Tolerance to Copper and to Salinity in Daphnia longispina: Implications within a Climate Change Scenario

Considering IPPC climate change scenarios, it is pertinent to predict situations where coastal ecosystems already impacted with chemical contamination became exposed to an additional stressor under a future scenario of seawater intrusion. Accordingly, the present study aimed at evaluating if a negative association between tolerance to a metal and to saltwater exists among genotypes of a freshwater organism. For this, five clonal lineages of the cladoceran Daphnia longispina O.F. Müller, exhibiting a differential tolerance to lethal levels of copper, were selected. Each clonal lineage was exposed to lethal and sublethal concentrations of sodium chloride (assumed as a protective surrogate to evaluate the toxicity of increased salinity to freshwater organisms). Mortality, time to release the first brood and total number of neonates per female were monitored and the somatic growth rate and intrinsic rate of natural increase were computed for each clonal lineage. Data here obtained were compared with their lethal responses to copper and significant negative correlations were found. These results suggest that genetically eroded populations of D. longispina, due to copper or salinity, may be particularly susceptible to a later exposure to the other contaminant supporting the multiple stressors differential tolerance.     

Human Left Ventral Premotor Cortex Mediates Matching of Hand Posture to Object Use

Visuomotor transformations for grasping have been associated with a fronto-parietal network in the monkey brain. The human homologue of the parietal monkey region (AIP) has been identified as the anterior part of the intraparietal sulcus (aIPS), whereas the putative human equivalent of the monkey frontal region (F5) is located in the ventral part of the premotor cortex (vPMC). Results from animal studies suggest that monkey F5 is involved in the selection of appropriate hand postures relative to the constraints of the task. In humans, the functional roles of aIPS and vPMC appear to be more complex and the relative contribution of each region to grasp selection remains uncertain. The present study aimed to identify modulation in brain areas sensitive to the difficulty level of tool object - hand posture matching. Seventeen healthy right handed participants underwent fMRI while observing pictures of familiar tool objects followed by pictures of hand postures. The task was to decide whether the hand posture matched the functional use of the previously shown object. Conditions were manipulated for level of difficulty. Compared to a picture matching control task, the tool object – hand posture matching conditions conjointly showed increased modulation in several left hemispheric regions of the superior and inferior parietal lobules (including aIPS), the middle occipital gyrus, and the inferior temporal gyrus. Comparison of hard versus easy conditions selectively modulated the left inferior frontal gyrus with peak activity located in its opercular part (Brodmann area (BA) 44). We suggest that in the human brain, vPMC/BA44 is involved in the matching of hand posture configurations in accordance with visual and functional demands.     

Learning Reflectance Confocal Microscopy of Melanocytic Skin Lesions through Histopathologic Transversal Sections

Histopathologic interpretation of dermoscopic and reflectance confocal microscopy (RCM) features of cutaneous melanoma was timidly carried out using perpendicular histologic sections, which does not mimic the same plane of the image achieved at both techniques (horizontal plane). The aim of this study was to describe the transverse histologic sections research technique and correlate main dermoscopic features characteristic of cutaneous melanoma (atypical network, irregular globules and pseudopods) with RCM and histopathology in perpendicular and transverse sections in order to offer a more precise interpretation of in vivo detectable features. Four melanomas and 2 nevi with different dermoscopic clues have been studied. Lesion areas that showed characteristic dermoscopic features were imaged by dermoscopy and confocal microscopy and directly correlated with histopathology in perpendicular and transverse sections. We presented the possibility to perform transverse sections as a new approach to understand RCM features. Atypical network showed different aspects in the 2 melanomas: in one case it was characterized by pleomorphic malignant melanocytes with tendency to form aggregates, whereas in the other elongated dendritic cells crowded around dermal papillae, some of them forming bridges that resembled the mitochondrial aspect at confocal and histopathology transversal sections. Pigment globules in melanomas and nevi differed for the presence of large atypical cells in the former, and pseudopods showed up as elongated nests protruded toward the periphery of the lesion. Transverse histologic research sections have a consistent dermoscopic and confocal correlate, and it may represent an help in confocal feature interpretation and an advance in improving melanoma diagnosis and knowledge of the biology of melanocytic lesions.     

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.     

Glocal Clinical Registries: Pacemaker Registry Design and Implementation for Global and Local Integration – Methodology and Case Study

Background

The ability to apply standard and interoperable solutions for implementing and managing medical registries as well as aggregate, reproduce, and access data sets from legacy formats and platforms to advanced standard formats and operating systems are crucial for both clinical healthcare and biomedical research settings.

Purpose

Our study describes a reproducible, highly scalable, standard framework for a device registry implementation addressing both local data quality components and global linking problems.

Methods and Results

We developed a device registry framework involving the following steps: (1) Data standards definition and representation of the research workflow, (2) Development of electronic case report forms using REDCap (Research Electronic Data Capture), (3) Data collection according to the clinical research workflow and, (4) Data augmentation by enriching the registry database with local electronic health records, governmental database and linked open data collections, (5) Data quality control and (6) Data dissemination through the registry Web site. Our registry adopted all applicable standardized data elements proposed by American College Cardiology / American Heart Association Clinical Data Standards, as well as variables derived from cardiac devices randomized trials and Clinical Data Interchange Standards Consortium. Local interoperability was performed between REDCap and data derived from Electronic Health Record system. The original data set was also augmented by incorporating the reimbursed values paid by the Brazilian government during a hospitalization for pacemaker implantation. By linking our registry to the open data collection repository Linked Clinical Trials (LinkedCT) we found 130 clinical trials which are potentially correlated with our pacemaker registry.

Conclusion

This study demonstrates how standard and reproducible solutions can be applied in the implementation of medical registries to constitute a re-usable framework. Such approach has the potential to facilitate data integration between healthcare and research settings, also being a useful framework to be used in other biomedical registries.     

Glycosaminoglycan remodeling during chondrogenic differentiation of human bone marrow−/synovial-derived mesenchymal stem/stromal cells under normoxia and hypoxia

Glycosaminoglycans (GAGs) are major components of cartilage extracellular matrix (ECM), which play an important role in tissue homeostasis not only by providing mechanical load resistance, but also as signaling mediators of key cellular processes such as adhesion, migration, proliferation and differentiation. Specific GAG types as well as their disaccharide sulfation patterns can be predictive of the tissue maturation level but also of disease states such as osteoarthritis. In this work, we used a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to perform a comparative study in terms of temporal changes in GAG and disaccharide composition between tissues generated from human bone marrow- and synovial-derived mesenchymal stem/stromal cells (hBMSC/hSMSC) after chondrogenic differentiation under normoxic (21% O₂) and hypoxic (5% O₂) micromass cultures. The chondrogenic differentiation of hBMSC/hSMSC cultured under different oxygen tensions was assessed through aggregate size measurement, chondrogenic gene expression analysis and histological/immunofluorescence staining in comparison to human chondrocytes. For all the studied conditions, the compositional analysis demonstrated a notable increase in the average relative percentage of chondroitin sulfate (CS), the main GAG in cartilage composition, throughout MSC chondrogenic differentiation. Additionally, hypoxic culture conditions resulted in significantly different average GAG and CS disaccharide percentage compositions compared to the normoxic ones. However, such effect was considerably more evident for hBMSC-derived chondrogenic aggregates. In summary, the GAG profiles described here may provide new insights for the prediction of cartilage tissue differentiation/disease states and to characterize the quality of MSC-generated chondrocytes obtained under different oxygen tension culture conditions.

     

On the release of fluoride from biofilm reservoirs during a cariogenic challenge: an in situ study

Abstract

Biofilm fluoride reservoirs may be a source of fluoride to the fluid phase during a sugar challenge reducing tooth mineral loss. However, the evidence for that is conflicting and has not been studied in biofilms containing different fluoride levels. In order to test fluoride release from biofilms with distinct fluoride concentrations, biofilms were grown in situ exposed to a combination of placebo, calcium and fluoride rinses forming biofilms with no (fluoride-free rinses), low (fluoride-only rinses) or high (calcium followed by fluoride rinses) fluoride concentrations, and collected before and 5?min after a sucrose challenge. Rinsing with fluoride increased fluoride concentration in the biofilm (p?<?0.05), mainly when a calcium pre-rinse was used before the fluoride (p?<?0.05). However, after a sugar challenge, no significant increase in the biofilm fluid fluoride concentration was observed, even in the fluoride-rich biofilms (p?>?0.05). Fluoride-rich biofilms do not release fluoride to the fluid phase during a sugar challenge.     

Water deficit tolerance in sugarcane is dependent on the accumulation of sugar in the leaf

Sugarcane productivity is severely affected by the occurrence of water deficit in the field, causing inhibition of growth and sugar production. Evaluating physiological responses of sugarcane under water deficit conditions is essential to understand physiological variables responsible for reaching homeostasis. Therefore, we analysed physiological traits of two sugarcane genotypes, RB835486 (Tolerant) and RB855453 (Susceptible), under water deficit conditions: well-watered (WW-Control), water deficit (WD) and rewatered (RW). The physiological response was evaluated using linear regression and multivariate analysis. Some characteristics such as water potential in leaves, photosynthesis, chlorophyll fluorescence, chlorophyll index, sucrose and starch contents did not show differences between the genotypes under water deficit conditions. However, the tolerant genotype showed increased reducing sugars content in the leaves, whereas the susceptible genotype had increased non-photochemical quenching (qN). After rewatering, the susceptible sugarcane genotype showed higher electron transport rate (ETR) and efficiency of PSII (Y). Multivariate analysis revealed that non-photochemical quenching and reducing sugars in the leaves were physiological variables responsible for reaching homestasis under water deficit conditions. Therefore, the reducing sugars concentration should be considered a physiological variable responsible for the adjustment made by the tolerant sugarcane genotype when submitted to water deficit.     

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The immunoglobulin G (IgG) molecule has a long circulating serum half-life (~3 weeks) through pH- dependent FcRn binding-mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non-antibody therapeutic proteins, we have evolved the ectodomain of a low-affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2–CH3 interface). High-throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32-fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10⁻⁷ M for wild type FcγRIIa and 2.82 × 10⁻⁸ M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD-L1 fused with engineered FcγRIIa (PD-L1–2A45.1) was compared with that of PD-L1 fused with wild type FcγRIIa (PD-L1–wild type FcγRIIa) and human PD-L1 in mice. PD-L1–2A45.1 showed 11.7- and 9.7-fold prolonged circulating half-life (t_1/2) compared to PD-L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUC_inf of PD-L1–2A45.1 was two-fold higher compared to that of PD-L1–wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half-life of therapeutic proteins.