Effect of Nk-lysin peptides on bacterial growth, MIC, antimicrobial resistance, and viral activities.

NK-lysins from chicken, bovine and human are used as antiviral and antibacterial agents. Gram-negative and gram-positive microorganisms, including Streptococcus pyogenes, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Shigella sonnei, Klebsiella pneumoniae and Salmonella typhimurium, are susceptible to NK-lysin treatment. The presence of dominant TEM-1 gene was noted in all untreated and treated bacteria, while TOHO-1 gene was absent in all bacteria. Importantly, ß-lactamase genes CTX-M-1, CTX-M-8, and CTX-M-9 genes were detected in untreated bacterial strains; however, none of these were found in any bacterial strains following treatment with NK-lysin peptides. NK-lysin peptides are also used to test for inhibition of infectivity, which ranged from 50 to 90% depending on NK-lysin species. Chicken, bo vine and human NK-lysin peptides are demonstrated herein to have antibacterial activity and antiviral activity against Rotavirus (strain SA-11). On the basis of the comparison between these peptides, potent antiviral activity of bovine NK-lysin against Rotavirus (strain SA-11) is particularly evident, inhibiting infection by up to 90%. However, growth was also significantly inhibited by chicken and human NK-lysin peptides, restricted by 80 and 50%, respectively. This study provided a novel treatment using NK-lysin peptides to inhibit expression of ß-lactamase genes in ß-lactam antibiotic-resistant bacterial infections.

Chicken ß-defensin-1 peptide as a candidate anticoccidial agent in broiler chickens.

The current study aimed to investigate the potentiality of using avian ß-defensin-1 peptide as a candidate agent against coccidiosis infection in broiler chicken.We employed an in-silico analysis to study the primary structure of ß-defensin-1 peptide as well as its 3-D and molecular dynamic structures. This will also enable obtaining adequate information about the mode of action of these peptides and the intra-cellular transduction pathways. The results revealed no significant difference among groups of broiler chicken in terms of body weight before the Eimeria challenge.The results of our study indicated a significant reduction in oocyst count in birds administered ß-defensin-1 peptide treatment, vis-a-vis healthy birds. The treated group showed a 2-3 times reduction in oocyst count, compared to the positive control group. The Eimeria oocysts count evaluated for birds administered with ß-defensin-1 after the Eimeria challenge showed a significant difference. The study indicated significant reduction and down-regulation in the level of expression of ß-defensin 1 and 4 in the control and treatment groups.This electrostatic profile and hydrophobicity regulate the functioning of this peptide. The results may help in the development of novel approaches that could be used as alternatives or adjunct to the existing means of coccidiosis control in broilers.

Anticancer activity of chicken cathelicidin peptides against different types of cancer.

BACKGROUND: This study served as the pioneer in studying the anti-cancer role of chicken cathelicidin peptides. METHODS AND RESULTS: Chicken cathelicidins were used as anticancer agent against the breast cancer cell line (MCF-7) and human colon cancer cell line (HCT116). In addition, the mechanism of action of the interaction of cationic peptides with breast cancer cell line MCF-7 was also investigated. An in vivo investigation was also achieved to evaluate the role of chicken cathelicidin in Ehrlich ascites cell (EAC) suppression as a tumor model after subcutaneous implantation in mice. It was found during the study that exposure of cell lines to 40 µg/ml of chicken cathelicidin for 72 h reduced cell lines growth rate by 90-95%. These peptides demonstrated down-regulation of (cyclin A1 and cyclin D genes) of MCF-7 cells. The study showed that two- and three-fold expression of both of caspase-3 and - 7 genes in untreated MCF-7 cells compared to treated MCF-7 cells with chicken cathelicidin peptides. Our data showed that chicken (CATH-1) enhance releasing of TNFα, INF-γ and upregulation of granzyme K in treated mice groups, in parallel, the tumor size and volume was reduced in the treated EAC-bearing groups. Tumor of mice groups treated with chicken cathelicidin displayed high area of necrosis compared to untreated EAC-bearing mice. Based on histological analysis and immunohistochemical staining revealed that the tumor section in Ehrlich solid tumor exhibited a strong Bcl2 expression in untreated control compared to mice treated with 10 & 20 µg of cathelicidin. Interestingly, low expression of Bcl2 were observed in mice taken 40 µg/mL of CATH-1. CONCLUSIONS: This study drive intention in treatment of cancer through the efficacy of anticancer efficacy of chicken cathelicidin peptides.

Characterization of transcription profile and structural properties of avian NK-lysin.

This study aimed to determine the transcription profile of NK-lysin gene in native chickens. Moreover, it was targeted toward determining the primary, three-dimensional, and molecular dynamic structures of NK-lysin and granulysin peptides to understand their mode of action and intracellular transduction pathways using in silico analysis. The results revealed that NK-lysin gene in native chickens and Gallus gallus were closely related to those of other avian species. However, there was a low sequence homology when aligned with the mammalian peptides. The coding region of NK-lysin peptide in native chickens encoded 140 amino acids as found in G. gallus with a homology of 98% that declined to 20%, particularly in mammalian species. The results revealed that the NK-lysin in native chickens was closely related to that in avian species at a range of 71-76%. However, it was different from that of other mammalians in terms of nucleotide and amino acid identities. The mRNA transcripts of NK-lysin had high and moderate expression levels in the testis and pancreas, respectively. Nonetheless, the small intestine, kidney, spleen, and liver had a low expression level. The NK-lysin peptides contained more than 50% of the total AA with a nonpolar feature, whereas polar AA constituted up to 30% of AA. The results also indicated that the hydrophilic regions and positively charged amino acids were predominant on the surface of the investigated peptides. The NK-lysin was folded in 4-5 helical units and 3-4 loop structures in their saposin domain. The third helical peptide was long in both avian and bovine species (104-123 residues). However, the fourth helical peptide was short in humans, pigs, and chimpanzees (101-123, 104-123, and 102-124 residues, respectively), with the helical unit residues of 95-97, 96-99, and 96-98, respectively. The obtained results can be helpful in developing novel approaches that could be used as alternatives or adjuncts to the existing means of control.

Molecular evolution of cytochrome C oxidase-I protein of insects living in Saudi Arabia.

The study underpins barcode characterization of insect species collected from Saudi Arabia and explored functional constraints during evolution at the DNA and protein levels to expect the possible mechanisms of protein evolution in insects. Codon structure designated AT-biased insect barcode of the cytochrome C oxidase I (COI). In addition, the predicted 3D structure of COI protein indicated tyrosine in close proximity with the heme ligand, depicted substitution to phenylalanine in two Hymenopteran species. This change resulted in the loss of chemical bonding with the heme ligand. The estimated nucleotide substitution matrices in insect COI barcode generally showed a higher probability of transversion compared with the transition. Computations of codon-by-codon nonsynonymous substitutions in Hymenopteran and Hemipteran species indicated that almost half of the codons are under positive evolution. Nevertheless, codons of COI barcode of Coleoptera, Lepidoptera and Diptera are mostly under purifying selection. The results reinforce that codons in helices 2, 5 and 6 and those in loops 2-3 and 5-6 are mostly conserved and approach strong purifying selection. The overall results argue the possible evolutionary position of Hymenopteran species among those of other insects.

On the potential of using peculiarities of the protein intrinsic disorder distribution in mitochondrial cytochrome b to identify the source of animal meats.

This study was conducted to identify the source of animal meat based on the peculiarities of protein intrinsic disorder distribution in mitochondrial cytochrome b (mtCyt-b). The analysis revealed that animal and avian species can be discriminated based on the proportions of the two groups of residues, Leu+Ile, and Ser+Pro+Ala, in the amino acid sequences of their mtCyt-b. Although levels of the overall intrinsic disorder in mtCyt-b is not very high, the peculiarities of disorder distribution within the sequences of mtCyt-b from different species varies in a rather specific way. In fact, positions and intensities of disorder/flexibility "signals" in the corresponding disorder profiles are relatively unique for avian and animal species. Therefore, it is possible to devise a set of simple rules based on the peculiarities of disorder profiles of their mtCyt-b proteins to discriminate among species. This intrinsic disorder-based analysis represents a new technique that could be used to provide a promising solution for identification of the source of meats.

Abundance and functional roles of intrinsic disorder in the antimicrobial peptides of the NK-lysin family.

NK-lysins are antimicrobial peptides (AMPs) that participate in the innate immune response and also have several pivotal roles in various biological processes. Such multifunctionality is commonly found among intrinsically disordered proteins. However, NK-lysins have never been systematically analyzed for intrinsic disorder. To fill this gap, the amino acid sequences of NK-lysins from various species were collected from UniProt and used for the comprehensive computational analysis to evaluate the propensity of these proteins for intrinsic disorder and to investigate the potential roles of disordered regions in NK-lysin functions. We analyzed abundance and peculiarities of intrinsic disorder distribution in all-known NK-lysins and showed that many NK-lysins are expected to have substantial levels of intrinsic disorder. Curiously, high level of intrinsic disorder was also found even in two proteins with known 3D-strucutres (NK-lysin from pig and human granulysin). Many of the identified disordered regions can be involved in protein-protein interactions. In fact, NK-lysins are shown to contain three to eight molecular recognition features; i.e. short structure-prone segments which are located within the long disordered regions and have a potential to undergo a disorder-to-order transition upon binding to a partner. Furthermore, these disordered regions are expected to have several sites of various posttranslational modifications. Our study shows that NK-lysins, which are AMPs with a set of prominent roles in the innate immune response, are expected to abundantly possess intrinsically disordered regions that might be related to multifunctionality of these proteins in the signal transduction pathways controlling the host response to pathogenic agents.

Identification of fraud (with pig stuffs) in chicken-processed meat through information of mitochondrial cytochrome b.

This study was conducted to find out the fraud in chicken-processed meat ingredients to protect consumers from commercial adulteration and authentication through a reliable way: direct amplification of conserved segment of cytochrome b gene of mitochondrial DNA, in addition, using species-specific primer assay for a certain cytochrome b. The results reported that chicken-processed meats were identified as a chicken meat based on amplification of conserved cytochrome b gene of mtDNA, while different fragments sizes were produced after the application of species-specific primer as follows: 227, 157, 274, 331, 389 and 439 bp for raw meat of chicken, goat, cattle, sheep, pig and horse, respectively. The results revealed that all chicken meat products are produced with 227 bp in size. While, an adulteration with pork stuffs was observed in some of the chicken meat products using a species-specific primer of cytochrome b gene, namely, chicken luncheon and chicken burger. This study represents a reliable technique that could be used to provide a promising solution for identifying the commercial adulteration and substitutions in processed meat in retail markets.

Chicken cathelicidins as potent intrinsically disordered biocides with antimicrobial activity against infectious pathogens.

This study was performed to identify the expression patterns of the cathelicidin genes in a local chicken breed and to evaluate the antimicrobial activities of the cathelicidin peptides against pathogenic bacteria. This analysis revealed that the coding regions of CATH-1, -2, and -3 genes contain 447 bp, 465 bp, and 456 bp, respectively, and encode proteins of 148, 154, 151 amino acids, respectively. The complete amino acid sequences of the cathelicidin peptides are similar to those found in Meleagris gallopavo, Phasianus colchicus, and Coturnix coturnix, and show high sequence identity to their Columba livia and Anas platyrhynchos counterparts. In contrast, these avian peptides shared a very low sequence identity with the mammalian cathelicidins. The analysis further revealed that the cathelicidin genes are expressed in various organ and tissues. We also show that the CATH peptides 1, 2, 3 and their amide-modified structures possess potent antimicrobial activities against both Gram-positive and Gram-negative pathogens, with these bacteria being affected to different extents. The antimicrobial activities of the peptides are slightly lower than those of their amide analogs. Computational analysis revealed that pre-pro-cathelicidins are hybrid proteins that contain ordered domains and functional intrinsically disordered regions. Furthermore, high structural and sequence variability of mature cathelicidins is a strong indication of their rather disordered nature. It is likely that intrinsic disorder is needed for the multifarious functionality of these antimicrobial peptides. Our analyses indicated that cathelicidin peptides require further study to better understand their full potentials in the treatment of diseases in both humans and animals. The data obtained for synthetic avian peptides will help elucidating of their potential applications in the pharmaceutical industry.

Biocidal activity of chicken defensin-9 against microbial pathogens.

In this study we identified the expression patterns of ß-defensin-9 in chickens from Saudi Arabia, evaluated the antimicrobial activities of synthetic chicken ß-defensin-9 (sAvBD-9) against pathogenic bacteria and fungi, and investigated the mode of action of sAvBD-9 on bacterial cells. The AvBD-9 gene of Saudi chickens encodes a polypeptide of 67 amino acids, which is highly similar to the polypeptide in duck, quail, and goose (97%, 86%, and 87%, respectively) and shares a low sequence similarity with the mammalian defensins. AvBD-9 is expressed in various organs and tissues of Saudi chickens and inhibits the growth of both Gram-negative and Gram-positive bacteria, as well as showing activity against unicellular and multicellular fungi (Aspergillus flavus, A. niger, and Candida albicans). sAvBD-9 completely inhibited the growth of both Gram-positive and Gram-negative bacterial strains as well as Candida albicans. The haemolytic effects of sAvBD-9 were limited. Morphological analysis by TEM revealed that sAvBD-9 induces shortening and swelling of Staphylococcus aureus and Shigella sonni cells, opens holes and deep craters in their envelopes, and leads to the release of their cytoplasmic content. Our data shed light on the potential applications of sAvBD-9 in the pharmaceutical industry.

Antimicrobial potentials and structural disorder of human and animal defensins.

Defensins are moonlighting peptides which are broadly distributed throughout all the living kingdoms. They play a multitude of important roles in human health and disease, possessing several immunoregulatory functions and manifesting broad antimicrobial activities against viruses, bacteria, and fungi. Based on their patterns of intramolecular disulfide bridges, these small cysteine-rich cationic proteins are divided into three major types, α-, ß-, and θ-defensins, with the α- and ß-defensins being further subdivided into a number of subtypes. The various roles played by the defensins in the innate (especially mucosal) and adoptive immunities place these polypeptides at the frontiers of the defense against the microbial invasions. Current work analyzes the antimicrobial activities of human and animal defensins in light of their intrinsic disorder propensities.

Antimicrobial activities of chicken ß-defensin (4 and 10) peptides against pathogenic bacteria and fungi.

Host Defense Peptides (HDPs) are small cationic peptides found in several organisms. They play a vital role in innate immunity response and immunomodulatory stimulation. This investigation was designed to study the antimicrobial activities of ß-defensin peptide-4 (sAvBD-4) and 10 (sAvBD-4) derived from chickens against pathogenic organisms including bacteria and fungi. Ten bacterial strains and three fungal species were used in investigation. The results showed that the sAvBD-10 displayed a higher bactericidal potency against all the tested bacterial strains than that of sAvBD-4. The exhibited bactericidal activity was significant against almost the different bacterial strains at different peptide concentrations except for that of Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus bovis (Str. bovis) strains where a moderate effect was noted. Both peptides were effective in the inactivation of fungal species tested yielding a killing rate of up to 95%. The results revealed that the synthetic peptides were resistant to salt at a concentration of 50 mM NaCl. However, they lost antimicrobial potency when applied in the presence of high salt concentrations. Based on blood hemolysis studies, a little hemolytic effect was showed in the case of both peptides even when applied at high concentrations. The data obtained from this study indicated that synthetic avian peptides exhibit strong antibacterial and antifungal activity. In conclusion, future work and research should be tailored to a better understanding of the mechanisms of action of those peptides and their potential use in the pharmaceutical industry to help reduce the incidence and impact of infectious agent and be marketed as a naturally occurring antibiotic.

Using cytochrome b gene of mtDNA as a DNA barcoding marker in chicken strains.

This was the second study to apply using of a cytochrome b gene as barcoding tool in distinguishing among chicken strains. We performed polymerase chain reaction (PCR) amplification using universal primer to amplify around 415 bp fragment of cytochrome b gene of mtDNA. The tree reported that both Saudi chicken strains (black and dark brown) are closely related and it might be separated from same origin rather than bronze ones. The phylogenetic tree also, exploited that native chicken strains were closely related to cluster of Ceylon jungle fowl, black Minorca egg chicken and Fayoumi egg chicken. The genetic divergence between these populations or types of chickens in Saudi Arabia was low (0.02) and it was very low (0.011), when compared to other species of Gallus. We confirmed that short fragment of cyt-b gene as a universal DNA barcode region. It was much more accurate and efficient tool to discriminate inter-species than intra-species. Applying cyt-b of mtDNA was successfully distinguished among native strains and other species of Gallus as in a previous study. However, applying this thought on different species of farm animal species is recommended.

Molecular detection of adulteration in chicken products based on mitochondrial 12S rRNA gene.

The aim of this study is to detect the fraudulent in chicken products constitutes in order to protect consumers in Saudi Arabia from illegal substitutions. Two different approaches were used in this study, direct sequencing of specific fragments of amplified mitochondrial 12S rRNA gene in addition to species-specific PCR primers for confirmation of the obtained Blast search results. The results showed that all processed chicken products were identified as chicken (Gallus gallus) by 90-98% homology depending on obtained sequence quality. Samples labeled with chicken luncheon (samples tested in this study) were identified as turkey meat (Meleagris gallopavo) by 98% homology, suggesting adulteration with inedible parts of turkey in chicken luncheon ingredients. The results showed also that not only chicken luncheon was mixed with inedible parts of turkey but also all chicken products tested in this study (chicken balls, chicken burger, chicken sausage and chicken mined meat) contained this turkey meat. Applying methods used in this study could be useful for accurate and rapid identification of commercial processed meat.

Novel mutations in the displacement loop of mitochondrial DNA are associated with acute lymphoblastic leukemia: a genetic sequencing study.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and represents approximately 25% of cancer diagnoses among those younger than 15 years of age. MATERIALS AND METHODS: This study investigated alterations in the displacement loop (d-loop) region of mitochondrial DNA (mtDNA) as a risk factor and diagnostic biomarker for early detection and diagnosis of acute lymphoblastic leukemia. Using mtDNA from 23 subjects diagnosed with acute lymphoblastic leukemia, the first 450 bp of the d-loop region were amplified and successfully sequenced. RESULTS: This revealed 132 mutations at 25 positions in this region, with a mean of 6 alterations per subject. The d-loop alterations in mtDNA in subjects were all identified as single nucleotide polymorphisms in a homoplasmic distribution pattern. Mutant alleles were observed in all subjects with individual frequency rates of up to 95%. Thirteen mutant alleles in the d-loop region of mtDNA occurred with a high frequency. Novel alleles and locations were also identified in the d-loop of mtDNA as follows: 89 G insertions (40%), 95 G insertions (13%), 182 C/T substitutions (5%), 308 C insertions (19%), and 311 C insertions (80%). The findings of this study need to be replicated to be confirmed. CONCLUSIONS: Further investigation of the relationship between mutations in mitochondrial d-loop genes and incidence of acute lymphoblastic leukemia is recommended.

Epigallocatechin-3-gallate inhibits tax-dependent activation of nuclear factor kappa B and of matrix metalloproteinase 9 in human T-cell lymphotropic virus-1 positive leukemia cells.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol molecule from green tea and is known to exhibit antioxidative as well as tumor suppressing activity. In order to examine EGCG tumor invasion and suppressing activity against adult T-cell leukemia (ATL), two HTLV-1 positive leukemia cells (HuT-102 and C91- PL) were treated with non-cytotoxic concentrations of EGCG for 2 and 4 days. Proliferation was significantly inhibited by 100 µM at 4 days, with low cell lysis or cytotoxicity. HTLV-1 oncoprotein (Tax) expression in HuT- 102 and C91-PL cells was inhibited by 25 µM and 125 µM respectively. The same concentrations of EGCG inhibited NF-kB nuclearization and stimulation of matrix metalloproteinase-9 (MMP-9) expression in both cell lines. These results indicate that EGCG can inhibit proliferation and reduce the invasive potential of HTLV-1- positive leukemia cells. It apparently exerted its effects by suppressing Tax expression, manifested by inhibiting the activation of NF-kB pathway and induction of MMP-9 transcription in HTLV-1 positive cells.

New haplotypes of the ATP synthase subunit 6 gene of mitochondrial DNA are associated with acute lymphoblastic leukemia in Saudi Arabia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and represents approximately 25% of cancer diagnoses among those younger than 15 years of age. AIM AND OBJECTIVES: This study investigated substitutions in the ATP synthase subunit 6 gene of mitochondrial DNA (mtDNA) as a potential diagnostic biomarker for early detection and diagnosis of acute lymphoblastic leukemia. Based on mtDNA from 23 subjects diagnosed with acute lymphoblastic leukemia, approximately 465 bp of the ATP synthase subunit 6 gene were amplified and sequenced. RESULTS: The sequencing revealed thirty-one mutations at 14 locations in ATP synthase subunit 6 of mtDNA in the ALL subjects. All were identified as single nucleotide polymorphisms (SNPs) with a homoplasmic pattern. The mutations were distributed between males and females. Novel haplotypes were identified in this investigation: haplotype (G) was recorded in 34% in diagnosed subjects; the second haplotype was (C) with frequency of 13% in ALL subjects. Neither of these were observed in control samples. CONCLUSIONS: These haplotypes were identified for the first time in acute lymphoblastic leukemia patients. Five mutations able to change amino acid synthesis for the ATP synthase subunit 6 were associated with acute lymphoblastic leukemia. This investigation could be used to provide an overview of incidence frequency of acute lyphoblastic leukemia (ALL) in Saudi patients based on molecular events.

DNA barcode through cytochrome b gene information of mtDNA in native chicken strains.

This study was carried out to figure out the potentiality of a cytochrome b gene as a barcoding tool in discriminating native chicken strains and other Gallus gallus species. We performed PCR amplification using universal primer to amplify around 415 bp fragment of cytochrome b gene of mtDNA. The results revealed that all Saudi chicken strains were identical to each other but when compared with other species of Gallus the differences were exciting. The phylogenetic tree revealed that there were seven clusters represented for native strains and were clustered together especially in black strain and dark brown ones. The results have confirmed that using cytochrome b gene to discriminate between Saudi chicken strains and other species of G. gallus fowl was a very sufficient tool. Moreover, we can consider short fragment of cytochrome b gene of mtDNA as a universal DNA barcode region. It was a much more accurate and efficient tool to discriminate interspecies than intraspecies. We think it needs more studies to confirm this concept, and we have to apply that tool for many species of vertebrate and invertebrate as well.