Designed leucine-rich repeat proteins bind two muramyl dipeptide ligands.

Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 µM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 µM, and lower affinity Kd2 values ranging from 3.1 to 227 µM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.

Using shared knowledge to determine ironic intent; a conversational response paradigm.

Mentalising has long been suggested to play an important role in irony interpretation. We hypothesised that another important cognitive underpinning of irony interpretation is likely to be children's capacity for mental set switching - the ability to switch flexibly between different approaches to the same task. We experimentally manipulated mentalising and set switching to investigate their effects on the ability of 7-year-olds to determine if an utterance is intended ironically or literally. The component of mentalising examined was whether the speaker and listener shared requisite knowledge.We developed a paradigm in which children had to select how a listener might reply, depending on whether the listener shared knowledge needed to interpret the utterance as ironic. Our manipulation of requisite set switching found null results. However, we are the first to show experimentally that children as young as seven years use mentalising to determine whether an utterance is intended ironically or literally.

Necrotizing Enterocolitis Pathophysiology: How Microbiome Data Alter Our Understanding.

Necrotizing enterocolitis is a major cause of mortality and morbidity in the preterm infant population. The gut microbiome is of particular interest in research surrounding necrotizing enterocolitis, because variations in the intestinal microbiota seem to correlate with the risk of inflammation and disease. Recent advances in non-culture-based genomic sequencing have also allowed for more intricate analyses of the intestinal microbiome. Its evolution seems to be influenced by intrauterine and extrauterine factors, ranging from antenatal antibiotic exposure to type of enteral feeds. Ultimately, these alterations in the gut microbiome have the potential to result in devastating diseases like necrotizing enterocolitis.

Modeling liver fluke transmission in northeast Thailand: Impacts of development, hydrology, and control.

Human infection with the Southeast Asian liver fluke Opisthorchis viverrini and liver fluke-associated cholangiocarcinoma cause significant disease burden in Southeast Asia. While there has been considerable work to understand liver fluke pathology and to reduce infection prevalence, there remains a limited understanding of the environmental determinants of parasite transmission dynamics to inform treatment and control programs. A particular setting where targeted control efforts have taken place is the Lawa Lake complex in northeast Thailand. Here, we describe the recent history of host infections, as well as the hydrologic characteristics of this floodplain ecosystem that influence the extent of snail habitat and fish mobility and the transport of human waste and parasite cercariae. Using mathematical modeling, we outline a framework for reconstructing environmental transmission of O. viverrini over the course of the Lawa Project control program from its inception in 2008 until 2016, using locally acquired but fragmentary longitudinal infection data for both humans and environmental hosts. The role of water flow in facilitating movement between snail, fish, human, and reservoir hosts is a particular focus with respect to its relevant scales and its impact on success of interventions. In this setting, we argue that an understanding of the key environmental drivers of disease transmission processes is central to the effectiveness of any environmental intervention.

Experimental and modelling investigations of Opisthorchis viverrini miracidia transmission over time and across temperatures: implications for control.

Transmissibility is a significant factor in parasite fitness. The rate and magnitude of parasite transmission affect prevalence and infection intensity in individual hosts and are influenced by environmental factors. In this context, the objectives of this study were: (i) to experimentally assess Opisthorchis viverrini miracidia survival and infectivity over time and across temperatures; and (ii) to combine these experimental results with environmental data to build a key component of a transmission model, identifying seasonal windows of transmission risk in hyper-endemic northeastern Thailand. Five replicates of 50 O. viverrini eggs were randomly distributed and maintained under four temperature conditions (25°C, 30°C, 35°C, 40°C). Microscopic observations were performed on all experimental units over a period of 3months to record miracidia motility and mortality trends. Six infection trials were also conducted to assess infectivity of miracidia over time and across temperatures, using observations of egg hatching success and infection rates. Upon completion of experiments, data were integrated into a transmission model to create a transmission risk index and to simulate seasonal transmission risk. Miracidia survival rate and motility decreased steadily with 50% mortality observed after 2weeks. Hatching and infection success also decreased significantly after 3weeks. Temperatures over 30°C were associated with increased mortality and decreased infectivity. When incorporating local environmental parameters into our model, we observed low transmission risk during the dry season and increasing transmission risk at the onset of the rainy season, culminating with the highest risk in September. We believe that our results provide the first estimates of O. viverrini miracidia survival and transmission potential under variable temperature conditions and suggest that high temperature treatment (>40°C) of fecal waste could be an efficient control strategy.

Context-driven expectations about focus alternatives.

What is conveyed by a sentence frequently depends not only on the descriptive content carried by its words, but also on implicit alternatives determined by the context of use. Four visual world eye-tracking experiments examined how alternatives are generated based on aspects of the discourse context and used in interpreting sentences containing the focus operators only and also. Experiment 1 builds on previous reading time studies showing that the interpretations of only sentences are constrained by alternatives explicitly mentioned in the preceding discourse, providing fine-grained time course information about the expectations triggered by only. Experiments 2 and 3 show that, in the absence of explicitly mentioned alternatives, lexical and situation-based categories evoked by the context are possible sources of alternatives. While Experiments 1-3 all demonstrate the discourse dependence of alternatives, only explicit mention triggered expectations about alternatives that were specific to sentences with only. By comparing only with also, Experiment 4 begins to disentangle expectations linked to the meanings of specific operators from those generalizable to the class of focus-sensitive operators. Together, these findings show that the interpretation of sentences with focus operators draws on both dedicated mechanisms for introducing alternatives into the discourse context and general mechanisms associated with discourse processing.

Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.

Syntactic priming without lexical overlap in reading comprehension.

Syntactic priming without lexical overlap is well-documented in language production. In contrast, reading-time comprehension studies, which typically use locally ambiguous sentences, generally find syntactic priming only with lexical overlap. This asymmetry has led some researchers to propose that distinct mechanisms underlie the comprehension and production of syntactic structure. Instead, we propose that methodological differences in how priming is assessed are largely responsible for the asymmetry: in comprehension, lexical biases in a locally ambiguous target sentence may overwhelm the influence of syntactic priming effects on a reader's interpretation. We addressed these issues in a self-paced reading study by (1) using target sentences containing global attachment ambiguities, (2) examining a syntactic structure which does not involve an argument of the verb, and (3) factoring out the unavoidable lexical biases associated with the target sentences in a mixed-effects regression model. Under these conditions, syntactic priming affected how ambiguous sentences were parsed, and facilitated reading times when target sentences were parsed using the primed structure. This resolves discrepancies among previous findings, and suggests that the same mechanism underlies syntactic priming in comprehension and production.

Biofunctionalization of α-zirconium phosphate nanosheets: toward rational control of enzyme loading, affinities, activities and structure retention.

Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand challenge. Here we show that preadsorption of cationized bovine serum albumin (cBSA) to α-Zr(IV) phosphate (α-ZrP) nanosheets promotes enzyme binding in a predictable manner, and surprisingly, the enzyme binding is linearly proportional to the number of residues present in the enzyme or its volume, providing a powerful, new predictable tool. The cBSA loaded α-ZrP (denoted as bZrP) was tested for the binding of pepsin, glucose oxidase (GOX), tyrosinase, catalase, myoglobin and laccase where the number of residues increased from the lowest value of ∼153 to the highest value of 2024. Loading depended linearly on the number of residues, rather than enzyme charge or its isoelectric point. No such correlation was seen for the binding of these enzymes to α-ZrP nanosheets without the preadsorption of cBSA, under similar conditions of pH and buffer. Enzyme binding to bZrP was supported by centrifugation studies, powder X-ray diffraction and scanning electron microscopy/energy-dispersive X-ray spectroscopy. All the bound enzymes retained their secondary structure and the extent of structure retention depended directly on the amount of cBSA preadsorbed on α-ZrP, prior to enzyme loading. Except for tyrosinase, all enzyme/bZrP biocatalysts retained their enzymatic activities nearly 90-100%, and biofunctionalization enhanced the loading, improved structure retention and supported higher enzymatic activities. This approach of using a chemically modified protein to serve as a glue, with a predictable affinity/loading of the enzymes, could be useful to rationally control enzyme binding for applications in advanced biocatalysis and biomedical applications.

Integration of cardiac proteome biology and medicine by a specialized knowledgebase.

RATIONALE: Omics sciences enable a systems-level perspective in characterizing cardiovascular biology. Integration of diverse proteomics data via a computational strategy will catalyze the assembly of contextualized knowledge, foster discoveries through multidisciplinary investigations, and minimize unnecessary redundancy in research efforts. OBJECTIVE: The goal of this project is to develop a consolidated cardiac proteome knowledgebase with novel bioinformatics pipeline and Web portals, thereby serving as a new resource to advance cardiovascular biology and medicine. METHODS AND RESULTS: We created Cardiac Organellar Protein Atlas Knowledgebase (COPaKB; www.HeartProteome.org), a centralized platform of high-quality cardiac proteomic data, bioinformatics tools, and relevant cardiovascular phenotypes. Currently, COPaKB features 8 organellar modules, comprising 4203 LC-MS/MS experiments from human, mouse, drosophila, and Caenorhabditis elegans, as well as expression images of 10,924 proteins in human myocardium. In addition, the Java-coded bioinformatics tools provided by COPaKB enable cardiovascular investigators in all disciplines to retrieve and analyze pertinent organellar protein properties of interest. CONCLUSIONS: COPaKB provides an innovative and interactive resource that connects research interests with the new biological discoveries in protein sciences. With an array of intuitive tools in this unified Web server, nonproteomics investigators can conveniently collaborate with proteomics specialists to dissect the molecular signatures of cardiovascular phenotypes.