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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]

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]

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]

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]

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]

Introduction: Identifying genomic regions under selection is the most challenging issue for improving important traits in animals. Few studies have focused on identifying genomic regions under selection in sheep. The aim of this study was to identify selective sweeps and to explore the relationship between these and quantitative trait loci (QTL) in both domestic and wild sheep species using single nucleotide polymorphism markers (SNPs)., Methods: Genomic data were obtained from the NextGen project, which included genotyping 20 domestic and 14 wild sheep using the Illumina Ovine SNP50K BeadChip. The XP-EHH, iHS, and RSB methods were employed to detect signatures of positive selection., Results: The results of the iHS method indicated 405 and 275 selective sweeps in domestic and wild sheep, respectively. Additionally, RSB and XP-EHH analyses revealed approximately 398 and 479 selective sweeps in domestic and wild sheep, respectively. Some of the genes associated with important QTL traits in domestic sheep include ADGRB3, CADM1, CAPN2, GALNT10, MTR, RELN , and USP25 , while in wild sheep, the relevant genes include ACAN, ACO1, GADL1, MGST3 , and PRDM16 . Selective sweeps identified in domestic sheep were associated with body weight, muscle weight, milk protein percentage, and milk yield. In contrast, selective sweeps found in wild sheep were linked to average daily gain, bone weight, carcass fat percentage, and dressing percentage., Discussion: These results indicate that selection by humans and the environment have largely progressed in harmony, highlighting the importance of both economic and environmental traits for survival. Additionally, the identification of potential candidate genes associated with economic traits and genomic regions that have experienced selection can be utilized in sheep breeding programs. However, due to the incomplete information regarding the functional annotation of genes in sheep and the limited sample size, further research with a larger sample group is essential to gain a deeper understanding of the candidate genes linked to economic traits in both domestic and wild sheep. Advancing knowledge in this area can significantly enhance the effectiveness of breeding strategies. The quantitative trait loci identified in this study have the potential to be incorporated into breeding plans for both domestic and wild sheep., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Taheri, Javadmanesh and Zerehdaran.)

Zinc (Zn) is a significant element of the reproductive system and is associated with several enzymes that regulate different metabolic pathways. Organic Zn can significantly affect polycystic ovarian syndrome (PCOS) pathogenesis. Insulin resistance (IR) is a common complication of PCOS. Mammalian target of rapamycin (mTOR), which controls crucial cell functions, is regulated by insulin and nutrients. It has two complexes, namely, mTORC1 and mTORC2. mTOR associates with its binding partner's regulatory associated protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (Rictor), which form these distinct complexes, respectively, and is activated in PCOS. This research aimed to evaluate the effect of Zn on the expression of mTOR signaling genes (Raptor and Rictor) and IR in PCOS model rats. Different Zn supplements, including standard diet (SD): (control - or + , SD without supplementation), Zn25, Zn75, and Zn175 (daily given three levels of 25, 75, and 175 mg Zn methionine (ZnMet)/kg for 6 weeks, respectively), were applied to the control and PCOS groups. Fasting glucose (FG), fasting insulin (FI), IR indices, and Raptor and Rictor expression levels were measured in both groups. The results showed that PCOS induction dramatically increased FG, FI, IR indices, and mTOR-related gene expression; however, different Zn supplementation concentrations, especially at 75 mg/kg, reduced the effects of PCOS induction. Organic Zn collectively exerted positive effects on Estradiol Valerate (EV)-induced PCOS rats by reducing IR and mTOR signaling gene (i.e., Raptor and Rictor) expression. Moreover, this study revealed a correlation between Zn and IR. Therefore, Zn supplementation could be a valuable therapeutic method for treating PCOS., Competing Interests: Declarations. Ethics Approval: All methods in the study were in accordance to the ethics guidelines on the animal experiments at the Ferdowsi University of Mashhad. The committee on the ethics of animal experiments approved the protocols by certificate number IR.UM.REC.1401.286. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The present study aimed to evaluate the effect of thymol on growth performance and apparent total tract digestibility of nutrients in severely feed-restricted lambs. Twenty-one male Baluchi lambs were randomly blocked by live weight into three groups: control without feed restriction (CON), feed restricted (FR), and feed restricted plus thymol (FR + T). The lambs underwent a four-week feed restriction period followed by four weeks of realimentation. Thymol was administered daily to the FR + T group during the feed restriction period. Average daily gain (ADG), average daily feed intake, feed efficiency (FE), partial efficiency of maintenance (PEM), and residual feed intake (RFI) were measured as growth performance parameters. Results showed that the severe feed restriction had adverse effects on ADG and FE, but improved PEM (P < 0.05). The effects of thymol administration on ADG, FE, PEM, and apparent total tract digestibility were not significant (P > 0.05). However, the lambs that received thymol during the feed restriction period showed a negative RFI during realimentation (P < 0.05). Overall, these findings suggest that feed restriction as well as thymol may have the potential to improve efficiency of feed utilization in growing lambs. However, this positive effect is independent of the improvement in nutrient digestibility., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Introduction: Sex differences in prenatal growth may contribute to sex-dependent programming effects on postnatal phenotype., Methods: We integrated for the first time phenotypic, histomorphological, clinico-chemical, endocrine and gene expression analyses in a single species, the bovine conceptus at mid-gestation., Results: We demonstrate that by mid-gestation, before the onset of accelerated growth, the female conceptus displays asymmetric lower growth compared to males. Female fetuses were smaller with lower ponderal index and organ weights than males. However, their brain:body weight, brain:liver weight and heart:body weight ratios were higher than in males, indicating brain and heart 'sparing'. The female placenta weighed less and had lower volumes of trophoblast and fetal connective tissue than the male placenta. Female umbilical cord vessel diameters were smaller, and female-specific relationships of body weight and brain:liver weight ratios with cord vessel diameters indicated that the umbilico-placental vascular system creates a growth-limiting environment where blood flow is redistributed to protect brain and heart growth. Clinico-chemical indicators of liver perfusion support this female-specific growth-limiting phenotype, while lower insulin-like growth factor 2 (IGF2) gene expression in brain and heart, and lower circulating IGF2, implicate female-specific modulation of key endocrine mediators by nutrient supply., Conclusion: This mode of female development may increase resilience to environmental perturbations in utero and contribute to sex-bias in programming outcomes including susceptibility to non-communicable diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Estrella, Gatford, Xiang, Javadmanesh, Ghanipoor-Samami, Nattrass, Shuaib, McAllister, Beckman, Thomsen, Clifton, Owens, Roberts, Hiendleder and Kind.)

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.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

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., (© 2023. The Author(s).)

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., (© 2023 Japanese Society of Animal Science.)

In the present study, kashk samples were collected from two regions of Iran, the Fars (Abadeh) and Razavi Khorasan (Kalat) provinces. Fifteen bacteria were isolated and physiological and biochemical assays were performed. After identification to the genus level, eight isolates were identified as lactic acid bacteria (LAB) and subjected to molecular identification and probiotic properties assays. The results revealed that the isolates were Enterococcus faecium KKP 3772 (KF1), Enterococcus faecium C1 (KF2), Pediococcus pentosaceus H11 (KF3), Pediococcus pentosaceus VNK-1 (KK4), Lactococcus lactis RSg (KK1), Enterococcus faecalis P190052 (KK2), Enterococcus mundtii CECT972T (KK3), and Lactiplantibacillus plantarum PM411 (KK5). Only the numbers of L. lactis RSg (KK1) and Lpb. Plantarum PM411 (KK5) decreased to below 9 Log CFU/mL after acidic conditions (pH = 3) and showed weak antibacterial activity. Enterococcus mundtii CECT972T (KK3) and E. faecium C1(KF2) were highly susceptible to bile salts, while P. pentosaceus VNK-1 (KK4) and P. pentosaceus H11 (KF3) showed the highest resistance. All of the isolates were resistant to tetracycline and sensitive to chloramphenicol and gentamicin. The antimicrobial activity of P. pentosaceus VNK-1 (KK4) and P. pentosaceus H11 (KF3) was higher than other isolates and consequently, their inhibition zones were larger. The adhesion capabilities of LAB isolates to intestinal epithelial cells were evaluated by examining the auto-aggregation factor and cell surface hydrophobicity. The highest and lowest cell surface hydrophobicity and auto-aggregation were obtained from P. pentosaceus VNK-1 (KK4) and E. mundtii CECT972T (KK3), respectively. In general, P. pentosaceus VNK-1 (KK4) and P. pentosaceus H11 (KF3) have shown better probiotic properties as compared to other isolates.

Background: Abuse of growth hormone (GH) is expanding in exercising populations due to its lipolytic and anabolic actions. The purpose of this study was to examine the interactive effect of exercise training and GH administration on histopathological and functional assessment in the liver of male Wistar rats., Methods: Forty-eight male Wistar rats were randomly divided into six groups including control + saline group (CS), GH injection group (GI), resistance training + saline group (RS), aerobic training + saline group (AS), resistance training + GH injection group (RG), aerobic training + GH injection group (AG). All groups were injected with either saline or GH 1 h before each training session. RT and AT were performed five days/week for a total of 8-weeks. At the end of the study, blood samples and liver tissue samples were taken to evaluate circulating AST, ALT, and ALP enzymes, as well as albumin protein. Histopathology of liver tissue was performed via qualitative microscopic evaluation., Results: Microscopic evaluation of liver tissue did not show any histopathologic changes. All the groups administered with GH showed a significant increase in ALT, ALP, and albumin protein (P<0.05). However, AST enzyme concentrations increased significantly only in the RG group (P=0.022). In addition, neither RS nor the AS groups showed significant AST, ALT, and ALP changes, but serum albumin concentration significantly increased in the AS group (P=0.033)., Conclusions: The elevation of liver enzymes showed that GH administration with or without exercise training might cause severe liver damage.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022. The Author(s).)

KRAS is one of the most widely prevalent proto-oncogenes in human cancers. The constitutively active KRAS oncoprotein contributes to both tumor onset and cancer development by promoting cell proliferation and anchorage-independent growth in a MAPK pathway-dependent manner. The expression of microRNAs (miRNAs) and the KRAS oncogene are known to be dysregulated in various cancers, while long noncoding RNAs (lncRNAs) can act as regulators of the miRNAs targeting KRAS oncogene in different cancers and have gradually become a focus of research in recent years. In this review article, we summarize recent advances in the research on lncRNAs that have sponging effects on KRAS-targeting miRNAs as crucial mediators of KRAS expression in different cell types and organs. A deeper understanding of lncRNA function in KRAS-driven cancers is of major fundamental importance and will provide a valuable clinical tool for the diagnosis, prognosis, and eventual treatment of cancers., (© 2021. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

This study was conducted to evaluate the effects of apple cider vinegar (ACV) administration on non-specific immunity of serum and skin mucus, growth indices, and activity of digestive enzymes (amylase, lipase, and protease) in Carassius auratus. For this purpose, 180 fish (weighing 7.35 ± 0.19 g) were allocated to 4 treatment groups with 3 replications in a completely randomized design. Fish were fed for 105 days using a basal diet supplemented with 0% (control), 1% (T 1), 2% (T 2), and 4% (T 3) ACV (contained 5% acetic acid). Results showed a significant increase in lysozyme activity, ACH50, and total immunoglobulin of skin mucus in fish fed with T2 diet (p < 0.05). Total immunoglobulin and lysozyme activity were significantly lower in the serum of fish fed with control diet than those fed with the mentioned treatment (p < 0.05). The highest value was observed in fish fed with T2 diet. Minimum (p < 0.05) complement activity (1.52 ± 0. 25 U ml -1 ) was observed in fish fed with control diet. The mean of the final weights (17.35 ± 1.39 g), daily growth (1.0 ± 0.01 g), and specific growth rate (2.19 ± 0.14) was significantly higher in T3 diet group than the controls (p < 0.05). While the highest amylase-specific activity was observed in the controls (p < 0.05), there was a significant increase in specific activity of protease, lipase, and alkaline phosphatase in T2 diet group (p < 0.05). According to the results of this study, the inclusion of a limited quantity of ACV (4%) into the diet can improve immunity and growth parameters in C. auratus.

Probiotics are beneficial microorganisms and have long been used in food production as well as health promotion products. Bioengineered probiotics are used to express and transfer native or recombinant molecules to the mucosal surface of the digestive tract to improve feed efficiency and promote health. Lactococcus lactis is a potential probiotic candidate to produce useful biological proteins. The aim of this investigation was to develop a recombinant Lactococcus lactis with the potential of producing phytase. To enhance the efficiency of expression and secretion of recombinant phytase, usp45 signal peptide was added to the expression vector containing phytase gene (appA2) derived from Escherichia coli. Sequencing of recombinant plasmid containing appA2 showed the correct construction of plasmid. Total length of the phytase insert was 1.25 kbp. A Blast search of the cloned fragment showed 99% similarity to the reported E. coli phytase sequence in the GenBank (accession number: AM946981.2). A plasmid containing usp45 and appA2 electrotransferred into Lactococcus lactis. Zymogram with polyacrylamide gel revealed that the protein extract from the supernatant and the cell pellet of recombinant bacteria had phytase activity. Enzyme activity of 4 U/ml was obtained in cell extracts, and supernatant maximal phytase activity was 19 U/ml. The recombinant L. lactis was supplemented in broiler chicken feed and showed the increase of apparent digestibility on phytate phosphorus in the digestive tract and it was same as performance of E. coli commercial phytase.

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene which is responsible for this disease has a 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 inherited to 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.