Penetration by Botryosphaeriaceae species in avocado,guava and persimmon fruit during postharvest

Botryosphaeriaceae species have a wide host range and a worldwide distribution. These fungal species can colonize several plant organs, such as the trunk, leaves and fruit. Some Botryosphaeriaceae species cause important diseases on persimmon, avocado and guava fruit. However, there is a lack of information regarding the mechanisms of penetration by Botryosphaeriaceae species on these tropical and subtropical fruits. This study aimed to better understand the mechanisms involved in fungal penetration, host specificity and aggressiveness of Botryosphaeria dothidea, Lasiodiplodia pseudotheobromae and Neofusicoccum parvum on avocado (Persea americana), guava (Psidium guajava) and persimmon (Diospyros kaki) fruit. Scanning electron microscopy (SEM) image analysis showed that in avocado fruit, the three studied Botryosphaeriaceae species penetrated through lenticels. In guava fruit, penetration through stomata was verified for Botryosphaeria dothidea and Neofusicoccum parvum. In persimmon fruit, an appressoria-like structure was observed for B. dothidea, which suggests direct penetration. Disease incidence in wounded fruit was 24% higher than in non-wounded fruit. L. pseudotheobromae and N. parvum showed differences in aggressiveness in guava fruit. The longest incubation period was observed for N. parvum inoculated on guava, with an average of 4.5 days, and the shortest incubation period was verified for B. dothidea inoculated on avocado, with an average of 2.8 days. The area under the disease progress curve (AUDPC) did not differ between Botryosphaeriaceae species on avocado, whereas on guava and persimmon fruit, the AUDPC was lower for B. dothidea. The information regarding penetration mechanisms and aggressiveness is important to improve postharvest disease control strategies.     

Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon

We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and ²H₂O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2–8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to ²H₂O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from ²H₂O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33–0.90% per day (half-life of 77–210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis. carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes     

Atlantic moonfishes: independent pathways of karyotypic and morphological differentiation

Fish of the genus Selene, known as lookdowns or moonfish, are one of the most morphologically derived groups of the family Carangidae, whose phylogenetic relationships are still largely unknown. In this study, we discuss karyoevolutionary aspects of three representatives of this genus from the Western Atlantic: Selene brownii (2n = 48; FN = 48), Selene setapinnis (2n = 46; FN = 48), and Selene vomer (2n = 48; FN = 50). Their body patterns were also investigated and compared to one another and in relation to two other species of different genera. Two mechanisms of karyotypic evolution seem to have acted in the diversification of this genus, namely pericentric inversions and centric fusions. Mapping of rDNA sequences showed that chromosome pairs bearing 5S rDNA sites are similar, whereas those bearing 18 rDNA sites are morphologically distinct while apparently also exhibiting interspecies synteny. Although the nucleolar organizer-bearing chromosomes are extremely efficient cytotaxonomic markers among Selene species, others cytogenetic patterns of these species are relatively conserved. Hybridization with telomeric probes (TTAGGG)_n did not exhibit interstitial telomeric sites (ITS), especially in S. setapinnis, where, along with a reduction in diploid number, a large metacentric pair derived from centric fusion is present. Data obtained by geometric morphometrics enable a clear morphological distinction among the three species, as well as in relation to two other species of the genus Caranx and Oligoplites. Data obtained suggest that morphologic evolution in Selene species was primarily dissociated from visible changes that occurred at the chromosomal level.     

Nurse effect of Bolax gummifera cushion plants in the alpine vegetation of the Chilean Patagonian Andes

Abstract. It has been proposed that in the harsh arctic and alpine climate zones, small microtopographic variations that can generate more benign conditions than in the surrounding environment could be perceived as safe sites for seedling recruitment. Cushion plants can modify wind pattern, temperature and water availability. Such modifications imply that cushion plants could act as ‘nurse plants’ facilitating the recruitment of other species in the community. This effect should be more evident under stressful conditions. We tested these hypotheses comparing the number of species that grow inside and outside Bolax gummifera cushions at two elevations (700 and 900 m a.s.l.) in the Patagonian Andes of Chile (50°S). At both elevations, and in equivalent areas, the number of species was registered within and outside cushions. A total of 36 and 27 plant species were recorded either within or outside B. gummifera cushions at 700 and 900 m a.s.l., respectively. At 700 m a.s.l., 33 species were recorded growing within cushions and 29 outside them, while at 900 m a.s.l. these numbers were 24 and 13 respectively. At both elevations there were significantly more species growing within than outside cushions, and the proportion of species growing within cushions increased with elevation. Thus there is a nurse effect of cushion plants and it is more evident at higher elevations. Shelter from wind and increased soil water availability seem to be the factors that increase plant recruitment within cushions.     

Mammalian RNAi: a practical guide

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Caenorhabditis elegans and Drosophila melanogaster genomes. Recent advances in the design and delivery of targeting molecules now permit efficient and highly specific gene silencing in mammalian systems as well. RNAi offers a simple, fast, and cost-effective alternative to existing gene targeting technologies both in cell-based and in vivo settings. Synthetic small interfering RNA (siRNA) and retroviral short hairpin RNA (shRNA) libraries targeting thousands of human and mouse genes are publicly available for high-throughput genetic screens, and knockdown animals can be rapidly generated by lentivirus-mediated transgenesis. RNAi also holds great promise as a novel therapeutic approach. This review provides insight into the current gene silencing techniques in mammalian systems.     

Mercury Levels in Barbadian Fisher Folk and Commonly Caught Fish Species

Ocean fish are an important staple food for many Caribbean persons. Some species, however, have high Hg concentration levels and represent a significant source of Hg exposure. This study assessed Hg concentrations in fish species commonly caught and consumed in Barbados as well as Hg exposure of those who work in the fishing industry. Total Hg concentrations were measured in 100 fish samples. Human Hg concentrations were determined from hair snippets taken from 46 fisher folk and fish consumption habits evaluated using interview-administered questionnaires. Mercury levels varied by fish species with predatory fish species having the highest concentrations. Low Hg concentrations were found in dolphinfish (Coryphaena hippurus) and flyingfish (Hirundichthys affinis). Reported fish consumption rates were high and varied significantly between men and women, 2675 ± 1600 and 1275 ± 600 (g/week), respectively (p = 0.01). The median hair Hg concentration was 3.1 ± 2.3 μg/g (range: 0.01–15.6 μg/g) and significantly associated with consumption of “high-risk” fish species (p = .02). These results show that it is possible to make informed choices concerning fish consumption habits in order to maximize benefits while minimizing the effect of Hg exposures, especially for sensitive groups such as pregnant women and infants.     

Computer simulation of hydrodynamic properties of semiflexible macromolecules: randomly broken chains, wormlike chains, and analysis of properties of DNA

The translational and rotational diffusion coefficients and the intrinsic viscosity of semiflexible, randomly broken, and wormlike chains have been obtained by Monte Carlo simulation in the context of the rigid-body treatment. Both approximate and rigorous rigid-body hydrodynamics are used, so that the error introduced by the approximate methods can be evaluated. A randomly broken chain and a wormlike chain having the same contour length and persistence length have the same radius of gyration but different values for any of the hydrodynamic properties. The two types of chains are compared in this regard. Considering that the cross section of the chain is represented by a cylinder better than by a string of spheres, we devise a cylindrical correction to be applied to the results simulated for chains of beads. Application is made to the analysis of experimental data for the translational and rotational coefficients of DNA fragments with up to 10(3) base pairs, obtaining the persistence length for each model. The values for the wormlike chain agree well with model-independent values obtained from radii of gyration and with other literature data at varying ionic strength. The randomly broken chain is equally able to reproduce the experimental length dependence of the properties, but the resulting persistence length may be too high.     

Glucose-induced activation of rubidium transport and water flux in sunflower root systems

Excised 20-d-old sunflower roots (Helianthus annuus L. cv. Sun-Gro 393) were used to study the effect of different sugars on rubidium and water fluxes. The roots sensed and absorbed glucose from the external medium inducing the activation of rubidium accumulated in the root (Rb(+) root), the flux of exuded rubidium (J(Rb)) and, to a lesser degree, the exudation rate (J(v)). These effects were also triggered by fructose, but not by 6-deoxyglucose (6-dG), a glucose analogue which is not a substrate for hexokinase (HXK). The effect of 2-deoxyglucose (2-dG), an analogue that is phosphorylated but not further metabolized, was complex, suggesting an inhibitory effect on solute transport to the xylem. The amounts of glucose required to activate rubidium and water fluxes were similar to those previously reported to regulate different processes in other plants (0.5--10 mM). When sorbitol was used instead of glucose, neither rubidium uptake (Rb(+) root plus J(Rb)) nor J(v) was activated. It is proposed that glucose present in the root plays an important signalling role in the regulation of Rb(+) (K(+)) and water transport in plant roots.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.