A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens × F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Transfer of members of the genus Falcivibrio to the genus Mobiluncus, and emended description of the genus Mobiluncus

It has long been thought that the genera Mobiluncus and Falcivibrio contain the same organisms. Using a polyphasic approach, it was found that Mobiluncus curtisii and Mobiluncus mulieris were the same as Falcivibrio vaginalis and Falcivibrio grandis, respectively. As the genus name Mobiluncus takes precedence, it is proposed that F. vaginalis and F. grandis be transferred to the genus Mobiluncus. In agreement with previous studies, results from phenotypic tests did not support the separation of M. curtisii strains into its two subspecies, M. curtisii subsp. curtisii and M. curtisii subsp. holmesii. Phenotypic complexity within M. curtisii dictates that the species should be treated as a complex until more in-depth analyses of the species have been performed. Phylogenetic analyses, based on 16S rRNA gene sequences, demonstrated that the genus Mobiluncus was associated with Varibaculum cambriense and the two subspecies of Actinomyces neuii, and that A. neuii is only distantly related to Actinomyces sensu stricto.

Differential induction of 12-O-tetradecanoylphorbol 13-acetate sequence gene expression in murine melanocytes and melanoma cells

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281–3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of 115 gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.

Exploring carrier agents in swarm-array computing

How can a bridge be built between autonomic computing approaches and parallel computing systems? The work reported in this paper is motivated towards bridging this gap by proposing a swarm-array computing approach based on ‘Intelligent Agents’ to achieve autonomy for distributed parallel computing systems. In the proposed approach, a task to be executed on parallel computing cores is carried onto a computing core by carrier agents that can seamlessly transfer between processing cores in the event of a predicted failure. The cognitive capabilities of the carrier agents on a parallel processing core serves in achieving the self-ware objectives of autonomic computing, hence applying autonomic computing concepts for the benefit of parallel computing systems. The feasibility of the proposed approach is validated by simulation studies using a multi-agent simulator on an FPGA (Field-Programmable Gate Array) and experimental studies using MPI (Message Passing Interface) on a computer cluster. Preliminary results confirm that applying autonomic computing principles to parallel computing systems is beneficial.

Adeno-associated virus-8-Mediated intravenous transfer of myostatin propeptide leads to systemic functional improvements of slow but not fast muscle

Myostatin is a member of the transformating growth factor-_ (TGF-_) superfamily of proteins and is produced almost exclusively in skeletal muscle tissue, where it is secreted and circulates as a serum protein. Myostatin acts as a negative regulator of muscle mass through the canonical SMAD2/3/4 signaling pathway. Naturally occurring myostatin mutants exhibit a ‘double muscling’ phenotype in which muscle mass is dramatically increased as a result of both hypertrophy and hyperplasia. Myostatin is naturally inhibited by its own propeptide; therefore, we assessed the impact of adeno associated virus-8 (AAV8) myostatin propeptide vectors when systemically introduced in MF-1 mice. We noted a significant systemic increase in muscle mass in both slow and fast muscle phenotypes, with no evidence of hyperplasia; however, the nuclei-to- cytoplasm ratio in all myofiber types was significantly reduced. An increase in muscle mass in slow (soleus) muscle led to an increase in force output; however, an increase in fast (extensor digitorum longus [EDL]) muscle mass did not increase force output. These results suggest that the use of gene therapeutic regimens of myostatin inhibition for age-related or disease-related muscle loss may have muscle-specific effects.

Two macrocyclic pentaaza compounds containing pyridine evaluated as novel chelating agents in copper(II) and nickel(II) overload

Two pentaaza macrocycles containing pyridine in the backbone, namely 3,6,9,12,18-pentaazabicyclo[12.3.1] octadeca-1(18),14,16-triene ([15]pyN(5)), and 3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1(19),15,17-triene ([16]pyN(5)), were synthesized in good yields. The acid-base behaviour of these compounds was studied by potentiometry at 298.2 K in aqueous solution and ionic strength 0.10 M in KNO3. The protonation sequence of [15]pyN(5) was investigated by H-1 NMR titration that also allowed the determination of protonation constants in D2O. Binding studies of the two ligands with Ca2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ metal ions were performed under the same experimental conditions. The results showed that all the complexes formed with the 15-membered ligand, particularly those of Cu2+ and especially Ni2+, are thermodynamically more stable than with the larger macrocycle. Cyclic voltammetric data showed that the copper(II) complexes of the two macrocycles exhibited analogous behaviour, with a single quasi-reversible one-electron transfer reduction process assigned to the Cu(II)/Cu(I) couple. The UV-visible-near IR spectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonal distorted coordination geometry for the metal centre. X-band EPR spectra of the copper(II) complexes are consistent with distorted square pyramidal geometries. The crystal structure of [Cu([15]pyN(5))](2+) determined by X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in a distorted square pyramidal environment.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Segmenting the Italian coffee market: marketing opportunities for economic agents working along the international coffee chain

Globalization, either directly or indirectly (e.g. through structural adjustment reforms), has called for profound changes in the previously existing institutional order. Some changes adversely impacted the production and market environment of many coffee producers in developing countries resulting in more risky and less remunerative coffee transactions. This paper focuses on customization of a tropical commodity, fair-trade coffee, as an approach to mitigating the effects of worsened market conditions for small-scale coffee producers in less developed countries. fair-trade labeling is viewed as a form of “de-commodification” of coffee through product differentiation on ethical grounds. This is significant not only as a solution to the market failure caused by pervasive information asymmetries along the supply chain, but also as a means of revitalizing the agricultural-commodity-based trade of less developed countries (LDCs) that has been languishing under globalization. More specifically, fair-trade is an example of how the same strategy adopted by developed countries’ producers/ processors (i.e. the sequence product differentiation - institutional certification - advertisement) can be used by LDC producers to increase the reputation content of their outputs by transforming them from mere commodities into “decommodified” (i.e. customized and more reputed) goods. The resulting segmentation of the world coffee market makes possible to meet the demand by consumers with preference for this “(ethically) customized” coffee and to transfer a share of the accruing economic rents backward to the Fair-trade coffee producers in LDCs. It should however be stressed that this outcome cannot be taken for granted since investments are needed to promote the required institutional innovations. In Italy FTC is a niche market with very few private brands selling this product. However, an increase of FTC market share could be a big commercial opportunity for farmers in LDCs and other economic agents involved along the international coffee chain. Hence, this research explores consumers’ knowledge of labels promoting quality products, consumption coffee habits, brand loyalty, willingness to pay and market segmentation according to the heterogeneity of preferences for coffee products. The latter was assessed developing a D-efficient design where stimuli refinement was tested during two focus groups.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Oscheius tipulae as an example of eEF1A gene diversity in nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

Changes in Apoptotic Gene Expression Induced by DNA Cross-Linkers

The Millard Research Laboratory is interested in the cytotoxic mechanisms of the bifunctional alkylators diepoxybutane (DEB), epichlorohydrin (ECH), and (1-chloroethenyl) oxirane (COX). Studies performed in the laboratory examine the dual nature of these DNA cross-linking compounds that can act as carcinogens or anti-cancer agents. The mechanisms through which these compounds induce cell death are explored in this study. Cells either undergo cell death due to necrosis or apoptosis. HL-60 cells were treated with varying concentrations of DEB, ECH, or COX. A caspase 3/7 assay was used to test for induction of apoptosis in the treated cells at varying incubation times. It was concluded that DEB induces apoptosis in HL-60 cells treated with 100 μM for 24 hours. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was then used to explore the changes in gene expression of various genes involved in apoptosis signaling. The results were inconclusive as to specific genes involved in DEB induced apoptosis, but the data does suggest that apoptosis is induced by a mitochondrial-mediated apoptosis signaling pathway.

Targeting the poor: a macroeconomic analysis of cash transfer programs

This paper introduces cash transfers targeting the poor in an incomplete markets model with heterogeneous agents facing idiosyncratic risk. These transfers change the degree of insurance in the economy and affect precautionary motives asymmetrically, leading the poorest households to decrease savings proportionally more than their richer counterparts. In a model economy calibrated to Brazil, once the cash transfer program is adopted, wealth inequality and social welfare increase, poverty decreases, while employment and income inequality remain about the same. Imperfect access to financial markets is important for these results, whereas whether the program is funded with lump sum or distortive taxes is not.

Chemically Assisted Enucleation Results in Higher G6PD Expression in Early Bovine Female Embryos Obtained by Somatic Cell Nuclear Transfer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)