Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number {"type":"entrez-nucleotide","attrs":{"text":"AY729671","term_id":"56718401","term_text":"AY729671"}}AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.     

Fucoidan and Derived Oligo-Fucoses: Structural Features with Relevance in Competitive Inhibition of Gastrointestinal Norovirus Binding

Norovirus infections belong to the most common causes of human gastroenteritis worldwide and epidemic outbreaks are responsible for hundreds of thousands of deaths annually. In humans, noroviruses are known to bind to gastrointestinal epithelia via recognition of blood-group active mucin-type O-glycans. Considering the involvement of l-α-fucose residues in these glycans, their high valency on epithelial surfaces far surpasses the low affinity, though specific interactions of monovalent milk oligosaccharides. Based on these findings, we attempted to identify polyfucoses (fucans) with the capacity to block binding of the currently most prevalent norovirus strain GII.4 (Sydney, 2012, {"type":"entrez-nucleotide","attrs":{"text":"JX459908","term_id":"409032931"}}JX459908) to human and animal gastrointestinal mucins. We provide evidence that inhibitory effects on capsid binding are exerted in a competitive manner by α-fucosyl residues on Fucus vesiculosus fucoidan, but also on the galacto-fucan from Undaria pinnatifida and their oligo-fucose processing products. Insight into novel structural aspects of fucoidan and derived oligosaccharides from low-mass Undaria pinnatifida were revealed by GCMS and MALDI mass spectrometry. In targeting noroviral spread attenuation, this study provides first steps towards a prophylactic food additive that is produced from algal species.     

Three New Asperentin Derivatives from the Algicolous Fungus Aspergillus sp. F00785

Three new asperentin-type compounds, 6-O-α-d-ribosylasperentin (1) and 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5–9), were isolated from a halotolerant Aspergillus sp. strain {"type":"entrez-nucleotide","attrs":{"text":"F00785","term_id":"707638","term_text":"F00785"}}F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.     

Assessment of bioflocculant production by Bacillus sp. Gilbert, a marine bacterium isolated from the bottom sediment of Algoa Bay

The bioflocculant-producing potentials of a marine bacteria isolated from the bottom sediment of Algoa Bay was investigated using standard methods. The 16S rDNA sequence analysis revealed 98% similarity to that of Bacillus sp. HXG-C1 and the nucleotide sequence was deposited in GenBank as Bacillus sp. Gilbert with accession number {"type":"entrez-nucleotide","attrs":{"text":"HQ537128","term_id":"312064208","term_text":"HQ537128"}}HQ537128. Bioflocculant was optimally produced when sucrose (72% flocculating activity) and ammonium chloride (91% flocculating activity) were used as sole sources of carbon and nitrogen, respectively; an initial pH 6.2 of the production medium; and Mg²⁺ as cation. Chemical analysis of the purified bioflocculant revealed the compound to be a polysaccharide.     

温度和药剂胁迫下黄翅绢野螟实时荧光定量PCR内参基因筛选

为筛选黄翅绢野螟(Diaphania caesalis)逆境胁迫下稳定表达的内参基因,使该虫抗逆基因的定量表达分析标准化.本研究根据黄翅绢野螟成虫转录组注释结果,利用qPCR技术分析了9个分别编码ACT、β-TUB、GAPDH、G6PDH、RPS3a、RPL13a、EF1α、EIF4A、/3-ACT的候选内参基因在温度...     

Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ ² P > 0.05), including only one common haplotype (GenBank access no. {"type":"entrez-nucleotide","attrs":{"text":"EF413075","term_id":"151936076","term_text":"EF413075"}}EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran     

不同氮处理茶树实时定量PCR内参基因筛选和验证

为筛选不同氮处理下茶树(Camellia sinensis)实时荧光定量PCR(q RT-PCR)试验体系中最佳内参基因,以茶树幼叶、成熟叶和幼根为材料,应用qRT-PCR分析GAPDH、18S rRNA、β-actin、RPL13、a-tubulin、Ru BP等6个内参基因在不同氮浓度和氮源处理下的表达变化,借助GeNorm和NormFinder程序对候选内参基因稳定性进行评价。结果表明,在不同氮浓度和氮源处理下,GAPDH、b-actin和RPL13表达稳定性较好,GAPDH+β-actin组合稳定性最佳,可用作茶树基因表达研究的内参基因;而a-tubulin和RuBP表达稳定性较差,不适合做内参基因。为进一步证实上述结果,分别以GAPDH、a-tubulin、GAPDH+β-actin组合为内参基因,分析茶树幼叶中硝酸根转运蛋白基因(CsNRT1.2)和铵根转运蛋白基因(Cs AMT1.1)的表达水平,发现以GAPDH和GAPDH+β-actin组合为内参基因时,目的基因的相对表达量随处理时间延长变化规律基本一致;而以a-tubulin为内参基因时,目的基因的变化规律与前两者存在明显差别。因此,根据特定试验体系选择合适的内参基因对于qRT-PCR定量结果的准确性和可靠性具有重要意义。     

Effect of Human Ovarian Tissue Vitrification/Warming on the Expression of Genes Related to Folliculogenesis

Background:

Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha (FIGLA), growth differentiation factor-9 (GDF-9), follicle-stimulating hormone receptor (FSHR), and KIT LIGAND after vitrification/warming of human ovarian tissue.

Methods:

Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group (control). Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF-9,, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups.

Results:

Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups (P > 0.05).

Conclusion:

Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes. Key Words: Vitrification, Gene expression, Humans     

Astaxanthin Alleviates Early Brain Injury Following Subarachnoid Hemorrhage in Rats: Possible Involvement of Akt/Bad Signaling

Apoptosis has been proven to play a crucial role in early brain injury pathogenesis and to represent a target for the treatment of subarachnoid hemorrhage (SAH). Previously, we demonstrated that astaxanthin (ATX) administration markedly reduced neuronal apoptosis in the early period after SAH. However, the underlying molecular mechanisms remain obscure. In the present study, we tried to investigate whether ATX administration is associated with the phosphatidylinositol 3-kinase-Akt (PI3K/Akt) pathway, which can play an important role in the signaling of apoptosis. Our results showed that post-SAH treatment with ATX could cause a significant increase of phosphorylated Akt and Bad levels, along with a significant decrease of cleaved caspase-3 levels in the cortex after SAH. In addition to the reduced neuronal apoptosis, treatment with ATX could also significantly reduce secondary brain injury characterized by neurological dysfunction, cerebral edema and blood-brain barrier disruption. In contrast, the PI3K/Akt inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, could partially reverse the neuroprotection of ATX in the early period after SAH by downregulating ATX-induced activation of Akt/Bad and upregulating cleaved caspase-3 levels. These results provided the evidence that ATX could attenuate apoptosis in a rat SAH model, potentially, in part, through modulating the Akt/Bad pathway.     

Phytotoxicity of Salvia spp. exudates

A study on thirteen Salvia species about the phytotoxicity of their aerial part exudates is reported. The selected species were: Salvia namaensis Schinz (1), Salvia fallax Fernald (2), Salvia disermas L. (3), Salvia chamaedryoides Cav. (4), Salvia confertiflora Pohl. (5), S. x jamensis J.Compton (6), Salvia buchananii Hedge (7), S. wagneriana Polak (8), Salvia scabra Linn.fil. (9), Salvia miniata Fernald (10), Salvia cacaliaefolia Benth.(11), Salvia adenophora Fernald (12), Salvia rutilans Carrière (13). For each species the inhibitory potential against Papaver rhoeas L. and Avena sativa L. was evidenced by dose-response studies and growth experiments performed both in Petri dish and in pot and also evaluated by means of germination indices and growth parameters. New indices useful for describing the phytotoxic response by a reduced number of testing solutions are proposed. Principal Component Analysis (PCA) of the germination data revealed that the exudate concentrations most significant in explaining the data variability were 1 and 2 μg/mL in Petri dish and 10 and 20 μg/mL in pot experiments. Root length and seedling weight were more important with respect to the other growth parameters.     

Astaxanthin Attenuates Early Acute Kidney Injury Following Severe Burns in Rats by Ameliorating Oxidative Stress and Mitochondrial-Related Apoptosis

Early acute kidney injury (AKI) is a devastating complication in critical burn patients, and it is associated with severe morbidity and mortality. The mechanism of AKI is multifactorial. Astaxanthin (ATX) is a natural compound that is widely distributed in marine organisms; it is a strong antioxidant and exhibits other biological effects that have been well studied in various traumatic injuries and diseases. Hence, we attempted to explore the potential protection of ATX against early post burn AKI and its possible mechanisms of action. The classic severe burn rat model was utilized for the histological and biochemical assessments of the therapeutic value and mechanisms of action of ATX. Upon ATX treatment, renal tubular injury and the levels of serum creatinine and neutrophil gelatinase-associated lipocalin were improved. Furthermore, relief of oxidative stress and tubular apoptosis in rat kidneys post burn was also observed. Additionally, ATX administration increased Akt and Bad phosphorylation and further down-regulated the expression of other downstream pro-apoptotic proteins (cytochrome c and caspase-3/9); these effects were reversed by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, the protective effect of ATX presents a dose-dependent enhancement. The data above suggested that ATX protects against early AKI following severe burns in rats, which was attributed to its ability to ameliorate oxidative stress and inhibit apoptosis by modulating the mitochondrial-apoptotic pathway, regarded as the Akt/Bad/Caspases signalling cascade.     

An Enterovirus-Like RNA Construct for Colon Cancer Suicide Gene Therapy

Background:

In gene therapy, the use of RNA molecules as therapeutic agents has shown advantages over plasmid DNA, including higher levels of safety. However, transient nature of RNA has been a major obstacle in application of RNA in gene therapy.

Methods:

Here, we used the internal ribosomal entry site of encephalomyocarditis virus and the 3’ non-translated region of Poliovirus to design an enterovirus-like RNA for the expression of a reporter gene (enhanced green fluorescent protein) and a suicide gene (thymidine kinase of herpes simplex virus). The expression of these genes was evaluated by flow cytometry and cytotoxicity assay in human colorectal adenocarcinoma cell line (SW480). We then armed RNA molecules with a target sequence for hsa-miR-143 to regulate their expression by microRNA (miRNA) mimics.

Results:

The results showed effective expression of both genes by Entrovirus-like RNA constructs. The data also showed that the restoration of hsa-miR-143 expression in SW480 leads to a significant translation repression of the introduced reporter and suicide genes.

Conclusion:

Collectively, our data suggest the potential use of Entrovirus-like RNA molecules in suicide gene therapy. Additionally, as a consequence of the possible downregulated miRNA expression in cancerous tissues, a decreased expression of gene therapy constructs armed with target sequences for such miRNA in cancer tissue is expected.Key Words: Thymidine kinase, Polio 3’NCR, miR-143     

Nutritional Value of Commercial Protein-Rich Plant Products

The goal of this work was to analyze nutritional value of various minimally processed commercial products of plant protein sources such as faba bean (Vicia faba), lupin (Lupinus angustifolius), rapeseed press cake (Brassica rapa/napus subsp. Oleifera), flaxseed (Linum usitatissimum), oil hemp seed (Cannabis sativa), buckwheat (Fagopyrum esculentum), and quinoa (Chenopodium quinoa). Basic composition and various nutritional components like amino acids, sugars, minerals, and dietary fiber were determined. Nearly all the samples studied could be considered as good sources of essential amino acids, minerals and dietary fiber. The highest content of crude protein (over 30 g/100 g DW) was found in faba bean, blue lupin and rapeseed press cake. The total amount of essential amino acids (EAA) ranged from 25.8 g/16 g N in oil hemp hulls to 41.5 g/16 g N in pearled quinoa. All the samples studied have a nutritionally favorable composition with significant health benefit potential. Processing (dehulling or pearling) affected greatly to the contents of analyzed nutrients.     

Identification and fine mapping of <Emphasis Type="Italic">Bph33</Emphasis>, a new brown planthopper resistance gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Background

Host-plant resistance is the most desirable and economic way to overcome BPH damage to rice. As single-gene resistance is easily lost due to the evolution of new BPH biotypes, it is urgent to explore and identify new BPH resistance genes.

Results

In this study, using F_2:3 populations and near-isogenic lines (NILs) derived from crosses between two BPH-resistant Sri Lankan rice cultivars (KOLAYAL and POLIYAL) and a BPH-susceptible cultivar 9311, a new resistance gene Bph33 was fine mapped to a 60-kb region ranging 0.91–0.97 Mb on the short arm of chromosome 4 (4S), which was at least 4 Mb distant from those genes/QTLs (Bph12, Bph15, Bph3, Bph20, QBph4 and QBph4.2) reported before. Seven genes were predicted in this region. Based on sequence and expression analyses, a Leucine Rich Repeat (LRR) family gene (LOC_Os04g02520) was identified as the most possible candidate of Bph33. The gene exhibited continuous and stable resistance from seedling stage to tillering stage, showing both antixenosis and antibiosis effects on BPH.

Conclusion

The results of this study will facilitate map-based cloning and marker-assisted selection of the gene.

     

Expression of ROR1 in patients with renal cancer--a potential diagnostic marker

Background: The ectopic expression of receptor tyrosine kinase Ror1 has been reported in patients with hematological malignancies such as chronic lymphocytic leukemia and acute lymphoblastic leukemia. Here we report, for the first time, expression of ROR1 gene in both tumor tissues and peripheral blood mononuclear cells (PBMC) from patients with renal cancer (RC). Methods: In the current study, the expression of ROR1 gene was semi-quantitatively measured in PBMC and tumor tissues from 16 RC patients as well as PBMC from 22 healthy individuals relative to the expression of the housekeeping gene phosphoglucomutase 1 by RT-PCR. Results: Our results showed that ROR1 was expressed at gene level in 81.3% of renal tumor tissues (13 out of 16) whereas it was expressed in 94% of PBMC from RC patients (15 out of 16). A weak expression of ROR1 was observed in PBMC of 4 out of 22 healthy individual. A significant expression of ROR1 was observed in PBMC from RC patients when compared to that in PBMC from normal healthy individuals (P<0.001). The expression of ROR1 in PBMC may reflect a shedding of tumor cells into blood stream. Conclusion: We conclude that detection of a high level of ROR1 expression in blood cells might assist in early detection of renal malignancies, providing taking into consideration the clinical symptoms of the disease. Key Words: ROR1, Ectopic expression, Renal cancer     

Molecular Genetic Analysis of the Variable Number of Tandem-Repeat Alleles at the Phenylalanine Hydroxylase Gene in Iranian Azeri Turkish Population

Background:

The variable numbers of tandem-repeat (VNTR) alleles at the phenylalanine hydroxylase (PAH) gene have been used in carrier detection and prenatal diagnosis in phenylketonuria families. This study was carried out to analyze VNTR alleles at the PAH gene in Iranian Azeri Turkish population.

Methods:

In this study, 200 alleles from general population were studied by PCR.

Results:

The frequencies of VNTR alleles were 45%, 46%, 2%, 3%, 1%, and 3% in studied group regarding 3, 8, 9, 11, 12, and 13 repeat copies, respectively. Statistically significant differences were not found between expected and observed frequencies of VNTR genotypes (P > 0.05).

Conclusions:

VNTR alleles with three and eight repeats were frequent, and the VNTR alleles with 13 repeats showed 3% frequency in the tested group. This study is the first report on tested population genetic structure using VNTR alleles at the PAH gene. Key Words: Phenylalanine hydroxylase, Population genetics, Variable numbers of tandem-repeat