Understanding Water Equilibration Fundamentals as a Step for Rational Protein Crystallization

Background: Vapor diffusion is the most widely used technique for protein crystallization and the rate of water evaporation plays a key role on the quality of the crystals. Attempts have been made in the past to solve the mass transfer problem governing the evaporation process, either analytically or by employing numerical methods. Despite these efforts, the methods used for protein crystallization remain based on trial and error techniques rather than on fundamental principles. Methodology/Principal Findings: Here we present a new theoretical model which describes the hanging drop method as a function of the different variables that are known to influence the evaporation process. The model is extensively tested against experimental data published by other authors and considering different crystallizing conditions. Aspects responsible for the discrepancies between the existing theories and the measured evaporation kinetics are especially discussed; they include the characterization of vapor-liquid equilibrium, the role of mass transfer within the evaporating droplet, and the influence of the droplet-reservoir distance. Conclusions/Significance: The validation tests show that the proposed model can be used to predict the water evaporation rates under a wide range of experimental conditions used in the hanging drop vapor-diffusion method, with no parameter fitting or computational requirements. This model combined with protein solubility data is expected to become a usefu

1-Hydr­oxy-3-(3-methyl­but-2-en­yloxy)xanthone

In the title compound, C18H16O4, a monoprenylated xanthone, the xanthone skeleton exhibits an essentially planar conformation (r.m.s. deviation 0.0072 Å) and the isoprenyl side chain remains approximately in the mean plane of the xanthone unit, making a dihedral angle of 4.5 (2)°. The hydroxyl group forms an intra­molecular O—H⋯O hydrogen bond. Moreover, there is a weak inter­molecular C—H⋯O inter­action between a ring C atom and the xanthene O atom. In the crystal structure, there are no inter­molecular hydrogen bonds and the crystallographic packing is governed by van der Waals forces, leading to an arrangement in which the mol­ecules assemble with their planes parallel to each other, having a separation of 3.6 (3) Å

Chemical Kinetic Strategies for High-Throughput Screening of Protein Aggregation Modulators

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti-amyloid and anti-oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high-throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation

Nutrition Day in nursing homes – risk factors for malnutrition

Introduction: Malnutrition in institutionalized older adults has a significant prevalence, and is associated with cognitive decline, higher morbidity and mortality rates. Objectives: The aim of this study was to assess risk factors for malnutrition in nursing home residents. Methods: Through Nutrition Day audit (2016–2018), demographic and anthropometric data was collected, as well as medical history and nutritional status according to Mini Nutritional Assessment. Results: Sixty-three individuals were included (57.1% male), with a mean age of 849 years. Mean Body Mass Index was 21.55.1 kg/m2. Malnutrition was identified in 47.6% of the patients and nutritional risk in 28.6%. Most of the sample (61.9%) was bedridden or in wheelchair. Most of the individuals showed dementia (82.5%) and depression (79.3%). A positive moderate significant correlation was found between nutritional status and cognitive status (r = 0.407;p=0.001); and between nutritional status and functional capacity (r = 0.474;p=0.000). Considering the diagnosis which caused institutionalization, brain pathologies were the most prevalent (79.4%), followed by cardiovascular diseases (49.2%) and skeletal muscle diseases (27.0%). Mean number of medications taken daily was 74 and 74.6% of the residents were polymedicated. The majority (61.9%) was taking antipsychotics and/or neuroleptics and 28.6% were taking antidepressives. Conclusions: In this sample, individuals are essentially older adults with a high prevalence of malnutrition. The high level of dependence and the presence of dementia are significant risk factors for the development of this condition. Disturbed emotional status and the use of multiple drugs associated with impaired appetite, although without statistically significant associations with nutritional status, are highly prevalent in this sample. Nutritional screening in this population is essential and timely necessary, aiming to prevent deterioration or improve nutritional status.info:eu-repo/semantics/publishedVersio

Contributo para a sua correta conservação

Na reabilitação e conservação de edifícios, as argamassas de revestimento desempenham um papel fundamental e devido à sua exposição e função protetora do substrato são os primeiros elementos do edifício onde é possível observar degradação. Para a sua correta conservação e preservação é essencial o adequado planeamento da intervenção a realizar, com base no conhecimento detalhado das caraterísticas que são inerentes às argamassas e às aplicações com elas efetuadas, como a constituição das camadas ou os produtos de reação e degradação presentes. O presente estudo, integrado no projeto DB-HERITAGE, consiste no levantamento e sistematização das características de argamassas antigas de edifícios portugueses, com base em numerosos estudos anteriores, organizados cronologicamente. A discussão das principais tendências ao longo das épocas e tipos de aplicação possibilita aferir alguns padrões. A identificação de grupos de argamassas de que existe pouca informação mostra a necessidade de, em fases posteriores, se complementarem esses estudos.publishersversionpublishe

Functional characterization of Arabidopsis thaliana transthyretin-like protein

<p>Abstract</p> <p>Background</p> <p><it>Arabidopsis thaliana </it>transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.</p> <p>Results</p> <p>The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities <it>in vitro</it>, in the full-length protein.</p> <p>Conclusions</p> <p>The <it>Arabidopsis thaliana </it>transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed <it>in vitro</it>.</p

The nucleation of protein crystals as a race against time with on- and off-pathways

High supersaturation levels are a necessary but insufficient condition for the crystallization of purified proteins. Unlike most small molecules, proteins can take diverse aggregation pathways that make the outcome of crystallization assays quite unpredictable. Here, dynamic light scattering and optical microscopy were used to show that the nucleation of lysozyme crystals is preceded by an initial step of protein oligomerization and by the progressive formation of metastable clusters. Because these steps deplete the concentration of soluble monomers, the probability of obtaining protein crystals decreases as time progresses. Stochastic variations of the induction time are thus amplified to a point where fast crystallization can coexist with unyielding regimes in the same conditions. With an initial hydrodynamic radius of similar to 100 nm, the metastable clusters also promote the formation of protein crystals through a mechanism of heterogeneous nucleation. Crystal growth (on-pathway) takes place in parallel with cluster growth (off-pathway). The Janus-faced influence of the mesoscopic clusters is beneficial when it accelerates the formation of the first precrystalline nuclei and is detrimental as it depletes the solution of protein ready to crystallize. Choosing the right balance between the two effects is critical for determining the success of protein crystallization trials. The results presented here suggest that a mild oligomerization degree promotes the formation of a small number of metastable clusters which then catalyze the nucleation of well differentiated crystals

What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests off-pathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include nonlinear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery