Urea’s Effect on the Ribonuclease A Catalytic Efficiency: A Kinetic, 1H NMR and Molecular Orbital Study

Understanding of protein–urea interactions is one of the greatest challenges to modern structural protein chemistry. Based in enzyme kinetics experiments and ¹H NMR spectroscopic analysis we proposed that urea, at low concentrations, directly interacts with the protonated histidines of the active center of RNase A, following a simple model of competitive inhibition. These results were supported by theoretical analysis based on the frontier molecular orbital theory and suggest that urea might establish a favorable interaction with the cationic amino acids. Our experimental evidence and theoretical analysis indicate that the initials steps of the molecular mechanism of Urea–RNase A interaction passes through the establishment of a three center four electron adduct. Also, our results would explain the observed disruption of the ¹H NMR signals corresponding to H12 and H119 (involved in catalysis) of the RNase A studied in the presence of urea. Our interaction model of urea–amino acids (cationic) can be extended to explain the inactivation of other enzymes with cationic amino acids at the active site.     

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called “portal region”, formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.     

Sedentary Behaviours in Mid-Adulthood and Subsequent Body Mass Index

Objectives

It is unclear whether sedentary behaviour, and the domain in which it occurs, is related to body mass index (BMI) change. We aim to elucidate whether sedentary behaviour is prospectively related to BMI change using markers from three domains (leisure, work and commuting).

Methods

Among employed 1958 British birth cohort members (n = 6,562), we analysed whether TV-viewing, work sitting (six categories: 0 h/d to >4 h/d) and motorised commuting (at 45 y) were related to BMI (at 45 y and 50 y) and BMI change 45–50 y, after adjusting for lifestyle and socioeconomic factors.

Results

Per category higher TV-viewing, 45 y and 50 y BMI were higher by 0.69 kg/m² (95% CI: 0.59,0.80) and 0.75 kg/m² (0.64,0.86) respectively. A category higher TV-viewing was associated with 0.11 kg/m² (0.06,0.17) increased BMI 45–50 y, attenuating to 0.06 kg/m² (0.01,0.12) after adjustment. There was no trend for work sitting with 45 y or 50 y BMI, nor, after adjustment, for BMI change. However, those sitting 2–3 h/d had greater BMI gain by 0.33 kg/m² (0.10,0.56) compared to those sitting 0–1 h/d. Associations between TV-viewing and BMI change were independent of work sitting. Motorised commuting was associated with 45 y, but not 50 y BMI or change.

Conclusions

TV-viewing is associated with BMI gain in mid-adulthood; evidence is weaker for other sedentary behaviours.     

Disability Affects the 6-Minute Walking Distance in Obese Subjects (BMI>40 kg/m2)

Introduction

In obese subjects, the relative reduction of the skeletal muscle strength, the reduced cardio-pulmonary capacity and tolerance to effort, the higher metabolic costs and, therefore, the increased inefficiency of gait together with the increased prevalence of co-morbid conditions might interfere with walking. Performance tests, such as the six-minute walking test (6MWT), can unveil the limitations in cardio-respiratory and motor functions underlying the obesity-related disability. Therefore the aims of the present study were: to explore the determinants of the 6-minute walking distance (6MWD) and to investigate the predictors of interruption of the walk test in obese subjects.

Methods

Obese patients [body mass index (BMI)>40 kg/m²] were recruited from January 2009 to December 2011. Anthropometry, body composition, specific questionnaire for Obesity-related Disabilities (TSD-OC test), fitness status and 6MWT data were evaluated. The correlation between the 6MWD and the potential independent variables (anthropometric parameters, body composition, muscle strength, flexibility and disability) were analysed. The variables which were singularly correlated with the response variable were included in a multivariated regression model. Finally, the correlation between nutritional and functional parameters and test interruption was investigated.

Results

354 subjects (87 males, mean age 48.5±14 years, 267 females, mean age 49.8±15 years) were enrolled in the study. Age, weight, height, BMI, fat mass and fat free mass indexes, handgrip strength and disability were significantly correlated with the 6MWD and considered in the multivariate analysis. The determination coefficient of the regression analysis ranged from 0.21 to 0.47 for the different models. Body weight, BMI, waist circumference, TSD-OC test score and flexibility were found to be predictors of the 6MWT interruption.

Discussion

The present study demonstrated the impact of disability in obese subjects, together with age, anthropometric data, body composition and strength, on the 6-minute walking distance.     

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.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Organic Codes: A Unifying Concept for Life

Although the knowledge about biological systems has advanced exponentially in recent decades, it is surprising to realize that the very definition of Life keeps presenting theoretical challenges. Even if several lines of reasoning seek to identify the essence of life phenomenon, most of these thoughts contain fundamental problem in their basic conceptual structure. Most concepts fail to identify either necessary or sufficient features to define life. Here, we analyzed the main conceptual frameworks regarding theoretical aspects that have been supporting the most accepted concepts of life, such as (i) the physical, (ii) the cellular and (iii) the molecular approaches. Based on an ontological analysis, we propose that Life should not be positioned under the ontological category of Matter. Yet, life should be better understood under the top-level ontology of “Process”. Exercising an epistemological approach, we propose that the essential characteristic that pervades each and every living being is the presence of organic codes. Therefore, we explore theories in biosemiotics and code biology in order to propose a clear concept of life as a macrocode composed by multiple inter-related coding layers. This way, as life is a sort of metaphysical process of encoding, the living beings became the molecular materialization of that process. From the proposed concept, we show that the evolutionary process is a fundamental characteristic for life’s maintenance but it is not necessary to define life, as many organisms are clearly alive but they do not participate in the evolutionary process (such as infertile hybrids). The current proposition opens a fertile field of debate in astrobiology, epistemology, biosemiotics, code biology and robotics.

     

Secretory structures at syconia and flowers of Ficus enormis (Moraceae): A specialization at ostiolar bracts and the first report of inflorescence colleters

The fig (Ficus L.) infructescence, called syconium, is a receptacle with an apical opening, the ostiole, closed by bracts. The ostiolar bracts produce an exudate, which is rather conspicuous in some species. It has not been histochemically analyzed yet, and the structures responsible for its production are still unknown. Some wild growing species of Ficus from Brazil produce high amounts of this ostiolar exudate. Ficus enormis (Mart. ex Miq.) Miq. grows as trees or shrubs in the Atlantic rainforest. Our goal was to identify the secretory structures present in the inflorescence and, to characterize histochemically the ostiolar tissues and exudates. Syconia samples of F. enormis were processed and stained according to the usual techniques in plant anatomy. The morphological analysis revealed different types of bracts, one type specialized in secretion, another showing transitional characteristics between secretory and non-secreting bracts, and a third one being non-secreting. They are designated as secretory ostiolar bracts, transitional bracts and wall bracts. The floral bracteoles, digital-shaped colleters present in the ostiole, at the syconium axis and at the flower receptacle, were also analyzed. All have similar structure, like finger-shaped secretory trichomes. The colleters present among ostiolar bracts may contribute to production and composition of the ostiole exudate.     

Dynamics of a biofouling community in finfish aquaculture: a case study from the South Adriatic Sea

Biofouling is one of the challenges that can strongly affect the finfish farm economy. Although several studies on biofouling in aquaculture have been conducted in the Mediterranean Sea, they focused on specific taxa or were limited to a particular period of sampling. The present study investigated for the first time the development, composition and variation in a biofouling community in a finfish farm with immersion time, season and depth. The results indicate that all these factors influence biofouling succession and recruitment. Moreover, the species that had a crucial role in structuring the community and in the farm cleaning activities were the ascidian Styela plicata and the bivalve Mytilus galloprovincialis. Compared with the literature data, the results highlight the heterogeneity in the composition of the biofouling present in the Mediterranean Sea. Moreover, such knowledge of the biofouling community could provide important information about management efforts and the costs that farmers will face when siting new fish farms.