The Value of Crop Production and Pollination Services in the Eastern Amazon.

Nature safeguards living organisms and the ecosystem functions and services delivered by them. Animal pollination is an important Ecosystem Service since it plays a key role for achieving the sustainable development goals by safeguarding worldwide food production. Thus, conservation of pollination services is a major priority for guaranteeing global food security in the long term. Here we evaluate the crop pollination services in Pará state (Eastern Amazon, Brazil) focusing on two questions: (1) What is the economic value of crop production and pollination service in Pará? (2) Which municipalities are most dependent on pollination services considering local economies? We found 36 crops produced in the state; 20 (55%) crops are dependent on animal pollinators. In 2016, crop production value (CPV) for Pará state was US$ 2.95 billion and total pollination service value (PSV) was US$ 983.2 million, corresponding to 33% of CPV in Pará. Highest PSV value crops were açaí palm (US$635.6 million), cocoa (US$187.6 million), soybean (US$98.4 million), and watermelon (US$26.1 million), accounting for 96% of Pará's PSV. Two municipalities (Medicilândia and Igarapé Miri) presented more than 50% of their GDP based on pollination services. In general, we found low crop diversity in the municipalities of Pará, suggesting an economic rural vulnerability for the state, mainly supported by the high productions of soy and açaí. Pollinator conservation and ecological intensified farming practices are urgent for supporting sustainable development for the state.

New self-assembled supramolecular hydrogels based on dehydropeptides.

Supramolecular hydrogels rely on small molecules that self-assemble in water as a result of the cooperative effect of several relatively weak intermolecular interactions. Peptide-based low molecular weight hydrogelators have attracted enormous interest owing to the simplicity of small molecules combined with the versatility and biocompatibility of peptides. In this work, naproxen, a well known non-steroidal anti-inflammatory drug, was N-conjugated with various dehydrodipeptides to give aromatic peptide amphiphiles that resist proteolysis. Molecular dynamics simulations were used to obtain insight into the underlying molecular mechanism of self-assembly and to rationalize the design of this type of hydrogelators. The results obtained were in excellent agreement with the experimental observations. Only dehydrodipeptides having at least one aromatic amino acid gave hydrogels. The characterization of the hydrogels was carried out using transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy and also rheological assays.

Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2013-31 March 2013.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross-tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.

Reconstitution of maturating and regulatory lymphocyte subsets after cord blood and BMT in children.

Some clinical characteristics of cord blood transplantation (CBT) might be explained by specificities in the reconstitution of immune subsets differing by their maturation stage or their implication in GVHD, tolerance or immune responses against tumor or infectious agents. Here, we compare the immune reconstitution of several of these subsets after CBT and BMT. B-cell count recovery was faster after CBT. There was no difference in the recovery of CD4(+) and CD8(+) cell counts. There was no difference either in the frequency of several subsets: CD45RO(+) memory, and CD45RA(+) naïve cells within the CD4(+) T-cell compartment, CD27(+) among B cells, CD56(bright), NKG2A(+), and KIR(+) cells among natural killer (NK) cells, CD25(+)FOXP3(+) regulatory T cells and invariant NKT cells. The proportion of the thymic naïve CD31(+)CD45RA(+)CD4(+) T cells was lower after CBT at 6 months post-transplant, and was still below normal at 1 year in both groups. NK-cell expansion was more sustained after CBT, with fewer double-negative NKG2A(-)KIR(-) hyporesponsive cells and more double-positive NKG2A(+)KIR(+) hyper-responsive NK cells. These results, therefore, indicate that further research to improve CBT outcome should try to improve thymopoieisis and take advantage of the sustained NK-cell reconstitution.

Expression of genes related to reproduction and pollen foraging in honey bees (Apis mellifera) narcotized with carbon dioxide.

It has been proposed that a honey bee (Apis mellifera) worker's preference for foraging for pollen or nectar is modulated by a gene network that was originally involved in regulating the reproductive cycles of an ancestral solitary species. We used carbon dioxide to induce narcosis in queens and workers. This treatment is known to initiate oogenesis in queens, reduce oogenesis in queenless workers and to change worker foraging preference. We then assessed changes in gene expression of genes suspected to be involved in either foraging behaviour or reproduction. We show that some genes change expression in the opposite direction between castes in response to treatment. Our results therefore support the hypothesis that reproductive and foraging traits are causally related in the honey bee.

Conformation study of HA(306-318) antigenic peptide of the haemagglutinin influenza virus protein.

Several HLA-DR alleles present the immunodominant HA(306-318) peptide of haemagglutinin of the influenza virus to T cells. NMR data of the peptide in various water solutions exclude any alpha-helix or turn conformations. Circular dichroism and Fourier transform infrared spectroscopies indicate an estimated beta-extended structure in water of 31% and 28%, respectively, with spectra shape similar to the ones observed for beta-sheet containing proteins. The H/D amide exchange suggests a stable length-dependent interchain hydrogen-bonding. The partially beta-extended conformation of HA(306-318) in solution might be close to the one found in HA(306-318)-HLA-DR1 complex. These results suggest different interconverting extended conformations of HA(306-318), depending on the microenvironment of the solution medium. This flexibility emphasizes the ability of some peptides to fit more easily the binding site of several HLA-DR molecules. Similar results were obtained on the HIV P25(263-277) peptide which has been previously shown to be a good DR1 binder. From a vibrational point of view, infrared Amide I frequencies of secondary structures in peptides were ascertained. As previously demonstrated for proteins in solution, Fourier transform infrared and circular dichroism spectroscopies appear to be valuable tools for conformational properties of peptides. Their use may contribute to the detection of peptide conformation-binding relationship which has to be further tested by biochemical and biological studies.

Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants.

The efficiency of wheat biolistic transformation systems strongly depends on the bombardment parameters, the condition of the donor plant, and the plant genotype chosen for the transformation process. This paper analyzes the transformation efficiency of the 129 wheat sister lines generically called 'Bobwhite', originally obtained from the cross 'Aurora'//'Kalyan'/'Bluebird 3'/'Woodpecker'. A number of factors influencing the transformation were examined, such as the ability to produce embryogenic callus, regeneration in selection medium, and overall transformation performance. Of the 129 genotypes evaluated, eight demonstrated transformation efficiencies above 60% (60 independent transgenic events per 100 immature embryos bombarded). Among the eight genotypes identified, we studied agronomic characteristics such as earliness to identify the most adaptable line(s) for different lab conditions. 'Bobwhite' SH 98 26 was identified as a super-transformable wheat line.

A STR mutation in a heteropaternal twin case.

A heteropaternal male twin case with two men being alleged fathers was investigated as requested by the Court. Up to 37 PCR-based polymorphic DNA systems were studied in this case which was complicated by a paternal ACTBP2 mutation detected in one twin. This is the first report on a STR mutation in a double paternity case where both biological fathers were indisputably identified. The STR systems enable the resolution of these complex genetic relationships even in a case where a mutation in one STR locus was encountered.

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants.

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.

The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage.

A unique feature of plant aspartic proteinase precursors is the presence of an internal domain, known as plant-specific insert, whose function is not completely understood. The three-dimensional structure of the plant-specific insert resembles that of saposin-like proteins, a group of lipid-binding proteins involved in a variety of physiological processes. Here we show that recombinant plant-specific insert is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH- and lipid-dependent manner. The leakage activity is higher at pH 4.5 and requires the presence of acidic phospholipids such as phosphatidylserine. To determine whether the same effect could be observed when the plant-specific insert is part of the precursor form, procardosin A and a mutant form lacking this specific domain were produced and characterized. Procardosin A displays a similar activity profile, whereas the mutant without the plant-specific insert shows only residual activity. These findings indicate that the plant-specific insert domain of plant aspartic proteinases mediates an interaction of their precursors with phospholipid membranes and induces membrane permeabilization. It is therefore possible that the plant-specific insert, alone or in conjunction with the proteolytic activity of plant aspartic proteinases, may function either as a defensive weapon against pathogens or in late autolysis of plant cells.

Sequence variation of new alleles at the short tandem repeat D19S253 locus.

This paper reports the sequences of two new alleles identified in a population database study on the short tandem repeat D19S253 locus. A Portuguese Caucasian population and a Portuguese African population were studied. Forty-four selected alleles were sequenced and 11 different alleles were found. All the sequenced alleles shown to possess a simple tetranucleotide GATA repeat region structure. The two new alleles, alleles 6 and 16, follow the simple repeat pattern. During paternity investigation casework, 1028 meiosis were analyzed and five isolated genetic incompatibilities detected. In one case, a non-detectable allele with the used set of primers could be the explanation. In the other four cases, single-step mutations could be considered. The mutation rate obtained for this locus was 3.89 x 10(-3).

Portuguese population and paternity investigation studies with a multiplex PCR--the AmpFlSTR Profiler Plus.

A Portuguese Caucasian population of 146 unrelated individuals was studied. DNA samples were amplified by multiplex PCR for D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 using the AmpFlSTR Profiler Plus PCR Amplification Kit (Perkin-Elmer). All loci met Hardy-Weinberg expectations. Forensic statistical parameters were according to those obtained by other authors. Statistical differences were observed concerning three loci when comparing the Portuguese Caucasian population and an Italian Caucasian population, although these differences mainly concern the less frequent alleles. Eighty-three paternity investigation cases were analysed. Exclusions in between three and nine loci were observed in all the 23 exclusion cases obtained. Most of the non-exclusion cases had probability of paternity > 99.9%. Two cases with an isolated genetic incompatibility between the alleged father and the child were detected, which may indicate probable mutation cases. These results demonstrate that the AmpFlSTR Profiler Plus is a suitable multiplex for paternity investigation in the Portuguese population.

Comparative calorimetric study of non-amyloidogenic and amyloidogenic variants of the homotetrameric protein transthyretin.

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary type of amyloidosis involving amino acid substitutions in transthyretin (TTR). V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of FAP. The thermal stability of the wild-type, V30M-TTR, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal unfolding of TTR is a spontaneous reversible process involving a highly co-operative transition between folded tetramers and unfolded monomers. All variants of transthyretin are very stable to the thermal unfolding that occurs at very high temperatures, most probably because of their oligomeric structure. The data presented in this work indicated that for the homotetrameric form of the wild-type TTR and its variants, the order of stability is as follows: wild-type TTR approximately > T119M-TTR > L55P-TTR > V30M-TTR, which does not correlate with their known amyloidogenic potential.

The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis.

In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett. 418, 297-300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions.

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos.

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests - corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta(TM) contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression.

South Portuguese population data on the loci HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and Gc.

Five South Portuguese Caucasian subpopulations were analyzed for the HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and Gc loci. Genotype distributions for these loci did not deviate from Hardy-Weinberg expectations. The allele and genotype frequencies found have been compared with previously published data from North and Central Portugal. A total of 11 out of 138 chi-square comparisons of allele frequencies between different Portuguese populations showed a certain degree of divergence. Alentejo, Algarve, Madeira Island and Azores Islands populations might be considered as different groups in a database. For forensic casework, a composite South Portuguese Caucasian population database was obtained for estimating multiple locus profile frequencies using the six PCR-based loci studied.

Molecular phylogeny of Cyprinidae inferred from cytochrome b DNA sequences.

Phylogenetic relationships between European cyprinids (Teleostei:Cypriniformes:Cyprinidae) were investigated by comparing cytochrome b gene sequences from 29 species, among which 20 were newly sequenced. Results were in general agreement with previous morphologically-based studies, but new interesting relationships were found. The classical barbelled/lacking barbels split is dubious. Genus Leuciscus appears paraphyletic. The phylogenetic location of some American cyprinid species was recovered; at least two distinct invasions of the New World are likely. Finally, the problem of intergeneric cyprinid hybrids is addressed. The genus rank for these interbreeding entities is supported and hybrids are seen as the consequence of a high genetic flexibility. This is the first molecularly based study of cyprinid diversity. It sheds light on the evolution and taxonomy of this major freshwater fish family.

The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution.

Amyloid fibril formation and deposition are the basis for a wide range of diseases, including spongiform encephalopathies, Alzheimer's and familial amyloidotic polyneuropathies. However, the molecular mechanisms of amyloid formation are still poorly characterised. In certain forms of familial amyloidotic polyneuropathy (FAP), the amyloid fibrils are mostly constituted by variants of transthyretin (TTR). V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of amyloidosis. Here, we report gel filtration chromatography experiments to characterise the aggregation states of WT-, V30M-, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, in solution, at nearly physiological pH. These studies show that all four protein tetramers dissociate to monomer upon dilution, in the submicromolar range, at pH 7.0. The amyloidogenic proteins V30M- and L55P-TTR show a complex equilibrium between monomers, tetramers and high molecular weight aggregate species. These aggregates dissociate directly to monomer upon dilution. This study shows that the tendency to form aggregates among the four studied proteins correlates with their known amyloidogenic potential. Thus, the amyloidogenic mutations could perturb the structure and/or stability of the monomeric species leading initially to the formation of soluble aggregates and at a later stage to insoluble amyloid fibrils.

Phylogenetic relationships within genus Leuciscus (Pisces, Cyprinidae) in Portuguese fresh waters, based on mitochondrial DNA cytochrome b sequences.

To investigate phylogenetic relationships among Leuciscus species occurring in Portuguese inland waters, the cytochrome b gene was sequenced from representatives of the main rivers. This study supports the recognition of the species level for L. pyrenaicus, including populations from the southern Portuguese drainages (Tejo, Sado, and Guadiana drainages), and for L. carolitertii, including populations from the northern Portuguese drainages. The existence of two new species occurring in the extreme southwestern drainages of Mira and Arade is also suggested. The present results support the monophyly of the Mira and the Arade populations, as well as an early divergence of these two lineages. The present-day distribution of Leuciscus species is seen as a consequence of Pliocene and Pleistocene events, such as river disjunctions and posterior confluence in epicontinental seas and river captures. A mixture of haplotypes was observed in the Mondego and the Tejo drainages, which could be a consequence of ancient river captures, with a possible mitochondrial DNA introgression in the Tejo drainage and a recent introduction by man in the Mondego drainage. The pattern of differentiation among mtDNA haplotypes and their geographic distribution is discussed in terms of evolutionary aspects.