Miracle-dz/UniFAR-FTUs huggingface dataset:
Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Low expression and instability during isolation are major obstacles preventing adequate structure‐function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C‐terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C‐termini (Cin) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C‐termini (Cout) to Cin ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted Cout topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein‐detergent complex was identified using an extended fluorescence‐detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure‐function studies. Five MPs were successfully cleaved from the GFP tag by site‐specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with Cout topology, yielding sufficient protein suitable for structure‐function studies and are superior to expression and purification in the absence GFP fusion tagging.
Exploring Liming and Ole Talk as a Culturally Relevant Methodology for Researching With Caribbean People

This article explores the necessity of developing a qualitative research methodology grounded in Caribbean peoples’ worldviews and interactions. It presents the epistemology and ontology of liming and ole talk to show their natural employment in qualitative research settings. Liming offers an opportunity for social engagement and provides a culturally relevant purpose, environment, and space in which ole talk can take place. Ole talk is presented as a uniquely Caribbean way of engaging with one another in small or large groups. The potential of liming and ole talk to create new ways to research and share knowledge is discussed. Through a brief analysis of two limes, this article proposes liming and ole talk as an authentic research methodology for researching Caribbean peoples and their contexts.
Thyroid status and its association with cognitive functioning in healthy boys at 10 years of age.

OBJECTIVE Thyroid hormones (THs) are crucial for the correct maturation of the CNS and the neurodevelopment of the child. We aimed to investigate the association of TSH and free thyroxine (FT4) levels with cognitive functioning in children from the INMA-Granada cohort studied during their follow-up at the age of 9-11 years. DESIGN We evaluated 300 children from the original cohort, which comprised 668 eligible mother-son pairs recruited at birth from 2000 to 2002 in Granada (Spain). METHODS FT4 and TSH concentrations were measured, and cognitive development was assessed using neuropsychological tests (n=187). Children with chronic disease related to thyroid function and/or cognitive development were excluded. RESULTS Median TSH and FT4 levels were 3.1 μIU/ml and 1.2 ng/dl respectively. In multivariable regression analyses adjusted for maternal and child characteristics, children with TSH levels in the top tertile had worse verbal comprehension and immediate and long-term recall. Children with FT4 levels in the top tertile had better attention and lower impulsivity and were at a lower risk of scoring below the 20th percentile in intelligence quotient (OR=0.24; 95% CI=0.08-0.74; P=0.013) and in abstract reasoning ability (OR=0.28; 95% CI=0.09-0.88; P=0.029). CONCLUSION Our findings indicate that circulating THs and TSH may in the top tertile have an impact on cognitive functions; thus, higher TSH slightly but significantly increased the risk of a lower score in certain neuropsychological tests.
5-Aminolevulinic Acid, but not 5-Aminolevulinic Acid Esters, is Transported into Adenocarcinoma Cells by System BETA Transporters

Abstract 5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis–Menten kinetics (Km = 8–10 mM and Vmax = 18–20 nmol·(mg protein × h)−1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32°C. The activation energy was 112 kJ·mol−1 in the temperature range 15°–32°C and 26 kJ·mol−1 above 32°C. Transport of 5-ALA was Na+ and partly Cl−-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65–75%, by the β-amino acids, β-alanine and taurine and by γ-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-β-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM β-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 μM) was not significantly influenced by β-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by β-amino acid and GABA carriers in this cell line.
Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP‐ and PACAP‐deficient mice

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are closely related neurotrophic peptides of the secretin/glucagon family. The two peptides are derived from a common ancestral gene and share many functional attributes in neuronal development/regeneration which occur not only from overlapping receptor subtype signaling but also through common mechanisms regulating their expression. Although PACAP or VIP null mice have been generated for study, it is unclear whether the expression of the complementary peptide or their receptor systems are altered in a compensatory manner during nervous system development. By radioimmunoassay and quantitative PCR measurements, we first show that PACAP and VIP have very different temporal patterns of expression in developing postnatal mouse brain. In wild‐type animals, PACAP transcript and peptide levels increased rapidly 2‐ and 5‐fold, respectively, within 1 week of age. These levels at 1 week of age were maintained through adulthood. VIP transcript and peptide levels, by contrast, increased 25‐ and 50‐fold, respectively, over a later time course. In parallel studies of development, there were no apparent compensatory increases in brain VIP expression in the PACAP knockout animals, PACAP expression in the VIP‐deficient animals, or receptor mRNA levels in either genotype. To the contrary, there was evidence for developmental delays in the expression of peptide and receptor transcripts in the knockout animals. A series of behavioral and neurological tests demonstrated differences between the knockout genotypes, revealing some functional distinctions between the two genes. These results suggest that the PACAP and VIP have evolved to possess distinct biological activities and intimate that the respective knockout phenotypes represent deficits unmitigated by the actions of the complementary related peptide.
Experimental investigation of the process of Arc propagation over an ice surface

In this paper, some of the characteristics of an arc propagating over an ice-covered insulator surface were investigated. Special attention was paid to the arc root and channel characteristics. All experiments were performed on a simplified physical model to simulate real ice-covered insulators. From the obtained results, based on a set of time-resolved measurements of a surface discharge, some important characteristics, namely arc foot geometry and channel diameter were observed. Possible mechanisms controlling these variations are discussed. An empirical model, accounting for arc propagation pattern on an ice surface, is proposed. The paper contains a substantial amount of new material with a view of closing some major gaps in the present state of knowledge of ice surface flashover.
Purification and Characterization of Heat‐Labile Toxin from Bordetella bronchiseptica

The heat‐labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer‐Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc Polyacrylamide gel electrophoresis and the gel diffusion‐test, and free of detectable hemagglutinin and endotoxin activity. A 386‐fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pi 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS‐polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.
Representation of wind power generation in economic models for long-term energy planning

The representation of wind power plants electricity generation in economic models for energy planning is problematic, since generation in wind power plants is variable and it is not possible to predict accurately enough wind fluctuations for more than a few days. Often, wind power plants generation patterns from a single historical year are repeated throughout the modelled time period. Typically, this method is used when analysing power system operation in hourly time intervals for all days in a year for each year. However, hourly time resolution is not feasible in large-scale models, which tend to require considerable amounts of computing power. Thus, some kind of time aggregation is needed. On the other hand, currently used methods for models with less than hourly temporal resolution are becoming inadequate because of increasing share of fluctuating electricity production from wind in total power generation. In this article, a methodology for evaluation of wind power plants stochastic electricity generation in power system development models is described. The methodology is based on evaluation of how much time single or multiple wind power plants generate a certain output range during a season or some time period within a year, modelling of output distribution for a typical day of the selected time period, and preparation of electricity output curves. These electricity output curves when modelling wind power plants in models with aggregated time allow to assess fluctuations in generation, observable regularities and enhance the objectivity of balancing power demand assessment, also ensure that electricity generated during a typical day corresponds to electricity generated during the selected time period. This methodology will help to determine the rational perspective power generation mix more accurately and make a better assessment of cost-effectiveness of wind power plants in economic models for energy planning, without significantly increasing the size of already large-scale models.
Kindlin-1 Is a Phosphoprotein Involved in Regulation of Polarity, Proliferation, and Motility of Epidermal Keratinocytes*

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.
Professional youth football academy injury data: collection procedures, perceived value, and use

ABSTRACT Purpose: There is a paucity of descriptive injury data relevant to professional academy football, with little to no evidence reporting how sports science/medicine staff within academies collect and use injury data. Materials and methods: An online survey relating to the perceptions surrounding injury data collection, its value and use was developed. Forty-seven applied practitioners working for different professional football academies completed the survey. Results: Injury data collection procedures conducted by appropriately trained medical staff are widespread among football academies. Injury data collection within academies was deemed worthwhile and important by 79% of practitioners. Similarly, 79% strongly agreed/agreed that using injury data for academic research is worthwhile. The engagement with and use of injury data by coaching staff appears to be relatively poor, with only 49% of practitioners stating coaches formally review data. Conclusions: Injury data are widely collected within academies and practitioners consider this information valuable. However, improving engagement with coaches and using the data for academic research could further improve applied practice. Practical implications: Applied practitioners should consider sharing injury data with both researchers and coaches. In doing so evidence-guided injury prevention interventions may be developed and subsequently applied in the field.
Robust Downlink Beamforming for Cognitive Radio Networks

We address the problem of worst-case robust downlink beamforming for a multi-antenna secondary network (SN) in a cognitive radio framework. An important issue is the interference leaked to the primary users (PUs) resulting from the transmission between the SN base station and the secondary users (SUs). Our aim is to provide the SUs with a minimum acceptable quality-of-service (QoS), while keeping the interference to the PUs below a given threshold. Previous solutions for this scenario involve several coarse approximations. Here we avoid these approximations and obtain an exact reformulation of the worst-case problem using Lagrange duality. Finally, we use semideﬁnite relaxation (SDR) to convert the resulting problem to a convex form. Computer simulations show that the SDR step does not involve any approximation as the resulting solution is always rank-one.
Evolutionary conservation of structure and function of the UVR8 photoreceptor from the liverwort Marchantia polymorpha and the moss Physcomitrella patens.

The ultraviolet-B (UV-B) photoreceptor UV RESISTANCE LOCUS 8 (UVR8) mediates photomorphogenic responses to UV-B in Arabidopsis through differential gene expression, but little is known about UVR8 in other species. Bryophyte lineages were the earliest diverging embryophytes, thus being the first plants facing the UV-B regime typical of land. We therefore examined whether liverwort and moss species have functional UVR8 proteins and whether they are regulated similarly to Arabidopsis UVR8. We examined the expression, dimer/monomer status, cellular localisation and function of Marchantia polymorpha and Physcomitrella patens UVR8 in experiments with bryophyte tissue and expression of green fluorescent protein (GFP)-UVR8 fusions in Nicotiana leaves and transgenic Arabidopsis. P. patens expresses two UVR8 genes that encode functional proteins, whereas the single M. polymorpha UVR8 gene expresses two transcripts by alternative splicing that encode functional UVR8 variants. P. patens UVR8 proteins form dimers that monomerise and accumulate in the nucleus following UV-B exposure, similar to Arabidopsis UVR8, but M. polymorpha UVR8 has weaker dimers and the proteins appear more constitutively nuclear. We conclude that liverwort and moss species produce functional UVR8 proteins. Although there are differences in expression and regulation of P. patens and M. polymorpha UVR8, the mechanism of UVR8 action is strongly conserved in evolution.
Development of a multiplex serological assay reveals a worldwide distribution of murine astrovirus infections in laboratory mice

Laboratory mice play a tremendous role in biomedical research in studies on immunology, infection, cancer and therapy. In the course of standardization of mice used in animal experiments, health monitoring constitutes an important instrument towards microbiological standardization. Infections with murine astroviruses (MuAstV) were only recently discovered and are, therefore, still relatively unknown in laboratory animal science. In rodent health monitoring viral infections within a population are commonly assessed in terms of specific antibodies by serological testing, as active infection and excretion of virus is often temporary and can easily be missed. So far only ongoing infections with astroviruses can be detected by PCR. The objective of this work was the development of a sensitive and specific MuAstV multiplex serological assay with a high-throughput capability to be used in routine testing of laboratory mice. Four different MuAstV proteins were recombinantly expressed and used as antigens. The best reacting antigen, the capsid spike protein VP27, was selected and tested with a panel of 400 sera of mice from units with a known MuAstV status. Assay sensitivity and specificity resulted in 98.5% and 100%, respectively, compared to RT-PCR results. Eventually this assay was used to test 5529 serum samples in total, during routine diagnostics at the German Cancer Research Center (DKFZ) in Heidelberg between 2015 and 2017. High sero-prevalence rates of up to 98% were detected in units with open cages indicating that the virus is highly infectious and circulates within these populations virtually infecting all animals regardless of the mouse strain. In addition, data collected from 312 mice purchased from commercial breeders and from 661 mice from 58 research institutes in 15 countries worldwide allowed the conclusion that MuAstV is widespread in contemporary laboratory mouse populations.
Pooled CRISPR interference screens enable high-throughput functional genomics study and elucidate new rules for guide RNA library design in Escherichia coli

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology provides potential advantages in high-throughput functional genomics analysis in prokaryotes over previously established platforms based on recombineering or transposon mutagenesis. In this work, as a proof-of-concept to adopt CRISPR/Cas9 method as a pooled functional genomics analysis platform in prokaryotes, we developed a CRISPR interference (CRISPRi) library consisting of 3,148 single guide RNAs (sgRNAs) targeting the open reading frame (ORF) of 67 genes with known knockout phenotypes and performed pooled screens under two stressed conditions (minimal and acidic medium) in Escherichia coli. Our approach confirmed most of previously described gene-phenotype associations while maintaining < 5% false positive rate, suggesting that CRISPRi screen is both sensitive and specific. Our data also supported the ability of this method to narrow down the candidate gene pool when studying operons, a unique structure in prokaryotic genome. Meanwhile, assessment of multiple loci across treatments enables us to extract several guidelines for sgRNA design for such pooled functional genomics screen. For instance, sgRNAs locating at the first 5% upstream region within ORF exhibit enhanced activity and 10 sgRNAs per gene is suggested to be enough for robust identification of gene-phenotype associations. We also optimized the hit-gene calling algorithm to identify target genes more robustly with even fewer sgRNAs. This work showed that CRISPRi could be adopted as a powerful functional genomics analysis tool in prokaryotes and provided the first guideline for the construction of sgRNA libraries in such applications. Importance To fully exploit the valuable resource of explosive sequenced microbial genomes, high-throughput experimental platform is needed to associate genes and phenotypes at the genome level, giving microbiologists the insight about the genetic structure and physiology of a microorganism. In this work, we adopted CRISPR interference method as a pooled high-throughput functional genomics platform in prokaryotes with Escherichia coli as the model organism. Our data suggested that this method was highly sensitive and specific to map genes with previously known phenotypes, potent to act as a new strategy for high-throughput microbial genetics study with advantages over previously established methods. We also provided the first guideline for the sgRNA library design by comprehensive analysis of the screen data. The concept, gRNA library design rules and open-source scripts of this work should benefit prokaryotic genetics community to apply high-throughput mapping of defined gene set with phenotypes in a broad spectrum of microorganisms.
A Process Model for Policy Analysis Within the Context of Political Caring

As the health care system in the United States is becoming increasingly more politically and economically oriented, the concept of political caring needs to be advanced in contemporary nursing practice (Ray, 1989, 2001; Turkel, 2001). The purpose of this article is to present a model outlining the process of policy analysis through a phenomenologica research study illuminating the life world descriptions of experiences of United States Air Force personnel with managed care in the military and the civilian health care system. This process shows how qualitative data are used to give voice to a moral crisis and contribute to health care policy.