Miracle-dz/UniFAR-FTUs huggingface dataset:
Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates.
Long-term effects of delayed-release dimethyl fumarate in multiple sclerosis: Interim analysis of ENDORSE, a randomized extension study

Background: Delayed-release dimethyl fumarate (DMF) demonstrated strong efficacy and a favorable benefit–risk profile for patients with relapsing–remitting multiple sclerosis (RRMS) in phase 3 DEFINE/CONFIRM studies. ENDORSE is an ongoing long-term extension of DEFINE/CONFIRM. Objective: We report efficacy and safety results of a 5-year interim analysis of ENDORSE (2 years DEFINE/CONFIRM; minimum 3 years ENDORSE). Methods: In ENDORSE, patients randomized to DMF 240 mg twice (BID) or thrice daily (TID) in DEFINE/CONFIRM continued this dosage, and those initially randomized to placebo (PBO) or glatiramer acetate (GA) were re-randomized to DMF 240 mg BID or TID. Results: For patients continuing DMF BID (BID/BID), annualized relapse rates were 0.202, 0.163, 0.139, 0.143, and 0.138 (years 1–5, respectively) and 63%, 73%, and 88% were free of new or enlarging T2 hyperintense lesions, new T1 hypointense lesions, and gadolinium-enhanced lesions, respectively, at year 5. Adverse events (AEs; serious adverse events (SAEs)) were reported in 91% (22%; BID/BID), 95% (24%; PBO/BID), and 88% (16%; GA/BID) of the patients. One case of progressive multifocal leukoencephalopathy was reported in the setting of severe, prolonged lymphopenia. Conclusion: Treatment with DMF was associated with continuously low clinical and magnetic resonance imaging (MRI) disease activity in patients with RRMS. These interim data demonstrate a sustained treatment benefit and an acceptable safety profile with DMF.
Contribution of Various Carbon Sources Toward Isoprene Biosynthesis in Poplar Leaves Mediated by Altered Atmospheric CO2 Concentrations

Biogenically released isoprene plays important roles in both tropospheric photochemistry and plant metabolism. We performed a 13CO2-labeling study using proton-transfer-reaction mass spectrometry (PTR-MS) to examine the kinetics of recently assimilated photosynthate into isoprene emitted from poplar (Populus × canescens) trees grown and measured at different atmospheric CO2 concentrations. This is the first study to explicitly consider the effects of altered atmospheric CO2 concentration on carbon partitioning to isoprene biosynthesis. We studied changes in the proportion of labeled carbon as a function of time in two mass fragments, M41+, which represents, in part, substrate derived from pyruvate, and M69+, which represents the whole unlabeled isoprene molecule. We observed a trend of slower 13C incorporation into isoprene carbon derived from pyruvate, consistent with the previously hypothesized origin of chloroplastic pyruvate from cytosolic phosphenolpyruvate (PEP). Trees grown under sub-ambient CO2 (190 ppmv) had rates of isoprene emission and rates of labeling of M41+ and M69+ that were nearly twice those observed in trees grown under elevated CO2 (590 ppmv). However, they also demonstrated the lowest proportion of completely labeled isoprene molecules. These results suggest that under reduced atmospheric CO2 availability, more carbon from stored/older carbon sources is involved in isoprene biosynthesis, and this carbon most likely enters the isoprene biosynthesis pathway through the pyruvate substrate. We offer direct evidence that extra-chloroplastic rather than chloroplastic carbon sources are mobilized to increase the availability of pyruvate required to up-regulate the isoprene biosynthesis pathway when trees are grown under sub-ambient CO2.
Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

Significance LacY catalyzes the coupled transport (symport) of a galactosidic sugar and an H+ and is the poster child for the major facilitator superfamily, the largest family of membrane transport proteins. A detailed mechanism has been postulated involving alternating access of sugar- and H+-binding sites to either side of the membrane that is driven by sugar binding and dissociation and independent of the H+ electrochemical gradient, which acts kinetically. To characterize structural intermediates in the transport cycle, stable conformers are essential, and camelid single-domain nanobodies (Nbs) are particularly useful in this context. Described herein is a structure of a LacY–Nb complex in which access to the sugar-binding site from the periplasmic cavity is diffusion-limited. The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside–H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.
A national evaluation of parenting programs in Sweden: The short-term effects using an RCT effectiveness design.

OBJECTIVE We evaluated the effectiveness of 4 parent-training programs for children with externalizing problems. We tested the effectiveness of 3 behavioral programs (Comet, Cope, and Incredible Years) and 1 nonbehavioral program (Connect) in reducing child behavior problems and attention-deficit/hyperactivity disorder (ADHD) symptoms, in improving positive parenting and parenting competence, and in decreasing negative parenting and parents' stress and depressive symptoms. METHOD This national study was designed as a randomized-controlled effectiveness trial (RCT). The treatments were carried out in 30 clinical and community-based practices. Parents of 908 children (ages 3-12 years) were randomly assigned to 1 of 2 parent training programs available at each practice, or to a wait-list condition, where parents had sought help from regular services. Before and after treatment, parents rated child behavior problems and parenting strategies. RESULTS At posttreatment, children whose parents had received interventions showed a strong decrease in child conduct problems and a moderate to strong decrease in ADHD symptoms. About half of parents whose children scored over the 95th percentile on the behavior measures (Eyberg Child Behavior Inventory, Swanson, Nolan, and Pelham Rating Scale), a clinically meaningful cutoff, reported that their children were no longer above the cutoff after the intervention. Parents showed considerably less negative behaviors toward their children at posttest compared with pretest; they increased in parental competence, and decreased in both stress and depressive symptoms. Overall, the behavioral programs were more effective than the nonbehavioral program. CONCLUSIONS The results support the general efficacy of parent training in a short-term perspective.
Dominating Biological Networks

Proteins are essential macromolecules of life that carry out most cellular processes. Since proteins aggregate to perform function, and since protein-protein interaction (PPI) networks model these aggregations, one would expect to uncover new biology from PPI network topology. Hence, using PPI networks to predict protein function and role of protein pathways in disease has received attention. A debate remains open about whether network properties of “biologically central (BC)” genes (i.e., their protein products), such as those involved in aging, cancer, infectious diseases, or signaling and drug-targeted pathways, exhibit some topological centrality compared to the rest of the proteins in the human PPI network. To help resolve this debate, we design new network-based approaches and apply them to get new insight into biological function and disease. We hypothesize that BC genes have a topologically central (TC) role in the human PPI network. We propose two different concepts of topological centrality. We design a new centrality measure to capture complex wirings of proteins in the network that identifies as TC those proteins that reside in dense extended network neighborhoods. Also, we use the notion of domination and find dominating sets (DSs) in the PPI network, i.e., sets of proteins such that every protein is either in the DS or is a neighbor of the DS. Clearly, a DS has a TC role, as it enables efficient communication between different network parts. We find statistically significant enrichment in BC genes of TC nodes and outperform the existing methods indicating that genes involved in key biological processes occupy topologically complex and dense regions of the network and correspond to its “spine” that connects all other network parts and can thus pass cellular signals efficiently throughout the network. To our knowledge, this is the first study that explores domination in the context of PPI networks.
Pausing of Chloroplast Ribosomes Is Induced by Multiple Features and Is Linked to the Assembly of Photosynthetic Complexes1[OPEN]

Ribosome pausing in chloroplasts is caused by multiple features of mRNA and nascent peptide chain and influences transmembrane protein folding and cofactor integration. Many mRNAs contain pause sites that briefly interrupt the progress of translation. Specific features that induce ribosome pausing have been described; however, their individual contributions to pause-site formation, and the overall biological significance of ribosome pausing, remain largely unclear. We have taken advantage of the compact genome of chloroplasts to carry out a plastid genome-wide survey of pause sites, as a basis for studying the impact of pausing on posttranslational processes. Based on ribosomal profiling of Arabidopsis (Arabidopsis thaliana) chloroplast mRNAs, we demonstrate that a combination of factors—mRNA secondary structure, internal Shine-Dalgarno sequences, and positively charged amino acids in the nascent peptide chain—explains 95% of the major pause sites on plastid mRNAs, whereas codon usage has little impact. The distribution of the pause sites is nonrandom and conforms to distinct patterns in the vicinity of sequences coding for transmembrane domains, which depend on their orientation within the membrane as well as being next to sequences coding for cofactor binding sites. We found strong indications that the mechanisms causing ribosomal pausing and at least some of the ribosomes pause sites are conserved between distantly related plant species. In addition, the positions of features that cause pausing are well conserved in photoautotrophic plants, but less so in their nonphotosynthetic, parasitic relatives, implying that the synthesis and assembly of photosynthetic multiprotein complexes requires localized ribosome pausing.
Complete Mitochondrial DNA Analysis of Eastern Eurasian Haplogroups Rarely Found in Populations of Northern Asia and Eastern Europe

With the aim of uncovering all of the most basal variation in the northern Asian mitochondrial DNA (mtDNA) haplogroups, we have analyzed mtDNA control region and coding region sequence variation in 98 Altaian Kazakhs from southern Siberia and 149 Barghuts from Inner Mongolia, China. Both populations exhibit the prevalence of eastern Eurasian lineages accounting for 91.9% in Barghuts and 60.2% in Altaian Kazakhs. The strong affinity of Altaian Kazakhs and populations of northern and central Asia has been revealed, reflecting both influences of central Asian inhabitants and essential genetic interaction with the Altai region indigenous populations. Statistical analyses data demonstrate a close positioning of all Mongolic-speaking populations (Mongolians, Buryats, Khamnigans, Kalmyks as well as Barghuts studied here) and Turkic-speaking Sojots, thus suggesting their origin from a common maternal ancestral gene pool. In order to achieve a thorough coverage of DNA lineages revealed in the northern Asian matrilineal gene pool, we have completely sequenced the mtDNA of 55 samples representing haplogroups R11b, B4, B5, F2, M9, M10, M11, M13, N9a and R9c1, which were pinpointed from a massive collection (over 5000 individuals) of northern and eastern Asian, as well as European control region mtDNA sequences. Applying the newly updated mtDNA tree to the previously reported northern Asian and eastern Asian mtDNA data sets has resolved the status of the poorly classified mtDNA types and allowed us to obtain the coalescence age estimates of the nodes of interest using different calibrated rates. Our findings confirm our previous conclusion that northern Asian maternal gene pool consists of predominantly post-LGM components of eastern Asian ancestry, though some genetic lineages may have a pre-LGM/LGM origin.
Enhanced Dynamics at the Free Surface of a Molecular Glass Film

In this thesis we describe two separate experiments involving the use of gold nanoparticles. The first experiment looks at the use of gold nanoparticles as a localized heat source and the potential application as a cancer treatment. The second experiment, which is the real focus of this thesis, applies gold nanoparticles in the study of the free surface dynamics of glassy thin films. Gold nanoparticles have the ability to strongly absorb the energy in an incident laser beam and convert that energy into heat. Photothermal therapy is a proposed cancer treatment which exploits this ability to irreparably damage cancerous tissues surrounding gold nanoparticles. In the first chapter of this thesis we explain an experiment designed to probe the local temperatures achieved in such a process. Gold nanoparticles are used to stabilize the boundary of an inverse micelle system which contains an aqueous fluorescent dye solution on it’s interior. A temperature dependent fluorescence intensity allows us to probe the temperature changes induced by laser irradiation. In the remainder of this thesis we describe a separate experiment involving the use of gold nanoparticles to study the free surface dynamics of thin glassy films. There is a growing body of evidence in the literature that thin polymer films in the glassy state exhibit heterogeneous dynamics; specifically that the first few nanometers from an air-polymer interface exhibit enhanced mobility relative to the interior of the film. The underlying mechanism responsible for this enhanced mobility remains elusive, however some believe it to be a direct consequence of the polymeric nature of these films. We describe in detail an experiment aimed at addressing this concern. We deposit gold nanoparticles onto the surface of a molecular (non-polymeric) glassy film and monitor their behaviour upon heating using atomic force microscopy. Our results clearly show the existence of enhanced surface mobility in the system studied and provide strong evidence that enhanced surface mobility should be expected in all glass forming systems.
Speech processing and production in two-year-old children acquiring isiXhosa: A tale of two children

We investigated the speech processing and production of 2-year-old children acquiring isiXhosa in South Africa. Two children (2 years, 5 months; 2 years, 8 months) are presented as single cases. Speech input processing, stored phonological knowledge and speech output are described, based on data from auditory discrimination, naming, and repetition tasks. Both children were approximating adult levels of accuracy in their speech output, although naming was constrained by vocabulary. Performance across tasks was variable: One child showed a relative strength with repetition, and experienced most difficulties with auditory discrimination. The other performed equally well in naming and repetition, and obtained 100% for her auditory task. There is limited data regarding typical development of isiXhosa, and the focus has mainly been on speech production. This exploratory study describes typical development of isiXhosa using a variety of tasks understood within a psycholinguistic framework. We describe some ways in which speech and language therapists can devise and carry out assessment with children in situations where few formal assessments exist, and also detail the challenges of such work.
Assessing Residents’ Competency at Baseline: How Much Does the Medical School Matter?

BACKGROUND Although there is some consensus about the competencies needed to enter residency, the actual skills of graduating medical students may not meet expectations. In addition, little is known about the association between undergraduate medical education and clinical performance at entry into and during residency. OBJECTIVE We explored the association between medical school of origin and clinical performance using a multi-station objective structured clinical examination for incoming residents at the University of Michigan Health System. METHODS Prior to assuming clinical duties, all first-year residents at the University of Michigan Health System participate in the Postgraduate Orientation Assessment (POA). This assesses competencies needed during the first months of residency. Performance data for 1795 residents were collected between 2002 and 2012. We estimated POA variance by medical school using linear mixed models. RESULTS Medical school predicted the following amounts of variance in performance-data gathering scores: 1.67% (95% confidence interval [CI] 0.36-2.93); assessment scores: 4.93% (95% CI 1.84-6.00); teamwork scores: 0.80% (95% CI 0.00-1.82); communication scores: 2.37% (95% CI 0.66-3.83); and overall POA scores: 4.19% (95% CI 1.59-5.35). CONCLUSIONS The results show that residents' medical school of origin is weakly associated with clinical competency, highlighting a potential source of variability in undergraduate medical education. The practical significance of these findings needs further evaluation.
A New Multidimensional Questionnaire to Assess Awareness of Age-Related Change (AARC).

BACKGROUND AND OBJECTIVES This two-study article describes the development and evaluation of a multidimensional questionnaire based on the subjective aging construct Awareness of Age-Related Change (AARC). AARC captures the inherent multidimensionality and complexity of aging attitudes, which are strongly linked to indicators of successful aging, including health and well-being. RESEARCH DESIGN AND METHODS In Study 1, we generated a large item pool related to subjective aging experiences and then evaluated the psychometric properties of a 189-item version of the AARC questionnaire in a sample of 396 adults aged 40-95 years. Based on findings from Study 1, we retained the best-performing items and arrived at a more parsimonious 50-item version (AARC-50). In Study 2, the psychometric properties of the 50-item version were examined in an expanded sample of 424 adults ages 40-98. RESULTS Factor analyses in Study 1 indicated a two-factor structure of the questionnaire, representing the awareness of positive (AARC-Gains) and negative (AARC-Losses) age-related changes across five behavioral domains. Confirmatory factor analysis in Study 2 further supported this two-factor structure. In both studies, the AARC questionnaire demonstrated strong psychometric properties, including scale and item reliability, convergent and divergent validity, and predictive validity. DISCUSSION AND IMPLICATIONS The availability of a reliable and valid assessment tool for measuring AARC-Gains and AARC-Losses allows researchers to capture detailed information about adults' positive and negative self-perceptions of aging across multiple behavioral domains, which are instrumental for promoting successful aging.
Targeting scFv-Fc-scTRAIL fusion proteins to tumor cells

Fusion proteins combining hexavalent TRAIL with antibody fragments allow for a targeted delivery and efficient apoptosis induction in tumor cells. Here, we analyzed scFv-Fc-scTRAIL molecules directed against EGFR, HER2, HER3, and EpCAM as well as an untargeted Fc-scTRAIL fusion protein for their potentials to induce cell death both in vitro and in a xenograft tumor model in vivo. The scFv-Fc-scTRAIL fusion protein directed against EGFR as well as the fusion protein directed against EpCAM showed targeting effects on the two tested colorectal carcinoma cell lines Colo205 and HCT116, while a fusion protein targeting HER3 was more effective than untargeted Fc-scTRAIL only on Colo205 cells. Interestingly, another anti-HER3 scFv-Fc-scTRAIL fusion protein exhibiting approximately 10-fold weaker antigen binding as well as the HER2-directed molecule were unable to increase cytotoxicity compared to Fc-scTRAIL. A comparison of EC50 values of cell death induction and antigen binding supports the assumption that high affinity antigen binding is one of the requirements for in vitro targeting effects. Furthermore, a minimal number of expressed target antigens might be required for increased cytotoxicity of targeted compared to non-targeted molecules. In a Colo205 s.c. xenograft tumor model, strongest antitumor activity was observed for the anti-HER3 scFv-Fc-scTRAIL fusion protein based on antibody 3-43, with complete tumor remissions after six twice-weekly injections. Surprisingly, a similar in vivo activity was also observed for untargeted Fc-scTRAIL in this tumor model, indicating that additional factors contribute to the potent efficacy of targeted as well as untargeted hexavalent Fc-scTRAIL fusion proteins in vivo.