Enhancement of C2C12 myoblast proliferation and differentiation by GASP-2, a myostatin inhibitor

BackgroundGASP-2 is a secreted multi-domain glycoprotein known as a specific inhibitor of myostatin and GDF-11. Here we investigate the role of GASP-2 on myogenesis and the effect of its glycosylation on its activity.MethodsGASP-2 overexpression or knockdown by shRNAs were carried out on C2C12 myoblasts cells. In silico analysis of GASP-2 protein was performed to identify its glycosylation sites. We produced a mouse recombinant GASP-2 protein in a prokaryotic system to obtain a fully deglycosylated protein allowing us to study the importance of this post-translational modification on GASP-2 activity.ResultsBoth mature and deglycosylated GASP-2 proteins increase C2C12 proliferation and differentiation by inhibiting the myostatin pathway. In silico and western-blot analyses revealed that GASP-2 presents one consensus sequence for N-glycosylation and six potential sites of mucin-type O-glycosylation.ConclusionsGASP-2 promotes myogenesis and thus independently of its glycosylation.General significanceThis is the first report demonstrating that GASP-2 promotes proliferation and differentiation of myoblasts by inhibiting the canonical pathway of myostatin.     

Assessing the sensitivity of divergence time estimates to locus sampling,calibration points,and model priors in a RAD-seq phylogeny of Carex section Schoenoxiphium

Restriction site-associated DNA sequencing (RAD-seq) and related methods have become relatively common approaches to resolve species-level phylogeny. It is not clear, however, whether RAD-seq data matrices are well suited to relaxed clock inference of divergence times, given the size of the matrices and the abundance of missing data. We investigated the sensitivity of Bayesian relaxed clock estimates of divergence times to alternative analytical decisions on an empirical RAD-seq phylogenetic matrix. We explored the relative contribution of secondary calibration strategies, amount of missing data, and the data partition analyzed to overall variance in divergence times inferred using BEAST MCMC analyses of Carex section Schoenoxiphium (Cyperaceae)—a recent radiation for which we have nearly complete species sampling of RAD-seq data. The crown node for Schoenoxiphium was estimated to be 15.22 (9.56–21.18) Ma using a single calibration point and low missing data, 11.93 (8.07–16.03) Ma using multiple calibration points and low missing data, and 8.34 (5.41–11.22) using multiple calibrations but high missing data. We found that using matrices with more than half of the individuals with missing data inferred younger mean ages for all nodes. Moreover, we have found that our molecular clock estimates are sensitive to the positions of the calibration(s) in our phylogenetic tree (using matrices with low missing data), especially when only a single calibration was applied to estimate divergence times. These results argue for sensitivity analyses and caution in interpreting divergence time estimates from RAD-seq data.     

Linkages between unpaved forest roads and streambed sediment: why context matters in directing road restoration

Unpaved forest roads remain a pervasive disturbance on public lands and mitigating sediment from road networks remains a priority for management agencies. Restoring roaded landscapes is becoming increasingly important for many native coldwater fishes that disproportionately rely on public lands for persistence. However, effectively targeting restoration opportunities requires a comprehensive understanding of the effects of roads across different ecosystems. Here, we combine a review and a field study to evaluate the status of knowledge supporting the conceptual framework linking unpaved forest roads with streambed sediment. Through our review, we specifically focused on those studies linking measures of the density of forest roads or sediment delivery with empirical streambed sediment measures. Our field study provides an example of a targeted effort of linking spatially explicit estimates of sediment production with measures of streambed sediment. Surprisingly, our review uncovered few studies (n = 8) that empirically tested the conceptual framework linking unpaved forest roads and streambed sediment, and the results varied considerably. Field results generally supported the conceptual model that unpaved forest roads can control streambed sediment quality, but demonstrated high‐spatial variability in the effects of forest roads on streambed sediment and the need to address hotspots of sediment sources. The importance of context in the effects of forest roads is apparent in both our review and field data, suggesting the need for in situ studies to avoid misdirected restoration actions.     

In vitro recombination and terminal elongation of RNA by Q beta replicase.

SV-11 is a short-chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV-11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV-11 contains an inverse duplication of the high-melting domain of MNV-11. SV-11 is thus a recombinant between the plus and minus strands of MNV-11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin-form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3' terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3' terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template-instructed synthesis. The reaction also adds nucleotides to the 3' terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.     

Agmatoploidy inCarex laevigata (Cyperaceae). Fusion and fission of chromosomes as the mechanism of cytogenetic evolution in Iberian populations

In this paper cytogenetic studies on 64 specimens from 20 Iberian populations ofCarex laevigata (Cyperaceae) are presented. Chromosome behaviour in meiosis suggests that the different chromosome numbers obtained (ranging from 2n = 69 to 2n = 80) were distributed according to an increasing geographic gradient of chromosome fission along the North South direction. Four relatively stable areas were also delimited according to chromosome numbers displayed by this species, i.e. 2n = c. 72, c. 74, c. 76, and 78. The meiotic behaviour ofCarex ×deserta (C. laevigata ×C. binervis) was also studied.     

The lymphocyte function-associated antigen (LFA)-1 and CD2/LFA-3 pathways of antigen-independent human T cell adhesion

Human cytotoxic T lymphocyte clones form conjugates with both antigen-positive and antigen-negative lymphoblastoid cells. Conjugates with antigen-negative targets form as rapidly, and are almost as frequent, as those with antigen-positive targets; both types are strong. Monoclonal antibodies against lymphocyte function-associated antigen (LFA)-1, CD2, and LFA-3 (or their Fab fragments) each consistently inhibit conjugate formation, but only partially; mixes of alpha LFA-1 with either CD2 monoclonal antibodies or alpha LFA-3 cause complete inhibition. Our previous studies have demonstrated two distinct pathways of antigen-independent conjugate (AIC) formation, one involving LFA-1 and the other involving CD2/LFA-3. The present studies showing supra-additive inhibition with mixes of Fab indicate that at least a major fraction of the conjugates involve T cells which utilize both pathways. Preincubation studies (and restricted expression for CD2) demonstrate that in the CD2/LFA-3 pathway, CD2 is critical on the effector and LFA-3 on the target and that in the LFA-1 pathway, LFA-1 is critical on the effector. Analysis of conjugate formation by primary allosensitized T cells confirms the critical findings made with T cell clones. Among a panel of antigen-negative "target" cell lines tested, there is wide variation in the number of AIC formed with cytotoxic T lymphocyte clones; this variation correlates partially with differences in level of expression of LFA-3. Both pathways of adhesion are utilized in AIC formation with all five targets tested, but there was variation between targets in the relative contribution by each pathway. Studies of inhibition of lysis (rather than conjugate formation) support the relevance of the two-pathway model to the lytic process as a whole. These studies demonstrate the general involvement of two pathways of adhesion in human T cell interactions: one involving T cell LFA-1 and the other involving T cell CD2 binding to target cell LFA-3.     

Probabilistic accounting of uncertainty in forecasts of species distributions under climate change

Forecasts of species distributions under future climates are inherently uncertain, but there have been few attempts to describe this uncertainty comprehensively in a probabilistic manner. We developed a Monte Carlo approach that accounts for uncertainty within generalized linear regression models (parameter uncertainty and residual error), uncertainty among competing models (model uncertainty), and uncertainty in future climate conditions (climate uncertainty) to produce site‐specific frequency distributions of occurrence probabilities across a species' range. We illustrated the method by forecasting suitable habitat for bull trout (Salvelinus confluentus) in the Interior Columbia River Basin, USA, under recent and projected 2040s and 2080s climate conditions. The 95% interval of total suitable habitat under recent conditions was estimated at 30.1–42.5 thousand km; this was predicted to decline to 0.5–7.9 thousand km by the 2080s. Projections for the 2080s showed that the great majority of stream segments would be unsuitable with high certainty, regardless of the climate data set or bull trout model employed. The largest contributor to uncertainty in total suitable habitat was climate uncertainty, followed by parameter uncertainty and model uncertainty. Our approach makes it possible to calculate a full distribution of possible outcomes for a species, and permits ready graphical display of uncertainty for individual locations and of total habitat.     

A review of the relationships between drought and forest fire in the United States

The historical and presettlement relationships between drought and wildfire are well documented in North America, with forest fire occurrence and area clearly increasing in response to drought. There is also evidence that drought interacts with other controls (forest productivity, topography, fire weather, management activities) to affect fire intensity, severity, extent, and frequency. Fire regime characteristics arise across many individual fires at a variety of spatial and temporal scales, so both weather and climate – including short‐ and long‐term droughts – are important and influence several, but not all, aspects of fire regimes. We review relationships between drought and fire regimes in United States forests, fire‐related drought metrics and expected changes in fire risk, and implications for fire management under climate change. Collectively, this points to a conceptual model of fire on real landscapes: fire regimes, and how they change through time, are products of fuels and how other factors affect their availability (abundance, arrangement, continuity) and flammability (moisture, chemical composition). Climate, management, and land use all affect availability, flammability, and probability of ignition differently in different parts of North America. From a fire ecology perspective, the concept of drought varies with scale, application, scientific or management objective, and ecosystem.     

Frontal sinus fractures: guidelines to management

The appropriate management of frontal sinus fractures is controversial. Experience with 78 frontal sinus fractures over a 9-year period was reviewed, and the fractures were classified into anterior wall, anterobasilar, and frontal skull fracture extensions. The presence of a concomitant CSF leak or an air-fluid level in the sinus was a diagnostic clue of posterior wall involvement. Ablation or obliteration of the fractured frontal sinus is not necessary. Primary reconstruction of the sinus and nasofrontal duct drainage constitute the preferred treatment. "Cranialization" of a severely damaged sinus is performed by excision of the posterior wall plugging of the nasofrontal duct and reconstruction of the anterior wall. Reconstruction of the anterior wall with primary bone grafting may be necessary in selected patients.