CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.     

Technical viability of the production,partial purification and characterisation of inulinase using pretreated agroindustrial residues

The present work aimed to study the viability of the use of sugarcane molasses and corn steep liquor (CSL) in a sequential inulinase production performing an up-stream pretreatment of these agroindustrial residues. A sequential strategy was used applying three central composite rotatable designs (CCRDs) to optimise medium composition, followed by a down-stream step. The medium containing 150 g L⁻¹ molasses, 50 g L⁻¹ CSL and 6 g L⁻¹ yeast extract, yielded a maximum inulinase production of 1,294 ± 7 U mL⁻¹, after 72 h of fermentation. A down-stream evaluation was carried out using an expanded bed of Streamline DAE resin (Pharmacia), with and without the up-stream treatment. The results showed that the enzyme could not be recovered from the non-pretreated medium, whereas a yield of 91% was obtained in the adsorption stage from the medium prepared with the up-stream treatment, showing the viability of producing the enzyme inulinase from agroindustrial residues using the integrated process.     

Life in Burrows Channelled the Morphological Evolution of the Skull in Rodents: the Case of African Mole-Rats (Bathyergidae,Rodentia)

African mole-rats are fossorial rodents that consist of five chisel-tooth digging genera (Heterocephalus, Heliophobius, Georychus, Fukomys, and Cryptomys) and one scratch digger (Bathyergus). They are characterized by striking physiological, morphological, and behavioral adaptations intimately related to their subterranean life. The influence of their mode of life in shaping the cranial morphology has yet to be evaluated in comparison to other Ctenohystrica, especially fossorial genera, which include the subterranean genera Spalacopus and Ctenomys. In our study, we seek to determine to what extent subterranean life affects the morpho-functional properties of the skull among fossorial ctenohystricans. 3D geometric morphometric analyses were performed on 277 skulls, encompassing 63 genera of Ctenohystrica, and complemented by biomechanical studies. African mole-rats and other subterranean Ctenohystrica, especially chisel-tooth diggers, have a short snout, a wide cranium with enlarged zygomatic arches, and a strongly hystricognathous mandible. Even if convergences are also manifest between most fossorial Ctenohystrica, subterranean rodents departed from the main ctenohystrican allometric trends in having a skull shape less size-dependent, but under stronger directional selection with intense digging activity as a major constraint. African mole-rats, notably chisel-tooth diggers, show important mechanical advantage for the temporalis muscles favoring higher forces at the bite point, while mechanical advantage of the superficial masseter muscles is lower compared to other Ctenohystrica. If subterranean species can be clearly discriminated based on their skull morphology, the intrinsic mosaic of anatomical characters of each genus (e.g., skull, teeth, and muscles) can be understood only in the light of their ecology and evolutionary history.     

Hexagonal Boron Nitride‐Based Electrolyte Composite for Li‐Ion Battery Operation from Room Temperature to 150 °C

Batteries for high temperature applications capable of withstanding over 60 °C are still dominated by primary cells. Conventional rechargeable energy storage technologies which have exceptional performance at ambient temperatures employ volatile electrolytes and soft separators, resulting in catastrophic failure under heat. A composite electrolyte/separator is reported that holds the key to extend the capability of Li‐ion batteries to high temperatures. A stoichiometric mixture of hexagonal boron nitride, piperidinium‐based ionic liquid, and a lithium salt is formulated, with ionic conductivity reaching 3 mS cm^?1, electrochemical stability up to 5 V and extended thermal stability. The composite is used in combination with conventional electrodes and demonstrates to be stable for over 600 cycles at 120 °C, with a total capacity fade of less than 3%. The ease of formulation along with superior thermal and electrochemical stability of this system extends the use of Li‐ion chemistries to applications beyond consumer electronics and electric vehicles.     

DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Molecular characterization of the rostral-most somites in early somitic stages of the chick embryo

Segmentation consists on the progressive formation of repetitive embryonic structures, named somites, which are formed from the most rostral part of the presomitic mesoderm. Somites are subdivided into anterior and posterior compartments and several genes are differentially expressed in either compartment. This has provided evidence for the importance of establishing the anterior-posterior polarity within each somite, which is critical for the correct segmented pattern of the adult vertebrate body. Although all somites appear morphologically similar, fate map studies have shown that the first 4 somites do not give rise to segmented structures, in contrast to more posterior ones. Moreover, in several somitogenesis-related mutants the anterior somites are not affected while posterior somites present clear defects or do not form at all. Altogether these data suggest relevant differences between rostral and caudal somites. In order to check for molecular differences between anterior and posterior somites, we have performed a detailed expression pattern analysis of several Notch signalling related genes. For the first time, we show that the somitic expression pattern profile is not the same along the anterior-posterior axis and that the differences are not observed always at the same somite level.     

Morphology of the stomach of the tropical house gecko Hemidactylus mabouia (Squamata: Gekkonidae)

Rodrigues Sartori, S. S., Nogueira, K. O. P. C., Rocha, A. S. and Neves, C. A. 2011. Morphology of the stomach of the tropical house gecko Hemidactylus mabouia (Squamata: Gekkonidae). —Acta Zoologica (Stockholm) 92 : 179–186. Hemidactylus mabouia is a common species in Brazil, which facilitates its use in research in several areas and allows display it as a benchmark for studies with reptiles. To study the morphology of the stomach of H. mabouia, we carried out anatomical, histological and histochemical analysis. The stomach of H. mabouia is ‘J’ shaped and can be divided into oral fundic (OF), aboral fundic (AF) and pyloric regions. The surface epithelium is composed of mucosecretory cells (MC) containing neutral mucins. In the lamina propria of the OF region, are large ramified tubulo‐acinar glands, which become smaller, less ramified and more tubular towards the AF region, and are simple tubular and short in the pyloric region. The fundic glands contain differentiated neck and pit. The neck is composed of MC containing neutral mucins and the pit is made of oxyntopeptic cells (OC). The OC of the OF region contained many zymogen granules, while those of the AF region contained few zymogen granules and many mitochondria, which suggests the existence of a gradient of pepsinogen and hydrochloric acid secretion. Pyloric glands consisted of MC containing neutral mucins and both argyrophil and argentaffin endocrine cells.     

THO2, a core member of the THO/TREX complex,is required for microRNA production in Arabidopsis

The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA‐dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA‐targeted mRNAs. Yeast two‐hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA‐processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine‐rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.     

Leptospirosis in Rio Grande do Sul,Brazil: An Ecosystem Approach in the Animal-Human Interface

Background

Leptospirosis is an epidemic-prone neglected disease that affects humans and animals, mostly in vulnerable populations. The One Health approach is a recommended strategy to identify drivers of the disease and plan for its prevention and control. In that context, the aim of this study was to analyze the distribution of human cases of leptospirosis in the State of Rio Grande do Sul, Brazil, and to explore possible drivers. Additionally, it sought to provide further evidence to support interventions and to identify hypotheses for new research at the human-animal-ecosystem interface.

Methodology and findings

The risk for human infection was described in relation to environmental, socioeconomic, and livestock variables. This ecological study used aggregated data by municipality (all 496). Data were extracted from secondary, publicly available sources. Thematic maps were constructed and univariate analysis performed for all variables. Negative binomial regression was used for multivariable statistical analysis of leptospirosis cases. An annual average of 428 human cases of leptospirosis was reported in the state from 2008 to 2012. The cumulative incidence in rural populations was eight times higher than in urban populations. Variables significantly associated with leptospirosis cases in the final model were: Parana/Paraiba ecoregion (RR: 2.25; CI_95%: 2.03–2.49); Neossolo Litolítico soil (RR: 1.93; CI_95%: 1.26–2.96); and, to a lesser extent, the production of tobacco (RR: 1.10; CI_95%: 1.09–1.11) and rice (RR: 1.003; CI_95%: 1.002–1.04).

Conclusion

Urban cases were concentrated in the capital and rural cases in a specific ecoregion. The major drivers identified in this study were related to environmental and production processes that are permanent features of the state. This study contributes to the basic knowledge on leptospirosis distribution and drivers in the state and encourages a comprehensive approach to address the disease in the animal-human-ecosystem interface.