Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system.

Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology

Advances and Pitfalls in the Analysis and Interpretation of Resting-State FMRI Data

The last 15 years have witnessed a steady increase in the number of resting-state functional neuroimaging studies. The connectivity patterns of multiple functional, distributed, large-scale networks of brain dynamics have been recognised for their potential as useful tools in the domain of systems and other neurosciences. The application of functional connectivity methods to areas such as cognitive psychology, clinical diagnosis and treatment progression has yielded promising preliminary results, but is yet to be fully realised. This is due, in part, to an array of methodological and interpretative issues that remain to be resolved. We here present a review of the methods most commonly applied in this rapidly advancing field, such as seed-based correlation analysis and independent component analysis, along with examples of their use at the individual subject and group analysis levels and a discussion of practical and theoretical issues arising from this data ‘explosion’. We describe the similarities and differences across these varied statistical approaches to processing resting-state functional magnetic resonance imaging signals, and conclude that further technical optimisation and experimental refinement is required in order to fully delineate and characterise the gross complexity of the human neural functional architecture

Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear.ObjectiveWe sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin.MethodsWe applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set.ResultsTwo thousand four hundred thirty differentially expressed genes (false discovery rate, P < .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response.ConclusionThese analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development

Model for Gravitational Interaction between Dark Matter and Baryons

We propose a phenomenological model where the gravitational interaction between dark matter and baryons is suppressed on small, subgalactic scales. We describe the gravitational force by adding a Yukawa contribution to the standard Newtonian potential and show that this interaction scheme is effectively suggested by the available observations of the inner rotation curves of small mass galaxies. Besides helping in interpreting the cuspy profile of dark matter halos observed in N-body simulations, this potential regulates the quantity of baryons within halos of different masses.Comment: 4 pages, 2 figures, final versio

Point-by-point inscription of apodized fiber Bragg gratings

We demonstrate apodized fiber Bragg gratings inscribed with a point-by-point technique. We tailor the grating phase and coupling amplitude through precise control over the longitudinal and transverse position of each laser-inscribed modification. This method of apodization is facilitated by the highly-localized, high-contrast modifications generated by focussed IR femtosecond laser inscription. Our technique provides a simple method for the design and implementation of point-by-point fiber Bragg gratings with complex apodization profiles.Comment: 6 pages, 4 figures, article in revie

The Kundu--Eckhaus equation and its discretizations

In this article we show that the complex Burgers and the Kundu--Eckhaus equations are related by a Miura transformation. We use this relation to discretize the Kundu--Eckhaus equation.Comment: 10 page

SMC in space and time: a project to study the evolution of the prototype interacting late-type dwarf galaxy

We introduce the SMC in space and time, a large coordinated space and ground-based program to study star formation processes and history, as well as variable stars, structure, kinematics and chemical evolution of the whole SMC. Here, we present the Colour-Magnitude Diagrams (CMDs) resulting from HST/ACS photometry, aimed at deriving the star formation history (SFH) in six fields of the SMC. The fields are located in the central regions, in the stellar halo, and in the wing toward the LMC. The CMDs are very deep, well beyond the oldest Main Sequence Turn-Off, and will allow us to derive the SFH over the entire Hubble tim

p53 is associated with high-risk and pinpointsTP53missense mutations in mantle cell lymphoma

Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from modern chemotherapy-based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high-risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients withTP53missense mutations and the prognostic impact of overexpression and mutations in a Swedish population-based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 andTP53status among 137 cases divided over the two-cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression andTP53mutations on survival (hazard ratio of 3 center dot 1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population-based setting.Peer reviewe