The as-synthesized samples were characterized by using field emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HRTEM), ultraviolet-visible (UV-vis) spectroscopy, Raman spectroscopy, X-ray diffraction (XRD), thermogravimetric-differential thermal analysis (TG-DTA) and gas-sensing test. The resistivity of the thick-film gas sensors based on sandwich-like graphene/WO3 nanocomposites can be controlled by varying the amount of graphene in the composites. Graphene/WO3 nanocomposites with graphene content higher than 1% show fast response, high selectivity and fine sensitivity to NOx.Graphene has attracted enormous attention owing to its extraordinary properties, while graphene-based nanocomposites hold promise for many applications. In this paper, we present a two-step exploitation method for preparation of graphene oxides and a facile solvothermal route for preparation of few-layer graphene nanosheets and graphene/WO Becau sensors ,14.3 is a well-known semiconductor material used for gas sensors to monitor toxic and explosive gases  [3 gas sensors is limited by poor selectivity, long response time, low sensitivity and high resistivity. Although methods, such as reduction of grain size, use of novel synthesis morphology, addition of dopants, and use of mixed sensing materials, have been adopted to enhance the sensing properties of WO3, fewer contributions have been made to reducing the high resistivity of WO3-based sensors [WOand NH3) ,18,19,20 sensors ,22.3 nanocomposites were prepared by a facile solvothermal method in an ethanol-distilled water medium. Material characterizations of the prepared samples were carried out by FE-SEM, HRTEM, UV-vis spectroscopy, Raman spectroscopy, XRD and TG-DTA. Gas-sensing properties of the sensors based on pure WO3, few-layer graphene nanosheets, and graphene/WO3 nanocomposites were investigated by using a HW-30A gas-sensing measurement system.In this paper, resistivity-controlled thick-film sensors based on few-layer graphene nanosheets and graphene/WOMorphologies of the first exploited graphite (EG) and graphite prepared from natural graphite by ultrasonication for 30 min are shown in −1, labeled as the D band, corresponds to breathing modes of rings or K-point phonons of A1g symmetry, whereas the G band at 1580 cm−1 corresponds to an E2g mode of graphite and is related to the vibration of sp2 bonded carbon atoms in a two-dimensional hexagonal lattice. The ratio of the intensity of the G band to that of the D band is related to the in-plane crystallite size, La. The in-plane crystallite size of FGO and FG, calculated by using the relationship La (nm) = 4.4 (I(G)/I(D)), about 4.51 and 3.7 nm, respectively, for which the corresponding I(G)/I(D) ratios are 1.0242 and 0.8315, respectively [−1, making it conceivable that the sample contained highly disordered and randomly arranged graphene sheets.Raman spectroscopy is a nondestructive and efficient approach to characterize graphitic materials, especially for determining ordered and disordered crystal structures of graphene. The Raman spectra of FGO and FG are shown in ectively . Compare3 nanocomposites can be indexed to hexagonal WO3(H2O)0.33 (JCPDS 87-1203), which is the same as the WO3 sample  to (002), which indicated that the preferred growth direction of WO3 was changed.XRD measurements were employed to investigate phase and structure of the synthesized samples. As shown in 3 sample d, and no3 and the FG (5%)/WO3 nanocomposites were further analyzed by FE-SEM and HRTEM. As shown in 3 sample exhibits unordered bundles with uniform strip size. TEM investigation can identify the bundled feature, giving evidence that each one-dimensional nanostructure bundle consists of a nanostrip with a thickness of about 100 nm, as shown in 3 nanocomposites. As displayed in 3 particles distributed randomly on the FG surface with different morphology are easily observed. The morphology change of WO3 indicates that the addition of FG has a significant effect on the WO3 particle growth, which is consistent with the XRD result, i.e., the preferred growth direction of WO3 was changed. The high-resolution TEM image of another area of the sample, given in 3(H2O)0.33. This is evidence for the existence of a sandwich-like structure between graphene and WO3. It is believed that both oxygen-containing defect sites and pristine regions of the GO favorably interact with W6+ via van der Waals interactions and chemisorptions, respectively. In addition, oxygen functional groups located at the surface of GO can act as anchor sites and effectively hinder diffusion, recrystallization, and growth of WO3 grains. The two morphologies of FG/WO3 nanocomposites may be formed via isolated growth of WO3 crystal species at the surface of GO and in situ growth of WO3 crystal species on the surface of GO, respectively.Morphologies of WO3 nanocomposites synthesized via the solvothermal method were characterized by TG-DTA, which was performed in air and N2 atmospheres with a heating rate of 15 °C·min−1. As shown in 2 atmosphere is significantly lower than that in air atmosphere especially at temperatures higher than 450 °C, which should result from the combustion of the carbon skeleton of graphene happened above 420 °C in air.Thermal properties and composition of the FG and FG (5%)/WO3 nanocomposites synthesized via the solvothermal method shows two abrupt weight losses occurring between 300 and 630 °C in air atmosphere. Correspondingly, the DTA curve shows an endothermic peak and an exothermal peak, which can be assigned to the loss of chemically bonded water and oxidation of graphene in air, respectively. As shown in 3 nanocomposites performed in N2 atmosphere exhibits a continuous weight loss, which can be assigned to the loss of chemically bonded water.From 3 sensors to different kinds of target gases were measured at different operating voltages. The results are shown in 3 sensors exhibit the largest response to NOx among all the tested gases at an operating voltage of 3.00 V. Furthermore, the optimal operating voltages are 3.00 V for NH3, 3.25 V for H2S and 4.00 V for acetone, dimethylbenzene, ethanol and trimethylamine. The response of the pure WO3 sensor to other target gases is extremely low at the tested operating voltage. As a result, the pure WO3 sensor is a very promising semiconductor for monitoring NOx at relatively low temperatures, but its high resistance limited its wider application.Selectivity is an important factor of gas sensors, so the responses of pure WO3-nanocomposite-based sensors with different amounts of FG  are shown in 3 nanocomposites with FG amount below 2%  is noticeably higher than that of the pure WO3 sensor . The sensors with few-layer graphene nanosheets and graphene/WO3 nanocomposites with graphene content higher than 1% have lower resistivity (below 135 kΩ) than that of the pure WO3 sensor. As we all know, WO3 is an n-type semiconductor and reduced graphene oxide is a p-type semiconductor, so it can form a p-n heterojunction between FG and WO3. The p-n heterojunction and the conduction of graphene play an important role in the resistance of FG/WO3 nanocomposites. As the amount of FG drops below 2%, the resistance of the FG/WO3 nanocomposites becomes controlled by the p-n heterojunction, so the resistance is really higher than that of WO3 and the sensors based on these are not suited for gas sensor application.Resistances of the FG/WO3 nanocomposites was studied at an operating voltage between 1.45 and 2.70 V. It was found that FG/WO3 nanocomposites only had gas sensitivity to NOx. The responses of FG/WO3 nanocomposite sensors with different amounts of FG to 100 ppm NOx at different operating voltages between 1.45 and 2.70 V are presented in x increased as the amount of FG decreased. Moreover, at operating voltages between 1.60 and 2.45 V, with the increase of operating voltage, the response of the FG/WO3 nanocomposite sensors was enhanced, and for operating voltages higher than 2.45 V, the response declined. As is well known, WO3 is very sensitive to NOx at relative higher operating voltage. As the operating voltage was increased to 2.45 V, the effect of WO3 on the response of the FG/WO3 nanocomposite sensors to NOx was enhanced. In other words, the counterbalance of the p-n heterojunction in the response of the FG/WO3 nanocomposite sensors to NOx cannot be ignored, so the response to NOx trends downward. Compared with the pure WO3 sensor, the sensitivity of FG/WO3 nanocomposite sensors was decreased, but both the resistance of the FG/WO3 nanocomposite sensors and the operating voltage were obviously lower.Gas sensitivity of the FG/WO3 nanocomposites, FG (3%)/WO3 nanocomposites, and FG (2%)/WO3 nanocomposites to 100 ppm NOx at an operating voltage of 2.45 V are shown in S = Rg/Ra, where Ra is a fixed value. The strong increase in Rg of the sensors upon exposure to 100 ppm NOx of the FG/WO3 nanocomposite sensors can be attributed to charge transfer between NOx molecules and the sensing material, where NOx act as an acceptor. It was clear that the response curves ascend or descend sharply with the inflow or outflow of NOx gas, indicating that the sensors have an excellent response-recovery property.The responses of FG, FG (5%)/WOAll the reagents used in the experiments were in analytic grade and used without further purification. Natural graphite, hydrogen peroxide, and ammonium persulfate were mixed at a weight ratio of 2:1:0.1 in a beaker at room temperature with ultrasonication for 5 min. Subsequently, the mixture was transferred to a crucible, and then it was placed in a microwave oven and irradiated at 500 W for 90 s . Under m2SO4 (98%). Then, KMnO4 (8.0 g) was added gradually under stirring and the temperature of the mixture was kept to below 10 °C by cooling. The reaction was maintained for 2 h below 10 °C. Then, the ice bath was removed, and the reaction mixture was stirred at 35 °C for 1 h, and diluted with 100 mL of distilled water. Because the addition of water in concentrated H2SO4 medium released a large amount of heat, the addition of water was performed to keep the temperature below 100 °C. After adding 100 mL of distilled water, the mixture was stirred for 1 h at 90 °C and further diluted to approximately 300 mL with distilled water. After that, 20 mL of 30% H2O2 was added to the mixture to reduce the residual KMnO4. Finally, the mixture was filtered and redispersed with 1 M HCl aqueous solution (300 mL) and left overnight. Several sets of filtering and redispersal processes were carried out to wash the SO42− completely. The resulting solid was dried at 50 °C for 24 h.Graphite oxides (GOs) were synthesized from EG using a modified Hummers method. In a typical procedure, 2.0 g of EG powder was put into 100 mL of cold (0 °C) concentrated H6) was dispersed in the mixture solution with continued stirring for 24 h. Then, the solution was transferred into a Teflon-line autoclave, which was put in an oven of 200 °C for 10 h. After the autoclave was naturally cooled to room temperature, the as-synthesized products were isolated by centrifugation, washed several times with deionized water and absolute ethanol to remove Cl−, respectively, and finally dried in a vacuum oven at 70 °C for 24 h. In addition, FG and WO3 were synthesized in the same way as the composites in the absence of WCl6 and GO, respectively.Graphite oxide was dispersed in 50 mL of distilled water with ultrasonication for 5 h (the second step of exfoliation of graphite), and the FGO solution was isolated by centrifugation, then 50 mL of ethanol was added into the FGO solution, and the pH value was adjusted to 8.0 with aqueous ammonia. One hundred and sixty mg of tungsten hexachloride  and a JEOL JEM-2100 (UHR) high-resolution transmission electron microscope (JEOL JEM-2100 (UHR) HRTEM). UV-vis spectroscopy measurements were performed on a UNICO UV-4802 UV-vis double-beam spectrophotometer  in ethanol dispersion. Raman spectra were recorded on a Renishaw inVia-Reflex confocal Raman microscope  with 532 nm laser excitation. Crystalline structure and crystallite size of the samples were characterized by x-ray diffraction , Cu Kα (λ = 0.15418 nm) radiation at 40 kV and 60 mA at room temperature. The TG-DTA measurement was carried out with a ZRY-1 instrument  at a heating rate of 15 °C/min with air and NS = Ra/Rg to the reducing gas and S = Rg/Ra to the oxidizing gas, where Ra and Rg are the resistances of a sensor in air and in a test gas, respectively.The fabrication process of gas sensors made from the as-prepared powder samples can be seen in . The gas3 nanocomposites were prepared via a solvothermal method in an ethanol-distilled water medium. It was found that the obtained few-layer graphene nanosheets have the intrinsic nature of graphene nanosheets. The preferred growth direction of WO3 was changed in the graphene/WO3 nanocomposites. The morphology of graphene/WO3 nanocomposites appeared as a sandwich-like structure. The few-layer graphene nanosheets and graphene/WO3 nanocomposite sensors with graphene content higher than 1% have obviously lower resistivity (below 135 kΩ) than that of a pure WO3 sensor , and they have the highest response to NOx at a relatively lower operating voltage (2.45 V).Graphene oxides were prepared by using a two-step exploitation method, and few-layer graphene nanosheets and graphene/WO


In addition, mutations at S409/410 of TDP-43 could inhibit its phosphorylation, attenuate nuclear loss, and abolish the increase in neuronal apoptosis in the subcortex. Inhibition of TDP-43 phosphorylation attenuated ICH-induced downregulation of mTOR activity and dynactin1 expression, which may relieve blocking of autophagosome-lysosome fusion and the increase of autophagosomal and lysosomal biogenesis induced by ICH. However, knockdown of TDP-43 could worsen ICH-induced SBI. Furthermore, TDP-43 could be dephosphorylated by calcineurin (CN), and CN activity was increased by OxyHb treatment. In conclusion, this study demonstrated that TDP-43 loss-of-function by phosphorylation at S409/410 may block autophagosome-lysosome fusion and induce elevation of LC3II and p62 levels by inhibiting the activity of mTOR and expression of dynactin1. This mechanism may play an important role in ICH-induced SBI, and TDP-43 may be a potential therapeutic target.This study aimed to determine the role of TAR DNA binding protein-43 (TDP-43) in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI) and its underlying mechanisms. After ICH, expression of TDP-43 in the nucleus was significantly decreased, and its expression in the cytoplasm increased both  Intracranial hemorrhage (ICH) is considered the most fatal subtype of stroke. It has a high mortality and accounts for 10%–15% of stroke cases  is a multifunctional DNA and RNA binding protein that participates in RNA transcription, alternative splicing, and regulation of mRNA stability in cells , phosphorylation, and ubiquitination. Phosphorylated TDP-43 plays important roles in formation of UPIs in FTLD and ALS  and increases autophagosomal and lysosomal biogenesis. In addition, loss of TDP-43 downregulates dynactin1 to impair fusion of autophagosomes with lysosomes  was provided by Novus Biologicals . Anti-mTOR (ab32028), anti-dynactin1 , anti-p62 (ab56416), anti-P-TDP43 (phospho S409 + S410), anti-NeuN (ab104224), and anti-histone H3 (ab1791) were purchased from Abcam . Anti-β-tubulin (sc-5274) was purchased from Santa Cruz .Adult male Sprague-Dawley rats weighing 280–300 g and pregnant rats were purchased from the Laboratory Animal Center, Medical College of Soochow University, Suzhou, Jiangsu, China. The animal experimental agreements were approved by the Experimental Animals Committee of the First Affiliated Hospital of Soochow University and complied with the ARRIVE guidelines. All rats were housed in a quiet environment maintained at 18–22°C with stable humidity and animals had free access to food and water. We strived to minimize the number of rats used and their pain.Rat (280–300 g) were fully anesthetized and then fixed in a stereotaxic apparatus frame . The rats were then placed supine on a heating pad that was maintained at approximately 27–35°C. The experimental ICH model was produced using stereotaxic insertion of a needle with a rounded tip and a side hole into the basal ganglia. The position of the basal ganglia was 3.5 mm lateral to the midline, 0.2 mm posterior to Bregma, and 5.5 mm ventral to the cortical surface. After the microinjector was in position, autologous blood was injected over 5 min, and the needle was left in place for an additional 5 min  and were randomly assigned to seven groups (six rats per group): sham group and six experimental groups arranged by time points of 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after ICH. At the appropriate time point, rats were killed, and the right hemisphere basal ganglia tissues  were separated and collected for analysis . Neurons were maintained in a 5% CO2 incubator at 37°C. Half of the culture medium was replaced every 2 days for 1 week. Cells were then transfected with siRNA. To mimic ICH and evaluate effects of TDP-43 in vitro, cells were exposed to OxyHb at a concentration of 10 μM. After these treatments, cellular morphology was observed using an inverted phase contrast microscope, and total cell protein was collected and stored at −80°C until tested.Whole brains of 17-day-old rat embryos were used to prepare primary neuron-enriched cultures. We tried to minimize the number of embryos used and their suffering. In brief, we removed blood vessels and the meninges, and then the brains were digested with 0.25% trypsin for 5 min. Next, we centrifuged the brain suspension at 500× Nuclear protein extraction was performed using a Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer’s instructions.g for 10 min at 4°C. The supernatant was collected, and a standard BCA method  was used to determine protein concentration. Then, protein samples (100 mg/lane) were loaded onto a 10% SDS polyacrylamide gel, separated and electrophoretically transferred to a PVDF membrane . The membrane was then blocked with 5% nonfat milk for 2 h at 37°C. Next, the membrane was incubated with the primary antibody overnight at 4°C, followed by incubation with the horseradish peroxidase-linked secondary antibody for 1.5 h at 37°C. The membrane was washed with PBST and visualized using enhanced chemiluminescence detection . Relative quantities of protein levels were analyzed using ImageJ software.Brain tissues around the hematoma, which had been stored in liquid nitrogen, were thoroughly ground on ice. RIPA lysis buffer (Beyotime) was used to lyse the brain tissue for 30 min. The lysates were then transferred to a centrifuge tube and centrifuged at 12,000 We performed double labeling for TDP-43 and NeuN to assess expression of TDP-43 in neurons. The rat brain samples were fixed in 4% paraformaldehyde and embedded in paraffin. Next, sections were incubated with the primary antibody  overnight at 4°C followed by incubation with the NeuN antibody  overnight at 4°C. Then, sections were incubated with the secondary antibodies, which included Alexa Fluor 488 donkey anti-rabbit IgG antibody, Alexa Fluor 555 donkey anti-mouse IgG antibody, Alexa Fluor 488 donkey anti-mouse IgG antibody, and Alexa Fluor 555 donkey anti-rabbit IgG antibody . Normal rabbit IgG and normal mouse IgG were used as negative controls (data not shown). Finally, sections were observed using a fluorescence microscope , and relative fluorescence intensity was analyzed using ImageJ software.in vitro and detection by western blots. The most efficient siRNAs were used in this study, and the TDP-43 target sequences were as follows:Specific siRNAs against TDP-43 were provided by Ribobio. Knockdown efficiency of siRNAs was determined by transfection GAGAGGACTTGATCATTAACAGCGTGCATATATCCAATTGCTGAACCTAAGCATAATThe coding region of rat TDP-43 cDNA was subcloned into a pEGFP-N2 expression vector to produce the pEGFPN2-TDP-43 construct (without an EGFP tag). In addition, a rat TDP-43 cDNA construct with mutations at a possible key phosphorylation site  was also subcloned into a pEGFP-N2 expression vector (without an EGFP tag). All constructs were confirmed by DNA sequencing.in vivo RNA transfection reagent (18668-11-1 Engreen) according to the manufacturer’s instructions. Briefly, 5 nmol TDP-43 siRNA and 5 nmol scramble siRNA were dissolved in 66.5 μL DEPC RNase-free water. Then, 5 μL Entranster-in vivo RNA transfection reagents and 5 μL normal saline were added immediately to 10 μL siRNA or 10 μL scramble siRNA. The solution was mixed for 15 min at room temperature. Finally, 20 μL Entranster-siRNA mixture was injected intracerebroventricularly 48 h before ICH.Transfection of siRNA in the rat brain was done using Entranster-in vivo DNA transfection reagent (18668-11-2 Engreen) according to the manufacturer’s instructions. Briefly, 10 μL Entranster-in vivo DNA transfection reagent was added to 5 μg plasmid or 5 μg empty vector. The solution was mixed for 15 min at room temperature. Finally, 15 μL Entranster-in vivo-plasmid mixture was injected intracerebroventricularly 48 h before ICH.Transfection of plasmid in the rat brain was performed using Entranster-® 3000 Transfection Reagent  according to the manufacturer’s instructions. Neurons were transfected with TDP-43 siRNA, empty vector, TDP-43 plasmid, or scramble siRNA. Next, 48 h after transfection, neurons were exposed to 10 μM OxyHb for an additional 48 h. After the indicated treatments, neurons were harvested for further analysis.Transfection of siRNA and plasmid in cultured neurons was performed using LipofectamineBrain tissues embedded in paraffin were used for TUNEL staining. The sections were deparaffinized, dehydrated by heating at 75°C in an oven for 60 min, and then rehydrated through xylenes and graded ethanol solutions to water. The sections were then incubated in Triton X-100 for 10 min. After washing three times in PBS (5 min per wash), sections were incubated with the TUNEL reaction mixture for 1 h at 37°C. The sections were washed again three times in PBS (5 min per wash). After the final wash, sections were coverslipped with an anti-fading mounting medium containing DAPI. The number of TUNEL-positive neurons per millimeter was determined for each sample. Cell counts from each brain sample were averaged to provide a mean value.Cell necrosis in brain tissue was assessed using FJB. FJB procedures were identical to those for TUNEL. The sections were deparaffinized, dehydrated in an oven, rehydrated through xylenes and graded ethanol solutions to water, and permeabilized in 0.04% Triton X-100. The sections were then incubated in FJB dye solution and visualized using a fluorescence microscope . FJB-positive cells were counted by an observer who was blind to the experimental groups. To evaluate the extent of cell necrosis, the necrotic index was defined as the average number of FJB-positive cells in each section counted in six microscopic fields (400× magnification).Lactate dehydrogenase (LDH) activity in cell culture supernatants was quantified using a specific LDH assay according to the manufacturer’s instructions . First, reaction wells, which included standard wells, sample wells, control wells, and empty wells, were set up, and reagents sufficient for the number of assays to be performed were prepared. Then, a master mix of the reaction mix was prepared to ensure consistency of concentration and added to the relative groups. Finally, activity of LDH was measured immediately at OD 450 nm using a microplate reader.Analysis of CN activity was performed using a CN activity quantification kit  according to the manufacturer’s instructions.post hoc test. The former was used for comparisons to a single control group, and the latter was used to compare with the preselected groups. All data are expressed as mean ± SEM. A value of p < 0.05 was considered statistically significant.GraphPad Prism 6 was used for all statistical analysis. Data were analyzed by one-way ANOVA followed by either a Dunnett’s or a Sidak’s in vivo confirmed that TDP-43 translocated from the nucleus to the cytoplasm, and this shift peaked at 48 h after induction of ICH. The relative fluorescence intensity of TDP-43 was significantly increased at 48 h compared with the sham group, and then it rebounded at 72 h after ICH, which suggests that TDP-43 returns to the nucleus after 48 h  group than in the WT group Figures . Howeverin vivo experiments, we found that protein levels of mTOR, which reflect activity of mTORC1, were significantly decreased in the ICH groups compared with the sham group. Nevertheless, compared with the ICH group, mTOR levels were significantly elevated after TDP-43 overexpression, and the TDP-43 mutation group showed higher levels than the WT group. However, the TDP-43 knockdown group showed an obvious decreasing trend when compared with the Ctrl RNA group after ICH (Figures It has been reported that TDP-43 can participate in autophagosomal and lysosomal biogenesis as well as in autophagosome-lysosome fusion in some neurodegenerative diseases (She et al.,  Figures . Next, p Figures . Moreove Figures .in vitro, neurons were treated with 10 μM OxyHb. Immunofluorescence staining was used to assess TDP-43 expression in neurons after OxyHb treatment. After OxyHb treatment, TDP-43 translocated from the nucleus to the cytoplasm. Compared with the control group, the shift peaked at 48 h and then rebounded by 72 h after OxyHb treatment, which was similar to results of the in vivo experiments (Figures in vivo experiments, TDP-43 mutations could inhibit LDH release, in contrast to an increase in release with TDP-43 knockdown (Figure To mimic ICH  Figures . Furthern Figure .Previous studies have indicated that CN can dephosphorylate P-TDP-43 (Liachko et al., in vivo and in vitro, resulting in neuronal apoptosis and SBI. It is known that phosphorylation of TDP-43 affects its subcellular localization in part by formation of aggregates that prevent its return to the nucleus in both ALS and FTLD (Nonaka et al., Numerous recent studies have shown that phosphorylated TDP-43 plays an important role in formation of UPIs in FTLD and ALS (Fatima et al., in vivo and in vitro, and we found that levels of P-TDP-43 were significantly reduced after mutations at S409/410. Furthermore, these mutations reduced nuclear loss of TDP-43. In addition, with the mutations of TDP-43, ICH-induced SBI was ameliorated as shown by decreased neuronal necrosis and apoptosis as well as reduced LDH release. However, TDP-43 knockout increased ICH-induced SBI, which is similar to effects of TDP-43 in ALS, showing that neuronal loss of TDP-43 can induce FTLD (Yousef et al., There are multiple potential phosphorylation sites within TDP-43, and S409/410 plays the most critical role (Neumann et al., This study revealed roles of mTORC1 dysfunction as well as abnormal autophagy and lysosomal biogenesis in TDP-43-mediated neurodegeneration (She et al., Since CN can dephosphorylate P-TDP-43 (Liachko et al., In conclusion, this study provided new insight into the cellular mechanisms underlying TDP-43 nuclear loss-induced brain injury after ICH. Phosphorylation-induced TDP-43 translocation may enhance biogenesis of autophagosomes and lysosomes and partly block fusion of autophagosomes and lysosomes, resulting in ICH-induced SBI Figure . Thus, TGC and ZY contributed to the conception of this study and designed the research. LS and KZ were responsible for conducting the experiments about the animal model and writing the manuscript. WZ and HS conducted the experiments about the cell culture. HL and HS helped to design the study’s analytic strategy and perform the analysis with constructive discussion. All authors have critically revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PIK3CA-mutated GCs. A high PD-L1/PD-1 expression was associated with a significantly better patient outcome, and PD-L1 turned out to be an independent survival prognosticator. The correlation of PD-L1/PD-1 expression with distinct clinico-pathological patient characteristics may serve as a surrogate marker of PD-L1-positive GCs and may direct the use of immune checkpoint treatment strategies.Targeting the PD-1/PD-L1 immune checkpoint signaling is a novel promising treatment strategy in several tumor entities, and it is suggested that PD-L1/PD-1 expression is predictive for a PD-1/PD-L1 checkpoint inhibitor treatment response. We investigated the expression of PD-L1 and PD-1 by immunohistochemistry in a large and well characterized gastric cancer (GC) cohort of Caucasian patients, consisting of 465 GC samples and 15 corresponding liver metastases. Staining results were correlated with clinico-pathological characteristics and survival. PD-L1 expression was found in tumor cells of 140 GCs (30.1%) and 9 liver metastases (60%) respectively in immune cells of 411 GCs (88.4%) and 11 liver metastases (73.3%). PD-1 was expressed in tumor infiltrating lymphocytes in 250 GCs (53.8%) and in 11 liver metastases (73.3%). PD-L1 expression was significantly more prevalent in men, GCs of the proximal stomach, unclassified, papillary, Her2/neu-positive, Epstein-Barr-virus-positive, microsatellite instable, and  The genetic complexity of gastric cancer (GC) has been recently shown in an integrative genomic analysis including whole-genome sequencing. A molecular classification was proposed, which categorizes four subtypes, i.e. Epstein-Barr-virus (EBV)-positive, microsatellite instable (MSI), chromosomal instable, and genomically stable GCs , 2. ThesPD-L1 (B7-H1) is a 290aa type I transmembrane surface glycoprotein encoded by the CD274 gene located on chromosome 9 and expressed by several cell types of the immune system, e.g. lymphocytes and dendritic cells, but also aberrantly on the surface of epithelial cells of a wide range of solid tumors. PD-L1 is the ligand of programmed cell death 1 (PD-1), which is a member of the immunoglobulin superfamily B7 and involved in immunomodulation . PD-1 isCurrently, more than 400 studies worldwide focus on PD-L1/PD-1 immune checkpoint signaling, including 65 studies for cancer of the gastrointestinal tract , and theThe clinico-pathological characteristics of our patient cohort are summarized in Table PD-L1 expression was observed in tumor, stromal and immune cells, but not in non-neoplastic gastric epithelium. 140 of 465 cases (30.1%) showed a membranous PD-L1 expression in tumor cells. The percentage of stained tumor cells ranged from 0 to 80% (median 0%), the staining intensity ranged from 0 to 3  also serving as a positive control for the negatively rated tumor cell cases. The percentage of stained immune cells ranged from 0 to 70% (median 5%), the intensity ranged from 0 to 3 (median 1). As the majority of cases with PD-L1 positive immune cells showed a weak staining intensity , the intensity of the immunostaining was neglected for further statistical analyses, and only the percentage of PD-L1 positive immune cells was considered. All cases were graded into four groups as 0 (negative), 1 (1-9% positive), 2 (10-20% positive), and 3 (>20% positive). For further statistical analyses, group 0 and 1 were summarized and classified as “PD-L1 negative in immune cells” , and group 2 and 3 were classified “PD-L1 positive in immune cells” .The PD-L1 expression in stroma cells was mainly very limited to single cells, hence only the presence or absence was evaluated; PD-L1 positive stroma cells were observed in 107 of 465 cases  are known to be associated with PD-L1 overexpression , 15. NexMoreover, four different PD-L1 expression patterns were observed: (1) EBV-positive GCs often showed a heterogeneous, “patchy” expression pattern with a striking accumulation of PD-L1 positive tumor cells around larger blood vessels. (2) MSI-GCs were mainly PD-L1-positive at the interface between neoplastic and non-neoplastic tissue, especially in areas of “pushing borders”. (3) Papillary type GCs often showed PD-L1 positivity within the fibrovascular connective tissue cores and the intratumoral necrosis. (4) Other cases mainly showed no distinct PD-L1 distribution pattern and were classified as “patternless” .PD-1 expression was observed neither in tumor nor stroma cells. PD-1 positive diffusely distributed TILs were present in 250 of 465 cases (53.8%). As the amount of PD-1 positive TILs was mainly restricted to single cells, a quantitative analysis was neglected. Apart from a cumulative appearance of PD-1 positive immune cells in intratumoral lymphocyte aggregates/lymph follicles  or immune cells (group 2 and 3) showed a significant better overall and tumor specific survival Figure ; Table 1s Figure . There ws Figure .Patients with PD-1 positive TILs showed a better tumor specific survival, which was not significant after Simes' multiple testing procedure Table .in=0.05 and pout=0.10. These were UICC-stage, lymph node ratio, R-status, MET-status and PD-L1 expression in immune cells , and PD-1-status (immune cells). Five parameters remained in the Cox model after running the backward LR method with pls Table .Tissue specimens from liver metastases were available in 15 cases. Nine metastases (60%) showed a membranous PD-L1 expression of tumor cells, which was mainly limited to single cells and ranged from 0.1 to 3% respectively from 1+ to 2+ Figure . DichotoPD-L1 expression in immune cells was found in 11 metastases (73.3%) and ranged from 1 to 10% Figure . DichotoPD-1 positive TILs were present in 11 metastases (73.3%). The expression in GCs and their corresponding metastases was concordant in 12 cases  with a membranous PD-L1 expression in tumor cells, the tumor surface could not be evaluated. In the remaining 118 cases, PD-L1 positive tumor cells were close to the mucosal surface and generally accessible by biopsy in 79 (66.9%) cases. In 39 cases (33.1%), PD-L1 positive tumor cells were localized only in the tumor center or near the invasion front Figure .The present study is the first evaluation of PD-L1 and PD-1 expression in a large and well-characterized Caucasian cohort of GC. PD-L1 and PD-1 were expressed in a substantial amount of GCs either in tumor and immune cells or immune cells only. PD-L1 expression was found to be an independent survival prognosticator and correlated with distinct clinico-pathological patient characteristics.Up to now, only five studies of smaller Asian patient cohorts investigated the PD-L1/PD-1 expression in GC –16. PD-LAccurate interpretation of immunohistochemical stains is crucial for the establishment of a valid, histology-based predictive biomarker. Although we aimed for the HistoScore, we recognized that the overall percentage of PD-L1-positive tumor cells was low and the assessment of different percentages of three different staining intensities was indiscernible and impractical. Applying our simplified IRS, we next used patient survival as surrogate marker for biological relevance. Interestingly, while dichotomization into completely negative and any positive staining of tumor cells did not show a significant correlation with patient survival , dichotoA significant number of patients is diagnosed at an advanced, inoperable stage . Only biIn patients with a metastatic and/or unresectable GC, biopsy specimens might be obtained from the liver metastases, rather than the primary tumor, and biomarker expression between the primary GC and the liver metastasis might also show divergent test results. For Her2/neu, a discordance rate between 9 and 16% is known . In our In view of non-representative tissue sampling issues, we were next interested to test the hypothesis whether other clinico-pathological patient characteristics may serve as surrogate markers for PD-L1/PD1-expression. Correlation of PD-L1/PD-1 expression with various clinico-pathological patient characteristics demonstrated that PD-L1 is enriched in men, intestinal, unclassified or papillary type GC of the proximal stomach, EBV-positive, and MSI GCs. The latter findings confirm independently previous observations , 20. EBVPIK3CA-mutational status and combination of PD-L1/PD-1-targeted therapies with mTOR-inhibitors merit consideration in GC.The simultaneous application of separate monoclonal antibodies (mAbs) or multi-specific mAbs is still in its infancies but might offer new treatment strategies for complex diseases . Stagg ePD-L1 expression in GC of Western patients correlates significantly with overall and tumor specific survival as well as distinct clinico-pathological patient characteristics. Our findings might help to develop valid predictive test algorithms, which do not rely only on immunostaining patterns and may also help to explore novel combination therapies, e.g. trastuzumab or everolimus and PD-1/PD-L1 checkpoint inhibitors.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1964 and later versions. Informed consent or substitute for it was obtained from all patients for being included in the study. Ethical approval was obtained from the local ethical review board (D 453/10).Epidemiological Cancer Registry of the state of Schleswig-Holstein, Germany. Follow-up data of those patients who were still alive were retrieved from hospital records and general practitioners. All patient data were pseudonymized prior to study inclusion.From the archive of the Institute of Pathology, University Hospital Kiel, we sought Caucasian patients who had undergone either total or partial gastrectomy for adenocarcinoma of the stomach or esophago-gastric junction between 1997 and 2009. The following patient characteristics were retrieved: type of surgery, age at diagnosis, gender, tumor size, tumor localization, tumor type, tumor grade, depth of invasion, residual tumor status, number of lymph nodes resected, and number of lymph nodes with metastases. Patients were included if an adenocarcinoma of the stomach or esophago-gastric junction was histologically confirmed. Exclusion criteria were defined as 1) histology identified a tumor type other than adenocarcinoma, and 2) patients had undergone a perioperative chemo- or radiotherapy. Each resected specimen had undergone gross sectioning and histological examination by trained and board certified surgical pathologists. Date of patient death was obtained from the th edition of the UICC guidelines [Tissue specimens were fixed in formalin and embedded in paraffin (FFPE). Deparaffinized sections were stained with hematoxylin and eosin. Histological re-examination of primary tissue sections was carried out for all cases to assure if inclusion criteria were met. Tumors were classified according to the Laurén classification  and re-eidelines .Immunostaining was carried out manually, using a rabbit monoclonal anti-PD-L1 antibody . Tissue sections were pretreated in citrate buffer for antigen retrieval and incubated with hydrogen peroxide block and Ultra V Block  to avoid unspecific reactions. For visualization the ImmPRESS-HRP-Universal–Antibody Polymer and the NovaRED substrate kit  were applied. Counterstaining was done with hematoxylin . Immunohistochemical PD-L1 stainings of liver metastases and PD-1 stainings of GCs and liver metastases were carried out with a Bondmax automated slide staining system , using the Polymer Refine Detection Kit  and the anti-PD-L1 antibody described above respectively a mouse monoclonal anti-PD-1-antibody . Germinal centers of lymph follicles served as internal positive control for both antibodies.Intensity and percentage of stained cells was evaluated separately for tumor, stroma and immune cells by two pathologists (CB and CR). For the evaluation of the PD-L1 expression in tumor cells, only the membranous staining was evaluated and the following immunoreactivity scoring system (IRS) was applied: Category A rated the percentage of immunoreactive cells and was graded as 0 (negative), 1 (≤1% positive), 2 (2 to 10% positive), 3 (11-50%) and 4 (>50%). Category B documented the intensity of immunostaining as 0 (no immunostaining), 1 (weak), 2 (moderate), or 3 (strong). The addition of category A and B resulted in an IRS ranging from 0 to 7. The evaluation of PD-L1 in stroma cells was rated as present or absent. For the evaluation of the PD-L1 expression in immune cells , only the percentage of positive cells was considered, and cases were graded as 0 (negative), 1 (1-5% positive), 2 (6-20%) and 3 (>20%). The immunostaining of PD-1 in immune cells was rated separately for tumor infiltrating lymphocytes (TILs) and intratumoral lymph follicles as present or absent.KRAS-, PIK3CA-, RHOA-, and GNAS-genotype was assessed as previously described [The mucin phenotype, Helicobacter pylori-, Her2/neu-, MET-, EBV-, MSI-status as well as the escribed , 26–29.Whole tissue sections from GCs and their liver metastases were stained with antibodies directed against PD-L1 and PD-1. The staining results were correlated with clinico-pathological and survival data.Statistical analyses were done using SPSS 20.0 . For the evaluation of PD-L1 in tumor cells and immune cells, marker expressions were first examined as raw score values and then dichotomized into positive and negative. Cross tabulations of clinical data and marker expressions were tested for independence using Fisher's exact test. The correlation between the PD-L1 and PD-1 expression in GCs and corresponding metastases was calculated by using Cohen's kappa. A kappa value of 0.20 was considered to be poor, of 0.21–0.40 to be fair, of 0.41–0.60 to be moderate, of 0.61–0.80 to be good, and of 0.81–1.00 to be very good. Median overall and tumour specific survival were calculated using the Kaplan-Meier method. Log-rank test was used to determine significance of differences between survival curves. Hazard ratios of variables were calculated by univariate Cox regression and those having p-values up to 0.1 were included in a multivariate Cox regression, combined with iterative backward LR method to identify independent prognostic variables. P-values up to 0.5 were considered as statistically significant. False discovery rate of correlations between clinical variables and biomarkers was controlled by applying the explorative Simes (Benjamini-Hochberg) procedure group-wise for each biomarker . P-value


This paper describes a study of the quantify surface roughness of experimentally manufactured particleboards and sandwiched panels having fibers on the surface layers. Surface quality of specimens before and after being overlaid with thin melamine impregnated papers was determined by employing profilometer equipment. Roughness measurements and Janka hardness were carried out on the specimens conditioned at 60% and 95% relative humidity levels. Based on the findings in this work, surface roughness of the specimens that were exposed two relative humidity exposure showed significant differences from each others. Data determined in this study could be beneficial to understand behavior of such panels exposed different humidity levels. In manufacture value-added products, wood based panels, such as fiberboards and particleboards, are used extensively as underlayment for thin overlays. These overlaid panels are main members in production of furniture and kitchen cabinets. As a well-known fact that wood-based composites are hygroscopic material and their dimensional stability varies with fluctuating as function of relative humidity. Especially, their surface quality depending on particle or fiber size on the face layer plays an important role on. Overall surface quality of panel is important so-called telegraphing effect of the roughness of the substrate shows through the overlay. Therefore, it is necessary to determine not only surface of the panel but also roughness of overlaid member with changing relative humidity so that they can be used more efficiency for different types of applications. Laminated and overlaid particleboard (PB) and medium density fiberboard (MDF) have been commonly used manufacturing indoor cabinets in Europa and the United States of America (USA) for over 35 years ,2. OverlIf surface irregularities on a composite panel are present, they may show through the overlays. Therefore, the overall quality of the final product can be affected by such irregularities on the substrate. In several previous studies, the stylus technique has been used to calculate the value of surface roughness of wood composites ,13,14,15In another previous work, medium density fiberboard surface roughness values were calculated using a fine stylus technique. During the sanding of the panels, surface roughness variations are introduced. In order to effectively evaluate this variation, previous studies used the profilometer technique . AlthougJuniperus virginiana L.) particles was supplied by a local sawmill in Oklahoma City. The particles contained both heartwood and softwood fractions of the trunk from eastern redcedar trees. Particles were dried to 2–3% moisture content in a laboratory type oven with a 1.0 m3 at the temperature of 67 ± 2 °C for 72 h. Dried particles were classified into two particle sizes, namely fine and coarse, on a 1 mm screen and 3 mm, respectively. After screening urea formaldehyde (UF) was blended with particles. Experimental panels were compressed at a temperature of 180 ± 2 °C and a pressure of 5.17 MPa for 5 min. All of the panels were pressed to a nominal thickness of 14 mm, and their target density was 0.70 g/cm3. Panels that were prepared with a length of 50 cm, width of 50 cm, and thickness of 14 mm. Manufacting process of composite panels is illustrated in Eastern redcedar (a), mean peak-to-valley height (Rz), and maximum roughness (Rmax), can be calculated from the digital information [Stylus method is a well accepted technique, resulting in quantitavie numerical values on the surface of sample. The profilometer consists of main unit and pick up which has a skid-type diamond stylus with 5 m tip radius and tracing span constant speed of 1 mm/s over 15.2 mm at a surface. Technical details and working principles of stylus type profilometer are presented in a past work . Roughneormation . a values of 3.02 μm. Samples with the size of 12 cm by 5 cm were used for random roughness measurements for test. A total of 20 samples were used for roughness measurements as illustrated in The calibration of the profilometer was checked every 100 measurements by using a standard reference plate with RHardness of the non-overlaid, overlaid particleboard, and non-overlaid, overlaid fiber sandwich panel specimens was tested by embedding a hemisphere steel having 11.2 mm diameter onto their tangential surface using a Comten 95 Series Universal Testing machine. Five measurements were taken from each sample and recorded in kg to evaluate their Janka hardness, as illustrated in 2. The test samples overlaid 50 s at Carver press with a 165 ± 5 °C under pressure of 2.3 MPa. Test samples were conditioned in a chamber with a temperature of 20 °C and a relative humidity of 65% until they reach to the equilibrium moisture content before any roughness measurements were taken from their surface. The overlaying process of the specimens is shown in A total of ten, five for each type of composites with dimensions of 12 cm by 5 cm were overlaid with melamine based decorative paper having weight of 7000 g/cmAfter initial measurements were taken from surface of samples, they were placed in chamber having 95% relative humidity and were kept for 10 days. In the next step individual samples were weighted at an accuracy of 0.1 g. Later roughness measurements from the surface of each overlaid samples. Roughness values of the samples were quantitatively evaluated at initial dry condition and as they were exposed to relative humidity levels of 60% and 95%. Penetration of humidity on roughness of overlaid and non-overlaid substrate is schematically illustrated in In order to analyze the significant differences of all the parameters that were used in this study, one-way analysis of variance (one-way ANOVA) was used. All results were computed employing IBM Statistical Package for the Social Sciences software (SPSS), version 21 .a value of 0.48 μm once they were exposed to 60% and 95% relative humidity levels, corresponding values were 0.62 μm and 1.67 μm, respectively. Measurements of average surface roughness values of fiber-sandwich composite panels are illustrated in z and Rmax as well.As expected, relative humidity effects all three measures of surface roughness in both fiber sandwich boards. It is seen in a values once they were exposed to 60% and 95% relative humidity levels, as illustrated in It seems that PB samples had rougher initial average surface values, however they had similar trend having higher RIn p < 0.05) on interaction effects between panel types and humidity level. Based on ANOVA, significant difference was observed between main effects such as composite panel types and humidity levels at a 95% confidence level. The average surface roughness values were found to be effective (After relative humidity exposure process, the surface roughness of composite panel specimens increased from 3.22 μm to 5.82 μm and 80% increase in the surface roughness was found. Both fiber-sandwich type and particleboard samples had higher surface roughness values at 95% humidity level. It appears that humidity level exposure effected surface quality of fiber-sandwich panels. In a had the greatest increase with a value of 83%, followed by Rmax 64%, and finally Rz the least 38% increase. If the trends are compared between the surface roughness characteristics, Ra had a steeper slope than that of other parameters. Therefore, it appears that an increase in relative humidity adversely influenced overall surface quality of the samples. It can be noticed that as percentage of relative humidity increase, all of the surface roughness values increase. Specifically, RIncreasing the percentage of relative humidity increased all the parameters of the surface roughness; this difference is due to the overlay, which protects the surfaces against relative humidity. Using overlay on the fiber sandwich board surfaces reduces the negative effects of relative humidity with an approximate value of nine times. Both MDF-Sandwich panel samples had a higher surface roughness values at 95% humidity level. Humidity levels effected MDF sandwich panels directly and surface quality deteriorated with humidity. In a had the greatest increase with a value of 58%, followed by Rmax 30%, and finally Rz, with the least 8% increase. If the trends are compared between the surface roughness characteristics, Ra had a steeper slope than that of other parameters. Therefore, it seems that increase in relative humidity adversely influenced the overall surface quality of the samples.The percentage of relative humidity increase, all surface roughness values of non-overlaid particleboard increase. Specifically, Ra had the greatest increase with a value of 143%, followed by Rmax 96%, and finally, Rz the least, with an 86% increase. If the trends are compared between the surface roughness characteristics, Ra had a steeper slope than that of other parameters. Therefore, it appears that the increase in relative humidity adversely influenced all the measures of surface quality for each sample evaluated. Increasing the percentage of relative humidity increased all parameters of the surface roughness, this difference is due to the overlay, which protects the surfaces against relative humidity. Using overlay on the particleboard surfaces reduces the negative effects of relative humidity, with an approximate value of six times. All of the surface roughness values of overlaid particleboard increase after increasing relative humidity. Specifically, RAs percentage of relative humidity increase, all Janka hardness values decrease. Specifically, non-overlaid particleboard had the minimum Janka harness value 171 kg overall. If the Janka hardness values are compared between the overlaid and non-overlaid specimens, overlaid samples have better Janka hardness values between 5% and 6%. Therefore, it appears that the increase in relative humidity adversely influenced the overall hardness values of the specimens. This study aimed to quantify the effect of humidity exposure on both surface quality and Janka hardness for different types of wood composite material, namely these were overlaid and non-overlaid composite panels. Based on the findings that are discussed in this work, as a conclusion, overlaid samples that were exposed to different levels of relative humidity had less overall surface roughness characteristics than the non-overlaid samples. Furthermore, the Janka hardness results were higher in the case of overlaid samples than in the non-overlaid samples. The data found in this work can inform future decisions to make more efficient use of overlaid and non-overlaid wooden composite boards. This work can be extended in further studies by evaluating other mechanical properties, such as bending and compression strength, of wood composite boards under different levels of relative humidity. Additionally, through future studies of dimensional stability, as effected by relative humidity, can gain a better understanding of the behavior of overlaid panels.


Autophagy is a catabolic mechanism where intracellular material is degraded by vesicular structures called autophagolysosomes. Autophagy is necessary to maintain the normal function of the central nervous system (CNS), avoiding the accumulation of misfolded and aggregated proteins. Consistently, impaired autophagy has been associated with the pathogenesis of various neurodegenerative diseases. The proteins TAR DNA-binding protein-43 (TDP-43), which regulates RNA processing at different levels, and chromosome 9 open reading frame 72 (C9orf72), probably involved in membrane trafficking, are crucial in the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Additionally, recent studies have identified a role for these proteins in the control of autophagy. In this manuscript, we review what is known regarding the autophagic mechanism and discuss the involvement of TDP-43 and C9orf72 in autophagy and their impact on neurodegenerative diseases. The population is aging worldwide. Indeed, between 2015 and 2030, the number of people in the world aged 60 years or over is projected to grow by 56 per cent, from 901 million to 1.4 billion; by 2050, the global population of older persons is projected to more than double in size, reaching nearly 2.1 billionNeurodegenerative diseases such as Alzheimer's, Parkinson's, Prion, Huntington's, and Motor neuron diseases (MND), as well as Spinal muscular atrophy, Spinocerebellar ataxia (SCA), Frontotemporal Lobar Degeneration (FTLD), and Amyotrophic lateral sclerosis (ALS) cause progressive damage in the central and peripheral nervous system, reducing the quality of life of affected patients , a form of dementia characterized by a progressive decline in behavior and/or language  genes are involved in the process of autophagic vacuole formation  in membrane microdomains where proteins with the PtdIns3P-binding (FYVE) and the conserved Phox-homology (PX) domains are recruited to form a protein elongation platform at the omegasome , which are considered cellular nutritional sensors able to modulate different signaling pathways  at Thr1227, which, together with TSC1, forms the TSC complex  is another negative regulator of autophagy  inhibits autophagy and promotes a neurodegenerative phenotype characterized by axonal degeneration and accumulation of aggregate-prone proteins in the cytosol of neurons without altering proteasome function  the gene encoding for Beclin 1 shows decreased mRNA levels in brain tissue, suggesting reduced autophagosome formation and increased development of AD , where omegasomes formation is inhibited due to the mislocalization of the protein ATG9, preventing the formation of new autophagosomes. This reduces the clearance of the protein α-synuclein, which accumulates in intra-neuronal inclusions called Lewy bodies, a feature of the brain tissue of PD patients . Autophagosomes move along microtubules in the direction of the perinuclear microtubule-organizing center of the neuron, which is enriched in lysosomes . Inefficient autophagosomal clearance can be associated with reduced lysosomal acidification, which reduces the activity of lysosomal hydrolases and promotes the accumulation of undigested byproducts. This is typical of the so-called “lysosomal storage disorders” (LSD), which comprise more than 50 diseases and represent the most common forms of neurodegenerative disease in childhood. While the proteins and mechanisms involved in these diseases differ, impaired autophagy is unfailingly present , hereafter called TDP-43, was initially described as a transcriptional repressor factor of HIV n]. These repeats locate in a non-coding region of C9orf72, specifically within the intron that separates exons 1a and 1b , whereas affected individuals incorporate between 30 and 250 hexanucleotide repeats (n ≥ 30) n repeats may involve both, a reduction in the expression of some C9orf72 variants and/or the aberrant formation of RNA foci n repeats, in all reading frames, and in the absence of an initiating ATG codon. Thus, this mechanism can generate polypeptides formed by different numbers of dipeptide units originated from one (G4C2)n repeat. These polypetides, known as DRPs (Dipeptide Repeat Proteins) or C9RANT proteins, have shown to be toxic for the cell, since they impair RNA biogenesis n repeats have been recently reviewed by Todd and Petrucelli  and CHMP2B. OPTN, which is involved in the mitophagy machinery, is proposed to impact on ALS as its mutations promote severe mitochondria degradation  as a new gene associated with ALS  and OPTN, which co-localize with TDP-43, p62/SQSTM1 and ubiquitin in spinal motor neurons of sporadic ALS patients . Indeed, it has been observed that HSP-90, cell division cycle 37 (CDC37) and TDP-43 form a protein complex that prevents TDP-43 degradation in HeLa cells. The disruption of this complex, by downregulation of HSP-90 or CDC37 promoted degradation of TDP-43 via induction of autophagy  are strictly coordinated to degrade WT and aggregated forms of TDP-43  , a master transcription factor that regulates the expression of genes controlling lysosomes biogenesis and autophagy induction , TBK1 Binding Protein 1 (SINTBAD) and Nucleosome Assembly Protein 1 (NAP1), forming a protein complex that activates the TANK-Binding Kinase 1 (TBK1). Active TBK1 phosphorylates SMCR8 at Ser 402 and Thr 796, stabilizing the C9orf72/SMCR8 complex and thus inducing autophagy. On the other hand, the interaction between C9orf72/SMCR8/WDR41 and ULK complex induced SMCR8 phosphorylation at Ser 400, 492, 562, and Thr 666/Ser 667. These modifications are less efficient in inducing autophagy compared with those performed by TBK1 (Sellier et al., in vivo model, the over-expression of Ataxin-2 Q30 aggregates induced cell toxicity when autophagy was inhibited by C9orf72 down-regulation (Sellier et al., In agreement with the role of C9orf72 in autophagy, different works have demonstrated that C9orf72 down-regulation impaired autophagy progression, as evidenced by the intracellular accumulation of p62/SQSTM1 (Farg et al., Autophagy dysfunction has been identified in several neurodegenerative diseases such as ALS and FTLD. TDP-43 and C9orf72 are also associated with these illnesses as well as other neurodegenerative disorders. TDP-43 aggregates are found in neurons of ALS and FTLD patients, and it has been proposed that they may act as a sink for the newly synthetized TDP-43 protein, thus leading to TDP-43 loss-of-function. Autophagy has been proposed to be crucial for the maintenance of TDP-43 protein levels and to avoid TDP-43 aggregation. Indeed, very recently, data indicate TDP-43 is able to regulate autophagy by stabilizing the mRNA of important components of the autophagic machinery, and, conversely, TDP-43 down-regulation/loss-of-function finally inhibits the autophagic flux.The association of C9orf72 with ALS and FTLD has recently been discovered, and the physiological functions of C9orf72 are still being investigated. Additionally, recent studies showed that C9orf72 is involved in membrane trafficking and autophagy. Specifically, it has been demonstrated that C9orf72 stimulates autophagy initiation by interacting with autophagic proteins involved in membrane trafficking. Consistently, in diseases such as ALS and FTLD, it has been observed a reduction in C9orf72 as well as decreased levels of autophagy.Thus, to date, evidences strongly support that both, TDP-43 and C9orf72 regulate autophagy and that loss-of-function of any of these factors blocks the autophagic flux. In the scenario of TDP-43-associated proteinopathies, TDP-43 loss-of-function caused by TDP-43 aggregates, can further increase cellular stress by blocking the autophagic process. In turn, autophagy inhibition provokes an exacerbated accumulation of TDP-43 aggregates, enhances cellular stress and induces cell death, leading to the onset or progression of neurodegenerative diseases.Consistently, in C9orf72-associated neuropathologies, two events lead to the onset of the disease. The first one is associated with the production of aberrant protein inclusions, such as TDP-43 or Ataxin aggregates, while the second one is characterized by the deregulation of autophagy caused by C9orf72 loss-of-function.Thus, the dysregulation of autophagy is key in the development of TDP-43- and C9orf72-associated neurodegenerative disorders. Even though different are the mechanisms that cause the impairment in autophagy, induction in the autophagic pathway can be considered as a general strategy to avoid these diseases, and research should focus on the identification of novel therapeutic strategies to stimulate autophagy.MB, EM, and AC wrote the article and made the figures. EB made important contributions, discussions and corrections to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The past few years have signaled a major breakthrough on the management of idiopathic pulmonary fibrosis (IPF). Finally, we have drugs in our arsenal able to slow down the inexorable disease natural course. On the other hand, the latter evidence has increased the responsibility for a timely and accurate diagnosis. Establishment of IPF diagnosis directly affects the choice of appropriate treatment. The current diagnostic guidelines represent a major step forward providing an evidence-based road map; yet, clinicians are encountering major diagnostic dilemmas that inevitably affect therapeutic decisions. This review article aims to summarize the current state of knowledge on the diagnostic procedure of IPF based on the current guidelines and discuss pragmatic difficulties and challenges encountered by clinicians with regards to their applicability in the everyday clinical practice. Idiopathic pulmonary fibrosis (IPF) represents the most common form of idiopathic interstitial pneumonia (IIPs) and is characterized by the gravest prognosis with a median survival of 3–5 years , irrespeHistologically, IPF is characterized by the pattern of usual interstitial pneumonia (UIP), which is denoted by spatial and temporal heterogeneity . It is oThe patient with IPF typically presents with progressive dyspnea on exertion of insidious onset and non-productive cough. The most characteristic clinical finding is the presence of Velcro rales that can be proven an extremely useful diagnostic tool for early disease diagnosis . ClubbinPulmonary function tests usually exhibit a restrictive pattern with concomitant reduction in diffusing capacity of carbon monoxide (DLco). However, the absence of restriction does not exclude the diagnosis of IPF, especially in the context of combined pulmonary fibrosis and emphysema, which is characterized by relatively preserved lung volumes with disproportionately reduced DLco . There aAccording to current diagnostic criteria , HRCT plThe presence of honeycombing is not synonymous with IPF, as it can be seen in other diseases as chronic HP, collagen tissue-related interstitial lung diseases, asbestosis, drug-induced lung toxicity, sarcoidosis, postradiation pneumonitis, and post ARDS. Minimal honeycombing can also be encountered in cases of fibrotic NSIP, which represents a major component of the differential list.The distribution of honeycombing can offer significant information. Typically, in IPF, it has a subpleural and basilar distribution. Chronic HP can be a great mimic of IPF. However, sometimes in chronic HP, honeycombing can be more marked in the upper/mid lung zones giving a hint to the actual diagnosis. The same upper lobe predominance of honeycombing can be seen in sarcoidosis as well. Furthermore, in sarcoidosis, the fibrotic process often follows the expected perilymphatic route, thus creating a “swath” of honeycombing extending from the hilum to the periphery of the lung. In patients who develop fibrosis post ARDS, it has a striking anterior distribution Table .Besides the distribution of honeycombing, there are other findings on HRCT that increase suspicion toward certain diagnoses. Silva et al.  studied In the appropriate clinical setting in patients with a UIP pattern, IPF diagnosis can be established without the need for surgical lung biopsy (SLB). In the cases of possible UIP and inconsistent with UIP, a SLB is recommended in order to reach a final diagnosis. When SLB is necessary, close cooperation with the thoracic surgeon is necessary in order to point the optimal sites for biopsy in order to increase the possibility of an accurate diagnosis. Areas of honeycombing should be avoided in lung sampling since they may reveal non-specific end-stage lung damage and absence of spatial and temporal heterogeneity suggestive of UIP. Samples should be obtained from at least two different lobes, because of the possibility of discordant findings (UIP in one lobe and NSIP in another). In such cases, the UIP pattern drives diagnosis and prognosis as well , 16.Regarding histopathology, current guidelines have classified patients into four categories: UIP, probable UIP, possible UIP, and not UIP . SpecifiMDT approach is acknowledged as a major advance in IPF diagnosis. It refers to the constructive exchange of views between a respiratory physician, radiologist, and pathologist with expertise in the field of ILDs. The added value of MDT diagnosis is its association with higher levels of diagnostic confidence and better interobserver agreement , 18. WalThe 2011 guidelines are a clear step forward considering that they provide clear guidance on an evidence-based approach. The most crucial caveat of these guidelines is that, in a significant percentage of “real-life” patients with IPF, lack clinical practicality regarding diagnosis, prognosis, and therapeutic decisions.High-resolution computed tomography plays a pivotal role in disease diagnosis and determines the need of SLB to establish a definite diagnosis. However, accurate identification of honeycombing is not straightforward even amongst thoracic radiologists. Interobserver agreement has been proven poor, and this has been validated in recent studies –24. Thisp = 0.01). Finally, in the INPULSIS trials, a significant proportion of patients  were enrolled based on possible UIP pattern (with traction bronchiectasis) and no confirmation by SLB. This group of patients exhibited the same progression of disease based on the annual decline of FVC compared to patients with honeycombing on HRCT and/or confirmation by SLB as well as similar treatment response to nintedanib  for interobserver variation in the evaluation of diffuse parenchymal lung disease . In the Surgical lung biopsy in patients with ILDs as it can trigger an acute exacerbation , 36, regvia video-assisted thoracoscopic surgery (VATS)] should be carefully considered on an individual basis, weighing risks vs diagnostic benefit.Two large series , 41 repoBy strictly adhering to current guidelines, a large number of patients  will need to be subjected to SLB. Clinical practice seems to have endorsed the facts that SLB carries a small but significant risk and that the possible UIP pattern in a patient with a high pretest probability of IPF retains sufficient PPV for UIP pathology. It is common practice that in patients with a high clinical probability of IPF , 38, 39 via VATS. A recent meta-analysis including 16 studies with BLC (642 patients) and 14 studies with VATS    reportedatients) , 50. In atients)  and prosAccording to the Bayesian diagnostic approach of IPF, it would be very useful to have biomarkers that would increase the pretest probability of IPF. Ideally, these biomarkers would not only have diagnostic value but would also offer clinically relevant prognostic information regarding not only the natural course of the disease but also response to therapy. While for pulmonary embolism, d-dimers are an established diagnostic indicator in a complex disease as IPF, it is unlikely that just one “diagnosticator” will suffice. Considering that IPF diagnosis represent the least critical question for clinicians (given the major improvements of HRCT), in the real-world clinical practice, an ideal biomarker would be the one who could fulfill the unmet need for timely prediction of disease progressiveness and treatment responsiveness. In line with this, most of the studied biomarkers were mostly used as disease prognosticators rather than disease-specific diagnostic tools. Matrix metalloproteinase-(MMP)-7 represents the most extensively studied molecular biomarker that showed promising prognostic value in several independent cohorts of patients with IPF. Despite the fact that elevated MMP-7 levels clearly discriminated patients with IPF from those with HP ; yet, thThe ILD community has made significant progress in understanding IPF. With the development of antifibrotic agents, accurate diagnosis is crucial. Guidance is needed to focus on practical implementation of current guidelines in a real-world clinical setting. An integral first step of the diagnostic process is the exclusion of alternative diagnoses. Equally important is the definition of the pretest probability for every patient with suspected IPF. The diagnostic significance of possible or even definite UIP pattern is completely different when facing a 45-year-old female or a 70-year-old male. Possible UIP pattern seems to carry sufficient PPV in patients with a high pretest probability of IPF. SLB should be considered in patients with inconsistent with UIP pattern after evaluating the individualized benefit risk ratio Table . BLC seeVT, AT, and SC wrote the manuscript. SP and DB revised the manuscript for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To test the feasibility and assess the uptake and acceptability of implementing a consumer questions programme, AskShareKnow, to encourage consumers to use the questions ‘1. What are my options; 2. What are the possible benefits and harms of those options; 3. How likely are each of those benefits and harms to happen to me?’ These three questions have previously shown important effects in improving the quality of information provided during consultations and in facilitating patient involvement.This single‐arm intervention study invited participants attending a reproductive and sexual health‐care clinic to view a 4‐min video‐clip in the waiting room. Participants completed three questionnaires: (T1) prior to viewing the intervention; (T2) immediately after their consultation; and (T3) two weeks later.A total of 121 (78%) participants viewed the video‐clip before their consultation. Eighty‐four (69%) participants asked one or more questions, and 35 (29%) participants asked all three questions. For those making a decision, 55 (87%) participants asked one or more questions, while 27 (43%) participants asked all three questions. Eighty‐seven (72%) participants recommended the questions. After two weeks, 47 (49%) of the participants recalled the questions.Enabling patients to view a short video‐clip before an appointment to improve information and involvement in health‐care consultations is feasible and led to a high uptake of question asking in consultations.This AskShareKnow programme is a simple and feasible method of training patients to use a brief consumer‐targeted intervention that has previously shown important effects in improving the quality of information provided during consultations and in facilitating patient involvement and use of evidence‐based questions. Shared decision making is considered key to supporting patients to understand treatment and possible options, express values and preferences to make good treatment decisions and better manage their health.To address these challenges, we previously conducted a crossover controlled trial to test whether three generic questions could facilitate communication about treatment options independently of the clinical context.Box 1What are my options?What are the possible benefits and harms of those options?How likely are each of those benefits and harms to happen to me? Including ‘What will happen if I do nothing?’In this prior study involving trained actors as unannounced standardized patients, we showed that these questions significantly increased facilitation of patient involvement in the consultations, as well increasing information given by physicians about options and their associated benefits and harms.®). The intervention included three components: a 4‐min video‐clip that participants were shown in the waiting room ; a pamphlet which incorporated a consultation summary sheet and website information (www.askshareknow.com.au); and a refrigerator magnet as a reminder for future use.This was a single‐arm intervention study to test the feasibility of the intervention, for which we developed a consumer questions training and support programme, now called AskShareKnow (ASK Patient–Clinician Communication ModelThe study was conducted at a metropolitan family planning clinic (part of a statewide non‐government organization) providing reproductive and sexual health‐care services, primarily to women. Patients >18 years of age, and with English fluency, attending appointments at the clinic were invited to participate in the study. Those with infants were excluded for logistical reasons.Family planning consultations are ideal for testing shared decision‐making interventions because they encompass a very wide range of treatment options  with near equipoise of benefits and harms, and are therefore very sensitive to consumer preference. Furthermore, women – the overwhelming consumers in this clinic – are usually the primary decision‐maker of health matters in society and have influence in over 80% of health‐care decisions.Recruitment took place between July and December 2012. The study was approved by the Human Research Ethics Committees of Family Planning NSW and the University of Sydney. Detailed study information was provided to participants and written consent obtained. The trial was registered with the Australian New Zealand Clinical Trials Registry no. ACTRN12610001055099.After providing consent, participants were provided with the three components of AskShareKnow described above, and asked to complete a questionnaire prior to their consultation (T1). They were then given a media tablet with headphones to watch the 4‐min AskShareKnow video‐clip (but not otherwise invited to ask the three questions featured). To avoid disrupting clinic flow, appointment scheduling took precedence over recruitment, so not all who were recruited were exposed to all the intervention. Those who watched the video‐clip prior to their consultation were invited to complete a second questionnaire immediately after the consultation (T2), and then a third questionnaire at 2 weeks (T3) . Questionnaire items included quantitative and qualitative responses. Quantitative responses were analysed using descriptive statistics. Participants were also invited to take part in a semi‐structured telephone interview at four to six weeks to obtain more detailed information about their experience and views of the AskShareKnow programme. Telephone Interviews were audio‐recorded transcribed verbatim and analysed using thematic analysis.The primary outcome was the use of the three questions in the health‐care consultation based on self‐report by the participants. Other outcomes focussed on key demand, acceptability and implementation aspects of AskShareKnowData were collected at three time points. The baseline questionnaire (T1) included demographic information, decision making and information preferencesA total of 197 participants consented to the study; however, participants who had not completed viewing the video‐clip before being called into their consultation see  were excSix study participants and four clinicians participated in semi‐structured telephone interviews exploring their experience of asking, or being asked, the questions.Thirty‐three women (27%) were born outside Australia, 73 (60%) university educated, 70 (58%) aged under 40 years Table . InformaDemographic information of those who consented but did not have sufficient time prior to their consultation to view the video‐clip was similar to the final sample included in the analysis.n = 58), 29 (50%) asked at least one of the Questions and 8 (14%) asking all three Questions. Of the three Questions, 1 and 2 were asked by 66 and 62 participants, respectively, while Question 3 was asked by 49  reported asking at least one of the questions during their consultation, with 35 29%) asking all three Questions (Table 9% askingThe other components of the ASK Patient–Clinician Communication model were less utilized by participants in the study. Thirty‐eight (31%) had time to view the AskShareKnow website prior to their consultation, and 58 (48%) reported reading the pamphlet prior to their consultation, with 19 (16%) reporting using the consultation summary sheet during the consultation.Several of the participants who were interviewed reported that the questions were similar to those they would normally ask, although commented the questions provided additional structure Box i.Box 2n = 4) interviewed commented that the questions covered information that would generally be covered during a consultation, but all stated it was useful for participants to be provided with the questions in the waiting room, although one did raise query whether they were necessary in all consultation types (Box The clinicians (ypes Box ii.n = 6) who were interviewed stated they would ask the questions again, although not necessarily verbatim , reported they would definitely recommend the questions. Two weeks later, 47 (83%) of those completing the final questionnaire would use the questions again. Each of the participants , or somewhat helpful 19 (31%). This reflected comments made by participants who were interviewed Box iv.Participants were asked whether they recalled the three questions two weeks after their consultation T3). Of the 95 participants who responded, 47 (49%) could; of 62 participants who provided details of the questions, 29 (47%) were able to recall all three questions accurately  programme used the three questions as one of its tools to promote SDM. Evaluation of this component was limited to the specific marketing campaign, and although this was positive (patients and clinicians liked it), the effectiveness of the campaign remains uncertain.Between October 2010 and August 2013, the UK Health Foundation through its MAGIC . While recall of the questions was high at 2 weeks, and participants reported that the questions were useful, and that they intended to recommend them to others, whether or not participants would repeat their use is unknown. Additionally, our exclusion of participants who did not have time to complete watching the 4‐min video‐clip reduced our sample; however, this was caused by the requirement in the research setting to gain consent and complete baseline questionnaires in addition to viewing the video‐clip.®) enabled consumers to ask the listed questions in health‐care consultations. Patients were able to view a short video‐clip to promote question asking immediately before their consultation, and most went on to ask at least one of the questions in that consultation.The brief consumer questions training and support programme, AskShareKnow (ASK Patient–Clinician Communication ModelThis brief intervention of three generic questions relating to health‐care decisions has shown important effects in improving the quality of information provided during clinical consultations and in facilitating patient involvement. It demonstrates the utility of the intervention through the ability of patients to use the questions following a brief waiting room video‐clip demonstration. While further evaluation to determine generalizability of study findings to other settings is needed and could add to the evidence by investigating the longer term impact of a larger campaign, should health systems proceed with implementing the AskShareKnow intervention at an institutional, regional or even national level as a simple way for patients and clinicians to share decisions in practice?HS conceived the study. HS, AB, DB, KC, LT, KM, CDM, PB, RM, VE and EW designed the study and obtained funding. AJ coordinated the running of the study and the data collection. KM carried out the statistical analysis. HS wrote the first draft of the manuscript. All authors contributed to the interpretation of the analysis and the writing of the manuscript. All authors are guarantors.This work was supported by Grant #0175‐1 from the Informed Medical Decisions Foundation. Dr. Shepherd received salary support from National Health and Medical Research Council Public Health Training Fellowship ID 568962. Dr. Shepherd had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.