Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.     

Incorporation of silicone oil into elastomers enhances barnacle detachment by active surface strain

Silicone-oil additives are often used in fouling-release silicone coatings to reduce the adhesion strength of barnacles and other biofouling organisms. This study follows on from a recently reported active approach to detach barnacles, which was based on the surface strain of elastomeric materials, by investigating a new, dual-action approach to barnacle detachment using Ecoflex®-based elastomers incorporated with poly(dimethylsiloxane)-based oil additives. The experimental results support the hypothesis that silicone-oil additives reduce the amount of substratum strain required to detach barnacles. The study also de-coupled the two effects of silicone oils (ie surface-activity and alteration of the bulk modulus) and examined their contributions in reducing barnacle adhesion strength. Further, a finite element model based on fracture mechanics was employed to qualitatively understand the effects of surface strain and substratum modulus on barnacle adhesion strength. The study demonstrates that dynamic substratum deformation of elastomers with silicone-oil additives provides a bifunctional approach towards management of biofouling by barnacles.     

Increased oxidative stress, the renin-angiotensin system, and sympathetic overactivation induce hypertension in kidney androgen-regulated protein transgenic mice

Gender differences in the incidence and severity of hypertension have suggested the involvement of a sex-dependent mechanism. Transgenic (Tg) mice overexpressing kidney androgen-regulated protein (KAP) specifically in kidney showed hypertension associated with oxidative stress. Reactive oxygen species (ROS) are strongly implicated in the pathological signaling leading to hypertension in a framework that includes renin-angiotensin system (RAS) activation, increased sympathetic activity, and cardiac remodeling. In this report, we observed that plasma levels of angiotensin II and catecholamines were increased in KAP Tg mice, compared with wild-type animals. Systemic administration of Tempol, a membrane-permeative superoxide dismutase mimetic, reduced arterial pressure as well as urinary excretion of oxidative stress markers and reduced both angiotensin II and norepinephrine plasma levels in KAP Tg mice. Intracerebroventricular administration of Tempol also reduced arterial pressure in Tg mice. Moreover, administration of apocynin and DPI, inhibitors of NADPH oxidase, a major source of ROS, also reduced arterial pressure and both angiotensin II and norepinephrine plasma levels in Tg mice. Thus, we analyzed the involvement of the RAS and sympathetic nervous system in KAP Tg mouse hypertension. Both captopril and losartan reduced arterial blood pressure in Tg mice, as also occurred after β-adrenergic blockade with atenolol. Also, intracerebroventricular losartan administration reduced arterial pressure in KAP Tg mice. Our data demonstrate that hypertension in male KAP Tg mice is based on increased oxidative stress, increased sympathetic activity, and RAS activation. Moreover, our results suggest a role for increased oxidative stress in the CNS as a major cause of hypertension in these animals.     

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion

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Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

The Penicillium echinulatum Secretome on Sugar Cane Bagasse

Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases). Glycoside hydrolase (GH) family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.     

An InCytes from MBC Selection: Pob1 Participates in the Cdc42 Regulation of Fission Yeast Actin Cytoskeleton

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1⁺ as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745–2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1ΔN) or the SAM domain (pob1ΔSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1ΔN, and pob1ΔSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.     

An APETALA3 homolog controls both petal identity and floral meristem patterning in Nigella damascena L. (Ranunculaceae)

Flower architecture mutants provide a unique opportunity to address the genetic origin of flower diversity. Here we study a naturally occurring floral dimorphism in Nigella damascena (Ranunculaceae), involving replacement of the petals by numerous sepal‐like and chimeric sepal/stamen organs. We performed a comparative study of floral morphology and floral development, and characterized the expression of APETALA3 and PISTILLATA homologs in both morphs. Segregation analyses and gene silencing were used to determine the involvement of an APETALA3 paralog (NdAP3–3) in the floral dimorphism. We demonstrate that the complex floral dimorphism is controlled by a single locus, which perfectly co‐segregates with the NdAP3–3 gene. This gene is not expressed in the apetalous morph and exhibits a particular expression dynamic during early floral development in the petalous morph. NdAP3–3 silencing in petalous plants perfectly phenocopies the apetalous morph. Our results show that NdAP3–3 is fully responsible for the complex N. damascena floral dimorphism, suggesting that it plays a role not only in petal identity but also in meristem patterning, possibly through regulation of perianth organ number and the perianth/stamen boundary.     

Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5αF′. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.