ZEB1 and ZEB2 gene editing mediated by CRISPR/Cas9 in A549 cell line.

OBJECTIVES: One of the best approaches for recognition of protein function is the induction of mutations for a gene knockout. In line with this strategy, gene editing tools allow researchers to induce these mutations. Lung cancer is one of the leading causes of death worldwide. ZEB1 and ZEB2 genes are the candidates for this disease. METHODS: The ZEB1 and ZEB2 knockout in the non-small cell lung cancer cell line (A549 cell) was investigated. Purification of recombination plasmids was performed from bacteria and then was transported to the A549 cell line. The deletion of ZEB1 and ZEB2 were examined by PCR. RESULTS: The results demonstrated the mutation and deletion in ZEB1 and ZEB2 genes. Based on the findings of this study, A549 cells were transfected with the vectors carrying the sgRNA/Cas9, simultaneously. The DNA fragment demonstrated the presence of indels in target sites as well as provided the potential of CRISPR/Cas9 system. CONCLUSION: CRISPR/Cas9 offers a great potential as an efficient technique for editing of ZEB1 and ZEB2 genes in A549 cell line (Tab. 1, Fig. 6, Ref. 44).

The effects of functionalization of carbon nanotubes on toxicological parameters in mice.

Carbon nanotubes (CNTs) have emerged as a new class of multifunctional nanoparticles in biomedicine, but their multiple in vivo effects remain unclear. Also, the impact of various functionalization types and duration of exposures are still unidentified. Herein, we report a complete toxicological study to evaluate the effects of single- and multiwalled carbon nanotubes (SWCNTs and MWCNTs) with either amine or carboxylic acid (COOH) surface functional groups. The results showed that significant oxidative stress and the subsequent cell apoptosis could be resulted in both acute and, mainly, in chronic intravenous administrations. Also, male reproductive parameters were altered during these exposures. The amino-functionalized CNTs had more toxic properties compared with the COOH functionalized group, and also, in some groups, the multiwalled nanotubes were more active in eliciting cytotoxicity than the single-walled nanotubes. Interestingly, the SWCNTs-COOH had the least alterations in most of the parameters. Evidently, it is concluded that the toxicity of CNTs in specific organs can be minimized through particular surface functionalizations.

Oxidative stress induced in the workers of natural gas refineries, no role for GSTM1 and GSTT1 polymorphisms.

Oxidative stress has an important role in the pathophysiology of many occupational diseases. In this controlled exposure study, the intensity of oxidative stress biomarkers was assessed in the workers of natural sweet and sour gas refineries (SwGR and SoGR, respectively) and compared with controls. In addition, the role of gluthatione S-transferase M1 (GSTM1)-null and GSTT1-null polymorphisms on the intensity of oxidative stress and liver function tests (LFTs) was investigated. Blood samples were taken and measured for lipid peroxidation (LPO) level, total antioxidant capacity (TAC) and total thiol molecules (TTMs). GSTM1- and GSTT1-null genotypes were determined using polymerase chain reaction. LPO was significantly (p < 0.05) higher in the workers of SoGR. TAC was significantly lower in SwGR subjects (p < 0.001). TTMs were significantly lower in SoGR and SwGR subjects. Among LFTs, activities of aspartate aminotransferase and alanine aminotransferase but not alkaline phosphatase were elevated significantly (p < 0.001) in SoGR subjects. Multivariate linear regression revealed no association between studied polymorphisms, oxidative stress biomarkers, and LFTs. These results indicate that working in the SoGR and SwGR can lead to oxidative stress and abnormal LFTs. Continuous monitoring of natural gas workers for probable ongoing problems is therefore suggested.

The expression of p38, ERK1 and Bax proteins has increased during the treatment of newly diagnosed acute promyelocytic leukemia with arsenic trioxide.

BACKGROUND: Promising reports exist regarding the use of arsenic trioxide (ATO) as first-line treatment in acute promyelocytic leukemia (APL). Although the in vitro effect of ATO is extensively studied, the in vivo mechanism(s) of ATO action is mostly unknown. PATIENTS AND METHODS: Newly diagnosed APL patients were involved and received ATO (0.15 mg.kg/day) for 28 days as induction followed by consolidation therapy. Bone marrow (BM) aspirates were obtained on days 0, 14 and 28 of treatment for further molecular studies. Clinical findings and white blood cell counts were recorded as well. RESULTS: Complete remission was observed in 17 (85%) patients with the median duration of 28 days (18-38) and cumulative dosage of median 280 mg (180-350). Hyperleukocytosis and APL differentiation syndrome (63%), gastrointestinal disorders (30%), liver enzyme elevation and night sweating (50%) were the most prevalent side-effects. The expression of Bax, ERK1 and p38 proteins and caspase-3 activity increased significantly in promyelocytes of BM aspirates at days 14 and 28 of induction therapy. CONCLUSION(S): These findings point toward the role of p38 and Bax in the induction of apoptosis, which was confirmed by increase in caspase-3 activity. However, the increase in ERK1 expression with regard to leukocytosis could translate to a proliferative/differentiation effect.

Effects of arsenic trioxide, all-trans-retinoic acid and dexamethasone on NB4 cell line.

BACKGROUND AND THE PURPOSE OF THE STUDY: Experimental and preclinical observations have indicated that combination therapy with all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) may strongly enhance their therapeutic effects in the treatment of acute promyelocytic leukemia (APL). Whilst dexamethasone (Dex) is routinely used for the control of APL- differentiation syndrome, its effect on the pharmacodynamics of ATO is not clear. Therefore, in this study, effects of therapeutic concentrations of ATO, ATRA and Dex and their sequential usages on the proliferation, differentiation and apoptosis in t(15;17)-positive NB4 cells was investigated. METHODS: Cells were treated with therapeutic concentrations of ATO, ATRA and Dex either as single or in combination and cell proliferation was assessed by XTT assay. Expression of CD11b as an indicator of cell differentiation and the percentage of 7-AAD positive cells as a marker of apoptosis were determined by flow cytometry. RESULTS: ATO, but not ATRA and Dex, decreased proliferation of the cells dose-dependently. Pre-treatment of the cells with any of the drugs did not alter the effects of other drugs on the proliferation. Pre-treatments with Dex blocked the apoptotic effect of ATO (1 µM). CONCLUSION: No improvement or antagonistic effects was observed with the pretreatment/ combination of the ATO and ATRA on the differentiation and apoptosis of the cells. It is possible that concomitant usage of Dex with apoptotic doses of ATO in APL patients counteract therapeutic effects of ATO.

A retrospective study of mushroom poisoning in Iran.

The objective of this study was to describe the pattern of mushroom poisoning in adults admitted to the Loghman-Hakim Hospital Poison Center from 1992 to 2002. All patients > or = 12 years of age were included in the study. The frequency of mushroom poisoning with respect to age, sex, season, reason, place of residence, latent phase, clinical and laboratory findings, treatment, and outcome of patients was investigated. Of the 72421 poisoning cases admitted to Loghman-Hakim Hospital Poison Center from 1992 to 2002, only 37 were poisoned by consumption of toxic mushrooms. As some of the patients' files were incomplete, only 25 files were included in the study. Of this number, 68% were male. The patients' age ranged between 12 and 65 years, with a mean of 31 years of age. All cases were accidental and mostly from Tehran (36%) and the northern provinces (rainy woodlands) of Iran (32%). Autumn was the most common season for poisoning with a frequency of 80%. The latent phase of poisonings was between 0.5 and 12 hours. The most frequently reported symptoms were vomiting (84%), nausea (60%), abdominal pain (60%) and diarrhea (40%). Jaundice was observed in 44% of cases, with a 50% rate of hepatic encephalopathy. A total of 66% of patients were discharged and the duration range of hospitalization was 1-12 days. In conclusion, people should be more informed of the dangers posed by wild mushrooms. Training of physicians and nurses in the accurate diagnosis and management of patients poisoned with poisonous mushrooms would improve the rate of survival.