Structure of a nanobody-stabilized active state of the β(2) adrenoceptor

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11?? outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.     

Measurement of Employees Subjective Well-Being as an Aim of Social Responsibility

Social responsibility (SR) includes well-being of all. Improving of SR depends also on measurement of well-being. In organizations it requires a hierarchy/network of requisitely holistic factors and indicators. We regarded several definitions of subjective well-being that take into account multiple dimensions and selected factors to be structured in the multi-criteria model: positive affect, negative affect, and life satisfaction. The article presents results of empirical research of subjective well-being in organizations in Slovenia. The factor analysis and theoretical foundations of the characteristics of subjective well-being factors, determined the importance of the criteria—multiple dimensions of subjective well-being. Data about positive and negative affection were obtained with the ‘Positive Affect Negative Affect Scale’; data about life satisfaction were obtained through the ‘Satisfaction with Life Scale’; thus the local values of alternatives—types of organizations in Slovenia—were measured by using value functions. The level of well-being—the aggregate value of well-being measure was assessed with multi-criteria decision-making methods. One can thus benchmark the well-being performance of organizations, determine the key success and failure factors and benefit from good examples. Results offer information for the interventions to increase the subjective well-being in organizations and thus to attain more SR in organization-internal relations.     

Biochemical,biophysical and molecular dynamics studies on the proteoglycan-like domain of carbonic anhydrase IX

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.     

Ongoing therapeutic trials and outcome measures for Duchenne muscular dystrophy

Muscular dystrophy is a heterogeneous group of genetic disorders characterised by progressive muscle tissue degeneration. No effective treatment has been discovered for these diseases. Preclinical and clinical studies aimed at the development of new therapeutic approaches have been carried out, primarily in subjects affected with dystrophinopathies (Duchenne and Becker muscular dystrophy). In this review, we outline the current therapeutic approaches and past and ongoing clinical trials, highlighting both the advantages and limits of each one. The experimental designs of these trials were based on different rationales, including immunomodulation, readthrough strategies, exon skipping, gene therapy, and cell therapy. We also provide an overview of available outcome measures, focusing on their reliability in estimating meaningful clinical improvement in order to aid in the design of future trials. This perspective is extremely relevant to the field considering the recent development of novel therapeutic approaches that will result in an increasing number of clinical studies over the next few years.     

A case of biodiversity overestimation in the Balkan Belgrandiella A. J. Wagner, 1927 (Caenogastropoda: Hydrobiidae): molecular divergence not paralleled by high morphological variation

Belgrandiella A. J. Wagner, 1927 is a genus comprising minute snails with conic or turriform shells that are facultative stygobionts, inhabiting subterranean waters and springs. Few specimens or even empty shells of this taxon can be found, so the taxonomy is based mostly on the shell morphology and geographic distribution. Anatomy is known for a few taxa, and its usefulness is restricted. This, coupled with a popular idea that isolation is the first factor shaping divergence and speciation of the spring/cave fauna, resulted in dozens of nominal species of Belgrandiella, known from shells at a given locality. Herein we present shells, anatomy and four molecular markers (nuclear H3, 18S and 28S and mitochondrial cytochrome oxidase subunit I) in Belgrandiella from 16 localities in Slovenia, northern Croatia, and Bosnia and Herzegovina. Five sites are type localities of Boleana umbilicata, Belgrandiella kusceri, Belgrandiella krupensis, Belgrandiella zermanica and Belgrandiella robusta, respectively. Considering the shell and geographic distribution, we have determined B. cf. robusta Belgrandiella cf. croatica, Belgrandiella cf. fontinalis, Belgrandiella cf. kuesteri, Belgrandiella cf. pageti and Belgrandiella cf. koprivnensis The shells of all, as well as penes and female reproductive organs of two putative, species have been illustrated. Molecular analysis indicates four distinct clades, most probably of species rank for B. kusceri, B. cf. kuesteri, B. cf. fontinalis and B. cf. koprivnensis. Four nominal species (B. krupensis, B. robusta, B. umbilicata and B. zermanica) and one genus (Boleana) must be synonymised with Belgrandiella kusceri, and there are no more than four valid, biological species in the studied Belgrandiella. The molecular divergence is completely unmirrored by high morphological variability. Hence the level of endemism is markedly overestimated in these snails.     

The positional identity of mouse ES cell-generated neurons is affected by BMP signaling

We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior–posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.     

The endocannabinoid system in rat gliosomes and its role in the modulation of glutamate release

The endocannabinoid system and endocannabinoid receptor-driven modulation of glutamate release were studied in rat brain cortex astroglial gliosomes. These preparations contained the endocannabinoids N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol, as well their major biosynthetic (N-acyl-phosphatidylethanolamines-hydrolyzing-phospholipase D and diacylglycerol-lipase) and catabolic (fatty acid amide-hydrolase and monoacylglycerol-lipase) enzymes. Gliosomes expressed type-1 (CB1R), type-2 (CB2R) cannabinoid, and type-1 vanilloid (TRPV1) receptors, as ascertained by Western blotting and confocal microscopy. Methanandamide, a stable analogue of anandamide acting as CB1R, CB2R, and TRPV1 agonist, stimulated or inhibited the depolarization-evoked gliosomal [³H]d-aspartate release, at lower and higher concentrations, respectively. Experiments with ACEA (arachidonyl-2′-chloroethylamide), JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]-pyran) and capsaicin, selective agonists at CB1R, CB2R and TRPV1, respectively, demonstrated that potentiation of [³H]d-aspartate release was due to CB1R while inhibition to CB2R and TRPV1 engagement. These findings were confirmed by using selective receptor antagonists. Furthermore, CB1R activation caused increase of intracellular IP3 and Ca²⁺ concentration, suggesting an involvement of phospholipase C.     

Local Statistical Modeling via a Cluster-Weighted Approach with Elliptical Distributions

Cluster-weighted modeling (CWM) is a mixture approach to modeling the joint probability of data coming from a heterogeneous population. Under Gaussian assumptions, we investigate statistical properties of CWM from both theoretical and numerical point of view; in particular, we show that Gaussian CWM includes mixtures of distributions and mixtures of regressions as special cases. Further, we introduce CWM based on Student-t distributions, which provides a more robust fit for groups of observations with longer than normal tails or noise data. Theoretical results are illustrated using some empirical studies, considering both simulated and real data. Some generalizations of such models are also outlined.