C9orf72 plays a central role in Rab GTPase-dependent regulation of autophagy

A GGGGCC hexanucleotide repeat expansion in the first intron of the C9orf72 gene is the most common genetic defect associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Haploinsufficiency and a resulting loss of C9orf72 protein function has been suggested as a possible pathogenic mechanism in C9ALS/FTD. C9ALS/FTD patients exhibit specific ubiquitin and p62/sequestosome-1 positive but TDP-43 negative inclusions in the cerebellum and hippocampus, indicating possible autophagy deficits in these patients. In a recent study, we investigated this possibility by reducing expression of C9orf72 in cell lines and primary neurons and found that C9orf72 regulates the initiation of autophagy. C9orf72 interacts with Rab1a, preferentially in its GTP-bound state, as well as the ULK1 autophagy initiation complex. As an effector of Rab1a, C9orf72 controls the Rab1a-dependent trafficking of the ULK1 initiation complex prior to autophagosome formation. In line with this function, C9orf72 depletion in cell lines and primary neurons caused the accumulation of p62/sequestosome-1-positive inclusions. In support of a role in disease pathogenesis, C9ALS/FTD patient-derived iNeurons showed markedly reduced levels of autophagy. In this Commentary we summarise recent findings supporting the key role of C9orf72 in Rab GTPase-dependent regulation of autophagy and discuss autophagy dysregulation as a pathogenic mechanism in ALS/FTD

Correction of EELS dispersion non-uniformities for improved chemical shift analysis

We outline a simple routine to correct for non-uniformities in the energy dispersion of a post-column electron energy-loss spectrometer for use in scanning transmission electron microscopy. We directly measure the dispersion and its variations by sweeping a spectral feature across the full camera to produce a calibration that can be used to linearize datasets post-acquisition, without the need for reference materials. The improvements are illustrated using core excitation electron energy-loss spectroscopy (EELS) spectra collected from NiO and diamond samples. The calibration is rapid and will be of use in all EELS analysis, particularly in assessments of the chemical states of materials via the chemical shift of core-loss excitations

Characterisation of the deuterium recycling at the W divertor target plates in JET during steady-state plasma conditions and ELMs

Experiments in the JET tokamak equipped with the ITER-like wall (ILW) revealed that the inner and outer target plate at the location of the strike points represent after one year of operation intact tungsten (W) surfaces without any beryllium (Be) surface coverage. The dynamics of nearsurface retention, implantation, desorption and recycling of deuterium (D) in the divertor of plasma discharges are determined by W target plates. As the W plasma-facing components (PFCs) are not actively cooled, the surface temperature (Tsurface) is increasing with plasma exposure, varying the balance between these processes in addition to the impinging deuteron fluxes and energies. The dynamic behaviour on a slow time scale of seconds was quantified in a series of identical L-mode discharges (JET Pulse Number (JPN) # - 81938 73) by intra-shot gas analysis providing the reduction of deuterium retention in W PFCs by 1/3 at a base temperature (Tbase) range at the outer target plate between 65 °C and 150 °C equivalent to a Tsurface span of 150 °C and 420 °C. The associated recycling and molecular D desorption during the discharge varies only at lowest temperatures moderately, whereas desorption between discharges rises significantly with increasing Tbase. The retention measurements represent the sum of inner and outer divertor interaction at comparable Tsurface. The dynamic behaviour on a fast time scale of ms was studied in a series of identical H-mode discharges (JPN # - 83623 83974) and coherent edge-localized mode (ELM) averaging. High energetic ELMs of about 3 keV are impacting on the W PFCs with fluxes of 3 ´ 10 D s m 23 1 +- -2 which is about four times higher than inter-ELM ion fluxes with an impact energy of about Eim = 200 eV. This intra-ELM ion flux is associated with a high heat flux of about 60 MW m−2 to the outer target plate which causes Tsurface rise by Δ T = 100 K per ELM covering finally the range between 160 °C and 1400 °C during the flat-top phase. ELM-induced desorption from saturated nearsurface implantation regions as well as deep ELM-induced deuterium implantation areas under varying baseline temperature takes place. Subsequent refuelling by intra-ELM deuteron fluxes occurs and a complex interplay between deuterium fuelling and desorption can be observed in the temporal ELM footprint of the surface temperature (IR thermography), the impinging deuteron flux (Langmuir probes), and the Balmer radiation (emission spectroscopy) as representative for the deuterium recycling flux. In contrast to JET-C, a pronounced second peak, ; 8 ms delayed with respect to the initial ELM crash, in the Dα radiation and the ion flux has been observed. The peak can be related to desorption of implanted energetic intra-ELM D+ diffusing to the W surface, and performing local recycling.EURATOM 63305

Magnetic phase separation in ordered alloys

We present a lattice model to study the equilibrium phase diagram of ordered alloys with one magnetic component that exhibits a low temperature phase separation between paramagnetic and ferromagnetic phases. The model is constructed from the experimental facts observed in Cu$_{3-x}$AlMn$_{x}$ and it includes coupling between configurational and magnetic degrees of freedom which are appropriated for reproducing the low temperature miscibility gap. The essential ingredient for the occurrence of such a coexistence region is the development of ferromagnetic order induced by the long-range atomic order of the magnetic component. A comparative study of both mean-field and Monte Carlo solutions is presented. Moreover, the model may enable the study of the structure of the ferromagnetic domains embedded in the non-magnetic matrix. This is relevant in relation to phenomena such as magnetoresistance and paramagnetism.Comment: 12 pages, 11 figures, accepted in Phys. Rev.

Reduced tubulin tyrosination as an early marker of mercury toxicity in differentiating N2a cells

The aims of this work were to compare the effects of methyl mercury chloride and Thimerosal on neurite/process outgrowth and microtubule proteins in differentiating mouse N2a neuroblastoma and rat C6 glioma cells. Exposure for 4 h to sublethal concentrations of both compounds inhibited neurite outgrowth to a similar extent in both cells lines compared to controls. In the case of N2a cells, this inhibitory effect by both compounds was associated with a fall in the reactivity of western blots of cell extracts with monoclonal antibody T1A2, which recognises C-terminally tyrosinated α-tubulin. By contrast, reactivity with monoclonal antibody B512 (which recognises total α-tubulin) was unaffected at the same time point. These findings suggest that decreased tubulin tyrosination represents a neuron-specific early marker of mercury toxicity associated with impaired neurite outgrowth

Bioinformatic design of dendritic cell-specific synthetic promoters

Next-generation DNA vectors for cancer immunotherapies and vaccine development require promoters eliciting predefined transcriptional activities specific to target cell types, such as dendritic cells (DCs), which underpin immune response. In this study, we describe the de novo design of DC-specific synthetic promoters via in silico assembly of cis-transcription factor response elements (TFREs) that harness the DC transcriptional landscape. Using computational genome mining approaches, candidate TFREs were identified within promoter sequences of highly expressed DC-specific genes or those exhibiting an upregulated expression during DC maturation. Individual TFREs were then screened in vitro in a target DC line and off-target cell lines derived from skeletal muscle, fibroblast, epithelial, and endothelial cells using homotypic (TFRE repeats in series) reporter constructs. Based on these data, a library of heterotypic promoter assemblies varying in the TFRE composition, copy number, and sequential arrangement was constructed and tested in vitro to identify DC-specific promoters. Analysis of the transcriptional activity and specificity of these promoters unraveled underlying design rules, primarily TFRE composition, which govern the DC-specific synthetic promoter activity. Using these design rules, a second library of exclusively DC-specific promoters exhibiting varied transcriptional activities was generated. All DC-specific synthetic promoter assemblies exhibited >5-fold activity in the target DC line relative to off-target cell lines, with transcriptional activities ranging from 8 to 67% of the nonspecific human cytomegalovirus (hCMV-IE1) promoter. We show that bioinformatic analysis of a mammalian cell transcriptional landscape is an effective strategy for de novo design of cell-type-specific synthetic promoters with precisely controllable transcriptional activities

Hydrocarbon-based statistical copolymers outperform block copolymers for stabilization of ethanol–water Foams

Well-defined block copolymers have been widely used as emulsifiers, stabilizers, and dispersants in the chemical industry for at least 50 years. In contrast, nature employs amphiphilic proteins as polymeric surfactants whereby the spatial distribution of hydrophilic and hydrophobic amino acids within the polypeptide chains is optimized for surface activity. Herein, we report that polydisperse statistical copolymers prepared by conventional free-radical copolymerization can provide superior foaming performance compared to the analogous diblock copolymers. A series of predominantly (meth)acrylic comonomers are screened to identify optimal surface activity for foam stabilization of aqueous ethanol solutions. In particular, all-acrylic statistical copolymers comprising trimethylhexyl acrylate and poly(ethylene glycol) acrylate, P(TMHA-stat-PEGA), confer strong foamability and also lower the surface tension of a range of ethanol–water mixtures to a greater extent than the analogous block copolymers. For ethanol-rich hand sanitizer formulations, foam stabilization is normally achieved using environmentally persistent silicone-based copolymers or fluorinated surfactants. Herein, the best-performing fully hydrocarbon-based copolymer surfactants effectively stabilize ethanol-rich foams by a mechanism that resembles that of naturally-occurring proteins. This ability to reduce the surface tension of low-surface-energy liquids suggests a wide range of potential commercial applications

Analysis of variance in soil research: let the analysis fit the design

Sound design for experiments on soil is based on two fundamental principles: replication and randomization. Replication enables investigators to detect and measure contrasts between treatments against the backdrop of natural variation. Random allocation of experimental treatments to units enables effects to be estimated without bias and hypotheses to be tested. For inferential tests of effects to be valid an analysis of variance (anova) of the experimental data must match exactly the experimental design. Completely randomized designs are usually inefficient. Blocking will usually increase precision, and its role must be recognized as a unique entry in an anova table. Factorial designs enable questions on two or more factors and their interactions to be answered simultaneously, and split-plot designs may enable investigators to combine factors that require disparate amounts of land for each treatment. Each such design has its unique correct anova; no other anova will do. One outcome of an anova is a test of significance. If it turns out to be positive then the investigator may examine the contrasts between treatments to discover which themselves are significant. Those contrasts should have been ones in which the investigator was interested at the outset and which the experiment was designed to test. Post-hoc testing of all possible contrasts is deprecated as unsound, although the procedures may guide an investigator to further experimentation. Examples of the designs with simulated data and programs in GenStat and R for the analyses of variance are provided as File S1

The anti-NGF antibody muMab 911 both prevents and reverses pain behaviour and subchondral osteoclast numbers in a rat model of osteoarthritis pain

Objective: Nerve growth factor (NGF) has a pivotal role in peripheral hyperalgesia and inflammation; anti-NGF antibodies attenuate pain responses in inflammatory pain models, and in people with osteoarthritis (OA) or low back pain. The aim of this study was to characterise the peripheral mechanisms contributing to the analgesic effects of anti-NGF antibody treatment in an established model of joint pain, which mimics key clinical features of OA. Design: Effects of preventative vs therapeutic treatment with an anti-NGF antibody (monoclonal antibody 911: muMab 911 (10 mg/kg, s.c.)) on pain behaviour (weight bearing asymmetry and hindpaw withdrawal thresholds (PWT)), cartilage damage, synovitis and numbers of subchondral osteoclasts were investigated in the monosodium iodoacetate (MIA) model. Potential direct effects of NGF on receptor activator of nuclear factor kappa-B ligand (RANKL) mediated osteoclastogenesis were investigated in cultured human osteoclasts. Results: Intra-articular MIA injection resulted in significant pain behaviour, cartilage damage, synovitis and increased numbers of subchondral osteoclasts. Both preventative and therapeutic treatment with muMab 911 significantly prevented, or reversed, MIA-induced pain behaviour, but did not alter cartilage or synovial pathology quantified at the end of the treatment period. NGF did not facilitate RANKL driven osteoclast differentiation in vitro, but preventative or therapeutic muMab 911 reduced numbers of TRAP positive osteoclasts in the subchondral bone. Conclusions: We demonstrate that anti-NGF antibody treatment attenuates OA pain behaviour despite permitting cartilage damage and synovitis. Indirec