Simultaneous impact of atorvastatin and mesenchymal stem cells for glioblastoma multiform suppression in rat glioblastoma multiform model

Glioblastoma multiform (GBM) is known as an aggressive glial neoplasm. Recently incorporation of mesenchymal stem cells with anti-tumor drugs have been used due to lack of immunological responses and their easy accessibility. In this study, we have investigated the anti-proliferative and apoptotic activity of atorvastatin (Ator) in combination of mesenchymal stem cells (MSCs) on GBM cells in vitro and in vivo. The MSCs isolated from rats and characterized for their multi-potency features. The anti-proliferative and migration inhibition of Ator and MSCs were evaluated by MTT and scratch migration assays. The annexin/PI percentage and cell cycle arrest of treated C6 cells were evaluated until 72 h incubation. The animal model was established via injection of C6 cells in the brain of rats and subsequent injection of Ator each 3 days and single injection of MSCs until 12 days. The growth rate, migrational phenotype and cell cycle progression of C6 cells decreased and inhibited by the interplay of different factors in the presence of Ator and MSCs. The effect of Ator and MSCs on animal models displayed a significant reduction in tumor size and weight. Furthermore, histopathology evaluation proved low hypercellularity and mitosis index as well as mild invasive tumor cells for perivascular cuffing without pseudopalisading necrosis and small delicate vessels in Ator?+?MSCs condition. In summary, Ator and MSCs delivery to GBM model provides an effective strategy for targeted therapy of brain tumor.

     

Deciphering the Metabolic Changes Associated with Diapause Syndrome and Cold Acclimation in the Two-Spotted Spider Mite Tetranychus urticae

Diapause is a common feature in several arthropod species that are subject to unfavorable growing seasons. The range of environmental cues that trigger the onset and termination of diapause, in addition to associated hormonal, biochemical, and molecular changes, have been studied extensively in recent years; however, such information is only available for a few insect species. Diapause and cold hardening usually occur together in overwintering arthropods, and can be characterized by recording changes to the wealth of molecules present in the tissue, hemolymph, or whole body of organisms. Recent technological advances, such as high throughput screening and quantification of metabolites via chromatographic analyses, are able to identify such molecules. In the present work, we examined the survival ability of diapausing and non-diapausing females of the two-spotted spider mite, Tetranychus urticae, in the presence (0 or 5°C) or absence of cold acclimation. Furthermore, we examined the metabolic fingerprints of these specimens via gas chromatography-mass spectrophotometry (GC-MS). Partial Least Square Discriminant Analysis (PLS-DA) of metabolites revealed that major metabolic variations were related to diapause, indicating in a clear cut-off between diapausing and non-diapausing females, regardless of acclimation state. Signs of metabolic depression were evident in diapausing females, with most amino acids and TCA cycle intermediates being significantly reduced. Out of the 40 accurately quantified metabolites, seven metabolites remained elevated or were accumulated in diapausing mites, i.e. cadaverine, gluconolactone, glucose, inositol, maltose, mannitol and sorbitol. The capacity to accumulate winter polyols during cold-acclimation was restricted to diapausing females. We conclude that the induction of increased cold hardiness in this species is associated with the diapause syndrome, rather than being a direct effect of low temperature. Our results provide novel information about biochemical events related to the cold hardening process in the two-spotted spider mite.     

Phylogenetic relationships of Iranian Allium sect. Allium (Amaryllidaceae,Allioideae) as inferred from nrDNA ITS,cpDNA rps16 and trnL–F sequences

Allium is a particularly species rich (more than 800 species) and economically important genus, with numerous taxonomic problems at all levels of classification. In this study, we try to uncover the phylogenetic relationships of the common leek Allium ampeloprasum based on selected samples of this species and its putative relatives in A. sect. Allium from Iran. The silica‐dried leaf samples of 56 accessions representing 23 species of Allium were sequenced; 53 sequences of nrDNA ITS, 35 sequences of plastid rps16 and 52 sequences of trnL–F were generated and additional accessions were extracted from GenBank in order to cover all recognized main lineages in the genus. Maximum Parsimony and Bayesian Inference generated similar trees, but the placement of A. ampeloprasum and its relatives differed slightly between the nuclear and the plastid phylogenies. In the nrITS tree, A. ampeloprasum is retrieved as a highly supported clade with A. iranicum, while in the combined plastid tree A. ampeloprasum formed a highly supported clade with A. vineale. This supports the hypothesis of a possible hybrid origin of A. ampeloprasum. Allium iranicum formed a clade in the plastid tree, but was resolved as paraphyletic in the nrITS tree, probably due to presence of multiple non‐concerted copies of nrITS. Close relationships are suggested between the following species: A. aznavense and A. wendelboi with A. talyschense, A. erubescens and A. rotundum with A. scorodoprasum and A. abbasii with A. phanerantherum.     

Cloning and expression of human IFN-γ in <Emphasis Type="Italic">Leishmania tarentolae</Emphasis>

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-γ), an antiviral pro-inflammatory cytokine, has broadened interest in optimizing methods for its production. We herein describe a unicellular eukaryotic system, Leishmania tarentolae, a Trypanosomatidae protozoan parasite of gecko Tarentola annularis, which has recently been introduced as a candidate for heterologous gene expression. In this study, the hIFN-γ cDNA was amplified from phyto-hemagglutinin-stimulated peripheral blood mononuclear cells of a healthy blood donor using RT–PCR. In order to express, the rhIFN-γ protein, the resulting cDNA was cloned in two expression cassettes (each containing one copy of hIFN-γ cDNA) and integrated into the small subunit of ribosomal RNA gene of L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Western blotting of rhIFN-γ and ELISA confirmed the expression and production of 9.5 mg of rhIFN-γ protein/l respectively.     

Erdj3 Has an Essential Role for Z Variant Alpha‐1‐Antitrypsin Degradation

Alpha‐1‐antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha‐1‐antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER‐associated degradation and autophagy. We hypothesized that alpha‐1‐antitrypsin (AAT)‐interacting proteins play critical roles in quality control of human AAT. Using co‐immunoprecipitation, we identified ERdj3, an ER‐resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin‐EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3‐mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD‐related liver disease. J. Cell. Biochem. 118: 3090–3101, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

The role of nitric oxide,reactive oxygen species,and protein kinase C in oxytocin-induced cardioprotection in ischemic rat heart

Ischemia–reperfusion injury is a common complication of heart disease that is the leading cause of death worldwide. Here, we plan to elucidate oxytocin cardioprotection effects against ischemia–reperfusion via nitric oxide (NO), reactive oxygen species (ROS), and protein kinase C (PKC) in anesthetized rat preconditioned myocardium. Forty-eight Sprague-Dawley rats were equally divided into eight groups. All animals were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin (OT), L-NAME (LNA, a nitric oxide synthase inhibitor), chelerythrine (CHE, a PKC enzyme inhibitor), and N-acetylcysteine (NAC, a ROS scavenger) were used prior to ischemia. Results showed that mean arterial pressure significantly reduced during the first 10 min of ischemia and reperfusion in IR, LNA, CHE, and NAC groups (p < 0.05). OT prevented mean arterial pressure decline during early phase of ischemia and reperfusion. Cardioprotective effects of OT in infarct size, plasma levels of creatine kinase-MB and lactate dehydrogenase, severity and incidence of ventricular arrhythmias were abolished by L-NAME, chelerythrine, and N-acetylcysteine (p < 0.05). The present study showed that OT pretreatment reduces myocardial infarct size and ventricular arrhythmias, and improves mean arterial pressure via NO production, PKC activation, and ROS balance. These findings provide new insight into therapeutic strategies for ischemic heart disease.     

Trichome micromorphology in Alcea L. and allied genera (Malvaceae) and its systematic implication

Trichomes of 26 species of Alcea (Malvaceae) were investigated using light (LM) and scanning electron microscopy (SEM). The trichomes show a great micromorphological variation, which provides data useful for species delimitation in Alcea. Two basic types of trichomes could be distinguished in Alcea and allied genera: glandular and eglandular. The glandular trichomes could in turn be subdivided into two subtypes: capitate and clavate. The eglandular trichomes could be subdivided into five subtypes: simple, fascicled, stellate, fascicled-stellate and pluri-radiate. Characters of taxonomic interest are: trichome density (glabrous to dense), number of arms per trichome, orientation relative to the epidermal surface (appressed to erect) and presence/absence of a stalk. According to the results the species of Alcea can be divided into four informal groups based on trichome types. The results further support the exclusion of the annual Althaea species from the perennial ones and their close relationship to Malva. In addition, a close relationship between perennial Althaea and basal Alcea lineages is supported. Based on the evolutionary framework provided by recent molecular phylogenetic investigations, the following trends can be observed in the Malva alliance: long and narrow-armed trichomes are primitive in relation to short and thick-armed trichomes; dense indumentum coverage is primitive in relation to moderately dense or glabrous ones; presence of simple hairs on stem (particularly on leaves) is more advanced than their absence; spreading villous-stellate and fascicled trichomes are more advanced than appressed stellate ones. Clavate trichomes, which were found exclusively in a few species of Alcea, should be considered as a derived state in relation to capitate ones, and they may provide a synapomorphy for the crown group of Alcea.     

The Neuroprotective Ability of Polyethylene Glycol is Affected by Temperature in Ex Vivo Spinal Cord Injury Model

Immediate membrane sealing after spinal cord injury (SCI) can prevent further degradation and result in ultimate functional recovery. It has been reported that polyethylene glycol (PEG) can repair membrane damage caused by mechanical insults to the spinal cord. Furthermore, membrane fluidity and its sealing process vary at different temperatures. Here, we have assessed the possible synergistic effects of PEG and temperature on the repair of neural membranes in an SCI model. The effects of PEGs (400, 1,000 and 2,000 Da) were studied at different temperatures (25, 37 and 40 °C) by means of compound action potential (CAP) recovery and a lactate dehydrogenase (LDH) assay. Isolated spinal cords were mounted in a double sucrose gap chamber, where the amplitude and area of CAPs were recorded after implementing injury, in the presence and absence of PEG. Moreover, the LDH assay was used to assess the effects of PEG on membrane resealing. Data showed that the least CAP recovery occurred at 25 °C, followed by 37 and 40 °C, in all treated groups. Moreover, maximum CAP amplitude recovery, 65.46 ± 5.04 %, was monitored in the presence of PEG400 at 40 °C, followed by 41.49 ± 2.41 % in PEG1000 and 37.36 ± 1.62 % in PEG2000. Furthermore, raising the temperature from 37 to 40 °C significantly increased CAP recovery in the PEG2000 group. Similar recovery patterns were obtained by CAP area measurements and LDH assay. The results suggest that application of low-molecular weight PEG (PEG400) in mild hyperthermia conditions (40 °C) provides the optimum condition for membrane sealing in SCI model.