342 research outputs found
Structural models of the CERV M domain.
No full textA. The model at left shows the AlphaFold structural prediction for the M domain of the CERV protein in C. elegans Cer1, with pLDDT coloring for confidence scores as above. The models at right show independent predictions using AlphaFold for M domains from Cer1 elements in each of the five Caenorhabditis species aligned in S3 Fig. Despite the low degree of sequence conservation in the M domain (S3 Fig), the models are closely similar with the principal difference being short beta-strands present in C.e, C.r, and C.l, but not in C.n, C.z, or C.i; models for each of the two groups are shown in superimposition and colored arbitrarily. B. The two top images show octamer models for the M domains of CERV from C.e and C.l, colored according to pLDDT scores. Space-filling models for each octamer (below) show the similar electrostatic potentials (red negative, blue positive; ChimeraX [132]). The M domains from each of the Caenorhabditis species are predicted to form similar ring-shaped multimers; rings form with a minimum of 5 subunits, and rings consisting of 5–8 subunits have high confidence scores. The inset at right is a high magnification ribbon view of the indicated 3 subunits, shown with arbitrary colors. The lines between the subunits represent residue contacts which are 3 Å or less and colored according to the AlphaFold pLDDT confidence scale (ChimeraX [132]. The asterisk indicates the S214 phosphorylation site described in the text. (TIF)</p
CERV structure and function in gRNA export.
No full textA. The left image is the AlphaFold-predicted structural model for the complete CERV protein (aa1-517), with the pLDDT color-coded confidence scores as in Fig 2A. The H, M, and G domains and the cysteine-rich loop described in the present study are indicated. Both images at right show different views of a space-filling model of the CERV hexamer; only the indicated amino acids are depicted in the model, and the subunit coloring is arbitrary. The central ring (top right, base view) is built from multimers of the M domain with the conserved Cys loops near the central axis (see also S6 Fig). The G domains extend radially from the M domains by flexible spokes (see also S4 Fig). H domains from adjacent subunits are predicted to bind together in coiled coils that project at variable angles from the ring (bottom right). B. Western blot of protein extracts from the indicated strains: cerv(stop) = WM746, gfp:cerv/gag = WM638, and gag:gfp = WM743 (see S1 Table for details). The blot was probed sequentially with α-GAG, mAbP3C6, and α-ACTIN. Note that the single band recognized by mAbP3C6 in the gfp:cerv/gag extract is GFP:CERV; this strain does not express GAG:GFP (see analysis in S7 Fig). C. Panel 1 (top row) shows A2 pachytene germ nuclei stained for CERV (mAbP3C6); the inset at right shows CERV, gRNA, and DNA (blue and cyan) at higher magnification. Panel 2 shows A4 pachytene germ nuclei stained for CERV. The intense, nuclear foci of CERV have disappeared; CERV is dispersed in the nucleoplasm and present in irregular aggregates at the center of nucleoli (arrows). Note that the level of gRNA in the nuclear foci (double arrows) has decreased and is comparable to the signal from cytoplasmic foci of gRNA (see also S8 Fig). D. Germ nuclei from A2 adults with a CERV-specific stop mutation. Panel 1 is an 8 micron Z-projection, showing there is no gRNA detectable in the cytoplasm (compare with the cytoplasmic gRNA visible in Fig 3G, which is a 3 micron Z-projection of similar germ nuclei). The lack of CERV expression is shown in the right half of panel 1. Panel 2 is a 3 micron projection showing the absence of GAG particles in the gonad core. E. Pachytene germ nuclei in a wild-type hermaphrodite cultured at the non-permissive temperature of 25°C. The gonad has prominent nuclear, but not cytoplasmic, foci of gRNA, and GAG is not expressed. Note that CERV is present in the nucleoplasm but not concentrated at the nuclear gRNA foci (arrows). F. A2 adult male gonad at 15°C. The gonad has nuclear, but not cytoplasmic, foci of gRNA, and GAG is not expressed. Note that CERV is present in the nucleoplasm but not concentrated on the nuclear gRNA (arrows). G. A2 adult hermaphrodite gonad at 15°C, showing the boundary (arrowhead) between the proliferation zone (inset 1) where gRNA is not exported, and the pachytene region (inset 2) where gRNA is exported and GAG is expressed. Note that the appearance of gRNA and GAG in the cytoplasm corresponds to where CERV first concentrates on the nuclear foci of gRNA. These images are 3-micron Z-projections, so signals from a few cytoplasmic foci of gRNA are artificially superimposed on the nuclei. Scale bars in microns = (A-G) 1.0.</p
CERV phosphorylation and the cysteine-rich loop are required for gRNA export.
No full textA.The peptide sequence at top left shows the beginning of the CERV M; residues in bold are invariant in diverse species of Caenorhabditis (see also S3 Fig). Most of this region is contained in a flexible cysteine-rich loop (AlphaFold model at right) which faces the central axis of the predicted ring multimers (Fig 3A; see S6 Fig for additional analysis of the M domain). The Cys loop brings multiple Cys residues into close proximity: predicted distances between the cysteine sulfur atoms are C195-C193 (3.3 Å), C193-C200 (3.8 Å), C200-C269 (3.9 Å), and C269-C195 (3.3 Å) (ChimeraX [132]). The blot (inset) shows protein extracts from the following strains: WT, WM746 [cerv(stop)], and JJ2706 [(cerv(S214A)]; see S1 Table for strain details. The blot was probed with mAbP3C6, which stains a prominent CERV band and a weaker GAG band in the wild-type extract. The cerv-specific STOP mutation eliminates both the CERV and GAG band, while the S214A substitution eliminates only the GAG band. B. Immunostained gonads from a homozygous mutant with a CERV S214A substitution (panel 1), and from a heterozygous strain with the same mutation plus a wild-type copy (panel 2). C. Immunoprecipitation assays of FLAG-tagged CERV and FLAG-tagged GAG followed by Western Blot analysis. Extracts are from WT worms, a strain with a FLAG tag on the N-terminus of CERV (WM894), and a strain with a FLAG tag on the C-terminus of GAG (WM895); see S1 Table for strain details. The extracts were immunoprecipitated with an anti-FLAG antibody and blotted; a duplicate blot is shown at right after treating the same extracts with phosphatase. Both blots were stained with the α-pS/T-P antiserum; the arrowhead points to a band at the predicted size of CERV that is absent after phosphatase treatment. D. Germ nuclei in an A2 wild-type gonad (panel 1) and a CERV S214A mutant (panel 2) stained for CERV and phospho-S/T-P. Note that the S214A mutant nuclei fail to stain with α-pS/T-P and CERV is present in the nucleoplasm but not concentrated into foci. E. Panel 1 shows different types of germ nuclei as listed after immunostaining for CERV and α-pS/T-P. Note that CERV only concentrates into foci after hermaphrodites are shifted to the export-permissive temperature of 15°C, where CERV becomes phosphorylated. Panel 2 shows the gonad boundary (arrowhead) where CERV first concentrates at the nuclear foci of gRNA, and panel 3 shows the subsequent, post-pachytene boundary where the CERV foci disappear. Some CERV in the post-pachytene region localizes to nucleolar inclusions (arrows) that stain positively for ubiquitin (panel 4, red). F. Mutant gonad with serine substitutions at each of the cysteines C193, C195, and C200 in the M domain of CERV (cer1(zu527) and strain JJ2700, Fig 4A). Panel 1 shows that nuclear, but not cytoplasmic foci of gRNA are present, and that CERV does not concentrate on the nuclear foci. Panel 2 shows that CERV appears to be phosphorylated at S214. Arrows indicate perinuclear foci of CERV that occur frequently in this strain. G. Mutant gonad with an R194A substitution in the M domain of CERV (cer1(zu531) and strain JJ2704). Panel 1 shows that gRNA is present in nuclear but not cytoplasmic foci, and that GAG is not expressed. Panel 2 shows that the nuclei contain bright foci of phosphorylated CERV. Panel 3 is a 4-micron Z-projection to visualize entire nuclear volumes, and shows that nuclei have more than the two expected CERV foci and that none of the CERV foci are coincident with the nuclear gRNA foci. Scale bars in microns (B,D-G) 1.0 micron.</p
Structural models for Cer1 GAG and CERV.
No full textA. Diagram comparing sizes and domains of GAG proteins from Ty3 [45]; HIV-1 [46]; and RSV [47] with the predicted GAG region of Cer1. For reference below, the CERVNTD is aligned with Cer1 GAG to show the position of in-frame peptides common to both (exons 1–3) and the unique peptide (exon 4). Some retroviral GAG proteins contain short, disordered regions between MA, CA, and NC that might function in particle morphogenesis or GAG-RNA interactions, but which are removed from mature virions [141]. Cer1 GAG is predicted to have three intrinsically disordered regions acids (grey boxes: aa1-31, 263–358, 564–630) as scored by IUPRED server (https://iupred3.elte.hu/) using "long disorder" parameters [142], but it has not been determined whether these represent processing sites. B. Structural prediction for Cer1 GAG residues 387–546 generated with AlphaFold and colored as per the AlphaFold pLDDT table, a per-residue estimate of confidence on a scale of 0–100 [131]. The DALI server was used to compare this model against experimentally determined structures in the PDB as described for S1 Fig. The model showed the highest similarity to CA proteins from diverse retroviruses and endogenous LTR retrotransposons, and particularly the C-terminal half of CA which mediates subunit multimerization [143]. CA domains from RSV (PDB:3g29) and PEG10 (PDB:7lga) are shown in the middle panel for comparison, along with a superimposition of those structures with the Cer1 model (ChimeraX Matchmaker [144]). The table shows quantitative data for representative structural alignments with DALI Z-scores and root-mean-square deviations (RMSD) from backbone in Angstroms; DALI Z-scores above 2 usually correspond to similar folds [145]. C. AlphaFold structural prediction for the large region of Cer1 GAG preceding the CA-like domain, colored as per the pLDDT table [131]. The model shows three long, anti-parallel alpha-helices, the longest of which contains a predicted leucine zipper (LZ). The red brackets mark the boundaries of the three peptides (e1-e3) that are spliced in-frame to make the CERVNTD. The model was searched against the PDB as above, but did not appear to have significant similarity to known proteins, including retroviral MA proteins. D. AlphaFold structural prediction for CERV. CERV residues here and below are numbered with respect to the CERV protein, rather than the entire Cer1 polyprotein. The complete CERV protein (aa1-517) was used for the structural prediction, but for clarity the terminal unstructured regions (1–20 and 470–517) are hidden. Red bars correspond to exon boundaries. The H domain consists of long, anti-parallel alpha-helices shared in part with GAG, and does not appear to have significant similarity to known proteins. The G and M domains are described below and in S6 Fig. E. The AlphaFold structural prediction for the G domain of C.e CERV is shown at top left, colored arbitrarily to indicate alpha-helices (salmon) and beta-strands (green). Highly similar structural models for the G domain were obtained in independent modeling for each of the Caenorhabditis species aligned in S1 Fig, shown here at smaller scale for C. zanzibari and C. inopinata. The G domain consists of five alpha helices (α1-α5) surrounding a five-stranded parallel β sheet; α1 and α5 are on one side of the sheet, and α2–4 are on the opposite side. This basic structure is termed a Rossmann-type fold, and is a common structural motif in nucleotide-binding proteins [146]. Most of those proteins have conserved motifs associated with ATP or GTP hydrolysis, such as the Walker A motif or P-loop (phosphate binding loop) [147], but some structurally similar proteins of unknown function have been identified, such as pseudoGTPases [148]. The G domain of CERV lacks critical residues in the P-loop and other motifs (bottom right), indicating that the G domain is not predicted to function in GTP hydrolysis. The DALI server was used as above to search for structures in the PDB with similarity to the G domain, and matches were found to numerous and diverse nucleotide-binding proteins, here shown for human Rag GTPase (PDB: 6wj2-F; DALI Z-score 9.2). The G domain also showed structural similarity to a few proteins that are not predicted to be NTP-binding proteins. One of these, TSR1 (PDB: 5wwn-A; DALI Z-score 6.2), is a pre-rRNA-processing protein that appears to be an inactive, structural mimic of a GTPase [149]. A second example is the TIR domain of the plant Arabidopsis immune receptor RRS1 (PDB: 4c6t-A; DALI Z-score 9.7). The TIR (Toll-interleukin-1 receptor) domain is thought to mediate protein heterodimerization in response to pathogen infection [150]. (TIF)</p
El Cuestionario de Experiencias Relacionadas con los Videojuegos (CERV) : un instrumento para detectar el uso problemático de videojuegos en adolescentes españoles
No full textEl objetivo del presente estudio es validar el Cuestionario de Experiencias Relacionadas con los Videojuegos (CERV). El cuestionario consta de 17 ítems, desarrollados a partir del CERI de Beranuy y cols., y valora el uso problemático de los videojuegos no masivos. Se ha validado para adolescentes que cursan estudios de secundaria obligatoria. Para la validación se ha realizado un análisis factorial confirmatorio (AFC) y un análisis de consistencia interna. La estructura factorial muestra dos factores a) Dependencia psicológica y uso para la evasión, y b) Consecuencias negativas del uso de videojuegos. Se ofrecen puntos de corte de la escala para sujetos sin problemas en el uso de videojuegos (SP), problemas potenciales en el uso de videojuegos (PP) y problemas severos en el uso de videojuegos (PS). Los resultados indican que se da una mayor prevalencia entre varones y que el uso problemático disminuye con la edad. El CERV parece ser un buen instrumento para el cribado de adolescentes con dificultades derivadas del uso de videojuegos. Estudios futuros deberían relacionar el uso problemático de videojuegos con dificultades en otros ámbitos de la vida, como el académicoThe aim of this study is to validate the Video Game-Related Experiences Questionnaire (CERV in Spanish). The questionnaire consists of 17 items, developed from the CERI (Internet-Related Experiences Questionnaire - Beranuy and cols.), and assesses the problematic use of non-massive video games. It was validated for adolescents in Compulsory Secondary Education. To validate the questionnaire, a confirmatory factor analysis (CFA) and an internal consistency analysis were carried out. The factor structure shows two factors: (a) Psychological dependence and use for evasion; and (b) Negative consequences of using video games. Two cut-off points were established for people with no problems in their use of video games (NP), with potential problems in their use of video games (PP), and with serious problems in their use of video games (SP). Results show that there is higher prevalence among males and that problematic use decreases with age. The CERV seems to be a good instrument for the screening of adolescents with difficulties deriving from video game use. Further research should relate problematic video game use with difficulties in other life domains, such as the academic fiel
El Cuestionario de Experiencias Relacionadas con los Videojuegos (CERV) : un instrumento para detectar el uso problemático de videojuegos en adolescentes españoles
No full textEl objetivo del presente estudio es validar el Cuestionario de Experiencias Relacionadas con los Videojuegos (CERV). El cuestionario consta de 17 ítems, desarrollados a partir del CERI de Beranuy y cols., y valora el uso problemático de los videojuegos no masivos. Se ha validado para adolescentes que cursan estudios de secundaria obligatoria. Para la validación se ha realizado un análisis factorial confirmatorio (AFC) y un análisis de consistencia interna. La estructura factorial muestra dos factores a) Dependencia psicológica y uso para la evasión, y b) Consecuencias negativas del uso de videojuegos. Se ofrecen puntos de corte de la escala para sujetos sin problemas en el uso de videojuegos (SP), problemas potenciales en el uso de videojuegos (PP) y problemas severos en el uso de videojuegos (PS). Los resultados indican que se da una mayor prevalencia entre varones y que el uso problemático disminuye con la edad. El CERV parece ser un buen instrumento para el cribado de adolescentes con dificultades derivadas del uso de videojuegos. Estudios futuros deberían relacionar el uso problemático de videojuegos con dificultades en otros ámbitos de la vida, como el académicoThe aim of this study is to validate the Video Game-Related Experiences Questionnaire (CERV in Spanish). The questionnaire consists of 17 items, developed from the CERI (Internet-Related Experiences Questionnaire - Beranuy and cols.), and assesses the problematic use of non-massive video games. It was validated for adolescents in Compulsory Secondary Education. To validate the questionnaire, a confirmatory factor analysis (CFA) and an internal consistency analysis were carried out. The factor structure shows two factors: (a) Psychological dependence and use for evasion; and (b) Negative consequences of using video games. Two cut-off points were established for people with no problems in their use of video games (NP), with potential problems in their use of video games (PP), and with serious problems in their use of video games (SP). Results show that there is higher prevalence among males and that problematic use decreases with age. The CERV seems to be a good instrument for the screening of adolescents with difficulties deriving from video game use. Further research should relate problematic video game use with difficulties in other life domains, such as the academic fiel
The Questionnaire of Experiences Associated with Video games (CERV): An instrument to detect the problematic use of video games in Spanish adolescents
No full textEl objetivo del presente estudio es validar el Cuestionario de Experiencias Relacionadas con los Videojuegos (CERV). El cuestionario consta de 17 ítems, desarrollados a partir del CERI de Beranuy y cols., y valora el uso problemático de los videojuegos no masivos. Se ha validado para adolescentes que cursan estudios de secundaria obligatoria. Para la validación se ha realizado un análisis factorial confirmatorio (AFC) y un análisis de consistencia interna. La estructura factorial muestra dos factores a) Dependencia psicológica y uso para la evasión, y b) Consecuencias negativas del uso de videojuegos. Se ofrecen puntos de corte de la escala para sujetos sin problemas en el uso de videojuegos (SP), problemas potenciales en el uso de videojuegos (PP) y problemas severos en el uso de videojuegos (PS). Los resultados indican que se da una mayor prevalencia entre varones y que el uso problemático disminuye con la edad. El CERV parece ser un buen instrumento para el cribado de adolescentes con dificultades derivadas del uso de videojuegos. Estudios futuros deberían relacionar el uso problemático de videojuegos con dificultades en otros ámbitos de la vida, como el académico.The aim of this study is to validate the Video Game-Related Experiences Questionnaire (CERV in Spanish). The questionnaire
consists of 17 items, developed from the CERI (Internet-Related Experiences Questionnaire - Beranuy and cols.), and assesses the problematic use of non-massive video games. It was validated for adolescents in Compulsory Secondary Education. To validate the questionnaire, a confirmatory factor analysis (CFA) and an internal consistency analysis were carried out. The factor structure shows two factors: (a) Psychological dependence and use for evasion; and (b) Negative consequences of using video games. Two cut-off points were established for people with no problems in their use of video games (NP), with potential problems in their use of video games (PP), and with serious problems in their use of video games (SP). Results show that there is higher prevalence among males and that problematic use decreases with age. The CERV seems to be a good instrument for the screening of adolescents with difficulties deriving from video game use. Further research should relate problematic video game use with difficulties in other life domains, such as the academic field
GDS (Geomagnetic Depth Sounding) in Italy: applications and perspectives
No full textThe analysis of geomagnetic field variations is a useful tool to detect electrical conductivity contrasts within the Earth. Lateral resolution of outlined patterns depends on the array dimensions and density of measurement sites over the investigated area. The inspection depth is constrained by the period of geomagnetic variations considered in data processing. Regions with significant geological features such as boundaries of continental plates, marginal areas of contact between tectonic units or other geodynamical processes, are of primary interest for the application of the MagnetoVariational (MV) method. In the last ten years, in the frame of the ElectroMagnetic (EM) sounding techniques in applied geophysics, this method has been applied in Italy by researchers of the Istituto Nazionale di Geofisica, Rome, the Dipartimento di Scienze della Terra, Universitá di Genova and the Czech Science Academy of Prague. The Ivrea body in the Northwestern Alps and their junction with the Apennine chain, the micro-plate of the Sardinian-Corsican system and, recently, the central part of the peninsula along Tyrrhenian-Adriatic lithospheric transects were investigated. Studies in time and frequency-domain used in the first investigations, have been followed by more refined analysis involving tests on the induced EM field dimension, computations of single site Transfer Functions (TFs) through Parkinson arrows' and Fourier maps in the Hypothetical Event technique (HE). It was possible to describe the electrical conductivity distribution in the inner part of the SW Alpine arc and to confirm the presence of lithospheric and asthenospheric anomalies obtained by other geophysical methods. For the Sardinia-Corsica system, 2D and 3D inversion models highlighted the existence of two major conducting bodies, one north of Corsica, and the other south of Sardinia. In Central Italy, the regional electrical conductivity distribution pointed out a deep conductive structure beneath the Apennines and a very resistive root for this part of the mountain chain
Stochastic interpretation of magnetotelluric data, comparison of methods
No full textGlobal optimization and stochastic approaches to the interpretation of measured data have recently gained particular
attraction as tools for directed search for and/or verification of characteristic structural details and quantitative
parameters of the deep structure, which is a task often arising when interpreting geoelectrical induction
data in seismoactive and volcanic areas. We present a comparison of three common global optimization and stochastic
approaches to the solution of a magnetotelluric inverse problem for thick layer structures, specifically the
controlled random search algorithm, the stochastic sampling by the Monte Carlo method with Markov chains
and its newly suggested approximate, but largely accelerated, version, the neighbourhood algorithm. We test the
algorithms on a notoriously difficult synthetic 5-layer structure with two conductors situated at different depths,
as well as on the experimental COPROD1 data set standardly used to benchmark 1D magnetotelluric inversion
codes. The controlled random search algorithm is a fast and reliable global minimization procedure if a relatively
small number of parameters is involved and a search for a single target minimum is the main objective of the
inversion. By repeated runs with different starting test model pools, a sufficiently exhaustive mapping of the parameter
space can be accomplished. The Markov chain Monte Carlo gives the most complete information for the
parameter estimation and their uncertainty assessment by providing samples from the posterior probability distribution
of the model parameters conditioned on the experimental data. Though computationally intensive, this
method shows good performance provided the model parameters are sufficiently decorrelated. For layered models
with mixed resistivities and layer thicknesses, where strong correlations occur and even different model classes
may conform to the target function, the method often converges poorly and even very long chains do not guarantee
fair distributions of the model parameters according to their probability densities. The neighbourhood resampling
procedure attempts to accelerate the Monte Carlo simulation by approximating the computationally expensive
true target function by a simpler, piecewise constant interpolant on a Voronoi mesh constructed over a
set of pre-generated models. The method performs relatively fast but seems to suggest systematically larger uncertainties
for the model parameters. The results of the stochastic simulations are compared with the standard
linearized solutions both for thick layer models and for smooth Occam solutions.JCR Journalope
Frequency and Volume of Resistance Training: Effect on Cervical Extension Strength
No full textQuantification of cervical extension (CERV EXT) strength is complicated by the inability to stabilize the torso and isolate the CERV EXT muscles. A newly developed machine designed to stabilize the torso and isolate the CERV EXT muscles was used to evaluate the effect of frequency and volume of resistance training on CERV EXT strength. Fifty men (age, 26 +/- 9 years; height, 174 +/- 16 cm; weight, 74 +/- 9 kg) and 28 women (age, 30 +/- 9 years; height, 152 +/- 32 cm; weight, 62 +/- 7 kg) volunteered to participate. Subjects were randomly stratified to one of four training groups or a control group (CONT, n = 19) that did not train. Each training group exercised for 12 weeks as follows; once per week using one set of dynamic exercise (DYN 1x/wk, n = 14), once per week using one set of DYN and one set of maximal isometric (IM) exercise at eight angles through a 126 degrees-range of CERV EXT (DYN-IM 1x/wk, n = 16), DYN 2x/wk (n = 19), or DYN-IM 2x/wk (n = 10). Maximal IM torque was measured at eight angles initially and after 12 weeks of training. All training groups improved CERV EXT strength (p \u3c or = 0.05) at all angles tested compared to the CONT except for DYN once per week at 0 degree of CERV flexion. A greater increase in strength was found when the groups that trained two times a week were compared to those that trained once per week
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