Pathobiology Research
Year:2016 | Volume:19 | Issue:3|Papers Count:7

Spermatogonial stem cells are foundation of the male reproductive system. These cells are the only conduit capable of transferring genetic traits from one generation to the next. Isolation and long-term preservation of spermatogonial stem cells for use in inducing spermatogenesis is one technique to preserve fertility in male patients who need chemotherapy. In vitro spermatogenesis is an alternative to achieve this goal. The use of an optimal model of human spermatogenesis is a major step in understanding the physiology and genetic pathways in the male reproductive system. In vitro spermatogenesis is crucial to reducing a complex process into smaller parts for experimentation, manipulation, and deriving cellular and molecular level knowledge. Is it possible to manipulate the paracrine environment and separately evaluate the effects of growth factors. Different in vitro culture systems are used to explore alternatives to spermatogenesis and obtain mature, functional spermatozoa for ultimate use in infertility treatment. In order to present a useful and practical method, this study provides an overview of different methods for the long-term preservation of spermatogonial stem cells and in vitro culture systems used in spermatogenesis.

Objective: Cell-derived micro vesicles are described as a new mechanism for cell-to-cell communication. Stem cell-derived exosomes have been described as a new mechanism for the paracrine effects of mesenchymal stem cells (MSCs). In this regard, exosomes may play a relevant role in the intercellular communication between MSCs and tumor cells.Methods: Exosomes were purified from the conditioned medium of MSCs by differential centrifugation. Exosome size and morphology were examined by scanning electron microscope and sized with dynamic light scattering (DLS). Western blot analysis confirmed the exosomes by using CD9 as a marker. Purified exosomes were labeled with a PKH26 red fluorescent labeling kit. The labeled exosomes were incubated with SKOV3 ovarian tumor cells for 12 h at 37°C, and we used an inverted fluorescence microscope to monitor cellular uptake.Results: Scanning electron microscopy revealed that the purified MSCs-derived exosomes had a spherical shape with a diameter of approximately 30-100 nm. Exosome size measurement by dynamic light scattering analysis also showed a single bell-shaped size distribution with a peak of ~80 nm. Western blot analysis also demonstrated the presence of CD9 (a representative marker of exosomes) in the purified exosomes. These data confirmed that the vesicles isolated from MSCs-conditioned media were the exosomes based on their size and presence of the protein marker CD9. Florescent microscopy showed that PKH26-labeled exosomes could be taken up by SKOV3 tumor cells with high efficiency.Conclusion: Our approach for isolation, characterization and cellular uptake of exosomes derived from MSCs is valuable and a prerequisite for future studies that intend to discover exosome function in tumor cells. The ability to study the biology of exosome uptake in cancer cells could provide opportunities for functional studies of these natural nano vesicles and their contents in cancer therapy.

Objective: The concern about the presence of aflatoxin and its risk for human and animal health has resulted in the introduction of different methods to eliminate or reduce this toxin. One of these methods is biological control of the fungus by other microorganisms.Methods: We isolated a strain of Bacillus amyloliquefaciens from the soil of a pistachio orchard, Damghan, Iran. This strain was applied as a biological control to examine the inhibition of growth and toxin production byAspergillus parasiticus NRRL2999. After 72 hours of incubation of the bacterium at 30°C, we separated the supernatant as a potential source for antifungal compounds. Different doses of the supernatant were co-cultured with the fungus suspension in glucose yeast extract broth (GYB) at 28°C for 4 days. After the incubation period, we measured the inhibition of fungal growth by dry weight assessment of the fungal mass. We performed qualitative and quantitative assessment to determine the amount of aflatoxin B1 by TLC and HPLC, respectively.Results: Increased bacterial culture supernatant as the antagonist in fungal growth medium resulted in decreased fungal growth. AFB1 production was 2.35 ppm in control samples, whereas the amount considerably decreased in samples treated by the bacterial supernatants. The toxin reduction was dose-dependent.Conclusion: The results of this study have shown that this bacterial strain, by taking into consideration its native origin, can be used as a biological control against aflatoxigenic fungi.

Objective: Alzheimer's disease (AD) is the most common form of dementia in the elderly. One of the major pathological hallmarks of AD is extracellular deposition of aggregated beta-amyloid (Aβ) peptide in the brain. Aβ causes neural cell death through several mechanisms. New treatments for AD focus on compounds that can modify disease progression and reduce symptoms through several mechanisms. Medicinal plants include several compounds with heterogeneous pharmacological effects that can be effective in the treatment of AD through different mechanisms. The aim of this study is to evaluate the protective effect of a methanolic extract of seven medicinal plants fromIran on Aβinduced toxicity in a cell culture model.Methods: We cultured PC12 cells according to standard protocols. The cells were incubated with Aβ alone or with different concentrations of extracts for 24 hours. Cell viability was measured using the MTT assay.Results: Sanguisorba minor, Cerasus microcarpa, Ferulago angulate, and Rosa canina significantly reduced Aβ-induced cell death in the PC12 cell culture. The observed protective effects of extracts were dose-dependent. We did not observe any protective effects with the Stachys pilifera, Amygdalus scoparia, and Alhagi pseudalhagi extracts.Conclusion: Sanguisorba minor, Cerasus microcarpa, Ferulago angulate, and Rosa caninaextracts could be considered for treatment of AD.

Objective: Candida albicans (C. albicans) is an opportunistic yeast that can lead to pathogenesis in immunocompromised individuals and under suitable conditions. Medicinal plants ingredients such as camphor can reduce the expressions of genes involved in virulence of the fungi through their antifungal properties. The products of INT1and EFG1 are implicated in inducing filamentous growth and adhesion of C. albicansto the host tissues. Both of these characteristics are very important in its virulence. The present study focuses on the evaluation of the effects of camphor on INT1 andEFG1 expressions at three time points of treatment (24, 48, and 72 hours) via realtime PCR.Methods: We prepared serial dilutions of camphor (1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 mg/ml) and co-cultured them with 1.5´103 cells/ml of a C. albicans ATCC 10231 suspension for 48 hours at 350C. Next, we determined the MIC50/90 and MFC. C. albicanscells were treated with the MIC50 concentration of camphor for 24, 48, and 72 hours. RNA from C. albicans was extracted before and after treatment, back translated into cDNA, and analyzed with real-time PCR.Results: MIC50, MIC90 and MFC of camphor were determined at 16 mg/ml, 32 mg/ml and 64 mg/ml, respectively. Evaluation of gene expression changes in yeast showed that camphor reduced theINT1 gene expression about 87% at 24 hours, 97% at 48 hours, and 86% at 72 hours after treatment compared to the untreated sample. EFG1 expression reduced about 58% at 24 hours, 93% at 48 hours, and 49% at 72 hours after treatment with camphor.Conclusion: In recent years, advancements have been seen in herbalism due to the increased drug resistance and adverse effects of chemical drugs. These plants may efficiently act as antifungal agents. The results of this study have shown that the use of camphor can significantly reduce the expression of virulence genesINT1 and EFG1 in C. albicans.

Objective: The human genome consists of protective genes, which contain a sequence known as the antioxidant responsive element (ARE) located in their promoter regions. ARE is specific to the transcription factor NF-E2 related factor2 (Nrf2). This signaling pathway is the major defense mechanism against oxidative stress that comprises the chemo protective response. The cell line that expresses the ARE reporter is sensitive for the detection of ARE activating compounds. It can help to identify toxicity risk and antioxidant activity of chemicals and drugs.Methods: We used a stable Huh7 ARE-reporter cell line in this study. Metabolic activity of these cells in different concentrations of lead (0 to 80 micromole) was evaluated by the MTT test. We assessed the effects of oxidative stress. We exposed the Huh7 ARE-reporter cell line to different concentrations of lead, an oxidative stress inducer, and nanocurcumin, an antioxidant compound, after which we investivated luciferase activity. Real-time PCR was used to detected ARE\KEAP1 pathway gene expression.Result: Lead, at 30 mM, suppressed 50% of the cells’ metabolic activity. Cells treated with both lead (30 mM) and nanocurcumin at 4 mM and 8 mM had decreased luciferase activity compared to those treated with only lead. This activity increased when we increased the nanocurcumin concentration to 16 mM. Real-time PCR analysis showed decreased NQO1 and NRF2 expressions in cells treated with both lead (30 mM) and nanocurcumin (4 mM and 8 mM) compared to just lead treated cells. However, NQO1 and NRF2 had increased expressions in cells treated with both lead (30 mM) and nanocurcumin (16 mM) compared to just lead treated cells.Conclusion: Nano curcumin, as an antioxidant, significantly reduced the toxic effects of lead toxic. This effect probably occurred through the ARE\KEAP1 pathway. Hence, nanocurcumin could be used as an antioxidant to reduce oxidative stress.