Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.     

Hypolipidemic and antiatherosclerotic potential of Pleurotus ostreatus, cultived by submerged fermentation in the high-fat diet fed rats

The ability of Pleurotus ostreatus biomass, cultived by submerged fermentation, to produce beneficial effect on lipid profile and macrophages activity during a high-fat diet (HFD) for a long-term intake was investigated. Blood samples were collected through cardiac puncture to measure the plasma cholesterol, triglycerides, low-density protein (LDL), high-density protein (HDL), aspartate aminotransferase (AST) activity, urea-blood urea nitrogen (BUN)/creatinine ratio of rats fed on an HFD for 4 months. Dosage of lipid hydroperoxides was carried out on methanolic extract of liver tissue. Peritoneal macrophages activity was evaluated in relation to the superoxide anion, hydrogen peroxide and nitric oxide production, phagocytosis and lysosomal volume. The administration of P. ostreatus significantly altered the lipid profile and oxidative stress as related to the LDL and triglycerides decrease and inhibitory effects on superoxide anion and hydrogen peroxide production. All findings of this study lead us to suggest that the P. ostreatus maybe a beneficial agent in the hyperlipidemia and atherosclerosis treatments.     

Putative local adaptations modulate the interactions of the carnivorous plant Drosera uniflora Willd (1809) (Droseraceae) with cushion and shrub nurse plants

Cushion and shrub plants are typical high mountain nurse plants. In Magellanic tundras of the Cordillera del Sarao on the coast of Chile, the carnivorous plant Drosera uniflora grows in association with cushions of Donatia fascicularis and the shrubs Chusquea montana var. nigricans and Lepidothamnus fonkii. The different microhabitats for recruitment, in addition to the limited gene flow of D. uniflora, enable us to hypothesise that this plant manifests putative local adaptations, expressed in seed germination and population abundance. Our aim was to evaluate the local adaptation of D. uniflora to cushion and shrub microhabitats by estimating germination and abundance. Local adaptation of seed germination was determined by means of reciprocal transplant experiments. Abundance was determined in small plots located inside, on the edge and outside the nurse plants. Seed origin and growth substrate play a decisive role in D. uniflora germination. Seeds that originate from cushions plant habitat and germinate in the same substrate do so in greater numbers than when those which originate from shrubby habitats. Conversely, seeds that originate from shrubby habitat and germinate in the same substrate do not exhibit significant differences. Abundance was always greater inside than outside of the nurse plants. We concluded that availability and quality of different microhabitats for seed germination and recruitment, together with a limited gene flow, would trigger putative local adaptations in D. uniflora, modulating a long history of interactions with the plants that act as nurse plants.     

<Emphasis Type="Italic">NmEXT</Emphasis> Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis>

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.     

Advantages of granivory in seasonal environments: feeding ecology of an arboreal seed predator in Amazonian forests

Specialized seed predators are uncommon in arboreal vertebrate assemblages, and the hypothesis that consuming seeds of immature fruits – which may be available for relatively long periods compared to mature fruit – could reduce seasonal food scarcity experienced by generalist frugivores remains largely untested. To test this hypothesis, we examined the diet and feeding ecology of bald‐faced saki monkeys Pithecia irrorata in a largely intact forest mosaic of southeastern Peru based on systematic monitoring of five habituated groups over a three‐year period and compared the relative availability of ripe and unripe fruits in their diet. Plant phenology data from individual tree crowns showed that, compared to ripe fruits, immature fruits were available in more tree species, in greater quantities, and for longer periods. Despite pronounced community‐wide seasonal changes in fruit production at our study area, feeding patterns of bald‐faced saki remained largely invariant: fruits comprised approximately 95% of the species’ monthly diet, with seeds alone accounting for 75%, with no major monthly dietary shifts. The flexible exploitation by this species of a consistently available food supply for which it faces little competition likely reduces foraging effort and consumption of less desirable foods, even during prolonged periods of overall fruit scarcity. The relative rarity of immature fruit specialists in tropical forests may reflect the fact that processing the hard pericarps and neutralizing the toxicities of immature seeds present substantial evolutionary hurdles that few arboreal vertebrate species have overcome.     

Variation in liana abundance and biomass along an elevational gradient in the tropical Atlantic Forest (Brazil)

Lianas play a key role in forest structure, species diversity, as well as functional aspects of tropical forests. Although the study of lianas in the tropics has increased dramatically in recent years, basic information on liana communities for the Brazilian Atlantic Forest is still scarce. To understand general patterns of liana abundance and biomass along an elevational gradient (0–1,100 m asl) of coastal Atlantic Forest, we carried out a standard census for lianas ≥1 cm in five 1-ha plots distributed across different forest sites. On average, we found a twofold variation in liana abundance and biomass between lowland and other forest types. Large lianas (≥10 cm) accounted for 26–35% of total liana biomass at lower elevations, but they were not recorded in montane forests. Although the abundance of lianas displayed strong spatial structure at short distances, the present local forest structure played a minor role structuring liana communities at the scale of 0.01 ha. Compared to similar moist and wet Neotropical forests, lianas are slightly less abundant in the Atlantic Forest, but the total biomass is similar. Our study highlights two important points: (1) despite some studies have shown the importance of small-scale canopy disturbance and support availability, the spatial scale of the relationships between lianas and forest structure can vary greatly among tropical forests; (2) our results add to the evidence that past canopy disturbance levels and minimum temperature variation exert influence on the structure of liana communities in tropical moist forests, particularly along short and steep elevational gradients.     

High-level secretion of growth hormone by retrovirally transduced primary human keratinocytes

A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/10⁶ cells/d) and mGH (11 μg/10⁶ cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice (lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase (p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy.     

Global diversity of crabs (Aeglidae: Anomura: Decapoda) in freshwater

The freshwater anomuran crabs of the family Aeglidae are all restricted to southern South America occurring in Chile, Brazil, Bolivia, Uruguay, Paraguay, and Argentina. The family consists of a single genus, Aegla, containing 63 currently described species. There are another 5–10 known yet undescribed species to complement this diversity. The aeglids occur in freshwater lakes, streams, rivers, and in caves with freshwater. The origin of the family appears to be from marine ancestors from the Pacific invading streams in Chile about 75 mya radiating both in Chile and again on the eastern side of the Andes, particularly in Brazil. Of the 63 species, 23 or 36.5% are considered under threat and are in need of conservation action. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.