Identification of Leishmania species causing cutaneous leishmaniasis using Random Amplified Polymorphic DNA (RAPD-PCR) in Kharve,Iran

Background:

Leishmaniasis, especially cutaneous leishmaniasis, is considered an important health problem in many parts of Iran including Kharve, Khorasan Razavi province. Cutaneous leishmaniasis is caused by various species of Leishmania, each having a different secondary host. Thus, identifying the parasites’ specie is of paramount importance for containment strategy planning. The morphological differentiation of Leishmania species is not possible, rendering the molecular methods as the sole means to this purpose. Therefore, to identify the causative agent of cutaneous leishmaniasis in Kharve, Random Amplified Polymorphic DNA-PCR (RAPD-PCR) was used.

Methods:

The disease was first confirmed by direct smears. Samples were gathered from 22 patients with established cutaneous leishmaniasis. The samples were immediately cultured in NNN medium, followed by sub-culture in RPMI-1640. Afterwards, DNA was extracted and amplified using RAPD-PCR. Electrophoresis patterns from each isolate were compared with reference strains of Leishmania major (L. major) and Leishmania tropica (L. tropica).

Results:

The results of this study indicated that the parasite causing cutaneous leishmaniasis in Kharve is L. tropica.

Conclusion:

It seems that L. tropica is the only causative agent of cutaneous leishmaniasis in Kharve, and RAPD-PCR is a suitable tool for Leishmania characterization in epidemiological studies.Key Words: Leishmania major, Leishmania tropica, RAPD-PCR, Khorasan, Kharve     

Substitutions of amino acids in α-helix-4 of gyrase A confer fluoroquinolone resistance on <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

DNA gyrase, an essential enzyme that regulates DNA topology in bacteria, is the target of fluoroquinolones. Three fluoroquinolone-resistant mutants derived from one strain of Clostridium perfringens had amino acid substitutions of glycine 81 to cysteine, aspartic acid 87 to tyrosine, or both, in α-helix-4 of gyrase A. The gyrase mutations affected the growth kinetics of mutants differently when the mutants were exposed to increasing concentrations of gatifloxacin and ciprofloxacin. Fluoroquinolone concentration-dependent effects observed during growth in the exponential and stationary phases depended on the presence of particular gyrA mutations. Introduction of a wild-type gyrA gene into the mutants enhanced their susceptibility to fluoroquinolones and decreased their growth rates proportional to increases in fluoroquinolone concentrations. Amino acid substitutions in α-helix-4 of gyrase A protected C. perfringens from fluoroquinolones, and a strain with two substitutions was the most resistant.     

Downregulation of Stemness Genes and Induction of Necrosis in Rat LA7 Cancer Stem Cells Induced Tumors Treated with Starved Fibroblasts Culture Supernatant

Background:Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy. Methods:The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining.Results:All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67⁺, cytokeratin⁺, vimentine⁺, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium.Conclusion:This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.Key Words: Breast cancer, Cancer Stem cells, Cell differentiation, Fibroblasts, Gene expression     

Pathophysiological roles of chronic low‐grade inflammation mediators in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common hormonal imbalance disease in reproductive‐aged women. Its basic characteristics are ovulatory dysfunction and ovarian overproduction of androgens that lead to severe symptoms such as insulin resistance, hirsutism, infertility, and acne. Notwithstanding the disease burden, its underlying mechanisms remain unknown, and no causal therapeutic exists. In recent years, further studies showed that inflammation processes are involved in ovulation and play a key role in ovarian follicular dynamics. Visceral adipose tissue can cause inflammatory response and maintenance of the inflammation state in adipocytes by augmented production of inflammatory cytokines, monocyte chemoattractant proteins, and recruitment of the immune cell. Therefore, the PCOS can be related to a low‐grade inflammation state and inflammatory markers. Investigating the inflammatory processes and mediators that contribute to the commencement and development of PCOS can be a critical step for better understanding the pathophysiology of the disease and its treatment through inhibition or control of related pathways. In the present review, we discuss the pathophysiological roles of chronic low‐grade inflammation mediators including inflammasome‐related cytokines, interleukin‐1β (IL‐1β), and IL‐18 in PCOS development.     

Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge

Biotechnology Letters - Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually...     

Improvement of cytotoxicity of mitoxantrone and daunorubicin by candidone,tephrosin, and bavachinin

Molecular Biology Reports - Flavonoids have been demonstrated to have the ability of sensitizing cancer cells to chemotherapy and inverse multidrug resistance via various mechanisms, such as...     

Association study of promoter polymorphisms of interferon alpha and beta receptor subunit 1 (IFNAR1) gene and therapeutic response to interferon-beta in patients with multiple sclerosis

Molecular Biology Reports - Multiple sclerosis (MS) is an autoimmune disease described by inflammatory neuronal losses and resultant failures. The disease could abate by interferon-beta...     

MAP30 transgenic tobacco lines: from silencing to inducing

Molecular Biology Reports - DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene...     

Utilizing a nano‐sorbent for the selective solid‐phase extraction of vanillic acid prior to its determination by photoluminescence spectroscopy

Vanillic acid (VA) is a phenolic acid, and acts as a natural antioxidant in fruits, vegetables and plants. The extraction and determination of trace levels of VA in plants is important, because stimulation of protein synthesis and activation of antioxidant enzymes occur in the presence of phenolic acids at trace levels. In this research, a photoluminescence spectroscopic method was developed for the quantification of VA in plant samples after separation and pre‐concentration. Selective extraction of VA from aqueous solution was performed using a solid‐phase extraction column packed with nickel–aluminum layered double hydroxide as a nano‐sorbent. After elution of extracted analyte from the column using 3 mL of a 3 mol/L NaOH solution, its concentration was determined spectrofluorometrically at λ_em = 357 nm with excitation at λ_ex = 280 nm. The spectrofluorometry method gave a linear response for VA within the range 20.0–900.0 µg/L, with a correlation coefficient of 0.9982. The limit of detection and sorption capacity were 7.6 µg/L and 66.2 mg/g, respectively. The method was validated by comparing the obtained results with gas chromatographic data. This method was used to determine VA in Chenopodium album and Prangos asperula plants. Copyright © 2014 John Wiley & Sons, Ltd.     

Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H⁺-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.