Impact of light quality on a native Louisiana Chlorella vulgaris/Leptolyngbya sp. co‐culture

Light effect on cultures of microalgae has been studied mainly on single species cultures. Cyanobacteria have photosynthetic pigments that can capture photons of wavelengths not available to chlorophylls. A native Louisiana microalgae (Chlorella vulgaris ) and cyanobacteria (Leptolyngbya sp.) co‐culture was used to study the effects of light quality (blue–467 nm, green–522 nm, red–640 nm and white–narrow peak at 450 nm and a broad range with a peak at 550 nm) at two irradiance levels (80 and 400 μmol m^?2 s^?1) on the growth, species composition, biomass productivity, lipid content and chlorophyll‐a production. The co‐culture shifted from a microalgae dominant culture to a cyanobacteria culture at 80 μmol m^?2 s^?1. The highest growth for the cyanobacteria was observed at 80 μmol μmol m^?2 s^?1 and for the microalgae at 400 μmol m^?2 s^?1. Red light at 400 μmol m^?2 s^?1 had the highest growth rate (0.41 d^?1), biomass (913 mg L^?1) and biomass productivity (95 mg L^?1 d^?1). Lipid content was similar between all light colors. Green light had the highest chlorophyll‐a content (1649 μg/L). These results can be used to control the species composition of mixed cultures while maintaining their productivity.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP31 and MIP32 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP31 and MIP32 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP31 and MIP32 contain four exons and three introns, and MIP31 and MIP32 shared the same intron/exon organization with that of human. The MIP31 and MIP32 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP32 gene was much lower than the Trsc-SCYA107 and MIP31 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP31 and MIP32 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP32 genes in PWBCs was observed at 1–12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP31 expression was observed at 3–12 h poststimulation only with PMA.     

家蝇幼虫消减文库的构建及差异表达基因的鉴定

为了鉴定家蝇Musca domestica免疫相关基因,应用抑制性消减杂交技术,构建刺激家蝇幼虫差异表达cDNA消减文库。以大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus诱导12 h的家蝇幼虫与未诱导的家蝇幼虫为消减杂交对象,获得了差异表达基因的cDNA片段,将其与T/A载体连接并转化大肠杆菌DH5α,构建了刺激家蝇幼虫cDNA消减文库。PCR检测发现,文库的阳性克隆中插入的cDNA片段大小在200~1 000 bp之间,随机挑选了161个含大小不等差异片段的克隆进行测序和同源性分析,鉴定了36种蛋白的基因片段,包括抗菌肽、酶、核糖体蛋白、其他功能蛋白以及功能不明的蛋白。用半定量RT-PCR分析了其中6种蛋白基因的表达,结果显示：防御素和攻击素基因在细菌刺激后24 h内明显上调表达,而溶菌酶、酚氧化酶原活化因子、糜蛋白酶和蛋白质合成起始因子基因在细菌刺激后0-4 h内表达受抑制,12 h后上调表达。该研究结果为家蝇免疫相关基因的克隆和家蝇免疫防御机制的探讨奠定了良好的基础。     

镍胁迫下安氏伪镖水蚤SSH文库的构建及铁蛋白cDNA的克隆与差异表达

为探讨镍胁迫下桡足类的分子响应机制,以安氏伪镖水蚤为研究对象,利用抑制性消减杂交(suppression subtractive hybridization,SSH)技术,构建了镍胁迫下安氏伪镖水蚤SSHcDNA文库,并随机挑取生长菌落140个克隆子,进行菌液PCR鉴定,计算文库重组率为98.6％,文库容量为1.12×106 cfu.将重组子测序,经BLAST一致性搜索比对分析发现,有一重组片段含有铁蛋白保守结构域,该片段大小为859 bp,连续编码170个氨基酸残基,Gen-Bank登录号为JK312601.半定量PCR证实,镍胁迫24 h后,安氏伪镖水蚤中铁蛋白表达显著上调.本文库的成功构建及铁蛋白cDNA片段的获得为进一步研究镍胁迫下桡足类的分子响应机制奠定了基础.     

Understanding resistant germplasm‐induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata

Curvularia lunata is an important pathogen causing Curvularia leaf spot in maize. Significant pathogenic variation has been found in C. lunata. To better understand the mechanism of this phenomenon, we consecutively put the selective pressures of resistant maize population on C. lunata strain WS18 (low virulence) artificially. As a result, the virulence of this strain was significantly enhanced. Using 2DE, 12 up‐regulated and four down‐regulated proteins were identified in virulence‐increased strain compared to WS18. Our analysis revealed that melanin synthesis‐related proteins (Brn1, Brn2, and scytalone dehydratase) and stress tolerance‐related proteins (HSP 70) directly involved in the potential virulence growth as crucial markers or factors in C. lunata. To validate 2DE results and screen differential genes at mRNA level, we constructed a subtracted cDNA library (tester: virulence‐increased strain; driver: WS18). A total of 188 unigenes were obtained this way, of which 14 were indicators for the evolution of pathogen virulence. Brn1 and hsp genes exhibited similar expression patterns corresponding to proteins detected by 2DE. Overall, our results indicated that differential proteins or genes, being involved with melanin synthesis or tolerance response to stress, could be considered as hallmarks of virulence increase in C. lunata.     

Investigation of ifosfamide nephrotoxicity induced in a liver–kidney co‐culture biochip

In this article, we present a liver–kidney co‐culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver–kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG‐MDCK) when compared to untreated co‐cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3‐dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG‐MDCK co‐cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG‐MDCK co‐culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3‐dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG‐MDCK co‐culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver–kidney micro fluidic co‐culture model using HepaRG‐MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a‐MDCK model. This study demonstrates the interest in the development of systemic organ–organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods. Biotechnol. Bioeng. 2013; 110: 597–608. © 2012 Wiley Periodicals, Inc.     

Label‐free and non‐invasive discrimination of HaCaT and melanoma cells in a co‐culture model by hyperspectral confocal reflectance microscopy

A novel hyperspectral confocal microscopy method to separate different cell populations in a co‐culture model is presented here. The described methodological and instrumental approach allows discrimination of different cell types using a non‐invasive, label free method with good accuracy with a single cell resolution. In particular, melanoma cells are discriminated from HaCaT cells by hyperspectral confocal imaging, principal component analysis and optical frequencies signing, as confirmed by fluorescence labelling cross check. The identification seems to be quite robust to be insensitive to the cellular shape within the studied samples, enabling to separate cells according to their cytotype down to a single cell sensitivity.

Set of hyperspectral images of melanoma‐keratinocytes co‐culture model (left), score plot of principal component analysis and spectral analysis of principal components coefficients (center), label‐free spectral identification of cell populations (right).     

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) by suppression subtractive hybridization

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5- and 3-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17–1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970–20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293–51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.The nucleotide sequences of OsGGT cDNA has been submitted to GenBank DDBJ under accession numbers AB164463.     

The putative‐farnesoic acid O‐methyl transferase (FAMeT) gene of Ceratitis capitata: characterization and pre‐imaginal life expression

Farnesoic acid O‐methyl transferase (FAMeT) is the enzyme involved in the penultimate step of insect juvenile hormone (JH) biosynthesis and is thus a key regulator in insect development and reproduction. We report the characterization of the putative‐FAMeT in the medfly or Mediterranean fruit fly, Ceratitis capitata. This gene was identified by suppressive subtractive hybridization and completely sequenced by the screening of a medfly cDNA library. The obtained sequence was analyzed for conserved protein domain identification and its expression profile was evaluated by quantitative Real‐Time PCR in medfly pre‐imaginal life. The tissue expression of the isolated gene was verified by in situ hybridization on third instar larvae sections. The characterization of the isolated gene pointed out several typical features of methyl transferase genes. The pre‐imaginal putative‐FAMeT expression levels were consistent with JH titer change in Diptera. As recognized in some crustaceans, this gene seems to be widely expressed in the medfly as well. Ceratitis capitata is one of the most relevant agricultural pests against which insecticides and the sterile insect technique (SIT) are extensively used in spite of the well‐known limitations of these approaches. Although results are not conclusive for the physiological role of the isolated gene, they suggest the characterization of a new gene in the Mediterranean fruit fly potentially involved in JH biosynthesis and may, therefore, have implications for pest control. © 2010 Wiley Periodicals, Inc.     

Interactions between Clostridium beijerinckii and Geobacter metallireducens in co‐culture fermentation with anthrahydroquinone‐2, 6‐disulfonate (AH2QDS) for enhanced biohydrogen production from xylose

To enhance biohydrogen production, Clostridium beijerinckii was co‐cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone‐2, 6‐disulfonate (AH₂QDS). In the co‐culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co‐culture fermentation were observed compared to a pure culture of C. beijerinckii without AH₂QDS. G. metallireducens grew in the co‐culture system, resulting in a decrease in acetate concentration under co‐culture conditions and a presumed regeneration of AH₂QDS from AQDS. These co‐culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production. Biotechnol. Bioeng. 2013; 110: 164–172. © 2012 Wiley Periodicals, Inc.     

Determination of amlodipine using terbium‐sensitized luminescence in the presence of europium(III) as a co‐luminescence reagent

A sensitive time‐resolved luminescence method for the determination of amlodipine (AM) in methanol and in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb³⁺) by formation of a ternary complex with AM in the presence of tri‐n‐octylphosphine oxide (TOPO) as co‐ligand, dodecylbenzenesulfate as surfactant and europium ion as a co‐luminescence reagent. The signal for Tb–AM–TOPO is monitored at λ_ex = 242 nm and λ_em = 550 nm. Optimum conditions for the formation of the complex in aqueous system were 0.015 m Tris (hydroxylmethyl) amino methane buffer, pH 9.0, TOPO (1.0 × 10^–4 m ), Eu³⁺ (2.0 × 10^–7 m ), dodecylbenzenesulfate (0.14%) and 6.0 × 10^–5 m of Tb³⁺, which allows the determination of 10–50 ppb of AM with a limit of detection of 1.2 ppb. The relative standard deviations of the method range between 0.1 and 0.2% indicated excellent reproducibility of the method. The proposed method was successfully applied for the assay of AM in pharmaceutical formulations and in plasma samples. Average recoveries of 98.5 ± 0.2% and 95.2 ± 0.2% were obtained for AM in tablet and plasma samples respectively. Copyright © 2013 John Wiley & Sons, Ltd.