Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase.

L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.

In vitro anticancer activity of Scrophularia amplexicaulis extracts on MCF-7 and WEHI-164 cell line.

Scrophularia amplexicaulis is an Iranian endemic plant belonging to the Scrophulariaceae family, which is used in traditional medicine to treat many diseases. The aim of this study was to evaluate the in vitro anticancer activity of S. amplexicaulis extracts against human breast carcinoma (MCF-7) and mouse fibrosarcoma (WEHI-164) cell lines. The ground aerial parts of S. amplexicaulis were soxhlet-extracted with n-hexane, dichloromethane and methanol. MTT assay exhibited that dichloromethane and methanol extracts remarkbly inhibited the growth of MCF-7 and WEHI-164 cancer cells in a dose-and time-dependent manner with little cytotoxicity on normal cell line HUVEC. Cell death ELISA, TUNEL assay, and the cleavage of poly ADP-ribose polymerase (PARP) uncovered that the cytotoxic effects of dichloromethane and methanol extracts were attributed to apoptosis in cancerous cells. Furthermore, quantitative real-time PCR revealed significant increases in the mRNA expression levels of p-53, caspase-3, caspase-9, Bax, and also a decrease in Bcl-2 expression. These results suggested that the extracts mainly induced apoptosis via a mitochondria-mediated intrinsic pathway. Notably, dichloromethane extract had higher cytotoxic and apoptotic activities than that of methanol extract, against both cancer cell lines, particularly MCF-7 cells. Our results indicate that S. amplexicaulis may serve as a promising source of potent agents for the treatment of human cancers.

Proteomics Analysis of Trastuzumab Toxicity in the H9c2 Cardiomyoblast Cell Line and its Inhibition by Carvedilol.

OBJECTIVE: Heart dysfunctions are the major complications of trastuzumab in patients with Human Epidermal growth factor Receptor-2 (HER2)-positive breast cancers. METHODS: In this study, the cytotoxicity of trastuzumab on H9c2 cardiomyoblasts was demonstrated, and the proteome changes of cells were investigated by a tandem mass tagging quantitative approach. The Differentially Abundant Proteins (DAPs) were identified and functionally enriched. RESULTS: We determined that carvedilol, a non-selective beta-blocker, could effectively inhibit trastuzumab toxicity when administrated in a proper dose and at the same time. The proteomics analysis of carvedilol co-treated cardiomyoblasts showed complete or partial reversion in expressional levels of trastuzumab-induced DAPs. CONCLUSION: Downregulation of proteins involved in the translation biological process is one of the most important changes induced by trastuzumab and reversed by carvedilol. These findings provide novel insights to develop new strategies for the cardiotoxicity of trastuzumab.

In Vivo Evaluation of Carvedilol Cardiac Protection Against Trastuzumab Cardiotoxicity.

Cardiac dysfunction is a major side effect of trastuzumab therapy for patients with HER2-positive breast cancer. Beta blockers, such as carvedilol, have been used for protection of trastuzumab cardiotoxicity but there is no definitive conclusive clinical report on their efficacy. In the present study, the preservability effects of carvedilol on trastuzumab-induced left ventricular (LV) dysfunction and the reversibility of trastuzumab-induced cardiotoxicity were evaluated in Wistar rats by echocardiography method. We showed that trastuzumab treatment of rats could induce the LV dysfunction through increasing the LV internal systolic diameter (LVIDs), increasing the end-systolic volume (ESV), decreasing the ejection fraction (EF), and decreasing the fractional shortening (FS). These parameters were not reversed after 14 days of stopping trastuzumab administration. Interestingly, carvedilol improved LVIDs, ESV, EF, and FS. Collectively, the results of this study have verified clinical observations which simultaneously administration of carvedilol may be considered as a possible therapeutic strategy to prevent trastuzumab-mediated LV dysfunction.

Phenotypic and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Clones Carrying the Panton-Valentine Leukocidin Genes Disseminating in Iranian Hospitals.

The spread of Panton-Valentine leukocidin (PVL)-carrying Staphylococcus aureus strains in both hospital and the community is a significant worldwide problem. The aim of the study was to investigate the clonal dissemination pattern of PVL-producing S. aureus strains isolated from hospitalized patients in Tehran, Iran. In this cross-sectional study, 70 PVL-carrying S. aureus strains were recovered from 240 clinical specimens and characterized by antibiotic susceptibility testing, agr typing, SCCmec typing, spa typing, multilocus sequence typing, and virulence and adhesion gene profiling. All the PVL-carrying S. aureus strains were confirmed as methicillin-resistant S. aureus (MRSA) and recovered from wounds (48.6%), blood (25.7%), exudate/pus (11.4%), sputum (8.6%), and body fluid (5.7%) samples. Among the 70 PVL-carrying S. aureus strains tested, 38 (54.3%) were positive for ant (4')-Ia gene, 27 (38.6%) for aac (6')-Ie/aph (2″), 13 (18.6%) for msr(A), 13 (18.6%) for erm(C), 13 (18.6%) for tet(M), 11 (15.7%) for erm(A), 10(14.3%) for msr(B), 9 (12.9%) for aph (3')-IIIa, 5 (7.1%) for mupA, and 2 (2.9%) for erm(B) genes. Five clonal complexes (CC) and nine different clones were detected in this study. The most frequent CC was CC22 (ST22) (42.8%) followed by CC30 (ST30) (21.5%), CC8 (ST8) (17.2%), CC1 (ST772) (11.4%), and CC80 (ST80) (7.1%). In this study, ST22-SCCmec IV/t852 was the predominant PVL-positive MRSA clone (20%), followed by ST8-SCCmec IV/t008 (17.2%), ST30-SCCmec IV/t019 (12.9%), ST22-SCCmec IV/t790 (11.4%), ST22-SCCmec IV/t005 (11.4%), ST30-SCCmec IV/t021 (8.6%), ST80-SCCmec IV/t044 (7.1%), ST772-SCCmec V/t657 (7.1%), and ST772-SCCmec V/t10795 (4.3%). Diversity in clonal types of PVL-carrying MRSA strains in our study supports the need to perform a systematic surveillance of PVL-positive MRSA strains.

G allele at -924 A > G position of FoxP3 gene promoter as a risk factor for tuberculosis.

BACKGROUND: Forkhead box protein 3 (FoxP3) is an important factor for development and function of Regulatory T cells (Treg). Studies have found an association between common gene polymorphisms in FoxP3 and some infectious diseases. The aim of this study was to evaluate possible associations between two Single nucleotide polymorphisms (SNPs) in the promoter of the FoxP3 gene to susceptibility to tuberculosis (TB) and the alteration of Foxp3 gene expression. METHODS: The pattern distribution of genotype at two position, -3279 A > C (rs3761548) and -924 A > G (rs2232365) on the promoter of FoxP3 gene was evaluated using polymerase chain reaction-single specific primer (PCR-SSP) method in 183 tuberculosis patients and 183 healthy control. In addition the quantity of FoxP3 gene expression at mRNA level was identified by the real-time PCR. RESULTS: The frequency of G allele at -924 A > G was significantly higher was higher in TB patients (59.5%) than control group (39.5%) (P ≤ 0.05). In addition, our data viewed approximately 5- folds more FoxP3 gene expression in female patients with GG genotype in comparison to female healthy cases with the same genotype (P ≤ 0.001). There was no statistically significant differences between the distribution pattern of -3279 A > C polymorphism in patients and healthy individuals along with it effect on the FoxP3 gene expression among both groups (P > 0.05). CONCLUSIONS: Our outcome suggests that the -924 A > G polymorphism leads to enhance FoxP3 gene expression and susceptibility to tuberculosis in the sex dependent manner. This event may rise the count of Treg cells and modulate the immune response against tuberculosis.

Single nucleotide polymorphisms of IFNγ (+874 A/T) and IFNγR1 (-56 C/T) in Iranian patients with TB.

BACKGROUND: Two important genes for controlling TB are IFNγ and IFNγR1. However, little information exists regarding genetic susceptibility of the Iranian TB population. METHODS: We investigated the single nucleotide polymorphisms (SNPs) in genes of IFNγ (+874 A/T) and IFNγR1 (-56 C/T) and serum level of IFNγ and their influence on TB in patients; 300 patients with TB and 300 healthy controls were enrolled in this study. PCR-restriction fragment length polymorphism was used to identify SNPs and serum level of IFNγ was measured by ELISA. RESULTS: The allelic and the genotypic form of IFNγ+874 A/T SNP of the studied population were not significant (p>0.05). Allele T frequencies of IFNγR1 -56 C/T promoter region in patients with pulmonary TB (PTB) or extrapulmonary TB (EPTB) were significantly greater than allele C. The -56 TT motif of IFNγR1 is associated with both forms of TB (p<0.05). The serum level of IFNγ was significantly higher in patients with TB than in controls, but there was no significant difference between serum level of IFNγ and the studied genotypes (p>0.05). CONCLUSIONS: The cause of active TB in the patients seems to be due to the lack of effective IFNγ function or the lack of effective signaling connection between IFNγ and its receptor in presence of -56 C/T polymorphism in promoter region of IFNγR1 gene.

The Study of HFE Genotypes and Its Expression Effect on Iron Status of Iranian Haemochromatosis, Iron Deficiency Anemia Patients, Iron-Taker and Non Iron-Taker Controls.

The role of HFE gene mutations or its expression in regulation of iron metabolism of hereditary haemochromatosis (HH) patients is remained controversial. Therefore here the correlation between two common HFE genotype (p.C282Y, p.H63D) and HFE gene expression with iron status in HH, iron deficiency anemia (IDA) and healthy Iranian participants was studied. For this purpose genotype determination was done by polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP). Real-Time PCR was applied for evaluation of HFE gene expression. Biochemical parameters and iron consumption were also assessed. Homozygote p.H63D mutation was seen in all HH patients and p.C282Y was not observed in any member of the population. A significant correlation was observed between serum ferritin (SF) level and gender or age of HH patients. p.H63D homozygote was seen to be able to significantly increase SF and transferrin saturation (TS) level without affecting on liver function. Our results also showed that iron consumption affects on TS level increasing. HFE gene expression level of IDA patients was significantly higher than other groups. Also the HFE gene expression was negatively correlated with TS. Finally, the main result of our study showed that loss of HFE function in HH is not derived from its gene expression inhibition and much higher HFE gene expression might lead to IDA. However we propose repeating of the study for more approval of our finding.

Increased expression of forkhead box protein 3 gene of regulatory T cells in patients with active tuberculosis.

Cell mediated immunity is the most important response against Mycobacterium tuberculosis (MTB). Regulatory T cells (Treg) play a vital role in suppressing the effector T cell response in tuberculosis (TB) patients. Forkhead box protein 3 (Foxp3) is an important regulator of Treg cells development and function. In this study, we showed that the expression of Foxp3 gene in Treg cells is increased in patients with active tuberculosis. In a case-control study, 183 TB patients and 183 controls were recruited according to ethnicity, gender and living area. Then, after isolation of peripheral blood mononuclear cells (PBMCs), FoxP3 gene expression was studied by real-time PCR. The expression of this gene in patients with pulmonary and extra-pulmonary tuberculosis was 2.8 fold higher than normal subjects (CI=1.29±2.37, P≤0.001). Also comparing the patients with pulmonary tuberculosis and the control group, a significant difference was observed (CI=1.81±2.96, P≤0.001). FoxP3 gene expression was 1.5 fold higher in women with pulmonary and extrapulmonary tuberculosis than in men with tuberculosis (CI=0.12±2.01, P=0.02). According to this study, the increased Foxp3 gene expression in patients with TB was observed and this may play as a contributing factor to suppression of Th1-type immune responses.