPP1R16A gene) for milk yield and fat percentage; rs109968515 (CYHR1 gene) for milk yield and fat percentage; rs134432442 (CPSF1 gene) for fat percentage; rs111018678 (TRAPPC9 gene) for protein percentage. Significant pathways involved in milk production traits through GO term enrichment analysis for biological process, cellular component, and molecular function included: regulation of cation channel activity (P = 1.6 × 10 − 2 ), ion channel complex (P = 1.4 × 10 − 2 ), and phosphoric diester hydrolase activity (P = 1.1 × 10 − 3 ) for milk yield; negative regulation of organ growth (P = 8.2 × 10 − 3 ), transmembrane transporter complex (P = 1.6 × 10 − 3 ), and potassium ion transmembrane transporter activity (P = 8.8 × 10 − 3 ) for fat percentage; mRNA polyadenylation (P = 1.2 × 10 − 2 ), mRNA cleavage factor complex (P = 9.8 × 10 − 4 ), and phosphoric diester hydrolase activity (P = 5.3 × 10 − 3 ) for protein percentage, respectively. Thus, the combination of GWAS summary statistics through a powerful methodology such as meta-analysis will assist us to accurately identify QTLs, potential candidate genes, and biological mechanisms. This kind of studies will help us to have better understanding of QTL regions and genome structure for milk production traits and improve genomic evaluations in Holstein cows. To the best of our knowledge, this is the first meta-analysis of GWAS and GO enrichment across countries for milk production traits in Holstein cows. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, including Bacillus subtilis, Staphylococcus epidermidis, Listeria innocua, Escherichia coli, Salmonella serovar Enteritidis, Aspergillus oryzae, Rhodotorula glutinis, and Geotrichum candidum. The results of MIC and MBC or minimum fungicidal concentration examinations showed that buforin I had higher antimicrobial activity than nisin on all microbial strains used in this study (P 0.5). E. coli was the most resistant to both antimicrobial agents, whereas L. innocua and S. 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 a synergistic effect, whereas it had no effect on Salmonella serovar Enteritidis and G. candidum. The combination of buforin I and nisin showed a partial synergistic effect on L. innocua, E. coli, and R. glutinis. Assessment of viability of the microorganisms under the antimicrobial agents alone and in combination with each other at MICs and fraction inhibitory concentrations 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 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 preservatives could be a promising option for the prevention of contamination, spoilage, and infestation of food and beverage products. [ABSTRACT FROM AUTHOR]

Three hundred and sixty 1-day-old male broiler chicks were randomly allocated to 4 treatments of 6 replicates to evaluate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on gut health attributes of broiler chickens under necrotic enteritis (NE) challenge. Treatments were as follows: (T1) unchallenged group fed with corn-soybean meal (CSM) without NE challenge and additives (NC); (T2) group fed with CSM and challenged with NE without any additives (PC); (T3) PC group supplemented with 20 mg cLFchimera/kg diet (AMP); (T4) PC group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds were sampled for villi morphology, ileal microbiota, and jejunal gene expression of cytokines, tight junctions proteins, and mucin. Results showed that AMP ameliorated NE-related intestinal lesions, reduced mortality, and rehabilitated jejunal villi morphology in NE challenged birds. While the antibiotic non-selectively reduced the count of bacteria, AMP restored microflora balance in the ileum of challenged birds. cLFchimera regulated the expression of cytokines, junctional proteins, and mucin transcripts in the jejunum of NE challenged birds. In conclusion, cLFchimera can be a reliable candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide. [ABSTRACT FROM AUTHOR]

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein–protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv‐engineered RNase A. Then, wild‐RNase A (14 kD), engineered RNase A (14 kD) and scFv‐engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni‐NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild‐RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv‐engineered RNase A, a dose–response cytotoxicity assay was performed on Her1‐positive and Her1‐negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1‐positive cell lines with an IC50 of 50 nM. It appears that scFv‐engineered RNase A can be a promising molecule for use. [ABSTRACT FROM AUTHOR]

Human T-cell lymphotropic virus (HTLV-1) and bovine leukemia virus (BLV) are oncogenic deltaretroviruses, which are the cause of adult T cell leukemia/lymphoma (ATLL) and enzootic bovine leukosis (EBL), respectively. In this study, to evaluate the virus-host interactions in the manifestation of the associated malignancy, four pooled RNA samples of each host (three RNAs in each sample) were applied to RNA-seq. Differential expression analyses were conducted separately between ATLL and EBL groups, in comparison with the healthy group, to identify functional Gene Ontology (GO) terms and hub genes, using DAVID database and MCODE plugin in Cytoscape software, respectively. A broad range of effective genes, involved in the ATLL and EBL, was up- and downregulated. In the virus side, in both malignancy, Tax was expressed very low, but the HTLV-1- HBZ and BVL- As2 transcripts were highly expressed. Some upregulated hub genes, IL2 , TOP2A , MKI67 , TP73 , MYC, and downregulated FOS gene family (FOS , FOSB, and FOSL2), are similarly activated in both human and bovine hosts, in related cell cycle and growth factors. Taken together, it seems that in preventing the infections and cell transformations, Tax must be targeted as a viral factor, and shared peptide in virological and immunological synapses as host factors. Therefore, in the malignant stages, HBZ and As2 transcripts along with growth factors, particularly IL-2R-γ and T-bet or TOP2A, and MKI67 should be targeted in both hosts. Additional studies at the protein level are necessary to elucidate the more useful targets for the therapy of these life-threatening diseases. • The manuscript is a comprehensive system biology study, using RNA-Seq technique. • Introducing a broad range of effective up and downregulated hub genes, involved in ATLL and EBL malignancies. • The genes IL2 , TOP2A , MKI67 , TP73 , MYC , Tbx21, and FOS gene family are novel target for therapy and diagnosis.. • In viral side, HBZ in HTLV-1 and As2 transcript in BLV should be targeted. [ABSTRACT FROM AUTHOR]

Nowadays, engineered mammalian cell lines such as CHO and HEK-293 are extensively used to express recombinant proteins which require post translation modifications. Post translation modifications completely depend on presence of an appropriate signal peptide in nascent proteins. Erythropoietin is a complex protein with post translation modifications that is widely used to treat anemia, hence production this protein as a recombinant protein needs an appropriate signal peptide. The objective of this study was prediction of the best signal peptide to express recombinant erythropoietin in eukaryote system. In order to predict the best signal peptide, first, 28 signal sequences were extracted from database, then, regions and signal peptide probability of them were evaluated by Signal P version 4.1. To investigate physical and chemical features of signal sequences such as isoelectric point, net positive charge, aliphatic index and amino acid length, Protparam was applied. The results revealed that among 28 signal sequences, only 15 of them can be applied as signal peptide to express erythropoietin. Eventually, among 15 signal peptides, SCGB1D1, APOB, ALB, ULBP2 and scrg1 (respectively) were theoretically introduced as the best signal peptides to employ. [ABSTRACT FROM AUTHOR]

Awassi breed is an important sheep in the Middle East, but its lower twinning potential is a limitation against improvement. Due to the dynamic role of oocyte-expressed protein (OOEP) gene in controlling folliculogenesis, this study was conducted to evaluate the possible association between OOEP and twinning rate, as well as body measurement traits, in Awassi ewes. A total of 232 Awassi ewes (123 ewes-producing singletons and 109 ewes-producing twins) were included in this study. Three PCR fragments (OOEP-1 to OOEP-3) were designed to cover the majority of OOEP sequences and genotyped by single-strand conformation polymorphism (SSCP) method. One nucleic acid variant was identified in the intron-2 of OOEP-3, or rs430263839:A>G. Ewes with rs430263839:AG genotype showed variable significant values of larger head length, chest and abdominal circumferences, and higher prolificacy and twinning rates than ewes with the rs430263839:AA genotype. The conducted computations predicted that the detected SNP had caused altered splicing mechanisms in ewes with rs430263839:AG genotype. The increased abdominal and chest diameters in ewes with the rs430263839:AG genotype were thought to help the uterus accommodate more lambs. The predicted splicing alteration caused by this SNP is the putative mechanism by which these ewes may improve reproductive and productive traits. This is the first study that demonstrated the importance of OOEP as a potential biomarker for the twinning rate, prolificacy, and some body measurement traits in Awassi ewes with an opportunity to be introgressed to other breeds using large-scale association analysis. • One nucleic acid variant was identified in the intron-2 of OOEP-3, or rs430263839:A>G. • Ewes with rs430263839:AG genotype showed higher values of several traits than ewes with the rs430263839:AA genotype. • The detected SNP had caused a putative altered splicing mechanisms in ewes with rs430263839:AG genotype. • The increased abdominal and chest diameters in ewes with rs430263839:AG may help the uterus accommodate more lambs. • Our study indicates the possible usefulness of the OOEP gene as a biomarker for several economic traits in Awassi ewes. [ABSTRACT FROM AUTHOR]

A total of 540 Japanese quail eggs were assigned to 9 treatments of 4 replicates to investigate the effect of in ovo injection of threonine (THR) on mucin2 (MUC2) mRNA expression and digestive enzyme activity. Treatments were (non-injected) eggs and those in ovo injected with saline (0.05 or 0.1 ml) with or without THR (5 mg/ml) in two sites (in or under the air sac). On hatch day, 0.05 ml in ovo injected (under the air sac: TUAS) hatchlings were divided into three groups based on NRC recommendations for THR, while all 0.1 ml in ovo injected chicks were removed due to low hatchability. The remaining treatments received the NRC recommended diet until day 10 post-hatch. Treatments had no effect on protease and amylase activities, while TUAS increased MUC2 gene expression. In conclusion, the in ovo injection of THR increased MUC2 gene expression but had no effect on enzyme activity. [ABSTRACT FROM AUTHOR]

This study focused on the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of poultry DNA in sausage. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. A DNA competitor differing by 83 bp in length from the poultry target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle and sheep. The results of QC-PCR showed that the percentage of contamination was in the range of 23.87-52.06%. [ABSTRACT FROM AUTHOR]

Abstract: The identification of animal species used in industrial meat products is very important in respect to economic considerations because European Union, which has implemented a set of very strict procedures to correctly label food. In this paper we present conserved region from mitochondrial 12S rRNA and 16S rRNA genes are powerful region for evaluate the presence of fraudulently added meat in compound food by multiplex polymerase chain reaction assay for the identification of most species (ruminant, poultry and porcine). For each food sources (ground meat, sausages and cold cut) 10 samples were collected and DNA extracted successfully. The results demonstrated that none of the samples were contaminated with porcine residuals, but 40% of sausages samples and 30% of cold cut samples were contaminated with poultry residuals. Also the ground meat samples were not contaminated with poultry residuals. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene, which is responsible for this disease, has recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein ( n = 30) and Brown Swiss ( n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101-bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation was inherited by him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI, and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program. [ABSTRACT FROM AUTHOR]

Cationic antimicrobial peptides are being developed as a promising class of antimicrobial sub-stances. The introduction of a new antibiotic component requires a comprehensive study of its properties so that it can be relied upon to continue laboratory procedures and clinical trials on laboratory animals or human volunteers. Antimicrobial activity of buforin I was evaluated against 15 of the most important pathogenic bacterial and fungal strains. This was followed by assessing anti-biofilm activity, time-dependent inhibitory, thermal stability, plas-ma stability, hemolysis, and cytotoxic activities. The range of obtained MICs was between 4 and 16 μg/mL. The most resistant and most sensitive microbial strains were S. salivarius and C. perfringens , respectively. Buforin I not only inhibited biofilm formation, but also showed a high biofilm radiation activity. Buforin I was stable in human plasma and also at different temperatures including 40, 60, and 80 °C. Although no significant anti-cancer properties were observed for buforin I, the lack of cytotoxicity as well as the lack of hemolytic activity confirm its safety. The high therapeutic index indicated that buforin I has a considerable pharmaceutical potential and can be a reasonable candidate to replace antibiotics or administered in combination with antibiotics to increase the effectiveness as well as reduce the dose of antibiotics. [Display omitted] • Antimicrobial activity of buforin I wasn't specific against gram-negative and gram-positive bacterial and fungal strains. • Buforin I has tendency to non-specifically bind to negative charged molecules like DNA as intracellular targets. • The antimicrobial effect of buforin I is seen due to translocating into the cell, without membrane permeabilization. • Buforin I is stable against different proteases in human plasma and it was stable by increasing temperature until 80 °C. • High antimicrobial activity, low cytotoxicity and negligible hemolysis ability of buforin I lead to high therapeutic index. [ABSTRACT FROM AUTHOR]

In most bacteria, Glutamate decarboxylase (GAD) activity significantly reduces at pH > 6. Since l -glutamate is neutral to slightly alkaline, a large amount of hydrochloric acid must be added to the reaction mixture. Acidic reaction conditions greatly increase the corrosion of production equipment and shorten the life of manufacturing equipment. Therefore, this study aims at studying the overexpression of recombinant GAD that is active at the neutral pH and high-level production of γ-aminobutyric acid. A gene sequence encoding GAD of Enterococcus faecium DO was cloned into PET21a (+) and was overexpressed in Escherichia coli BL21 (DE3). The construct was confirmed by colony PCR and double digested with BamHI and XhoI. The result from Western blot analysis showed a signal band at the molecular mass of 54 kDa. The specific activity of the recombinant GAD was 78.17 U mg−1 at 45 °C and pH = 6.6. It was reduced rapidly when pH > 7.8. The K m , V max , and k cat values of recombinant GAD were 1.8 mM, 12.9 μmol L−1 min−1, and 0.43 s−1, respectively. Therefore, recombinant GAD is a potent candidate for GABA production in food and pharmaceutical industries, because it increases the efficiency of GABA synthesis, especially in foods with a neutral pH range. • Overexpression of recombinant Glutamate decarboxylase in Escherichia coli BL21 (DE3). • Production of γ-aminobutyric acid by the recombinant Glutamate decarboxylase at the neutral pH. • A desirable activity level for the recombinant GAD was observed. • The GAD enzyme has potential industrial application. [ABSTRACT FROM AUTHOR]

Mastitis is one of the costliest diseases in dairy farms caused by infection of different microorganisms such as Escherichia coli , Streptococcus uberis and Staphylococcus aureus. Promoters are significantly involved in regulating gene expression and shedding light on the mechanisms of transcriptional regulation in physiological and immunological processes of the infections. Exploiting regulatory elements such as transcription factor binding sites (TFBSs modules) on the promoter region could reveal co-regulated genes, which allow screating regulatory models and executing a cross-sectional analysis on several databases. In this study, the promoter regions of 11 genes associated with contagious mastitis including CCL4, CXCL8, STAT3, IKBKB, MAPK14, NFKBIA, NFKB1, TNF, IL18, IL6, and HCK were investigated to predict the activating regulatory modules on promoters and to discover the key related transcription factors. By exploring the promoter regions, 228 genes were discovered comprising the same transcription factors modules. Out of 228 genes, 36 were validated using five microarray datasets. The promoter research of these genes revealed that as many as 7 down-regulated and 12 up-regulated genes are predictable in the network. The genes whose functions were associated with the initial gene list (11 genes), were identified by DAVID queries with TFBSs models implying that the approach provides a clear image of the underlying regulatory mechanism of gene expression profile and offers a novel approach in designing gene networks in cattle. • Better understand the functions of Mastitis in dairy cattle. • Comprehensive bioinformatics approaches: microarray and transcriptomic analysis. • Gene Ontology and pathways analysis. • Identification of the genes to elucidate mechanisms responsible for Mastitis disease. [ABSTRACT FROM AUTHOR]

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics. [ABSTRACT FROM AUTHOR]