The laminin-binding domain of agrin is structurally related to N-TIMP-1

Agrin is the key organizer of postsynaptic differentiation at the neuromuscular junction. This organization activity requires the binding of agrin to the synaptic basal lamina. Binding is conferred by the N-terminal agrin (NtA) domain, which mediates a high-affinity interaction with the coiled coil domain of laminins. Here, we report the crystal structure of chicken NtA at 1.6 A resolution. The structure reveals that NtA harbors an oligosaccharide/oligonucleotide-binding fold with several possible sites for the interaction with different ligands. A high structural similarity of NtA with the protease inhibition domain in tissue inhibitor of metalloproteinases-1 (TIMP-1) supports the idea of additional functions of agrin besides synaptogenic activity.     

Analysis of longitudinal marginal structural models

In this article we construct and study estimators of the causal effect of a time-dependent treatment on survival in longitudinal studies. We employ a particular marginal structural model (MSM), proposed by Robins (2000), and follow a general methodology for constructing estimating functions in censored data models. The inverse probability of treatment weighted (IPTW) estimator of Robins et al. (2000) is used as an initial estimator and forms the basis for an improved, one-step estimator that is consistent and asymptotically linear when the treatment mechanism is consistently estimated. We extend these methods to handle informative censoring. The proposed methodology is employed to estimate the causal effect of exercise on mortality in a longitudinal study of seniors in Sonoma County. A simulation study demonstrates the bias of naive estimators in the presence of time-dependent confounders and also shows the efficiency gain of the IPTW estimator, even in the absence such confounding. The efficiency gain of the improved, one-step estimator is demonstrated through simulation.     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Localized expression of the seizure-related gene SEZ-6 in developing and adult forebrains

Despite its initial identification in neurons exposed to the seizure-inducing drug pentylentetrazole (PTZ), the function of the seizure-related gene SEZ-6 remains obscure. Expression analysis indicates specific expression in the adult brain and testis, and the structure of the predicted protein suggests putative roles in cell-cell recognition and signalling. We report here that type I SEZ-6 mRNA is strongly expressed in highly specific regions in the developing forebrain. Specifically, the pattern of SEZ-6 expression is closely tied with the emergence of the neocortical layers and hippocampus, and implies a forebrain-specific role for this gene during development. In the adult hippocampus, SEZ-6 appears to be a CA1-specific regional marker.     

Prediction of proton chemical shifts in RNA – Their use in structure refinement and validation

An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct., 76, 81–92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341–346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable analysis could be made of the structural dependencies of the ¹H chemical shifts revealing their physical origin. For example, a down-field shift of either H2 or H3 or both indicates a high-syn/syn -angle. In an A-helix it is essentially the 5-neighbor that affects the chemical shifts of H5, H6 and H8 protons. The H5, H6 and H8 resonances can therefore be assigned in an A-helix on the basis of their observed chemical shifts. In general, the chemical shifts were found to be quite sensitive to structural changes. We therefore propose that a comparison between calculated and observed ¹H chemical shifts is a good tool for validation and refinement of structures derived from NOEs and J-couplings.     

Protochlorophyllide-independent import of two NADPH:Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plastid. In barley and Arabidopsis thaliana , the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse-grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse-grown plants were incubated with Δ -aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB. Assays performed on stroma-enriched fractions from chloroplasts and etioplasts of barley indicated that no post-import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.     

Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

IntroductionThe clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing.Material and methodsIndividual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes.ResultsThe prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients.Conclusion and recommendationOur findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.