Genetic manipulation, a feasible tool to enhance unique characteristic of Chlorella vulgaris as a feedstock for biodiesel production

Developing a reliable technique to transform unicellular green algae, Chlorella vulgaris, could boost potentials of using microalgae feedstock in variety of applications such as biodiesel production. Volumetric lipid productivity (VLP) is a suitable variable for evaluating potential of algal species. In the present study, the highest VLP level was recorded for C. vulgaris (79.08 mg l^?1 day^?1) followed by 3 other strains studied; C. emersonii, C. protothecoides, and C. salina by 54.41, 45 and 18.22 mg l^?1day^?1, respectively. Having considered the high productivity of C. vulgaris, it was selected for the preliminary transformation experiment through micro-particle bombardment. Plasmid pBI 121, bearing the reporter gene under the control of CaMV 35S promoter and the kanamycin marker gene, was used in cells bombardment. Primary selection was done on a medium supplemented by 50 mg l^?1 kanamycin. After several passages, the survived cells were PCR-tested to confirm the stability of transformation and then were found to exhibit β-glucuronidase (GUS) activity in comparison with the control cells. Southern hybridization of npt II probe with genomic DNA revealed stable integration of the cassette in three different positions in the genome. The whole process was successfully implemented as a pre-step to transform the algal cells by genes involved in lipid production pathway which will be carried out in our future studies.     

T cells from indolent CLL patients prevent apoptosis of leukemic B cells in vitro and have altered gene expression profile

T cells may have a role in sustaining the leukemic clone in chronic lymphocytic leukemia (CLL). In this study, we have examined the ability of T cells from CLL patients to support the survival of the leukemic B cells in vitro. Additionally, we compared global gene expression of T cells from indolent CLL patients with healthy individuals and multiple myeloma (MM) patients. Apoptosis of purified leukemic B cells was inhibited in vitro when co-cultured with increasing numbers of autologous T cells (p < 0.01) but not autologous B and T cells of normal donors. The anti-apoptotic effect exceeded that of the anti-apoptotic cytokine IL-4 (p = 0.002) and was greater with CD8+ cells (p = 0.02) than with CD4+ cells (p = 0.05). The effect was depended mainly on cell–cell contact although a significant effect was also observed in transwell experiments (p = 0.05). About 356 genes involved in different cellular pathways were deregulated in T cells of CLL patients compared to healthy individuals and MM patients. The results of gene expression profiling were verified for 6 genes (CCL4, CCL5 (RANTES), XCL1, XCL2, KLF6, and TRAF1) using qRT–PCR and immunoblotting. Our results demonstrate that CLL-derived T cells can prevent apoptosis of leukemic B cells and have altered expression of genes that may facilitate the survival of the leukemic clone.     

Effects of nonylphenol on hematological parameters and immune responses in immature rainbow trout (Oncorhynchus mykiss)

ABSTRACT

This study investigated the effects of nonylphenol (NP) on hematological and immunological parameters in both male and female rainbow trout (Oncorhynchus mykiss). Fish were randomly distributed into six groups and administered with NP (10, 50 and 100 μg g^-1 week^-1 BW) and a single dose of 17-β estradiol (E2; 2 μg g^-1 week^-1 BW, positive control). The solvent controls received ethanol and coconut oil as a vehicle, while the controls were not injected. Red blood cells (RBCs) count, hematocrit (Hct), hemoglobin (Hb), white blood cells (WBCs), and lymphocytes demonstrated a NP dose-dependent decrease, whereas mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), monocytes, and neutrophils showed an increasing trend in both male and female fish 21 days post-treatment. Also, RBCs, Hb, MCHC, WBCs, and lymphocytes were significantly reduced (p<0.05) in E2 treated fish. Lysozyme, complement components (C3 and C4) and immunoglobulin M (IgM) were increased in fish sera subjected to 10 and 50 μg g^-1 NP, while these decreased in groups administered with 100 μg g^-1 NP and 2 μg g^-1 E2. Except for C4 level at 10 μg g^-1 NP, no significant differences were observed in hematological and immunological parameters of male and female in each treatment. Overall, a frequent exposure to NP could lead to adverse effects on fish immune-physiological functions which may cause serious ecological threats of fish natural population sustainability.     

Perturbations of amino acid metabolism associated with glyphosate-dependent inhibition of shikimic acid metabolism affect cellular redox homeostasis and alter the abundance of proteins involved in photosynthesis and photorespiration

The herbicide glyphosate inhibits the shikimate pathway of the synthesis of amino acids such as phenylalanine, tyrosine, and tryptophan. However, much uncertainty remains concerning precisely how glyphosate kills plants or affects cellular redox homeostasis and related processes in glyphosate-sensitive and glyphosate-resistant crop plants. To address this issue, we performed an integrated study of photosynthesis, leaf proteomes, amino acid profiles, and redox profiles in the glyphosate-sensitive soybean (Glycine max) genotype PAN809 and glyphosate-resistant Roundup Ready Soybean (RRS). RRS leaves accumulated much more glyphosate than the sensitive line but showed relatively few changes in amino acid metabolism. Photosynthesis was unaffected by glyphosate in RRS leaves, but decreased abundance of photosynthesis/photorespiratory pathway proteins was observed together with oxidation of major redox pools. While treatment of a sensitive genotype with glyphosate rapidly inhibited photosynthesis and triggered the appearance of a nitrogen-rich amino acid profile, there was no evidence of oxidation of the redox pools. There was, however, an increase in starvation-associated and defense proteins. We conclude that glyphosate-dependent inhibition of soybean leaf metabolism leads to the induction of defense proteins without sustained oxidation. Conversely, the accumulation of high levels of glyphosate in RRS enhances cellular oxidation, possibly through mechanisms involving stimulation of the photorespiratory pathway.     

Amelioration of altered serum,liver, and kidney antioxidant enzymes activities by sodium selenite in alloxan-induced diabetic rats

Background:

The aim of this study was to evaluate the possible protective effect of sodium selenite on serum, liver, and kidney antioxidant enzymes activities in alloxan-induced type 1 diabetic rats.

Methods:

Forty Sprague-Dawley male rats were randomly divided into four groups; Group one as control, Group two as sham-treated with sodium selenite by 1 mg/kg intraperitoneal (i.p.) injections daily, Group three as diabetic untreated, and Group four as diabetic treated with sodium selenite by 1 mg/kg i.p. injections daily .Diabetes was induced in the third and fourth groups by subcutaneous alloxan injections. After eight weeks the animals were euthanized and livers and kidneys were immediately removed and used fresh or kept frozen until analysis. Before the rats were killed blood samples were also collected to measure glutathione peroxidase (GPX) and catalase (CAT) activities in sera.

Results:

Glutathione peroxidase and CAT activities serum, liver, and kidney were all significantly less in the diabetic rats than in the controls. Sodium selenite treatment of the diabetic rats resulted in significant increases in GPX activity in the kidneys and livers, and CAT activity in the sera and livers.

Conclusions:

Our results indicate that sodium selenite might be a potent antioxidant that exerts beneficial effects on both GPX and CAT activities in alloxan-induced type 1 diabetic rats. Key Words: Diabetes, Rat, Sodium selenite, Antioxidant enzymes activity     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.