Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.Peer reviewe

Joining Together to Build More: The New England Software Carpentry Library Consortium

In 2017 a group of academic library and information technology staff from institutions across New England piloted a process of joining The Carpentries, an organization developed to train researchers in essential computing skills and practices for automating and improving their handling of data, as a consortium. The New England Software Carpentry Library Consortium (NESCLiC) shared a gold-level tier membership to become a Carpentries member organization. NESCLiC members attended a Software Carpentry workshop together and then participated in instructor training as a cohort, collaborating on learning the material, practicing, and beginning to host and teach workshops as a group. This article describes both the successes and challenges of forming this new consortium, suggests good practices for those who might wish to form similar collaborations, and discusses the future of this program and other efforts to help researchers improve their computing and data handling skills

Translation and psychometric assessment of the mastectomy module of the BREAST-Q questionnaire for use in Nigeria

Background: The majority of non-metastatic breast cancer patients in sub-Saharan Africa are recommended to have mastectomy. The impact of mastectomy on a predominantly young African patient population requires evaluation. The BREAST-Q is a validated patient-reported outcome measure of quality-of-life following breast surgery that has been translated into 30 languages-none in Africa. This study aimed to translate and assess the psychometric properties of the mastectomy module of the BREAST-Q for use in Nigeria. Methods: The BREAST-Q mastectomy module was translated from English to Yoruba and its psychometric properties assessed using best practice guidelines. Translation was performed in 4 steps: forward translation (x2), back translation, back translation review, and cognitive interviews with post-mastectomy patients. The translated BREAST-Q instrument was administered to post-mastectomy patients (n = 21) alongside the EORTC-QLQ BR23 to evaluate construct validity. Test-retest reliability was evaluated using intraclass correlation coefficients (ICC); surveys were re-administered 4 weeks apart. Results: The translation process identified English phrases not amenable to direct translation, including “emotionally healthy” and descriptions of pain (“nagging,” “throbbing,” “sharp”). Translations were amended to reflect local context and question intent. During cognitive interviews, patients provided suggestions to simplify complex phrases, e.g. “discomfort in your breast area.”. Internal consistency within scales was over 0.70 for psychosocial wellbeing (α = 0.84–0.87), sexual wellbeing (α = 0.98–0.99), physical wellbeing in chest (α = 0.84–0.86), and satisfaction with care (α = 0.89–0.93). ICC for test-retest reliability was moderate (0.46–0.63). Conclusions: The Yoruba version of the BREAST-Q mastectomy module presents a unique opportunity to adequately capture the experiences of Nigerian women post mastectomy. This instrument is being used in a pilot study of Nigerian patients to identify targets for intervention to improve the patient experience and compliance with breast cancer surgery

Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond

Structural and Functional Features of a Developmentally Regulated Lipopolysaccharide-Binding Protein

ABSTRACT Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses. IMPORTANCE Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a reliable biomarker of chronic inflammation. Here, we describe a symbiosis-induced invertebrate LBP whose tertiary structure and LPS-binding characteristics are similar to those of mammalian LBPs; however, the primary structure of this distantly related squid protein (EsLBP1) differs in key residues previously believed to be essential for LPS binding, suggesting that an alternative strategy exists. Surprisingly, symbiotic expression of eslbp1 is induced by peptidoglycan derivatives, not LPS, a pattern converse to that of RegIIIγ, an important mammalian immunity protein that binds peptidoglycan but whose gene expression is induced by LPS. Finally, EsLBP1 occurs along the apical surfaces of all the host’s epithelia, suggesting that it was recruited from a general defensive role to one that mediates specific interactions with its symbiont

Clinical, radiologic, pathologic, and molecular characteristics of long-term survivors of diffuse intrinsic pontine glioma (DIPG): a collaborative report from the International and European Society for Pediatric Oncology DIPG registries

Purpose Diffuse intrinsic pontine glioma (DIPG) is a brainstem malignancy with a median survival of < 1 year. The International and European Society for Pediatric Oncology DIPG Registries collaborated to compare clinical, radiologic, and histomolecular characteristics between short-term survivors (STSs) and long-term survivors (LTSs). Materials and Methods Data abstracted from registry databases included patients from North America, Australia, Germany, Austria, Switzerland, the Netherlands, Italy, France, the United Kingdom, and Croatia. Results Among 1,130 pediatric and young adults with radiographically confirmed DIPG, 122 (11%) were excluded. Of the 1,008 remaining patients, 101 (10%) were LTSs (survival ≥ 2 years). Median survival time was 11 months (interquartile range, 7.5 to 16 months), and 1-, 2-, 3-, 4-, and 5-year survival rates were 42.3% (95% CI, 38.1% to 44.1%), 9.6% (95% CI, 7.8% to 11.3%), 4.3% (95% CI, 3.2% to 5.8%), 3.2% (95% CI, 2.4% to 4.6%), and 2.2% (95% CI, 1.4% to 3.4%), respectively. LTSs, compared with STSs, more commonly presented at age < 3 or > 10 years (11% v 3% and 33% v 23%, respectively; P < .001) and with longer symptom duration ( P < .001). STSs, compared with LTSs, more commonly presented with cranial nerve palsy (83% v 73%, respectively; P = .008), ring enhancement (38% v 23%, respectively; P = .007), necrosis (42% v 26%, respectively; P = .009), and extrapontine extension (92% v 86%, respectively; P = .04). LTSs more commonly received systemic therapy at diagnosis (88% v 75% for STSs; P = .005). Biopsies and autopsies were performed in 299 patients (30%) and 77 patients (10%), respectively; 181 tumors (48%) were molecularly characterized. LTSs were more likely to harbor a HIST1H3B mutation (odds ratio, 1.28; 95% CI, 1.1 to 1.5; P = .002). Conclusion We report clinical, radiologic, and molecular factors that correlate with survival in children and young adults with DIPG, which are important for risk stratification in future clinical trials

Planetary Candidates Observed by Kepler, III: Analysis of the First 16 Months of Data

New transiting planet candidates are identified in sixteen months (May 2009 - September 2010) of data from the Kepler spacecraft. Nearly five thousand periodic transit-like signals are vetted against astrophysical and instrumental false positives yielding 1,091 viable new planet candidates, bringing the total count up to over 2,300. Improved vetting metrics are employed, contributing to higher catalog reliability. Most notable is the noise-weighted robust averaging of multi-quarter photo-center offsets derived from difference image analysis which identifies likely background eclipsing binaries. Twenty-two months of photometry are used for the purpose of characterizing each of the new candidates. Ephemerides (transit epoch, T_0, and orbital period, P) are tabulated as well as the products of light curve modeling: reduced radius (Rp/R*), reduced semi-major axis (d/R*), and impact parameter (b). The largest fractional increases are seen for the smallest planet candidates (197% for candidates smaller than 2Re compared to 52% for candidates larger than 2Re) and those at longer orbital periods (123% for candidates outside of 50-day orbits versus 85% for candidates inside of 50-day orbits). The gains are larger than expected from increasing the observing window from thirteen months (Quarter 1-- Quarter 5) to sixteen months (Quarter 1 -- Quarter 6). This demonstrates the benefit of continued development of pipeline analysis software. The fraction of all host stars with multiple candidates has grown from 17% to 20%, and the paucity of short-period giant planets in multiple systems is still evident. The progression toward smaller planets at longer orbital periods with each new catalog release suggests that Earth-size planets in the Habitable Zone are forthcoming if, indeed, such planets are abundant.Comment: Submitted to ApJS. Machine-readable tables are available at http://kepler.nasa.gov, http://archive.stsci.edu/kepler/results.html, and the NASA Exoplanet Archiv

Life Beyond the Solar System: Remotely Detectable Biosignatures

For the first time in human history, we will soon be able to apply to the scientific method to the question "Are We Alone?" The rapid advance of exoplanet discovery, planetary systems science, and telescope technology will soon allow scientists to search for life beyond our Solar System through direct observation of extrasolar planets. This endeavor will occur alongside searches for habitable environments and signs of life within our Solar System. While these searches are thematically related and will inform each other, they will require separate observational techniques. The search for life on exoplanets holds potential through the great diversity of worlds to be explored beyond our Solar System. However, there are also unique challenges related to the relatively limited data this search will obtain on any individual world

Structure and Age Jointly Influence Rates of Protein Evolution

What factors determine a protein's rate of evolution are actively debated. Especially unclear is the relative role of intrinsic factors of present-day proteins versus historical factors such as protein age. Here we study the interplay of structural properties and evolutionary age, as determinants of protein evolutionary rate. We use a large set of one-to-one orthologs between human and mouse proteins, with mapped PDB structures. We report that previously observed structural correlations also hold within each age group – including relationships between solvent accessibility, designabililty, and evolutionary rates. However, age also plays a crucial role: age modulates the relationship between solvent accessibility and rate. Additionally, younger proteins, despite being less designable, tend to evolve faster than older proteins. We show that previously reported relationships between age and rate cannot be explained by structural biases among age groups. Finally, we introduce a knowledge-based potential function to study the stability of proteins through large-scale computation. We find that older proteins are more stable for their native structure, and more robust to mutations, than younger ones. Our results underscore that several determinants, both intrinsic and historical, can interact to determine rates of protein evolution