DEVELOPMENT OF MICROSATELLITE PRIMERS FOR JATROPHA CURCAS (EUPHORBIACEAE) AND TRANSFERABILITY TO CONGENERS

Premise of the study: Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. Methods and Results: Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. Conclusions: The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.

Comparative study and characterization of starches isolated from unconventional tuber sources

Some properties of canna (Canna indica L.) and bore (Alocasia macrorrhiza) starches were evaluated and compared using cassava starch (Manihot esculenta Crantz) as a reference. Proximate analysis, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and viscosity measurements were performed. Canna and bore starches showed a similar degree of purity as that of the cassava starch. Canna starch exhibited higher thermal stability and viscosity of solution values than those of bore and cassava starches. XRD spectra showed that canna starch crystallizes as a B-type structure; however, bore and cassava starches crystallize as an A-type structure. Results proved that canna and bore starches are promising bio(materials), obtained from unconventional sources, to be used for industrial applications, as their physicochemical properties are similar to those of cassava starch, which it is known has potential applications in this area.

Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source

Endophytic fungi live inside plants, apparently do not cause any harm to their hosts and may play important roles in defense and growth promotion. Fungal growth is a routine practice at microbiological laboratories, and the Potato Dextrose Agar (PDA) is the most frequently used medium because it is a rich source of starch. However, the production of potatoes in some regions of the world can be costly. Aiming the development of a new medium source to tropical countries, in the present study, we used leaves from the guarana (a tropical plant from the Amazon region) and the olive (which grows in subtropical and temperate regions) to isolate endophytic fungi using PDA and Manihot Dextrose Agar (MDA). Cassava (Manihot esculenta) was evaluated as a substitute starch source. For guarana, the endophytic incidence (EI) was 90% and 98% on PDA and MDA media, respectively, and 65% and 70% for olive, respectively. The fungal isolates were sequenced using the ITS- rDNA region. The fungal identification demonstrated that the isolates varied according to the host plant and media source. In the guarana plant, 13 fungal genera were found using MDA and six were found using PDA. In the olive plant, six genera were obtained using PDA and 4 were obtained using MDA. The multivariate analysis results demonstrated the highest fungal diversity from guarana when using MDA medium. Interestingly, some genera were isolated from one specific host or in one specific media, suggesting the importance of these two factors in fungal isolation specificity. Thus, this study indicated that cassava is a feasible starch source that could serve as a potential alternative medium to potato medium.

Hemiklonale Vererbung im hybridogenetischen Rana grafi-Komplex (Anura: Ranidae)

Hemiklonale Vererbung im hybridogenetische Rana grafi-Komplex (Anura: Ranidae)Im Rahmen der vorliegenden Studie wurde eine großflächige Untersuchung an südfranzösischen Wasserfröschen durchgeführt. Es wurden 31 Populationen mit 918 Tieren beprobt und mit Referenzproben genetisch verglichen. Die Genotypen der Tiere wurden mittels Allozymelektrophorese an sieben diagnostischen Loci bestimmt. Für Teilproben wurde das Alter, die Fläche der Erythrocyten und der DNA-Gehalt bestimmt. Die wichtigsten Ergebnisse der unterschiedlichen methodischen Ansätze lassen sich wie folgt zusammenfassen:(1) Es wurden neben einem großen Anteil von R. grafi (452 Tiere), R. perezi (200 Tiere) und R. ridibunda (254 Tiere) auch ein R. esculenta und zwei R. lessonae in der Camargue nachgewiesen.(2) Die Geschlechterverhältnisse der einzelnen Taxa waren stark gestört. Dies gilt besonders für den Hybriden R. grafi mit einem Männchenanteil von nur 7,3%. Das Geschlechterverhältnis von R. ridibunda ist besonders in PGR-Population zu Gunsten von Weibchen verschoben. Eine Modellierung von Populationen zeigt, daß wahrscheinlich R. grafi-Männchen durch eine gestörte Gametogenese für die gestörten Geschlechtsverhältnisse seiner Parentalformen verantwortlich ist.(3) Die genetische Analyse zeigt eine unerwartet hohe genetische Variabilität von R. ridibunda im Vergleich zu R. perezi und eine geographische Varianz der genetischen Variabilität in allen untersuchten Taxa. Die genetische Variabilität zeigte bei allen Wasserfroschformen in der Camargue ein Maximum.(4) Die hohe genetische Variabilität geht hauptsächlich, neben Einführung von verschiedenen genetischen R. ridibunda-Linien, auf Introgression zurück.

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Working on a baseline for the Kongsfjorden food web: production and properties of macroalgal particulate organic matter (POM), supplementary material

Macroalgae, in particular kelps, produce a large amount of biomass in Kongsfjorden, which is to a great extent released into the water in an annual cycle. As an example, the brown alga Alaria esculenta loses its blade gradually, 3 ± 0.8 % of the blade area per day (August 2012), thereby adding to the pool of particulate organic matter (POM) in the fjord. Upon release small thallus pieces are "aging" in that they are prone to leaching and serving as substrate for microorganisms, thus turning into palatable food for suspension and bottom feeders. In order to define a macroalgal baseline for the Kongsfjorden food web, stable isotopes d14C and d15N were measured in individuals of A. esculenta, Saccharina latissima and Laminaria digitata directly sampled after collection and in artificially produced POM (aPOM) of A. esculenta that was allowed to age under experimental conditions. In aPOM from this species sampled in August 2012 the C/N ratios decreased between d1 and d8 of a 14-day culture period in parallel to the fading photosynthetic activity of the algal fragments as demonstrated by use of an Imaging-PAM. Microscopic observations of the aPOM in August 2012 and 2013 revealed the frequent occurrence of small brown algal endo- and epiphytes. First feeding experiments with Mysis oculata (Mysids) and Hiatella arctica (Bivalves) showed that these species can ingest macroalgal POM. The importance of kelp-derived POM for the food web is subject of the current research.

Neogene floras and palaeoclimatic estimates from various sites of South-West China

Neogene climates and vegetation history of western Yunnan are reconstructed on the basis of known fossil plants using the Coexistence Approach (CA) and Leaf Margin Analysis (LMA). Four Neogene leaf floras from Tengchong, Jianchuan and Eryuan in southwestern China are analyzed by the CA, and the paleoclimatic data of one Miocene carpoflora from Longling and three Pliocene palynofloras from Longling, Yangyi and Eryuan are used for comparison. The Miocene vegetation of the whole of West Yunnan is subtropical evergreen broad-leaved forest, and a similar mean annual precipitation is inferred for Tengchong, Longling and Jianchuan. However, by the Late Pliocene a large difference in vegetation occurred between the two slopes of Gaoligong Mountain, western Yunnan. The region of Tengchong retained a subtropical evergreen broad-leaved forest vegetation, whereas in Yangyi and Eryuan a vertical vegetation zonation had developed, which consists, in ascending order, of humid evergreen broad-leaved, needle and broad-leaved mixed evergreen, and coniferous forests. Distinctively, the Late Pliocene vegetational patterns of West Yunnan were already very similar to those of the present, and the Pliocene mean annual precipitation in Tengchong was markedly higher than that of Yangyi and Eryuan. Considering that the overall vegetation of West Yunnan and the precipitation at Yangyi and Eryuan have undergone no distinct change since the Late Pliocene, we conclude that the Hengduan Mountains on the northern boundary of West Yunnan must have arisen after the Miocene and approached their highest elevation before the Late Pliocene. Furthermore, the fact of the eastern portion of the Tibetan Plateau underwent a slight uplift after the Late Pliocene is also supported.

Spontaneous heterosis in larval life-history traits of hemiclonal frog hybrids

European water frog hybrids Rana esculenta (Rana ridibunda × Rana lessonae) reproduce hemiclonally, transmitting only their ridibunda genome to gametes. We compared fitness-related larval life-history traits of natural R. esculenta from Poland with those of the two sympatric parental species and of newly generated F1 hybrids. Compared with either parental species, F1 hybrid offspring had higher survival, higher early growth rates, a more advanced developmental stage by day 49, and earlier metamorphosis, but similar mass at metamorphosis. R. esculenta from natural lineages had trait values intermediate between those of F1 offspring and of the two parental species. The data support earlier observations on natural R. esculenta that had faster larval growth, earlier metamorphosis, and higher resistance to hypoxic conditions compared with either parental species. Observing larval heterosis in F1 hybrids in survival, growth rate, and time to metamorphosis, however, at an even higher degree than in hybrids from natural lineages, demonstrates that heterosis is spontaneous and results from hybridity per se rather than from subsequent interclonal selection; in natural lineages the effects of hybridity and of clonal history are confounded. This is compelling evidence for spontaneous heterosis in hybrid clonals. Results on hemiclonal fish hybrids (Poeciliopsis) showed no spontaneous heterosis; thus, our frog data are not applicable to all hybrid clonals. Our data do show, however, that heterosis is an important potential source for the extensively observed ecological success of hybrid clonals. We suggest that heterosis and interclonal selection together shape fitness of natural R. esculenta lineages.

Cyanogenesis in Cassava1 : The Role of Hydroxynitrile Lyase in Root Cyanide Production

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 mu g IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans. (c) 2005 Elsevier B.V. All rights reserved.

Analysis of plant microfossils in archaeological deposits from two remote archipelagos: The Marshall Islands, Eastern Micronesia, and the Pitcairn Group, Southeast Polynesia

Pollen and starch residue analyses were conducted on 24 sediment samples from archaeological sites on Maloelap and Ebon Atolls in the Marshall Islands, eastern Micronesia, and Henderson and Pitcairn Islands in the Pitcairn Group, Southeast Polynesia. The sampled islands, two of which are mystery islands (Henderson and Pitcairn), previously occupied and abandoned before European contact, comprise three types of Pacific islands: low coral atolls, raised atolls, and volcanic islands. Pollen, starch grains, calcium oxylate crystals, and xylem cells of introduced non-Colocasia Araceae (aroids) were identified in the Marshalls and Henderson (ca. 1,900 yr B.P. and 1,200 yr B.P. at the earliest, respectively). The data provide direct evidence of prehistoric horticulture in those islands and initial fossil pollen sequences from Pitcairn Island. Combined with previous studies, the data also indicate a horticultural system on Henderson comprising both field and tree crops, with seven different cultigens, including at least two species of the Araceae. Starch grains and xylem cells of Ipomoea sp., possibly introduced 1. batatas, were identified in Pitcairn Island deposits dated to the last few centuries before European contact in 1790.

Evaluation of catalytic pyrolysis of cassava rhizome by principal component analysis

Rhizome of cassava plants (Manihot esculenta Crantz) was catalytically pyrolysed at 500 °C using analytical pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) method in order to investigate the relative effect of various catalysts on pyrolysis products. Selected catalysts expected to affect bio-oil properties were used in this study. These include zeolites and related materials (ZSM-5, Al-MCM-41 and Al-MSU-F type), metal oxides (zinc oxide, zirconium (IV) oxide, cerium (IV) oxide and copper chromite) catalysts, proprietary commercial catalysts (Criterion-534 and alumina-stabilised ceria-MI-575) and natural catalysts (slate, char and ashes derived from char and biomass). The pyrolysis product distributions were monitored using models in principal components analysis (PCA) technique. The results showed that the zeolites, proprietary commercial catalysts, copper chromite and biomass-derived ash were selective to the reduction of most oxygenated lignin derivatives. The use of ZSM-5, Criterion-534 and Al-MSU-F catalysts enhanced the formation of aromatic hydrocarbons and phenols. No single catalyst was found to selectively reduce all carbonyl products. Instead, most of the carbonyl compounds containing hydroxyl group were reduced by zeolite and related materials, proprietary catalysts and copper chromite. The PCA model for carboxylic acids showed that zeolite ZSM-5 and Al-MSU-F tend to produce significant amounts of acetic and formic acids.

Água residuária da mandioca como fertilizante orgânico em pasto de Brachiaria brizantha cv. Marandu

Brazil is the world's second largest producer of cassava, which most of the production is used to make flour and starch, generating large amounts of waste, cassava. In general, this waste is disposed of directly into the soil and waterways, causing serious environmental impacts. In view of this, the aim of this work was to evaluate the use of cassava wastewater water (cassava) as organic fertilizer in Brachiaria brizantha pasture. Marandu. The experiment was conducted at the Campus Macaíba the Federal University of Rio Grande do Norte. The treatments were increasing rates of cassava, applied to the soil as organic fertilizer. The experimental design was a randomized block design with six treatments and four replications. The treatments consist of cassava doses (0, 15, 30, 60 and 120 m³ ha- 1 ) and a treatment with mineral fertilizer (AM) in the form of NPK (140: 30: 120 kg ha-1 ). Three cuts with an interval of 60 days were carried out. The variables evaluated were: plant height; accumulation of morphological components of fodder; Trapping Light (IL); Leaf Area Index (LAI); Total chlorophyll (CT); Feature Production Seca (PMS). The dry matter production at a dose of 120 m³ha-1 had a quantitative increase, with a total production in 2796 kg ha-1 DM in the second cut, providing an increase of 493% compared to control, and the residual effect observed in the third cut caused a 100% increase compared to 0 m³ ha-1 . Comparing the PMS obtained with the use of AM and other treatments it was observed that it was the second cut equivalent to a dose of 120 m³ ha-1 and the third equivalence has been cut at doses 60 and 120 m³ha-1. For the variables plant height, IL, IAF, CT and leaves Mass adding cassava in the soil promoted a positive linear increase for the three cuts. However, with the AM the IAF was superior to the other treatments. The thatched mass reached its highest production (838 kg ha-1 DM) in the second cut when using a dose of 120 m³ha-1 . In dead material mass in the second and third sections, there was increased linearly increased total of 322 and 452% respectively, compared to a dose of 0 m³ha-1 . The use of cassava showed herbicidal effect for the variable mass of the undesirable negative linear response resulting in decreasing the amount of residue with increasing doses. Manipueira can be used as organic fertilizer in Brachiaria brizantha cv. Marandu for improvements in the productive characteristics, as promoted significant increases in 8 most of the variables studied, especially at a dose of 120 m³ha-1 . This benefits the environment by being alternative for disposal of cassava.