Isolation, characterization, and genomic analysis of vB_PaeS_TUMS_P81, a lytic bacteriophage against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that can lead to nosocomial infections which are in turn life threatening. The increase in antibiotic resistance, at an alarming rate, has resulted in a pressing need for alternative therapeutic approaches such as phage therapy, which hold promise according to several studies. This study featured the isolation and characterization of vB_PaeS_TUMS_P81, a new lytic Pseudomonas phage. The whole-genome sequencing indicated that it has a genome of 73,167 bp containing 93 predicted coding sequences. Genes involved in virulence or lysogeny pathway were nowhere to be found in the genome, so it is potentially safe when it comes to therapeutic applications. Genomic and phylogenetic analysis indicated that vB_PaeS_TUMS_P81 is a member of the genus Litunavirus, belonging to Schitoviridae family. The present study lays the groundwork for further research on treatment of P. aeruginosa infections.

Isolation, characterization, and genomic analysis of vB_PaeP_TUMS_P121, a new lytic bacteriophage infecting Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause life-threatening nosocomial infections. The alarming increase in antibiotic resistance has led to an urgent need for alternative therapeutic approaches, such as phage therapy, which has shown promising results in many studies. In this study, P121, a new lytic Pseudomonas phage, was isolated and characterized. Whole-genome sequencing showed that it has a genome of 73,001 bp that contains 91 predicted coding sequences. No genes involved in virulence or lysogeny were found in the genome, thus making it potentially safe for therapeutic applications. Genomic and phylogenetic analysis indicated that P121 is a member of the genus Litunavirus, family Schitoviridae. The present study provides some basic information for further research on treatment of P. aeruginosa infections.

Nanobodies in Human Infections: Prevention, Detection, and Treatment.

Despite the existence of vaccination, antibiotic therapy, and antibody therapies, infectious diseases still remain as one of the biggest challenges to human health all over the world. Among the different methods for treatment and prevention of infectious diseases, antibodies are well known but poorly developed. There is a new subclass of antibodies calledheavy-chain antibodies that belong to the IgG isotype. However, they are low in molecular weight and lost the first constant domain (CH1). Their single-domain antigen-binding fragments, identified as nanobodies, have unique characteristics, which make them superior in comparison with the conventional antibodies. Low molecular weight and small size, high stability and solubility, ease of expression, good tissue penetration, and low-cost production make nanobodies an appropriate alternative to use against infectious disease. In this research, we review the properties of nanobodies and their potential applications in controlling human infections and inflammations.

PerioVax3, a key antigenic determinant with immunoprotective potential against periodontal pathogen.

Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.

Comparison of SYBR Green and TaqMan real-time PCR methods for quantitative detection of residual CHO host-cell DNA in biopharmaceuticals.

The Chinese hamster ovary (CHO) cell line is one of the predominant hosts used in the bioproduction of pharmaceutical proteins. There have been many concerns about the use of animal cell lines in biopharm industries, and one of the most important concerns has been residual host-cell DNA. Improper integration of residual DNA into the recipient genomes could activate oncogenes or deactivate tumor suppressor genes. Real-time polymerase chain reaction (PCR) is a routine assay method used in the quantification of DNA. In this study, genomic CHO DNA was purified and subjected to real-time PCR. The efficiency of the reaction was calculated, and the limit of detection (LOD) was determined. The calculated efficiency for the primers using the SYBR Green method was 94.3% (r(2) = 0.998). A melting curve analysis showed neither unspecific products nor primer dimers. The calculated efficiency for the TaqMan assay was 96.6% (r(2) = 1). The results showed that the LOD of the SYBR Green and TaqMan assays were 100 fg and 10 fg, respectively. Since the LOD of the TaqMan assay showed a better sensitivity than the SYBR Green, this method could be used directly on the final products for the quantification of residual DNA, without prior DNA extraction.

Human cytochrome b5 reductase: structure, function, and potential applications.

Cytochrome b5 reductase is a flavoprotein that is produced as two different isoforms that have different localizations. The amphipathic microsomal isoform, found in all cell types with the exception of erythrocytes, consists of one hydrophobic membrane-anchoring domain and a larger hydrophilic flavin catalytic domain. The soluble cytochrome b5 reductase isoform, found in human erythrocytes, is a truncated protein that is encoded by an alternative transcript and consists of the larger domain only. Cytochrome b5 reductase is involved in the transfer of reducing equivalents from the physiological electron donor, NADH, via an FAD domain to the small molecules of cytochrome b5. This protein has received much attention from researchers due to its involvement in many oxidation and reduction reactions, such as the reduction of methemoglobin to hemoglobin. Autosomal cytochrome b5 reductase gene deficiency manifests with the accumulation of oxidized Fe+3 and recessive congenital methemoglobinemia in humans. In this article, we provide a comprehensive overview of the structure and function of cytochrome b5 reductase from different eukaryotic sources and its potential use in the food industry, biosensor, and diagnostic areas.

Modulation of hepatitis C virus core DNA vaccine immune responses by co-immunization with CC-chemokine ligand 20 (CCL20) gene as immunoadjuvant.

Plasmid DNA vaccination is a promising vaccine platform for prevention and treatment of infectious disease. Enhancement of the DNA vaccine potency by co-inoculation of immunoadjuvant has been shown to be an effective strategy. Modulation of dendritic cells and T-cells locomotion and trafficking to prime an immune response is mediated by distinct chemokines. The recent study was designed to elucidate the adjuvant activity of plasmid expressing CC-chemokine ligand 20 (pCCL20) in co-inoculation with hepatitis C virus (HCV) core DNA vaccine immunization. pCCL20 was constructed and evaluated for its functional expression. Sub-cutaneous inoculation of pCCL20 with HCV core DNA vaccine was performed via electroporation in BALB/c mice on day 0 and 14 and a HCV core protein booster was applied on day 28. On week after final immunization, both humoral and cell-mediated immune responses were assessed by indirect ELISA for core specific antibodies, lymphocyte proliferation, cytokine ELISA/ELISpot and cytotoxic Grenzyme B (GrzB) release assays. Mice were co-immunized with pCCL20 developed higher levels of core specific IFN-γ/IL-4 ratio and IL-2 release, IFN-γ producing cells, lymphocyte proliferation and cytotoxic Grenzyme B release in both draining lymph nodes and spleen cells of immunized mice. The core-specific serum total IgG and IgG2a/IgG1 ratio were significantly higher when the pCCL20 was co-inoculated. These results suggest the potential of CCL20 chemokine as vaccine adjuvant to enhance Th1 mediated cellular and humoral immune responses in HCV core DNA immunization.

Genome expression analysis by suppression subtractive hybridization identified overexpression of Humanin, a target gene in gastric cancer chemoresistance.

BACKGROUND: In cancer cells, apoptosis is an important mechanism that influences the outcome of chemotherapy and the development of chemoresistance. To find the genes involved in chemoresistance and the development of gastric cancer, we used the suppression subtractive hybridization method to identify the genes that are overexpressed in gastric cancer tissues compared to normal gastric tissues. RESULTS: In the suppression subtractive hybridization library we constructed, the most highly overexpressed genes were humanin isoforms. Humanin is a recently identified endogenous peptide that has anti-apoptotic activity and has been selected for further study due to its potential role in the chemoresistance of gastric cancer. Upregulation of humanin isoforms was also observed in clinical samples by using quantitative real-time PCR. Among the studied isoforms, humanin isoform 3, with an expression level of 4.166 ± 1.44 fold, was the most overexpressed isoform in GC. CONCLUSIONS: The overexpression of humanin in gastric cancer suggests a role for chemoresistance and provides new insight into the biology of gastric cancer. We propose that humanin isoforms are novel targets for combating chemoresistance in gastric cancer.

Assessment of cytokine expression profile in acute myeloid leukemia patients before and after chemotherapy.

OBJECTIVE: One of the major goals of cancer treatment is the monitoring of chemotherapeutic protocols. Quantitative and comparative cytokine expression profiling could be reliable to be used for biomarkers in deadly and fast-growing cancers such as acute myeloid leukemia (AML). The present study aims to assess and further validate cytokines with probable effects on proliferation and maturation of blood cells in AML. MATERIALS AND METHODS: Gene expression levels of IL-1ß, IL-10, IL-8, TNF-α, and IFN-γ were analyzed before and after chemotherapy and after granulocyte colony-stimulating factor (G-CSF) therapy in 46 AML patients by an in-house quantitative comparative RT-PCR method. RESULTS: Our findings indicated that although the gene expression level of TNF-α was almost constant in all 3 samples, IL-1ß, IL-8, and IL-10 expression levels showed a decrease after chemotherapy and an increase after G-CSF therapy. On the other hand, the expression level of IFN-γ had a different pattern with an increase after chemotherapy and a decrease after G-CSF therapy. CONCLUSION: Taken together, the results of this study are in support of the idea that the analyzed cytokines could be useful biomarkers for AML treatment monitoring. However, further molecular epidemiological investigations are suggested to elaborate more cancer monitoring biomarkers.

Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 : H7.

Bacteriophages are considered as promising biological agents for the control of infectious diseases. Sequencing of their genomes can ascertain the absence of antibiotic resistance, toxin or virulence genes. The anti-O157 : H7 coliphage, PhaxI, was isolated from a sewage sample in Iran. Morphological studies by transmission electron microscopy showed that it has an icosahedral capsid of 85-86 nm and a contractile tail of 115×15 nm. PhaxI contains dsDNA composed of 156 628 nt with a G+C content of 44.5 mol% that encodes 209 putative proteins. In MS analysis of phage particles, 92 structural proteins were identified. PhaxI lyses Escherichia coli O157 : H7 in Luria-Bertani medium and milk, has an eclipse period of 20 min and a latent period of 40 min, and has a burst size of about 420 particles per cell. PhaxI is a member of the genus 'Viunalikevirus' of the family Myoviridae and is specific for E. coli O157 : H7.

Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1.

DNA ligases join 3' hydroxyl and 5' phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.

Isolation, characterization, and genome analysis of a broad host range Salmonella phage vB_SenS_TUMS_E4: a candidate bacteriophage for biocontrol.

Salmonella enteritidis is one of the most important foodborne pathogens that cause numerous outbreaks worldwide. Some strains of Salmonella have become progressively resistant to antibiotics, so they could represent a critical threat to public health and have led to the use of alternative therapeutic approaches like phage therapy. In this study, a lytic phage, vB_SenS_TUMS_E4 (E4), was isolated from poultry effluent and characterized to evaluate its potential and efficacy for bio-controlling S. enteritidis in foods. Transmission electron microscopy revealed that E4 has a siphovirus morphotype, with an isometric head and non-contractile tail. Determining the host range showed that this phage can effectively infect different motile as well as non-motile Salmonella enterica serovars. The biological characteristics of E4 showed that it has a short latent period of about 15 min and a large burst size of 287 PFU/cell, and is also significantly stable in a broad range of pHs and temperatures. The E4 whole genome contains 43,018 bp and encodes 60 coding sequences (CDSs) but no tRNA genes. Bioinformatics analysis revealed that the genome of E4 lacks any genes related to lysogeny behavior, antibiotic resistance, toxins, or virulence factors. The efficacy of phage E4 as a bio-control agent was assessed in various foodstuffs inoculated with S. enteritidis at 4°C and 25°C, and the resulting data indicated that it could eradicate S. enteritidis after a very short time of 15 min. The findings of the present study showed that E4 is a hopeful candidate as a bio-control agent against S. enteritidis and has the potential to be used in various foodstuffs.

A suggested new bacteriophage genus: "Viunalikevirus".

We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.

Melatonin may play a role in modulation of bax and bcl-2 expression levels to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis.

The close relationship between free radicals effects and apoptosis process has been proved. Melatonin has been reported as a direct free radical scavenger. We investigated the capability of melatonin in the modification of radiation-induced apoptosis and apoptosis-associated upstream regulators expression in rat peripheral blood lymphocytes. Rats were irradiated with a single whole body Cobalt 60-gamma radiation dose of 8Gy at a dose rate of 101cGy/min with or without melatonin pretreatments at different concentrations of 10 and 100mg/kg body weight. The rats were divided into eight groups of control, irradiation-only, vehicle-only, vehicle plus irradiation, 10mg/kg melatonin alone, 10mg/kg melatonin plus irradiation, 100mg/kg melatonin alone and 100mg/kg melatonin plus irradiation. Rats were given an intraperitoneal (IP) injection of melatonin or the same volume of vehicle alone 1h prior to irradiation. Blood samples were taken 4, 24, 48 and 72h after irradiation for evaluation of flow cytometric analysis of apoptotic lymphocytes using Annexin V/PI assay and measurement of bax and bcl-2 expression using quantitative real-time PCR (RT(2)qPCR). Irradiation-only and vehicle plus irradiation showed an increase in the percentage of apoptotic lymphocytes significantly different from control group (P<0.01), while melatonin pretreatments in a dose-dependent manner reduced it as compared with the irradiation-only and vehicle plus irradiation groups (P<0.01) in all time points. This reduced apoptosis by melatonin was related to the downregulation of bax, upregulation of bcl-2, and therefore reduction of bax/bcl-2 ratio. Our results suggest that melatonin in these doses may provide modulation of bax and bcl-2 expression as well as bax/bcl-2 ratio to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis.

Prediction of the pho regulon in Streptomyces clavuligerus DSM 738.

Negative control of clavulanic acid by phosphate in Streptomyces clavuligerus DSM 738 suggests that a pho regulon may exist in this bacterium. S. clavuligerus PhoP was expressed with a C-terminal His-tag in Escherichia coli and purified. Binding of PhoP-His 6 to promoter fragments of phoRP/phoU and pstS was demonstrated in gel retardation experiments. These fragments contained direct 11 bp repeats resembling PHO boxes. The tentative consensus sequence, GKTCRHBBNSV, was used to search other potential PhoP target genes in the genomic sequence of S. clavuligerus. In total, the putative PHO binding sequence was found in promoter regions of 31 S. clavuligerus genes. Binding of PhoP- His 6 to the PHO box present in the promoter region of the phosphate transporter gene SSCG_07547 of S. clavuligerus was demonstrated. Furthermore, it was shown by real time PCR that decreased concentrations of phosphate do affect increased expressions of genes to which PhoP binds. These findings confirm that a pho regulon exists in S. clavuligerus.

RNA-mediated gene silencing in Candida albicans: inhibition of hyphae formation by use of RNAi technology.

The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 µM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 µM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.

A recombinant Plasmodium vivax apical membrane antigen-1 to detect human infection in Iran.

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.

Comparison of Cytokine Expression in Human PBMCs Stimulated with Normal and Heat-Shocked Lactobacillus plantarum Cell Lysate.

Regulation of immune responses is among the beneficial effects of probiotic bacteria on human health. In this study, we aim to investigate the effect of normal and heat-shocked Lactobacillus plantarum PTCC 1058 cell lysate on cytokine expression by human PBMCs. The mid-exponential phase L. plantarum (108 CFU/mL) were used to prepare cell lysate. Isolated PBMCs were stimulated with 100 µg/mL of each normal and heat-shocked L. plantarum cell lysate for 72 h. Non-stimulated PBMCs were also evaluated as negative control. The mRNA expression of IL-6, IL-10, IFN-É£, TNF-α, and TGF-ß genes was determined by quantitative RT-PCR amplification of total RNA extracted from PBMCs. Both types of cell lysate were able to increase pro-inflammatory cytokines and decrease anti-inflammatory cytokines. However, this effect was significantly stronger in heat-shocked cell lysate-treated PBMCs. Moreover, comparison of IFN-É£/IL-10, IFN-É£/TGF-ß, IL-6/IL-10, IL-6/TGF-ß, and TNF-α/IL-10 ratios in both conditions demonstrated that in the heat-shocked group, all of the above ratios were significantly higher than normal lysate treatment (p˂0.001), suggesting that heat-shocked probiotics are a potent inducer of the immune system in comparison to intact probiotics. Regarding these results, it may be possible to develop a new postbiotic product for the stimulation of immune responses of cancer patients or individuals who suffer from an immune defect.

Reporting a Transcript from Iranian Viola Tricolor, Which May Encode a Novel Cyclotide-Like Precursor: Molecular and in silico Studies.

The cyclotides are the largest known family of cyclic proteins, which are found in several plant families including Violaceae. They are circular bioactive peptides consisting of 28-37 amino acids, which possess a cyclic cystine knot (CCK) motif and could be useful in biotechnology and drug design as scaffolds for peptide-based drugs. This study describes our finding of a potentially novel gene transcript from the petals of the Iranian Viola tricolor (V. tricolor) flowers. This study is based on the cDNA screening method employed for isolation of cyclotide precursor genes and in silico analysis. Our study resulted in the finding of a novel cyclotide-like precursor from V. tricolor, which is documented in the NCBI by GenBank accession number: KP065812. The in silico analysis revealed that there are lots of similar sequences in many other plant families and they all exhibit some different features from previously discovered cyclotide precursors. The differences occur particularly in the main cyclotide domain that exists without the usual CCK structure. All of these hypothetical precursors have a conserved ER-signal sequence, a Cysteine (C)-rich sequence forming two zinc finger motifs and a cyclotide-like region containing several conserved elements including two highly conserved C residues. In conclusion, using the cDNA screening method we found a potentially new cyclotide-like precursor gene and in silico studies revealed its significant characteristics that may open up a new research line on the distribution and evolution of cyclotides.