Escherichia coli expression, refolding and characterization of human laforin

Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5–7 mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI

Expression of the functional carbohydrate-binding module (CBM) of human laforin

Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.Fundação para a Ciência e a Tecnologia (FCT, Portugal)

Dextrin nanoparticles as a protein delivery system: the interleukin-10 case study

(undefined

Efficient Online Timed Pattern Matching by Automata-Based Skipping

The timed pattern matching problem is an actively studied topic because of its relevance in monitoring of real-time systems. There one is given a log $w$ and a specification $\mathcal{A}$ (given by a timed word and a timed automaton in this paper), and one wishes to return the set of intervals for which the log $w$, when restricted to the interval, satisfies the specification $\mathcal{A}$. In our previous work we presented an efficient timed pattern matching algorithm: it adopts a skipping mechanism inspired by the classic Boyer--Moore (BM) string matching algorithm. In this work we tackle the problem of online timed pattern matching, towards embedded applications where it is vital to process a vast amount of incoming data in a timely manner. Specifically, we start with the Franek-Jennings-Smyth (FJS) string matching algorithm---a recent variant of the BM algorithm---and extend it to timed pattern matching. Our experiments indicate the efficiency of our FJS-type algorithm in online and offline timed pattern matching

Visualization of Directional Beaming of Weakly Localized Raman from a Random Network of Silicon Nanowires

Disordered optical media are an emerging class of materials that can strongly scatter light. These materials are useful to investigate light transport phenomena and for applications in imaging, sensing and energy storage. While coherent light can be generated using such materials, its directional emission is typically hampered by their strong scattering nature. Here, the authors directly image Rayleigh scattering, photoluminescence and weakly localized Raman light from a random network of silicon nanowires via real-space microscopy and Fourier imaging. Direct imaging enables us to gain insight on the light transport mechanisms in the random material, to visualize its weak localization length and to demonstrate out-of-plane beaming of the scattered coherent Raman light. The direct visualization of coherent light beaming in such random networks of silicon nanowires offers novel opportunities for fundamental studies of light propagation in disordered media. It also opens venues for the development of next generation optical devices based on disordered structures, such as sensors, light sources, and optical switches

Estimativas de parâmetros genético e fenotipicos dos pesos ao nascimento e a desmama e do ganho de peso pre-desmama em um rebanho Canchim.

O objetivo do presente estudo foi estimar as herdabilidades e as correlações genética, fenotipica e ambiental, dos pesos ao nascimento e a desmama e ganho de peso diário do nascimento a desmama de animais da raça Canchim, criados em regime de pasto na região Oeste do Estado de São Paulo.Resumo expandido

Cluster analysis of breeding values for milk yield and lactation persistency in Guzerá cattle.

Abstract The aim of this study was to explore the pattern of genetic lactation curves of Guzerá cattle using cluster analysis. Test-day milk yields of 5,274 first-lactation Guzerá cows were recorded in a progeny test. A total of 34,193 monthly records were analyzed with a random regression animal model using Legendre polynomials to fit additive genetic and permanent environmental random effects and mean trends. Hierarchical and non-hierarchical cluster analyses were performed based on the EBVs for monthly test-day milk yield, peak yield, lactation persistency, and partial cumulative and 305-day yields. The heritability estimates for testday milk yields ranged from 0.24 to 0.52. Cluster analysis identified animals in the population that belong to different groups according to milk production level and lactation persistency