Utilization of Whole Exome Sequencing in Non-Syndromic Premature Ovarian Failure: Ficolin-3 Gene Mutation in an Iranian Family

Background:Premature ovarian failure is a heterogeneous disorder, leading to early menopause. Several genes have been identified as the cause of non-syndromic POF. Our aim was to explore the genetic defects in Iranian patients with POF. Methods:We studied a family with three females exhibiting non-syndromic POF. WES was performed for one of the affected individuals after ruling out the presence of CGG repeat expansion at FMR1 gene in the family. Sanger sequencing was used to confirm the candidate sequence variants in the proband, and screening of the detected mutation was performed for the other affected and unaffected members of the family. Results:A homozygous frameshift mutation, c.349delC, was identified in FCN3 gene in the proband and two other patients. The parents and two healthy brothers were heterozygous for the mutation, and an unaffected sister was homozygous for wild type. Conclusion:This is the first report of a mutation in FCN3 gene in a family with POF. Our findings can lead to the enhancement of genetic databases of patients with POF, specifically for families with high-risk background. Key Words: Ficolin-3, Premature ovarian failure, Whole exome sequencing     

Distal Renal Tubular Acidosis in an Iranian Patient with Hereditary Spherocytosis

Background:Hereditary spherocytosis and hereditary dRTA are associated with mutations in the SLC4A1 gene encoding the AE1. In this study, some patients with clinical evidence of congenital HS and renal symptoms were investigated. Methods:Twelve patients with congenital HS and renal symptoms were recruited from Ali-Asghar Children’s Hospital (Tehran, Iran). A patient suspected of having dRTA was examined using WES method, followed by Sanger sequencing. Results:One patient (HS03) showed severe failure to thrive, short stature, frequent urinary infection, and weakness. A homozygote (rs571376371 for c.2494C>T; p.Arg832Cys) and a heterozygote (rs377051298 for c.466C>T; p.Arg156Trp) missense variant were identified in the SLC4A1 and SPTA1 genes, respectively. The compound heterozygous mutations manifested as idRTA and severe HS in patient HS03. Conclusion:Our observations, for the first time, revealed clinical and genetic characteristics of idRTA and severe HS in an Iranian patient HS03. Key Words: Erythrocyte membrane protein, Hereditary spherocytosis, Hemolytic anemia, Whole-exome sequencing     

Investigating the CYP2B6 rs3745274 and rs3211371 Polymorphisms in Methadone-Responder and Non-Responder Addicts in Iran

Background:Methadone therapy is a major protocol in opioid addiction cases in many health care systems. Population-based studies have shown that in addicted people, the genetic profile affects their response to methadone therapy. Therefore, this study designed to examine the frequency of two SNPs of the CYP2B6 gene (rs3745274 and rs3211371) in addicted cases in two methadone-responders and methadone non-responders groups. Methods:A total of 199 opioid-addicted individuals and 117 unaffected control subjects were genotyped for rs3745274 and rs3211371 polymorphisms of the CYP2B6 gene using the tetra-primer ARMS-PCR. Results:Results of this study revealed the significant association of rs3745274 GG (p < 0.001; OR = 0.027; 95% CI = 0.14-0.49) and GT (p < 0.001; OR = 4.04; 95% CI = 2.26-7.21) genotypes with the risk of addiction in methadone-responders. Also, a significant association between rs3745274 GG (p < 0.001; OR = 0.28; 95% CI = 0.15-0.51) and GT (p < 0.001; OR = 5.1; 95% CI = 2.8-5.28) genotypes and addiction relapse was found in methadone non-responders. Conclusion:Based on our findings, we can conclude that rs3745274 variant of CYP2B6 gene could serve as a potential biomarker, to evaluate the prognosis of addicted people fate under treatment with methadone. Key Words: Addiction, Biomarker, Methadone, Single-nucleotide polymorphism     

Expression and Characterization of a Cold-Adapted Alginate Lyase with Exo/Endo-Type Activity from a Novel Marine Bacterium Alteromonas portus HB161718T

The alginate lyases have unique advantages in the preparation of alginate oligosaccharides and processing of brown algae. Herein, a gene alg2951 encoding a PL7 family alginate lyase with exo/endo-type activity was cloned from a novel marine bacterium Alteromonas portus {"type":"entrez-nucleotide","attrs":{"text":"HB161718","term_id":"239323637"}}HB161718^T and then expressed in Escherichia coli. The recombinant Alg2951 in the culture supernatant reached the activity of 63.6 U/mL, with a molecular weight of approximate 60 kDa. Alg2951 exhibited the maximum activity at 25 °C and pH 8.0, was relatively stable at temperatures lower than 30 °C, and showed a special preference to poly-guluronic acid (polyG) as well. Both NaCl and KCl had the most promotion effect on the enzyme activity of Alg2951 at 0.2 M, increasing by 21.6 and 19.1 times, respectively. The TCL (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) analyses suggested that Alg2951 could catalyze the hydrolysis of sodium alginate to produce monosaccharides and trisaccharides. Furthermore, the enzymatic hydrolysates displayed good antioxidant activity by assays of the scavenging abilities towards radicals (hydroxyl and ABTS+) and the reducing power. Due to its cold-adapted and dual exo/endo-type properties, Alg2951 can be a potential enzymatic tool for industrial production.     

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate

Background:There is limited information on the 3D prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of PmrA/B in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods:The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and MIC assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results:Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 β-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three β-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. {"type":"entrez-protein","attrs":{"text":"WP_071210493.1","term_id":"1098418292"}}WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 β-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: TheDNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii. Key Words: Acinetobacter baumannii, Amino acid substitution, Colistin, Mutation     

Identification and Expression of Two Novel Cytochrome P450 Genes,CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.     

Identification of a Novel Stop Loss Mutation in P2RX2 Gene in an Iranian Family with Autosomal Nonsyndromic Hearing Loss

Background:Hearing loss, a congenital genetic disorder in human, is difficult to diagnose. WES is a powerful approach for ethiological disgnosis of such disorders. Methods:One Iranian family with two patients were attented in the study. Sequencing of known NSHL genes was carried out to recognize the genetic causes of HL. Results:Molecular analyses identified a novel stop loss mutation, c.1048T>G (p.Term350Glu), whitin the P2RX2 gene, causing a termination-site modification.This event would lead to continued translation into the 3'' UTR of the gene, which in turn may result in a longer protein product. The mutation was segregating with the disease phenotype and predicted to be pathogenic by bioinformatic tools. Conclusion:This study is the first Iranian case report of a diagnosis of ADNSHL caused by P2RX2 mutation. The recognition of other causative mutations in P2RX2 gene more supports the probable function of this gene in causing ADNSHL. Key Words: Autosomal dominant 41, Deafness, Mutation, P2RX2, Whole exome sequencing     

Exon Sequencing of PKD1 Gene in an Iranian Patient with Autosomal-Dominant Polycystic Kidney Disease

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic kidney disorders with the incidence of 1 in 1,000 births. ADPKD is genetically heterogeneous with two genes identified: PKD1 (16p13.3, 46 exons) and PKD2 (4q21, 15 exons). Eighty five percent of the patients with ADPKD have at least one mutation in the PKD1 gene. Genetic studies have demonstrated an important allelic variability among patients, but very few data are known about the genetic variation among Iranian populations. Methods: In this study, exon direct sequencing of PKD1 was performed in a seven-year old boy with ADPKD and in his parents. The patient’s father was ADPKD who was affected without any kidney dysfunction, and the patient’s mother was congenitally missing one kidney. Results: Molecular genetic testing found a mutation in all three members of this family. It was a missense mutation GTG>ATG at position 3057 in exon 25 of PKD1. On the other hand, two novel missense mutations were reported just in the 7-year-old boy: ACA>GCA found in exon 15 at codon 2241 and CAC>AAC found in exon 38 at codon 3710. For checking the pathogenicity of these mutations, exons 15, 25, and 38 of 50 unrelated normal cases were sequenced. Conclusion: our findings suggested that GTG>ATG is a polymorphism with high frequency (60%) as well as ACA>GCA and CAC>AAC are polymorphisms with frequencies of 14% and 22%, respectively in the population of Southwest Iran. Key Words: Autosomal dominant polycystic kidney disease (ADPKD), Polycystic kidney diseases (PKD), PKD1 gene, Iran     

A Novel Splice Site Variant in the LDLRAP1 Gene Causes Familial Hypercholesterolemia

Background:FH, a hereditary disorder, is caused by pathogenic variants in the LDLR, APOB, and PCSK9 genes. This study has assessed genetic variants in a family, clinically diagnosed with FH. Methods:A family was recruited from MASHAD study in Iran with possible FH based on the Simon Broom criteria. The DNA sample of an affected individual (proband) was analyzed using WES, followed by bioinformatics and segregation analyses. Results:A novel splice site variant (c.345-2A>G) was detected in the LDLRAP1 gene, which was segregated in all affected family members. Moreover, HMGCR rs3846662 g.23092A>G was found to be homozygous (G/G) in the proband, probably leading to reduced response to simvastatin and pravastatin. Conclusion:LDLRAP1 c.345-2A>G could alter the PTB, which acts as an important part of biological pathways related to lipid metabolism. Key Words: Genetic research, LDLRAP1, Hypercholesterolemia, Hydroxymethylglutaryl-CoA Reductase Inhibitors     

Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ ² P > 0.05), including only one common haplotype (GenBank access no. {"type":"entrez-nucleotide","attrs":{"text":"EF413075","term_id":"151936076","term_text":"EF413075"}}EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran     

Inhibitory Potential of Acroptilon repens against Key Enzymes involved in Alzheimer and Diabetes,Phytochemical Profile,Radical Scavenging,and Antibacterial Activity

Background:This study was devoted to assessing the inhibitory potential of acetone, methanol, and ethanol extracts of Acroptilon repens against disease-associated enzymes, as well as their antioxidant/antibacterial activity and phytochemical composition. Methods:Comparative assessment using various antioxidant evaluation methods, including FRAP, scavenging ability on DPPH radical and hydrogen peroxide, and RP, indicated that the acetone extract presented the highest antioxidant activity, due to its highest total antioxidant content. Results:The TPC and TFC of these extracts were 3.44 ± 0.32 mg GAE/g DW and 2.09 ± 0.2 mg QE/g DW, respectively. The hydrodistillation essential oil from A. repens was analyzed by GC/MS, and 17 compounds were identified. All extracts showed good inhibitory activities against disease-related enzyme acetylcholinesterase and α-amylase, with the lowest IC₅₀ for acetonic extract. Extracts of A. repens exhibited inhibiting activities against the Gram-positive bacteria, with the most effect of acetone extract. Conclusion:Our findings suggest A. repens as a promising source of natural antioxidant, antimicrobial, anti-cholinesterase and anti-amylase agents for the management of oxidative damage, and pharmaceutical, food, and cosmeceutical purposes. Key Words: Acroptilon repens, Antioxidants, Phytochemicals     

Strain-Specific Behavior of Mycobacterium tuberculosis in Interruption of Autophagy Pathway in Human Alveolar Type II Epithelial A549 Cells

Background:Autophagy induction has been shown to differ in magnitude depending on the mycobacterial species. However, few studies have investigated the specific autophagic capacity of different Mtb strains in ATs. This study aimed to elucidate the host autophagic response to different Mtb strains in ATs responsible for TB in the capital of Iran, Tehran. Methods:A549 cells were infected with three different Mtb clinical isolates (Beijing, NEW1, and CAS1/Delhi) and the reference strain H37Rv. Following RNA extraction, the expression of eight ATG genes, four mycobacterial genes, and three miRNAs was evaluated using quantitative RT-PCR. Results:The results revealed that all four strains influenced the autophagy pathway in various ways at different magnitudes. The Beijing and H37Rv strains could inhibit autophagosome formation, whereas the CAS and NEW1 strains induced autophagosome formation. The expression of genes involved in the fusion of autophagosomes to lysosomes (LAMP1) indicated that all the studied strains impaired the autophagolysosomal fusion; this result is not unexpected as Mtb can block the autophagolysomal fusion. In addition, the Beijing and H37RV strains prevented the formation of autophagic vacuoles, besides mycobacterial targeting of lysosomes and protease activity.Conclusion:This preliminary study improved our understanding of how Mtb manages to overcome the host immune system, such as autophagy, and evaluated the genes used by specific strains during this process. Further studies with a large number of Mtb strains, encompassing the other main Mtb lineages, are inevitable.Key Words: A549 cells, Autophagy, MicroRNA, Mycobacterium tuberculosis     

Frequency of Efficient Circulating Follicular Helper T Cells Correlates with Dyslipidemia and WBC Count in Atherosclerosis

Background:The significance of cTfh cells and their subsets in atherosclerosis is not well understood. We measured the frequency of cTfh subsets in patients with different degrees of stenosis using flow-cytometry. Methods:Participants included high (≥50%; n = 12) and low (<50%; n = 12) stenosis groups, as well as healthy controls (n = 6). Results: The frequency of CCR7^loPD-1^hiefficient-cTfh was significantly higher in patients with high stenosis compared to healthy controls (p = 0.003) and correlated with LDL (p = 0.043), cholesterol (p = 0.043), triglyceride (p = 0.019), neutrophil count (p = 0.032), platelet count (p = 0.024), NLR (p = 0.046), and PLR (p = 0.025) in high stenosis group. The frequency of CCR7^hiPD-1^lo quiescent-cTfh was higher in healthy controls compared to the high-stenosis group (p = 0.001) and positively correlated with HDL (p = 0.046). The frequency of efficient-cTfh cells was correlated with platelet count (p = 0.043), NLR (p = 0.036), and PLR (p P = 0.035) in low-stenosis group, while that of quiescent-cTfh cells was negatively correlated with LDL (p = 0.034), cholesterol (p = 0.047), platelet count (p = 0.032), and PLR (p = 0.041). Conclusion:High percentages of cTfh and efficient-cTfh cells in patients with advanced atherosclerosis and their correlation with dyslipidemia and WBC counts suggests an ongoing cTfh subset deviation, towards efficient phenotype in the milieu of inflammation and altered lipid profile. Efficient cTfh cells have an effector phenotype and could in turn contribute to atherosclerosis progression. Key Words: Atherosclerosis, Blood Platelets, Neutrophils, Dyslipidemias     

Investigation of TNF-α and IL-6 Levels in the Sera of Non-Melanoma Skin Cancer Patients

Background:TNF-α and IL-6 are both pleiotropic cytokines playing major roles in cancer-associated cytokine networks. They have previously been investigated for their function in skin malignancies, mostly melanomas, and studies on NMSC patients are relatively rare. In this study, we aimed to assess the associations of serum levels of IL-6 and TNF-α with NMSCs and its clinicopathological features. Methods:This cases-control study was carried out to investigate the serum levels of TNF-α and IL-6 in 70 NMSC patients, in comparison with 30 healthy individuals, by means of flow cytometric bead-based immuneoassay. Results:Serum levels of both TNF-α and IL-6 were significantly higher in NMSC patients (6.470 vs. 4.355 pg/ml; p = 0.0468, respectively), compared to healthy individuals (3.205 vs. 0.000 pg/ml; p = 0.0126, respectively). In the subgroup analysis, SCC patients had higher serum levels of IL-6 compared to healthy individuals (3.445 vs. 0.000 pg/ml; p = 0.0432). No other significant differences were observed in the serum levels of these two cytokines among different clinicopathological subgroups of the patients. Conclusion:The increased levels of TNF-α and IL-6 in NMSC patients can be introduced as an epiphenomenon of a complex cancer-induced cytokine cascade. Key Words: Biomarkers, Cytokines, Interleukin-6, Tumor Necrosis Factor-alpha     

Identification of New CTX Analogues in Fish from the Madeira and Selvagens Archipelagos by Neuro-2a CBA and LC-HRMS

Ciguatera Poisoning (CP) is caused by consumption of fish or invertebrates contaminated with ciguatoxins (CTXs). Presently CP is a public concern in some temperate regions, such as Macaronesia (North-Eastern Atlantic Ocean). Toxicity analysis was performed to characterize the fish species that can accumulate CTXs and improve understanding of the ciguatera risk in this area. For that, seventeen fish specimens comprising nine species were captured from coastal waters inMadeira and Selvagens Archipelagos. Toxicity was analysed by screening CTX-like toxicity with the neuroblastoma cell-based assay (neuro-2a CBA). Afterwards, the four most toxic samples were analysed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Thirteen fish specimens presented CTX-like toxicity in their liver, but only four of these in their muscle. The liver of one specimen of Muraena augusti presented the highest CTX-like toxicity (0.270 ± 0.121 µg of CTX1B equiv·kg⁻¹). Moreover, CTX analogues were detected with LC-HRMS, for M. augusti and Gymnothorax unicolor. The presence of three CTX analogues was identified: C-CTX1, which had been previously described in the area; dihydro-CTX2, which is reported in the area for the first time; a putative new CTX m/z 1127.6023 ([M+NH₄]⁺) named as putative C-CTX-1109, and gambieric acid A.     

Impact of ABCB1 Gene Polymorphisms and Smoking on the Susceptibility Risk of Chronic Myeloid Leukemia and Cytogenetic Response

Background:IM, a strong and selective TKI, has been approved as the front line of treatment in CML patients. In spite of satisfactory results of imatinib in the treatment of patients with CML, patients with treatment failure or suboptimal response developed resistance that might be because of pharmacogenetic variants. This study attempted to evaluate the influence of ABCB1 gene polymorphisms and smoking on CML risk and resistance to imatinib. Methods: ABCB1 (c.1236C>T, c.3435C>T) polymorphisms were genotyped in 98 CML patients and 100 sex- and age-matched healthy subjects by PCR-RFLP method, followed by sequencing. The patients were evaluated for cytogenetic response by the standard chromosome banding analysis in regular intervals. Results:Our results showed that c.1236CC genotype was significantly associated with imatinib resistance (OR = 3.94; p = 0.038). Analysis of the joint of SNP-smoking combination showed that smokers with c.1236TT/CT and c.1236CC genotypes had the increased risk of CML (OR = 6.04; p = 0.00 and OR = 4.95, p = 0.005) and treatment failure (OR = 5.36, p = 0.001 and OR = 15.7, p = 0.002), respectively. Smokers with c.3435TT/CT and c.3435CC genotypes also displayed the elevated risk of CML development (OR = 6.01, p = 0 and OR = 4.36, p = 0.011) and IM resistance (OR = 5.61, p = 0.001 and OR = 13.58, p = 0.002), respectively. Conclusion:Our findings suggest that c.1236CC genotype has clinical importance in the prediction of treatment outcome with IM, and smoking could have a synergistic role in CML risk and IM resistance. Key Words: ATP binding cassette transporter subfamily B, Imatinib mesylate, Smoking     

Recombinant Production of a Novel Fusion Protein: Listeriolysin O Fragment Fused to S1 Subunit of Pertussis Toxin

Background:Some resources have suggested that genetically inactivated PTs bear a more protective effect than chemically inactivated products. This study aimed to produce new version of PT, by cloning an inactive PTS1 in a fusion form with N-terminal half of the LLO pore-forming toxin. Methods:Deposited pdb structure file of the PT was used to model an extra disulfide bond. Codon-optimized ORF of the PTS1 was used to make recombinant constructs of PTS1 and LLO-PTS1 in the pPSG-IBA35 vector. The recombinant PTS1 and LLO-PTS1 proteins were expressed in BL21 DE3 and SHuffle T7 strains of E. coli and purified by affinity chromatography. Cytotoxic effects of the recombinant proteins were examined in the MCF-7 cell line. Results:The purity of the products proved to be more than 85%, and the efficiency of the disulfide bond formation in SHuffle T7 strain was higher than BL21 DE3 strain. No cytotoxicity of the recombinant proteins was observed in MCF-7 cells. Soluble recombinant PTS1 and LLO-PTS1 proteins were produced in SHuffle T7 strain of E. coli with high efficiency of disulfide bonds formation. Conclusion:The LLO-PTS1 with corrected disulfide bonds was successfully expressed in E. coli SHuffle T7 strain. Due to the safety for human cells, this chimeric molecule can be an option to prevent pertussis disease if its immunostimulatory effects would be confirmed in the future. Key Words: Adjuvant, Cloning, Fusion protein, Pertussis toxin     

Interaction of Thalassia testudinum Metabolites with Cytochrome P450 Enzymes and Its Effects on Benzo(a)pyrene-Induced Mutagenicity

The aim of the present work was to evaluate the effects of Thalassia testudinum hydroethanolic extract, its polyphenolic fraction and thalassiolin B on the activity of phase I metabolizing enzymes as well as their antimutagenic effects. Spectrofluorometric techniques were used to evaluate the effect of tested products on rat and human CYP1A and CYP2B activity. The antimutagenic effect of tested products was evaluated in benzo[a]pyrene (BP)-induced mutagenicity assay by an Ames test. Finally, the antimutagenic effect of Thalassia testudinum (100 mg/kg) was assessed in BP-induced mutagenesis in mice. The tested products significantly (p < 0.05) inhibit rat CYP1A1 activity, acting as mixed-type inhibitors of rat CYP1A1 (Ki = 54.16 ± 9.09 μg/mL, 5.96 ± 1.55 μg/mL and 3.05 ± 0.89 μg/mL, respectively). Inhibition of human CYP1A1 was also observed (Ki = 197.1 ± 63.40 μg/mL and 203.10 ± 17.29 μg/mL for the polyphenolic fraction and for thalassiolin B, respectively). In addition, the evaluated products significantly inhibit (p < 0.05) BP-induced mutagenicity in vitro. Furthermore, oral doses of Thalassia testudinum (100 mg/kg) significantly reduced (p < 0.05) the BP-induced micronuclei and oxidative damage, together with an increase of reduced glutathione, in mice. In summary, Thalassia testudinum metabolites exhibit antigenotoxic activity mediated, at least, by the inhibition of CYP1A1-mediated BP biotransformation, arresting the oxidative and mutagenic damage. Thus, the metabolites of T. testudinum may represent a potential source of chemopreventive compounds for the adjuvant therapy of cancer.