Preliminary Analysis of Methamphetamine Detection in Lucilia sericata (Diptera: Calliphoridae) Reared in Methamphetamine-Treated Meat at Various Developmental Stages.

Background: It is possible to identify drugs and poisons present in cadavers by analyzing blowfly larvae and pupae collected during forensic autopsies. The main purpose of this study was to use Lucilia sericata's larvae and pupae to identify drugs and poisons present in human cadavers. Methods: In an investigation, immature L. sericata fed meat treated with methamphetamine (MA) at various concentrations (45, 90, and 180 ng/mg) were analyzed to detect MA. Acetylation derivatization and liquid-liquid extraction (LLE) were used as sample preparation methods prior to gas chromatography-mass spectrometry (GC-MS) analytical instrumentation to find MA. Results: According to this study, L. sericata can be used in toxicological testing to identify MA in a host body. All L. sericata larval stages, particularly the third stage larva, pupa and empty pupa tested were positive for MA. Larvae in their first instar produced weak peaks. The L. sericata post-feeding instar following the 45 ng/mg treatment showed the highest MA concentration. For the first time, derivatization using the acetylation approach was used to prepare samples, and successfully, excellent results were obtained. Conclusion: Low quantities of MA can be easily found in immature fly samples using GC-MS. It is important to analyze all samples including human tissues and insect samples, for postmortem drug testing. They can be utilized to find entire MA before they are excreted in excretory samples such as urine. Also, third instar larvae are a great and reliable sample for toxicological study.

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.

Correction to: The Molecular Mechanism of Aluminum Phosphide poisoning in Cardiovascular Disease: Pathophysiology and Diagnostic Approach.

The article "The Molecular Mechanism of Aluminum Phosphide poisoning in Cardiovascular Disease: Pathophysiology and Diagnostic Approach", written by Seyed Farzad Hosseini · Mehdi Forouzesh · Mohsen Maleknia · Samira Valiyari · Mahmood Maniati · Azin Samimi, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 25th July 2020 with open access. With the author(s)' decision to step back from Open Choice, the copyright of the article changed on 29th July 2020 to © Springer Science+Business Media, LLC, part of Springer Nature 2020 and the article is forthwith distributed under the terms of copyright. The original article has been corrected.

The Molecular Mechanism of Aluminum Phosphide poisoning in Cardiovascular Disease: Pathophysiology and Diagnostic Approach.

Nowadays, poisoning with metal phosphides, especially aluminum phosphide (ALP), is one of the main health threats in human societies. Patients suffer from significant complications due to this type of poisoning, and the heart is one of the main organs targeted by ALP. Therefore, in this study, we discussed the effect of phosphine on cardiac function. This study is based on data obtained from PubMed, between 2002 and 2020. The key keywords included "Aluminum phosphide," "Oxidative Stress," "Mitochondria," "Cardiovascular disease," and "Treatment." The results showed that ALP produced reactive oxygen species (ROS) due to mitochondrial dysfunction. ROS production leads to red blood cell hemolysis, decreased ATP production, and induction of apoptosis in cardiomyocytes, which eventually results in cardiovascular disease. Since ALP has the most significant effect on cardiomyocytes, the use of appropriate treatment strategies to restore cell function can increase patients' survival.

Epidemiologic study of traffic crash mortality among motorcycle users in Iran (2011-2017).

PURPOSE: Motorcycle accident is a major cause of road traffic injuries and the motorcyclists are considered as vulnerable road users. The present study aimed to determine the epidemiological characteristics of fatal motorcycle crashes in Iran. METHODS: In this cross-sectional study, a total of 28,356 motorcycle traffic fatalities registered in the Legal Medicine Organization of Iran were analyzed during the period between March 2011 and March 2017. The examined variables included demographic characteristics, helmet use, crash mechanisms, crash location, position state, type of counterpart vehicle, cause of death and place of death. In the study, road traffic mortalities involving drivers and/or passenger of motorcycles were included. Cases or events registered without these conditions were excluded from the study. To analyse the data, SPSS statistics 25 and GraphPad Prism 8 softwares were used. RESULTS: Of the 122,682 fatal traffic injury cases, 28,356 (23.1%) were motorcycle users, of whom 95.3% were male and 4.7% were female. Most of the motorcycle fatalities belonged to the age group of 18-24 years (29.1%). Head trauma was the major cause of death (59.0%). Also, the overall proportion of safety helmet use among motorcycle crash victims was estimated at 37.4%. Most of the road traffic crash cases (46.8%) happened out of city and half of people (49.9%) died in hospital. About 77.4% of the victims were motorcycle riders and 21.1% were pillion passengers. The highest rate of mortality belonged to the self-employed (38.4%) and then workers (21.8%) and students (10.2%). In addition, most fatalities occurred in people with low education (77.5%) and the least occurred in university graduates (5.5%). Among 31 provinces of Iran, Fars had the highest (9.3%) occurrence rate and Kohgiluyeh and Buyer-Ahmad had the lowest (0.5%). Most of the crash mechanisms were due to motorcycle-vehicle crashes (80.2%), followed by rollover (9.8%). CONCLUSION: Comprehensive public education and special rules are needed to reduce the rate of deaths in motorcycle crashes.

Novel fusion protein NGR-sIL-24 for targetedly suppressing cancer cell growth via apoptosis.

Pro-apoptotic peptides have attracted much attention as promising anticancer agents due to their high activity. However, poor cellular uptake of the peptides is often associated with limited therapeutic application. Cell-penetrating homing peptides (CPHPs) were found to increase cell internalization as well as anticancer efficacy of the peptide conjugates. In this study, we developed a novel recombinant fusion protein composed of sIL-24 peptide as a pro-apoptotic moiety and asparagine-glycine-arginine (NGR) motif as a CD13-targeting CPHP component. In silico analysis demonstrated that flexible GGGGS linker provided the best structure and stability for our designed fusion protein. Cell adhesion experiments showed a significant binding affinity toward high CD13-expressing cells (U937 and A549) for NGR-sIL-24. Moreover, confocal microscopy revealed that NGR strongly facilitated the binding and cellular uptake of sIL-24 in U937 and A549 cancer cells. NGR-sIL-24 treatment markedly inhibited the growth of U937 and A549 cancer cells in a dose and time-dependent manner, without affecting the normal cell line MRC-5. Flow cytometric analysis and Hoechst 33342 staining exhibited potent apoptosis induction in U937 and A549 cells treated with NGR-sIL-24. Further mechanism elucidation uncovered that apoptotic death promoted by NGR-sIL-24 was attributed to upregulation of BiP/GRP78, Bax/Bcl-2, GADD34, cytochrome c release, and cleavage of caspase-3, suggesting NGR-sIL-24 penetration into cancerous cells and subsequent apoptosis induction, mainly through endoplasmic reticulum (ER) stress-dependent and mitochondria-dependent signaling pathways. Our results indicate that the designed recombinant fusion protein NGR-sIL-24 may serve as a potential targeted therapy agent for cancers with high expression of CD13.

A-NGR fusion protein induces apoptosis in human cancer cells.

The NGR peptide is one of the well-known peptides for targeting tumor cells. It has the ability to target aminopeptidase N (CD13) on tumor cells or the tumor vascular endothelium. In this study, the NGR peptide was used for targeting A subunit of the Shiga toxin to cancer cells. The cytotoxic effect of the A-NGR fusion protein was assessed on HT1080, U937, HT29 cancer cells and MRC-5 normal cells. For this purpose, cells were treated with different concentrations of A-NGR (0.5-40 µg/ml). The evaluation of cell viability was achieved by MTT assay. Apoptosis was determined by annexin-V/PI double staining flow cytometry. Alterations in the mRNA expression of apoptosis - related genes were assessed by real time RT- PCR. The results showed that A-NGR fusion protein effectively inhibited the growth of HT1080 and U937 cancer cells in comparison to negative control (PBS) but for CD13-negative HT-29 cancer cells, only at high concentrations of fusion protein was inhibited growth recorded. On the other hand, A-NGR had little cytotoxic effect on MRC-5 normal cells. The flow cytometry results showed that A-NGR induces apoptosis. Furthermore, the results of real time RT-PCR revealed that A-NGR significantly increases the mRNA expression of caspase 3 and caspase 9. Conclusively, A-NGR fusion protein has the ability of targeting CD13-positive cancer cells, the cytotoxic effect on CD13-positive cancer cells as well as has low cytotoxic effect on normal cells.

sIL-24 peptide, a human interleukin-24 isoform, induces mitochondrial-mediated apoptosis in human cancer cells.

PURPOSE: Interleukin-24 (IL-24) is a unique cytokine in the IL-10 family that reveals tumor-suppressive activity against a broad range of cancers. Alternative splicing of human IL-24 generates several isoforms with different pro-apoptotic activities. In the current study, we aimed to investigate the cytotoxic properties of a recombinant smallest isoform of IL-24 (sIL-24) and the underlying molecular mechanisms in PC-3, A549, U937, and Raji cancer cells as well as normal cell line MRC-5. METHODS: Following treatment of the cells with recombinant sIL-24 peptide and full-length IL-24 protein, cytotoxicity was determined by MTT assay. Apoptosis induction was evaluated using annexin-V/PI double staining flow cytometry and Hoechst 33342 staining. The expression of Bax, Bcl-2, cytochrome c, and caspase-3 was analyzed by Western blotting. RESULTS: MTT assay exhibited that sIL-24 dose and time dependently inhibited the proliferation of IL-24 receptor-positive PC-3, U937, and Raji cells more effectively than full-length IL-24. In contrast, sIL-24 had little cytotoxic effect on A549 cells lacking the IL-24 receptor, or on MRC-5 normal cells. Flow cytometric analysis and morphological observation revealed an efficient apoptosis induction in the receptor-positive cells. Furthermore, Western blot assay demonstrated that cell death induced by sIL-24 was associated with upregulation of the Bax/Bcl-2 ratio, cytochrome c release, and the expression of cleaved caspase-3, suggesting that sIL-24 induced apoptosis mainly through the mitochondrial pathway. Notably, among the tested cells, induction of apoptosis was more significant in PC-3 cells. CONCLUSION: Our results suggest that the sIL-24 peptide is a promising candidate for potential treatment of human cancers.

A Survey on Phytochemical Composition and Biological Activity of Zygophyllum fabago from Iran.

Purpose: Zygophyllum fabago L. (Z. fabago) is a widespread perennial herb which is used as a medicinal plant in traditional medicine of Iran, Turkey and China. The present study was a survey on phytochemical constituents and biological activities of this plant. Methods: Methanolic extract of the roots was fractionated over a C-18 pre-packed cartridge (Sep-pak) and chromatographic separation was performed on a reversed-phase preparative HPLC. Structural elucidation of the isolated compounds was carried out using UV, 1H-NMR and 13C-NMR spectral analyses. Furthermore, the chemical compositions of the essential oil of the aerial parts were identified by GC-MS analysis. Antiproliferative and antioxidant activities of all extracts from aerials were determined by MTT and DPPH assays, respectively. Results: Phytochemical investigation on the plant roots led to the isolation and identification of two the 60% methanol-water Sep-pak fraction, a prenylated flavone glycoside, 6-C-prenyl-7-O-[ ß -D-4'''-O-acetyl-glucopyranosyl-(1'''→2'')-ß-D-glucopyranosyl] apigenin, which was named as a Zygocaperoside and also, other flavonoid, was named as the Isorhamnetin -3-O glucoside. None of the extracts showed antiproliferative effect against cancerous cells. However, among the extracts, methanolic extract indicated antioxidant activity. Moreover, essential oils of flowers and leaves of plant have high amounts of sesquiterpene hydrocarbons and diterpenoides. Conclusion: The results of present study introduce Z. fabago roots as a new source of flavonoid glycosides and suggest it as an appropriate candidate for further pharmacological studies.

Expression, Purification and Functional Assessment of Smallest Isoform of Human Interleukin-24 in Escherichia coli

ABSTRACT Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.

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.

Methanolic Fractions of Ornithogalum cuspidatum Induce Apoptosis in PC-3 Prostate Cancer Cell Line and WEHI-164 Fibrosarcoma Cancer Cell Line.

PURPOSE: The present study, was aimed to assess the cytotoxic effects of Ornithogalum cuspidatum methanolic fractions on PC-3, prostate cancer cells and WEHI-164, Fibrosarcoma cells. METHODS: Methanolic fractions of O. cuspidatum were prepared using solid phase extraction and the cells were treated with different concentrations for 12 and 24 hours. Cytotoxicity and cell viability were measured by MTT assay. ELISA was also employed to assess the histone-associated DNA fragments and the involvement of apoptotic mechanisms. RESULTS: 10 and 20% fractions had not significant cytotoxic effects (p>0.05) but other fractions exerted growth inhibition on both cancer cell lines (p<0.05). After 24h of incubation with 40, 60, 80 and 100% fractions, the IC50 values were: 165, 85, 65 and 45µg/ml on PC-3 cells and 200, 96, 76 and 73µg/ml against WEHI-164 cell line, respectively. ELISA results also revealed that, both cell lines had undergone apoptosis. CONCLUSION: It is deduced that, 80% and 100% methanolic fractions had significant anti-proliferative and apoptotic impacts on PC-3 and WEHI-164 cells in vitro and could be considered for developing chemo-preventive substances.

Inhibitory and cytotoxic activities of salvia officinalis L. Extract on human lymphoma and leukemia cells by induction of apoptosis.

PURPOSE: Salvia officinalis L., also known as Maryam Goli, is one of the native plants used to Persian medicinal herbs. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized crude methanol extracts prepared from Salvia officinalis L., on a non-Hodgkin's B-cell lymphoma (Raji) and human leukemic monocyte lymphoma (U937), Human acute myelocytic leukemia (KG-1A) and Human Umbilical Vein Endothelial (HUVEC) cell lines. METHODS: The effect of methanolic extract on the inhibition of cell proliferation and cytotoxic activity was evaluated by Dye exclusion and Micro culture tetrazolium test (MTT) cytotoxicity assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determined whether the mechanism involves induction of apoptosis or necrosis. RESULTS: The present results demonstrated that methanolic extract at 50 to 800 µg/ml dose and time-dependently suppressed the proliferation of KG-1A, U937 and Raji cells by more than 80% (p<0.01), with ascending order of IC50 values in 24: KG-1A (214.377 µg/ml), U937 (229.312 µg/ml) and Raji (239.692 µg/ml) when compared with a chemotherapeutic anticancer drug, paclitaxel (Toxol), confirming the tumour-selective cytotoxicity. The crude extract however did not exert any significant cytotoxic effect on normal cell line HUVEC (IC50>800 Ag/ml). Nucleosome productions in KG-1A, Raji and U937 cells were significantly increased respectively upon the treatment of Salvia officinalis L. extract. CONCLUSION: The Salvia officinalis L. extract was found dose and time-dependently inhibits the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.

Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis.

Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 µg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.

Dichloromethane and Methanol Extracts of Scrophularia oxysepala Induces Apoptosis in MCF-7 Human Breast Cancer Cells.

PURPOSE: Breast cancer is the most common cause of cancer-related death in women worldwide. Therefore, there is an urgent need to identify and develop therapeutic strategies against this deadly disease. This study is the first to investigate the cytotoxic effects and the mechanism of cell death of Scrophularia oxysepala extracts in MCF-7 human breast cancer cells. METHODS: Three extracts of Scrophularia oxysepala including the n-hexane, dichloromethane and methanol extracts were examined. MTT (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Trypan-blue assays were performed in MCF-7 cells as well as Human umbilical vein endothelial cells (HUVEC) to analyze the cytotoxic activity of the extracts of Scrophularia oxysepala. Further, the apoptosis inducing action of the extracts was determined by TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) test and cell death assay. RESULTS: The results showed that the n-hexane extract had no cytotoxic effects but dichloromethane and methanol extracts significantly inhibited cell growth and viability in a dose and time dependent manner without inducing damage to non-cancerous cell line HUVEC. In addition, Cell death assay and DNA fragmentation analysis using TUNEL indicated induction of apoptosis by dichloromethane and methanol extracts of Scrophularia oxysepala in MCF-7 cells. CONCLUSION: Our studies suggest that this plant may contain potential bioactive compound(s) for the treatment of breast cancer.