The Primer Specificity is Critical to Getting Right Results in Real Time PCR

Not ha

Comparing the Cytotoxic Effects of Clove Essential Oil (Syzygium Aromaticum) and its Major Compound (Eugenol) Against Some Human Breast Cancer Cell Lines

Background & Objective: Breast cancer is the most common cancer among women, especially in developed countries. Because of resistance to chemotropic drugs, the development of new green drugs is crucial. Essential oils with a broad range of bioactivities such as antioxidant and anticancer activities are great resources for research and development. In this study, anticancer effects of clove (Syzygium aromaticum) essential oil as the common medicinal plant against some human breast cancer cell lines was investigated. Materials & Methods: Components of clove essential oil were identified. The anticancer effect of the essential oil and its major ingredient (eugenol) was investigated on four human breast cancer cell lines. Results: Ninety-two percent of clove essential oil was included by eugenol (65%), trans-caryophyllene (12%), eugenol acetate (10%), caryophyllene oxide (3%), and a-humulene (2%). Both clove essential oil and eugenol showed proper effect (IC50 µg.mL-1) on targeted cell lines, MCF-7 (151.94 and 86.13), MDA-MB-175 (162.92 and 33.25), MDA-MB-231 (180.61 and 69.75), and MDA-MB-468 (211.11 and 53.91). Conclusions: Regarding the proper anticancer effects of both samples, they could be considered as anticancer agents for further investigation

Suicide gene therapy for breast cancer with a suicide-inducing vector carrying the mammaglobin-1 promoter

BACKGROUND: Breast carcinoma is the most frequent cancer among women and finding a therapeutic approach is necessary for the treatment of this carcinoma. Gene therapy is a promising therapeutic approach for cancer. Targeted expression of the desired therapeutic proteins within the tumor cells is the best approach to reduce toxicity and improve survival. The mammaglobin-1 gene encodes a novel, breast cancer-associated glycoprotein. Mammaglobin-1 expression is restricted to the mammary gland and the function of the mammaglobin-1 protein is unknown. No mammaglobin-1 expression has been reported in various types of benign tissue or neoplasia other than the breast carcinomas. Over expression of mammaglobin-1 gene was reported in breast cancer tissues by some scientists. In this study, for the first time, the mammaglobin-1 promoter was introduced as a cancer specific promoter with a high efficacy. METHODS: A suicide-inducing vector that expresses the BAX suicide gene under the transcriptional control of the mammaglobin-1 promoter was constructed and evaluated against breast cancer in vitro. RESULT S: These data show that the mammaglobin-1 promoter is active in the breast cancer cell lines. Under the control of the mammaglobin-1 promoter, BAX expression in MD-MBA 438 cell line was five-fold higher than in human embryonic kidney (HEK) cell line and no expression was found in A549 cell line. With the help of mammaglobin-1 promoter, a wonderful enhancement was observed in the apoptosis in breast cancer cell lines. CONCLUSIONS: Concerning the expression of BAX gene under the control of mammaglobin-1, it can be used to specify suicide gene therapy in the treatment of breast tumor. © 2017 EDIZIONI MINERVA MEDICA

A Case Report of COVID-19 and Subacute Thyroiditis in Fars, Iran

Background & Objective: COVID-19 can affect thyroid gland and causes subacute thyroiditis. Case Presentation: We introduced a 60-year-old woman with an initial symptom of anterior cervical pain without any other constitutional symptoms. Nasopharyngeal and oropharyngeal swabs were detected positive for COVID-19 using RT-PCR assay. According to ultrasonographic,  laboratory (lowered TSH, elevated CRP and ESR), and physical findings, subacute thyroiditis was found following the SARS-CoV-2 infection. Conclusion: As subacute thyroiditis associated with SARS-Cov-2 may be represented without any fever, ruling out this infection in these patients is considerable

Validation of a Simple and Rapid Method for Assessment of Intracellular Bacterial Asparaginase: Validation of a Simple Method for Assessment of Bacterial Asparaginase

L-Asparaginase has remarkable properties which make it useful in dual pharmaceutical and food industries.In this study, simple and advantageous methods have been validated for rapid and precisedetermination of intracellular L-Asparaginasein bacterial species. A suspension of bacterial cells was used instead of cell extract and incubated by substrate (asparagine) after simple wash and centrifugation steps. Due to loss of enzyme activity which could be caused by cell distruption methods such as sonication or enzymatic treatment, cell suspension was used instead of the cell extract. Thus, this method not only is cost effective but also speeds up the screening process and leasd to higher measurement accuracy. To validate this method, two species of bacteria; E.coli ATCC 8739 and Halomonas H28 were used. After cultivation, the cells were harvested and washed. Then, 5 serial dilutions were prepared from each bacterium, and the asparaginase activity in each of them was measured by methods including sonication, enzymatic lyses, and the cell suspension. The results have showed that the changes in asparaginase activity in all 5 serial dilutions are linear and there is good agreement between the sonication and the cell suspension methods. Also, it was shown that activities measured by the enzymatic method were significantly higher than the other two methods

Nano-scaled emulsion and nanogel containing Mentha pulegium essential oil: cytotoxicity on human melanoma cells and effects on apoptosis regulator genes

Abstract Background Topical drug delivery using nanoemulsions and nanogels is a promising approach to treating skin disorders such as melanoma. Methods In this study, the chemical composition of Mentha pulegium essential oil with five major compounds, including pulegone (68.11%), l-menthone (8.83%), limonene (2.90%), iso-pulegone (2.69%), and iso-menthone (1.48%) was first identified using GC-MS (Gas chromatography–Mass Spectrometry) analysis. Afterward, a nano-scaled emulsion containing the essential oil with a droplet size of 7.70 ± 1 nm was prepared. Nanogel containing the essential oil was then prepared by adding (2% w/v) carboxymethyl cellulose to the nano-scaled emulsion. Moreover, the successful loading of M. pulegium essential oil in the nano-scaled emulsion and nanogel was confirmed using ATR-FTIR (Attenuated total reflectance-Fourier Transform InfraRed) analysis. Then, human A375 melanoma cells were treated with different concentrations of samples, the MTT assay evaluated cell viability, and cell apoptosis was confirmed by flow cytometry. In addition, the expression of apoptotic and anti-apoptotic genes, including Bax and Bcl-2, was evaluated using the qPCR (quantitative Polymerase Chain Reaction) technique. Results The results showed that cell viability was reduced by 90 and 45% after treatment with 300 μg/mL of the nanogel and nano-scaled emulsion. As confirmed by flow cytometry, this effect was mediated by apoptosis. Furthermore, gene expression analysis showed up-regulation of Bax and down-regulation of Bcl-2 genes. Therefore, the prepared nanogel, with high efficacy, could be considered a potent anticancer agent for supplementary medicine and in vivo research

Analysis of polychlorinated biphenyls (PCBs) in dairy products by modified QuEChERS/GC‐QqQ‐MS/MS method: A risk assessment study

Abstract Polychlorinated biphenyls (PCBs) are harmful chemicals that are persistent in the environment and can accumulate in the food chain. The purpose of the present research was to assess non‐dioxin‐like polychlorinated biphenyls (NDL‐PCBs) in some dairy products (yogurt, doogh, and kashk) using modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) technique and gas chromatography–triple‐quadrupole mass spectrometry (GC‐QqQ‐MS/MS) method and risk assessment study. The LOQs (limit of quantifications), LODs (limit of detections), recovery, and RSD for the PCB analytes were 0.180–0.360, 0.06–0.12 ng/g fat, 97.45–102.63%, and 6.33–8.86%, respectively. The results revealed that the mean concentrations of Ʃ6‐NDL‐PCBs in samples were 15.17 ± 3.44 ng/g fat, which was lower than the standard level established by European Union (EU, 40 ng/g fat). The maximum mean level was PCB 180 (9.98 ± 2.04 ng/g fat) and the minimum mean level of PCBs in samples was PCB 28 (0.09 ± 0.06 ng/g fat). Also, results showed that kashk samples had a maximum mean level of 6‐NDL‐PCBs (18.66 ± 2.42 ng/g fat) and doogh samples had a minimum mean level of 6‐NDL‐PCBs (12.21 ± 2.22 ng/g fat). The mean level of 6‐NDL‐PCBs in yogurt samples was 14.65 ± 2.02 ng/g fat. The heat map results showed the correlation between the spectral indices of 6‐NDL‐PCBs in different dairy products. According to the Monte Carlo method, risk assessment was done using calculating the Estimated Daily Intake (EDI) and Incremental Life Cancer Risk (ILCR). The EDI values of 6 NDL‐PCBs based on the 95th percentile in yogurt, doogh, and kashk were 14.3, 1.49, and 0.5 ng/kg.day, respectively. Considering that the contaminant level in the samples is lower than the EU limit, it can be concluded that dietary exposure to 6 NDL‐PCBs may not pose a risk to the health of consumers

A Novel Multiplex LAMP Assay for the Rapid and Accurate Diagnosis of Visceral Leishmaniasis Caused by Leishmania infantum from Iran

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per μL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per μL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries

Evaluation of the topical gel and oral administration of Punica Granatum Var Pleniflora on oral mucositis induced by 5-Fluorouracil in golden hamsters

Abstract Background Oral mucositis (OM), an acute inflammation of the oral cavity, is a common complication in patients undergoing invasive myeloblastic chemotherapy or radiation therapy. 5-fluorouracil (5-FU) is one of the most effective therapeutic drugs, but one of the common side effects of 5-FU administration is OM. Unfortunately, no suitable treatment has been found, so far to control its side effects. Studies showed that herbal medicine like Punica granatum var pleniflora (PGP) has medicinal properties such as anti-inflammatory and antibacterial and can be an alternative for the treatment of fungal infection. Accordingly, we decided to investigate the therapeutic effect of PGP in the treatment of OM caused by 5-FU in golden hamsters. Methods Sixty male golden hamsters were divided into six main group. Chemotherapy with 5-FU at dose of 60 mg/kg was performed at a ten-day duration. Then, cheek pouches of the hamsters were scratched with an 18-gauge sterile needle to induce oral mucositis in animals. On the twelfth day, as a day of intensification of OM, treatment with PGP including topical gel with concentrations of 5% and 10% and oral administration of hydro-alcoholic extract with doses of 125 mg/kg and 250 mg/kg for three- and five-day therapeutic duration were separately started. Finally, samples of cheek pouches in hamsters were collected on 14th and 17th days and histopathologic score (HPS), malondialdehyde (MDA), and myeloperoxidase (MPO) levels were assayed. Results A significant (p < 0.05) decrease in histopathologic score was observed in G10%−, P125-treated groups in comparison to the Ctrl group. Our data showed that treatment with G10% is more potent than P125-treated group. In contrast, histopathologic score in G10%, P125, and P250 treated groups demonstrated almost similar values On the 17th day. However, the levels of MDA and MPO in the treatment groups were enhanced compared with control group (p < 0.05). Conclusions It is possible that PGP can play protective role in the healing of tissue damage caused by chemotherapy with 5-FU due to the presence of its natural compounds and antioxidant properties