Dynamic tandem proximity-based proteomics-Protein trafficking at the proteome-scale.

TransitID is a new methodology based on proximity labeling allowing for the study of protein trafficking a the proteome scale.

An orthology-based methodology as a complementary approach to retrieve evolutionarily conserved A-to-I RNA editing sites.

Adar-mediated adenosine-to-inosine (A-to-I) mRNA editing is a conserved mechanism that exerts diverse regulatory functions during the development, evolution, and adaptation of metazoans. The accurate detection of RNA editing sites helps us understand their biological significance. In this work, with an improved genome assembly of honeybee (Apis mellifera), we used a new orthology-based methodology to complement the traditional pipeline of (de novo) RNA editing detection. Compared to the outcome of traditional pipeline, we retrieved many novel editing sites in CDS that are deeply conserved between honeybee and other distantly related insects. The newly retrieved sites were missed by the traditional de novo identification due to the stringent criteria for controlling false-positive rate. Caste-specific editing sites are identified, including an Ile>Met auto-recoding site in Adar. This recoding was even conserved between honeybee and bumblebee, suggesting its putative regulatory role in shaping the phenotypic plasticity of eusocial Hymenoptera. In summary, we proposed a complementary approach to the traditional pipeline and retrieved several previously unnoticed CDS editing sites. From both technical and biological aspects, our works facilitate future researches on finding the functional editing sites and advance our understanding on the connection between RNA editing and the great phenotypic diversity of organisms.

Transition Metal-Free Synthesis of meta-Bromo- and meta-Trifluoromethylanilines from Cyclopentanones by a Cascade Reaction.

Anilines are key constituents in biologically active compounds and often obtained from transition metal-catalyzed coupling of an aryl halide with an amine. In this work, we report a transition metal-free method for the synthesis of meta-bromo- and meta-trifluoromethylanilines starting from 3-tribromomethylcyclopentanone or 3-(2-bromo-2-chloro-1,1,1-trifluoroethyl)cyclopentanone, respectively. The scope of the transformation is shown by application of primary, secondary and aromatic amines. The reaction proceeds in acceptable to high yields (20-81 %), and allows for the synthesis of anilines with substitution patterns otherwise difficult to access.

Steps towards the international regulatory acceptance of non-animal methodology in safety assessment.

The current animal-based paradigm for safety assessment must change. In September 2016, the UK National Centre for Replacement, Refinement and Reduction of Animals in Research (NC3Rs) brought together scientists from regulatory authorities, academia and industry to review progress in bringing new methodology into regulatory use, and to identify ways to expedite progress. Progress has been slow. Science is advancing to make this possible but changes are necessary. The new paradigm should allow new methodology to be adopted once it is developed rather than being based on a fixed set of studies. Regulatory authorities can help by developing Performance-Based Standards. The most pressing need is in repeat dose toxicology, although setting standards will be more complex than in areas such as sensitization. Performance standards should be aimed directly at human safety, not at reproducing the results of animal studies. Regulatory authorities can also aid progress towards the acceptance of non-animal based methodology by promoting "safe-haven" trials where traditional and new methodology data can be submitted in parallel to build up experience in the new methods. Industry can play its part in the acceptance of new methodology, by contributing to the setting of performance standards and by actively contributing to "safe-haven" trials.

Embryotoxicity Induced by Triclopyr in Zebrafish (Danio rerio) Early Life Stage.

Triclopyr, an auxin-like herbicide that is widely employed for managing weeds in food crops and pastures, has been identified in various environmental settings, particularly aquatic ecosystems. Limited understanding of the environmental fate of this herbicide, its potential repercussions for both the environment and human health, and its insufficient monitoring in diverse environmental compartments has caused it to be recognized as an emerging contaminant of concern. In this study, we have investigated how triclopyr affects zebrafish, considering a new alternative methodology. We focused on the endpoints of developmental toxicity, neurotoxicity, and behavior of zebrafish embryos and larvae. We determined that triclopyr has a 96 h median lethal concentration of 87.46 mg/L (341.01 µM). When we exposed zebrafish embryos to sublethal triclopyr concentrations (0.5, 1, 5, 10, and 50 µM) for up to 144 h, we found that 50 µM triclopyr delayed zebrafish egg hatchability. Yolk sac malabsorption was significant at 0.5, 1, 5, and 10 µM triclopyr. In zebrafish larvae, uninflated swim bladder was significant only at 50 µM triclopyr. Furthermore, zebrafish larvae had altered swimming activity after exposure to 10 µM triclopyr for 144 h. In summary, these comprehensive results indicate that even low triclopyr concentrations can elicit adverse effects during early zebrafish development.

A new methodology for sporogony research of avian haemoproteids in laboratory-reared Culicoides spp., with a description of the complete sporogonic development of Haemoproteus pastoris.

BACKGROUND: Haemosporidian parasites of the genus Haemoproteus (Haemoproteidae) are widespread and cause haemoproteosis in birds and therefore, their diversity, ecology and evolutionary biology have become subjects of intensive research. However, the vectors and transmission patterns of haemoproteids as well as the epidemiology of haemoproteosis remain insufficiently investigated. Several species of Culicoides (Ceratopogonidae) support complete sporogony of haemoproteids belonging to the subgenus Parahaemoproteus. However, experimental research with these fragile insects is difficult to design in the field, particularly because their abundance markedly depends on seasonality. This is an obstacle for continuous sampling of sufficient numbers of naturally infected or experimentally exposed midges from wildlife. We developed simple methodology for accessing sporogonic development of haemoproteids in laboratory-reared Culicoides nubeculosus. This study aimed to describe the mosaic of methods constituting this methodology, which was applied for investigation of the sporogonic development of Haemoproteus (Parahaemoproteus) pastoris, a widespread parasite of the common starling Sturnus vulgaris. METHODS: The methodology consists of the following main stages: (i) laboratory rearing of C. nubeculosus from the egg stage to adult insects; (ii) selection of naturally infected birds, the donors of mature gametocytes to expose biting midges; (iii) experimental exposure of insects and their laboratory maintenance; and (iv) dissection of exposed insects. Biting midges were exposed to H. pastoris (cytochrome b lineage hLAMPUR01) detected in one naturally infected common starling. Engorged insects were dissected at intervals in order to follow sporogony. Microscopic examination and PCR-based methods were used to identify the sporogonic stages and to confirm the presence of the parasite lineage in infected insects, respectively. RESULTS: Culicoides nubeculosus females were successfully reared and exposed to H. pastoris, which completed sporogonic development 7-9 days post-infection when sporozoites were observed in the salivary glands. CONCLUSIONS: The new methodology is easy to use and non-harmful for birds, providing opportunities to access the sporogonic stages of Parahaemoproteus parasites, which might be used in a broad range of parasitology and genetic studies. Culicoides nubeculosus is an excellent experimental vector of subgenus Parahaemoproteus and is recommended for various experimental studies aiming investigation of sporogony of these pathogens.

Interfacial biomechanical properties of a dual acid-etched versus a chemically modified hydrophilic dual acid-etched implant surface: an experimental study in Beagles.

BACKGROUND: The high survival clinical success rates of osseointegration are requisites for establishing a long-term biomechanical fixation and load-bearing potential of endosseous oral implants. The objective of this preclinical animal study was to evaluate the effect of surface microtopography and chemistry on the early stages of biomechanical rigidity with a sandblasted, dual acid-etched surface, with or without an additional chemical modification (SAE-HD and SAE, respectively), in the tibia of Beagle dogs. METHODS: Two pairs of implants, with the same macrogeometry but different surface technology ((a) dual acid-etched surface treatment with hydrochloric and sulfuric acid followed by microwave treatment and insertion in isotonic saline solution to increase hydrophilicity (SAE-HD) (test, n = 12) and (b) dual acid-etched surface (SAE) (control, n = 12)), were installed bilaterally in the proximal tibia of six Beagle dogs. In order to determine the effect of surface modification on biomechanical fixation, a test protocol was established to assess the torque and a complete set of intrinsic properties. Maximum removal torque (in N cm) was the primary outcome measure, while connection stiffness (N cm/rad) and removal energy (× 10-2J) were the secondary outcome measures and were assessed after 2 and 4 weeks in vivo. A general linear statistical model was used and performed for significant differences with the one-way ANOVA followed by Tukey post hoc test (P < 0.05). RESULTS: The removal torque values did not reveal significant statistical differences between SAE-HD and SAE implants at any observation times (P = 0.06). Although a slight increase over time could be observed in both test and control groups. SAE-HD showed higher removal energy at 4 weeks (999.35 ± 924.94 × 10- 2 J) compared to that at 2 weeks (421.94 ± 450.58 × 10- 2 J), while SAE displayed lower values at the respective healing periods (P = 0.16). Regarding connection stiffness, there were no significant statistical differences neither within the groups nor over time. There was a strong, positive monotonic correlation between removal torque and removal energy (=0.722, n = 19, P < 0.001). CONCLUSIONS: In this study, no significant differences were observed between the specific hydrophilic (SAE-HD) and hydrophobic (SAE) surfaces evaluated, in terms of biomechanical properties during the early osseointegration period.

Use of Frozen Leaves for Morpho-anatomical Characterization of Nectandra megapotamica (Spreng) Mez, Lauraceae

Abstract Nectandra megapotamica (Spreng.) Mez. it is a native tree species of the Atlantic Forest, commonly known as canela-preta. The species has some anti-inflammatory, antitumor and antirheumatic properties among others. In this work the use of frozen plant material for microscopy analysis was tested. In addition, the leaf morpho-anatomy of the species was characterized which allowed to perform a structural description and to identify structures of secretion and storage of essential oil. The plant material was prepared for analyzes in optical, fluorescence and scanning electron microscopy. The leaf anatomy shows glabrous epidermis, unistratified, paracytic stomata, absence of trichomes, polyhedral epidermal cells. Some typical family characteristics were observed as a closed arc-shaped bicollateral bundle vascular system and dorsiventral mesophyll. The structures of secretion and storage of essential oil were identified as secretory cavities.

Doppler ultrasound venous mapping of the lower limbs.

BACKGROUND: The study aim was to test the accuracy (intra and interobserver variability), sensitivity, and specificity of a simplified noninvasive ultrasound methodology for mapping superficial and deep veins of the lower limbs. METHODS: 62 consecutive patients, aged 62 ± 11 years, were enrolled. All underwent US-examinations, performed by two different investigators, of both legs, four anatomical parts, and 17 veins, to assess the interobserver variability of evaluation of superficial and deep veins of the lower limbs. RESULTS: Overall the agreement between the second versus the first operator was very high in detecting reflux (sensitivity 97.9, specificity 99.7, accuracy 99.5; P = 0.80 at McNemar test). The higher CEAP classification stages were significantly associated with reflux (odds ratio: 1.778, 95% confidence interval: 1.552-2.038; P < 0.001) as well as with thrombosis (odds ratio: 2.765, 95% confidence interval: 1.741-4.389; P < 0.001). Thus, our findings show a strict association between the symptoms of venous disorders and ultrasound evaluation results for thrombosis or reflux. CONCLUSION: This study demonstrated that our venous mapping protocol is a reliable method showing a very low interobserver variability, which makes it accurate and reproducible for the assessment of the morphofunctional status of the lower limb veins.