In this article, we report a facile approach to enhance the electrochemical performance of Li-rich oxides with vulnerable phase stability. The Li-rich oxide nanoparticles were attached to the surface of graphene; the graphene surface acted as a matrix with high electronic conductivity that compensated for the low conductivity and enhanced the rate capability of the oxides. Our novel approach constitutes a direct assembly of two materials via electrostatic interaction, without a high-temperature heat treatment. The inevitable deterioration in phase integrity of previous composites between carbon and Li-rich oxides resulted from the reaction of oxygen in the structure with carbon during the heat-treatment process. However, our new method successfully attached Li-rich nanoparticles to the surface of graphene, without a phase change of the oxides. The resulting graphene/Li-rich oxide composites exhibited superior capacity and rate capability compared to their pristine Li-rich counterparts. Our novel strategy for the composition relies on the electrostatic interaction of the two species. As illustrated in Fig. 0.2Li0.2Mn0.6]O2 nanoparticles were prepared via the general combustion method using a dispersing agent to control the particle size of the cathode powder [0.2Li0.2Mn0.6]O2 nanoparticles were composited with graphene via electrostatic interactions. The commercial graphene (AMG Graphite/Graphit Kropfmühl) was immersed into the 1-N acidic solution (nitric acid/sulfuric acid = 3:1) for 2 h for surface modification and washed several times using distilled water. Then the Li[Ni0.2Li0.2Mn0.6]O2 and graphene particles were dispersed in an ethanol-based solution by ultra-sonication; the weight % of the graphene to the Li[Ni0.2Li0.2Mn0.6]O2 was controlled to amounts of 0.5, 2.0, and 5.0. The pH of the solution was adjusted to 4 using buffer solution (pH 4) in order to control the surface potential of the graphene and the Li[Ni0.2Li0.2Mn0.6]O2 particles. After a 10-min stirring for homogeneous mixing, the solution was vacuum filtered and the resulting samples were dried at 200 °C for 2 h under an air atmosphere. X-ray diffraction (XRD) measurements were then performed on the samples with a Rigaku X-ray diffractometer using monochromatized Cu-Kα radiation (λ = 1.5406 Å). In addition, the surface of the Li[Ni0.2Li0.2Mn0.6]O2 nanoparticle/graphene composite was examined using a transmission electron microscope  and X-ray photoelectron spectroscopy . The C content of the composites was determined via thermogravimetric analysis  by heating from 200 to 750 °C under an air atmosphere.Li[Nie powder . The pre0.2Li0.2Mn0.6]O2 nanoparticle/graphene composite (or pristine Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles) and carbon black (Super P) with polyvinylidene fluoride (PVDF) in a weight ratio of 80 (cathode):10 (super P):10(PVDF). After 24 h of ball-mill processing, the viscous slurry was coated onto an Al foil using a doctor blade and subsequently dried at 90 °C in an oven. A coin-type cell (2032) consisting of a cathode, Li-metal anode, separator , and an electrolyte (1 M LiPF6 in EC/DMC (1:1 vol%)) was used. The cells were subjected to galvanostatic cycling in the voltage range of 4.8–2.0 V and at various charge-discharge rates, using a WonATech voltammetry system. In addition, impedance measurements were performed by applying an AC voltage at an amplitude of 5 mV and a frequency range of 0.1 Hz to 100 KHz, using an electrochemical workstation .For electrochemical testing, a cathode slurry was prepared by mixing Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles via electrostatic interaction. Therefore, the appropriate pH value of the surface charges of graphene and the Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles was determined via zeta potential measurements. As Fig. 0.2Li0.2Mn0.6]O2 nanoparticle switched from positive (over 20 mV) to negative (−20 mV) with increasing pH value. The graphene and Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles should be oppositely charged in order to trigger the mutual assembly via electrostatic interaction, and hence the pH value should be lower than 4. Figure 0.2Li0.2Mn0.6]O2 nanoparticles.An appropriate pH value must be selected in order to directly assemble composites of graphene and LiO2 nanoparticles and composites of the graphene and Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles. Hereafter, we refer to the Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles attached to 0.5 wt.% graphene, 2.0 wt.% graphene, and 5.0 wt.% graphene (wt.% means weight percent of the Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles) as composite 0.5, composite 2.0, and composite 5.0, respectively. The Li[Ni0.2Li0.2Mn0.6]O2 powder consists of 200~500-nm-sized nanoparticles  surface area. In contrast, the graphene surface of composite 2.0 was appropriately covered with Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles, as shown in Fig. 0.2Li0.2Mn0.6]O2 nanoparticles are well-attached to, but only sparsely populate graphene; i.e., the surface area of the graphene far exceeded that of the nanoparticlesFigure les Fig. a, e. Fig0.2Li0.2Mn0.6]O2 particles in our composites to retain their structural integrity and phase; this assumption was deemed reasonable since the composites were assembled via electrostatic interaction, without the use of a high-temperature heat-treatment process that leads to oxygen loss and phase changes. To determine the validity of this assumption, the pristine sample and composites were evaluated via XRD and XPS measurements. Figure 2 structure (space group R-3m); the peaks occurring at angles of 20°–25° are associated with superlattice ordering in the transition metal layers. The pattern was examined in further detail by enlarging the peaks occurring at angles of 18°–20°, 20°–30°, and 40°–50°. These peaks are associated with the (003), superlattice ordering and graphene, and (104) and (105) reflections, which are shown in Fig. 0.2Li0.2Mn0.6]O2). As shown in Fig. As mentioned previously, we expected the LiO2 nanoparticles; i.e., the high electronic conductivity of the graphene matrix compensates for the low conductivity of the Li[Ni0.2Li0.2Mn0.6]O2 nanoparticles, thereby leading to the enhanced electrochemical performance of the composite.The effect of graphene as a matrix material was determined by evaluating the electrochemical properties of the pristine samples and the composites. Figure 0.2Li0.2Mn0.6]O2 cathode. Furthermore, the enhanced rate capability  are, in general, susceptible to phase changes and deterioration of the phase integrity; this deterioration results from the reaction of carbon with oxygen and consequent loss of oxygen from the structure of the vulnerable Li-rich oxide phase. However, we successfully fabricated graphene/Li[Ni0.2Li0.2Mn0.6]O2 composites using electrostatic interaction without deterioration of the phase integrity of the oxides, as confirmed by XRD and XPS analysis. The optimized graphene/Li[Ni0.2Li0.2Mn0.6]O2 composites (composite 2.0) exhibited higher discharge capacity and improved rate capability compared with those of pristine Li[Ni0.2Li0.2Mn0.6]O2. This improvement is attributed to the high conductivity of graphene, which compensates for the low conductivity of the pristine Li[Ni0.2Li0.2Mn0.6]O2.Li[Ni


Identification of serum proteins that track with disease course in sarcoidosis may have clinical and pathologic importance. We previously identified up-regulated transcripts for interferon-inducible chemokines CXCL9, and CXCL10, in blood of sarcoidosis patients compared to controls. The objective of this study was to determine whether proteins encoded by these transcripts were elevated in serum and identified patients with remitting vs. chronic progressive sarcoidosis longitudinally.et al.Serum levels of CXCL9, CXCL10, and proteins associated with inflammation and/or disease activity  were measured in a prospective cohort of sarcoidosis subjects and controls. Comparisons were made between groups and clinical course using pulmonary function measures and a severity score developed by Wasfi In a cross-sectional analysis of 36 non-immunosuppressed sarcoidosis subjects, serum CXCL9, CXCL10, and sIL2R were significantly elevated compared to 46 controls (p < 0.0001). CXCL9 and CXCL10 were strongly inter-correlated (p = 0.0009). CXCL10 and CXCL9 were inversely correlated with FVC% predicted and DLCO% predicted, respectively. CXCL10 and CXCL9 significantly correlated with sarcoidosis severity score. sIL2R, ESR, CRP, and ACE serum levels did not correlate with pulmonary function measures or severity score. In the longitudinal analysis of 26 subjects, changes in serum CXCL10 level over time corresponded with progression versus remission of disease.Interferon-γ–inducible chemokines, CXCL9 and CXCL10, are elevated in sarcoidosis and inter-correlated with each other. Chemokine levels correlated with measures of disease severity. Serial measurements of CXCL10 corresponded to clinical course. IRF1), which correlated with the severity of lung disease, suggesting a concordance of gene regulation in both blood and lung tissue compartments , we, we13], et al. found a 12-fold elevation of circulating CXCL10 but non-significant elevation CXCL9 in sarcoidosis subjects compared to controls ; ho; ho38];  disease , which o disease . In contpression ,41, sincpression . Finallypression . Taken tet al.. Th. Th21]. et al. since ouIn summary, we found that serum CXCL9 and CXCL10 levels correlated with sarcoidosis disease severity as assessed by FVC and DLCO, as well as the Wasfi severity score, which incorporates additional clinical phenotypes of sarcoidosis. Changes in serum CXCL10 levels over time correlated with both chronic progressive disease (associated with sustained elevations of CXCL10) or remitting sarcoidosis (associated with a reduction over time in CXCL10). Our findings in the peripheral blood, along with data from several other cross-sectional BALF studies, suggest that CXCL9 and CXCL10 may play important roles in the pathogenesis of sarcoidosis. Larger replicate studies are needed to validate our findings.sIL2R: Soluble interleukin-2 receptor; IFN: Interferon; ACE: Angiotensin converting enzyme; CRP: C-reactive protein; ESR: Erythrocyte sedimentation rate; UCSF: University of California San Francisco; ATS: American Thoracic Society; FVC: Forced vital capacity; FEV1: Forced expiratory volume in 1 second; DLCO: Diffusing capacity.The authors declare that they have no competing interests.LK, RS, MLN, ADG, and PGW contributed to the study design, data acquisition and analysis, and writing of the manuscript. MA contributed to the data acquisition and statistical analyses. CK, CPN, JR, ED, and MH contributed to patient recruitment, study design, pulmonary function testing, blood collection, and data generation. All authors read and approved the final manuscript.Patient selection flow diagram for cross-sectional versus longitudinal cohorts. This diagram depicts the selection criterion for sarcoidosis subjects in the cross-sectional and longitudinal analyses and, within the longitudinal cohort, the definitions of chronic versus remitting sarcoidosis.Click here for fileCharacteristics of Cross-Sectional Sarcoidosis Subjects with Normal versus Low Pulmonary Function Testing. This table compares the characteristics of cross-sectional sarcoidosis subjects with low pulmonary function testing (as defined by a FVC or DLCO of less than 80 percent predicted) with sarcoidosis subjects with normal pulmonary function testing.Click here for file


Plastic debris pervades in our oceans and freshwater systems and the potential ecosystem-level impacts of this anthropogenic litter require urgent evaluation. Microbes readily colonize aquatic plastic debris and members of these biofilm communities are speculated to include pathogenic, toxic, invasive or plastic degrading-species. The influence of plastic-colonizing microorganisms on the fate of plastic debris is largely unknown, as is the role of plastic in selecting for unique microbial communities. This work aimed to characterize microbial biofilm communities colonizing single-use poly (PET) drinking bottles, determine their plastic-specificity in contrast with seawater and glass-colonizing communities, and identify seasonal and geographical influences on the communities. A substrate recruitment experiment was established in which PET bottles were deployed for 5–6 weeks at three stations in the North Sea in three different seasons. The structure and composition of the PET-colonizing bacterial/archaeal and eukaryotic communities varied with season and station. Abundant PET-colonizing taxa belonged to the phylum Bacteroidetes  and diatoms . The PET-colonizing microbial communities differed significantly from free-living communities, but from particle-associated (>3 μm) communities or those inhabiting glass substrates. These data suggest that microbial community assembly on plastics is driven by conventional marine biofilm processes, with the plastic surface serving as raft for attachment, rather than selecting for recruitment of plastic-specific microbial colonizers. A small proportion of taxa, notably, members of the Cryomorphaceae and Alcanivoraceae, were significantly discriminant of PET but not glass surfaces, conjuring the possibility that these groups may directly interact with the PET substrate. Future research is required to investigate microscale functional interactions at the plastic surface. Plastic pollution was first reported in remote, offshore basins of the north Atlantic ocean over forty years ago , 2, twenAs the spatial distribution of marine plastic debris continues to be better resolved, research is increasingly focused on assessing its effects on environmental and public health. The impacts of plastic pollution range from organismal, such as morbidity and mortality due to entanglement and intestinal blockage  to food Plastic surfaces in seawater can form microbial biofilms visible by eye within one week and cause a physical change, with a significant increase in plastic hydrophilicity and a shift from positive towards neutral buoyancy after 2 weeks . Yet, thArcobacter and Colwellia, which have previously been shown to affiliate with hydrocarbon contamination . Sterile PCR grade water served as negative control and known DNA as positive control , taxonomy assignment  and picking of operational taxonomic units, OTUs, (label = 0.03) were carried out using Mothur . Chloropth percentile of reads  were removed from analyses, proportions of OTUs per sample were calculated , proportions were square root-transformed, and scaled by the minimum library size per subset. This work was performed in R  or calculating Monte-Carlo tests [‘p(MC)’]. Unique perm, unique permutations. The results  of the PERMDISP tests calculated to centroids are also provided.(XLSX)Click here for additional data file.S4 TableDiscriminant OTUs  identified, using class and subclass distinctions, in comparisons of: (a) PET-associated, particle-attached (>3 μm) and free-living (0.2 μm-3 μm) seawater communities in summer (lefse class: treatment) across all stations (lefse subclass: station), (b) PET- and glass-attached communities in spring (lefse class: treatment) across all stations (lefse subclass: station), (c) PET-associated communities from summer, spring, winter (lefse class: season) across all stations (lefse subclass: station), (d) PET-associated communities from Dowsing, Warp, Gabbard (lefse class: station) across all seasons (lefse subclass: season), (e) attached (PET and >3μm seawater fraction) and free-living (3–0.2 μm seawater fraction) communities  across all stations (lefse subclass: station).(XLSX)Click here for additional data file.S5 TableDiscriminant OTUs  identified, using class and subclass distinctions, in comparisons of: (a) PET-associated, particle-attached (>3 μm) and free-living (0.2 μm-3 μm) seawater communities in summer (lefse class: treatment) across all stations (lefse subclass: station), (b) PET- and glass-attached communities in spring (lefse class: treatment) across all stations (lefse subclass: station), (c) PET-associated communities from summer, spring, winter (lefse class: season) across all stations (lefse subclass: station), (d) PET-associated communities from Dowsing, Warp, Gabbard (lefse class: station) across all seasons (lefse subclass: season), (e) attached (PET and >3μm seawater fraction) and free-living (3–0.2 μm seawater fraction) communities  across all stations (lefse subclass: station).(XLSX)Click here for additional data file.


Drosophila melanogaster and TARDBP in mammals) is the main protein component of the pathological inclusions observed in neurons of patients affected by different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The number of studies investigating the molecular mechanisms underlying neurodegeneration is constantly growing; however, the role played by TDP-43 in disease onset and progression is still unclear. A fundamental shortcoming that hampers progress is the lack of animal models showing aggregation of TDP-43 without overexpression. In this manuscript, we have extended our cellular model of aggregation to a transgenic Drosophila line. Our fly model is not based on the overexpression of a wild-type TDP-43 transgene. By contrast, we engineered a construct that includes only the specific TDP-43 amino acid sequences necessary to trigger aggregate formation and capable of trapping endogenous Drosophila TDP-43 into a non-functional insoluble form. Importantly, the resulting recombinant product lacks functional RNA recognition motifs (RRMs) and, thus, does not have specific TDP-43-physiological functions (i.e. splicing regulation ability) that might affect the animal phenotype per se. This novel Drosophila model exhibits an evident degenerative phenotype with reduced lifespan and early locomotion defects. Additionally, we show that important proteins involved in neuromuscular junction function, such as syntaxin (SYX), decrease their levels as a consequence of TDP-43 loss of function implying that the degenerative phenotype is a consequence of TDP-43 sequestration into the aggregates. Our data lend further support to the role of TDP-43 loss-of-function in the pathogenesis of neurodegenerative disorders. The novel transgenic Drosophila model presented in this study will help to gain further insight into the molecular mechanisms underlying neurodegeneration and will provide a valuable system to test potential therapeutic agents to counteract disease.Transactive response DNA-binding protein 43 kDa (TDP-43, also known as TBPH in  Summary: An engineered TDP-43 construct can be used to induce TDP-43 aggregation in Drosophila, providing a model that could be useful for characterization of pathogenetic mechanisms and drug screening. Drosophila melanogaster and TARDBP in mammals), resulting in nuclear clearance of the protein  and amyotrophic lateral sclerosis (ALS) are two neurodegenerative diseases that can exist both as distinct clinical entities and as a clinical continuum with overlapping pathogenic pathways . A commo protein . TDP-43  protein  that are protein . By expl protein , differedeficits , indicatdeficits . In accoregation . The C-tregation , and it regation . The Q/No models , 2015. Io models ,b.Drosophila melanogaster line expressing the construct EGFP-12×Q/N in the eye under the control of the GMR-Gal4 driver, it has been shown that EGFP-12×Q/N is able to trigger aggregation similarly to that observed in cells and that there is no intrinsic toxicity of the aggregates  in a cell line is able to trigger the aggregation of endogenous TDP-43, resulting in its loss of function as determined by POLDIP3 exon 3 alternative splicing . In thisTo test the aggregation efficiency of the transgene, we produced a HEK293 AggIn stable cell line. After tetracycline induction, anti-Flag staining showed the presence of many, prevalently cytosolic, aggregates . SimilarBudini et al., 2012bFurthermore, we also performed the splicing assay on the two additional endogenous transcripts BIM and MADD, whose splicing profile is known to be affected upon TDP-43 depletion . For botin vivo in Drosophila melanogaster.Taken together, these experiments provide evidence of the endogenous TDP-43 loss of function upon AggIn expression and indicate that AggIn is a promising construct to model TDP-43 aggregation To create a novel animal model for TDP-43 aggregation, we cloned the AggIn construct in the pUASTattB vector, under the control of the upstream activating sequence (UAS). After embryo injection, five different fly lines were obtained and screened for transgene expression by using the GMR-Gal4 driver. Although four of these lines expressed the transgene at comparable levels, the fifth demonstrated a higher expression level (data not shown). Therefore, in the subsequent steps of our study, we focused our attention on two of these transgenic lines: the one expressing the transgene at the top expression level (UAS_5A) and one out of the four expressing the transgene at comparable level (UAS_2B) A.Fig. 3Drosophila phenotype, we expressed the construct selectively in neurons, by crossing the UAS_5A and UAS_2B flies with a fly containing the pan-neuronal elav-Gal4 driver (the flies were grown at 25°C). As expected, elav-Gal4>UAS_5A flies demonstrated a transgene expression level twofold higher than the one observed in elav-Gal4>UAS_2B, as calculated from normalized expression values , and a second transgenic fly line expressing the irrelevant protein EGFP . As clearly shown in Fig. S1A), the expression of the AggIn transgene did not substantially alter the eye anatomy, although there was a modest change in visual ability, particularly in the high-expression UAS_5A line . This suggests that AggIn expression also does not induce obvious signs of eye-structure degeneration during aging. Therefore, both fly lines were considered suitable for further studies of the phenotypic effects of aggregation.The intrinsic toxicity of the protein aggregates was low. In fact, whereas the expression of a wild-type dTDP-43 transgene using the GMR-Gal4 driver resulted in the formation of large necrotic patches in the eye of newly eclosed flies . Both elav-Gal4>UAS_5A and elav-Gal4>UAS_2B flies already demonstrated a statistically significant impairment of the climbing ability at the first time-point (day 3). As expected, at each time point analyzed, the elav-Gal4>UAS_5A showed a more severe impairment than the elav-Gal4>UAS_2B . Indeed,Taken together, these results suggest that the AggIn product affects the motility of flies in an expression-dependent manner.Fig. S2). Whereas the elav-Gal4>UAS_2B line did not show differences in larval lethality compared to controls  and revealed only a slight increase in pupal lethality, elav-Gal4>UAS_5A animals demonstrated a higher larval and pupal lethality compared to both the controls and to the elav-Gal4>UAS_2B line. Nonetheless, we were able to analyze the phenotype of the animals during the larval stage. To this aim, we assayed third-instar larvae movement by counting the number of their peristaltic waves in 2 min on a suitable solid substrate . In addition, as a negative control of the experiment, we used a transgenic line expressing the irrelevant protein EGFP in neurons  and the wild-type control line w1118. We also, as a positive control, analyzed the movement of TBPHΔ23 larvae, the first-discovered dTDP-43-null allele fly line, which shows a severe neurodegenerative phenotype with a locomotion defect in larval stages and dramatic locomotive defects after eclosion (Δ23)  A. TherefThe creation of the HEK293 AggIn stable cell line has shown that transgene expression efficiently triggers the formation of aggregates able to recruit and trap the endogenous TDP-43 protein and give rise to a TDP-43 loss-of-function effect .To show that AggIn expression also induces the formation of insoluble aggregates able to trap endogenous dTDP-43 in flies, we performed solubility experiments on transgenic fly heads co-expressing AggIn and a Flag-tagged form of dTDP-43 under the control of the GMR-Gal4 driver. As a control experiment, Flag-dTDP-43 was co-expressed with the unrelated protein EGFP. This experiment clearly shows that AggIn expression results in the formation of insoluble aggregates  presynaptic boutons in muscle 6 and 7 of third-instar larvae  and Cysteine-string protein (CSP) are two presynaptic vesicular proteins. It has been recently reported that their downregulation is an early event of dTDP-43 dysfunction r larvae . Startinic model A. IntereIn order to support the hypothesis that endogenous dTDP-43 loss of function was also responsible for the phenotype observed during the larval stage in elav-Gal4>UAS_5A line, we analyzed SYX protein expression in the NMJ presynaptic boutons in muscle 6 and 7 of these transgenic larvae by immunohistochemistry. As expected, SYX levels were strongly reduced in the synaptic terminals of elav-Gal4>UAS_5A third-instar larvae compared to elav-Gal4>UAS_Egfp controls . Interestingly, whereas the phenotype onset in elav-Gal4>UAS_Egfp-12×Q/N flies matches with a strong decrease of endogenous dTDP-43 at day 10 , although it is significantly higher than at day 10.Consistently, the observation that dTDP-43 functional loss is detected in elav-Gal4>UAS_5A but not in elav-Gal4>UAS_Egfp-12×Q/N larvae correlates with the ability of aggregates to sequester endogenous dTDP-43. Indeed, we recently demonstrated that the climbing impairment in elav-Gal4>UAS_Egfp-12×Q/N flies overlaps with an age-related physiological drop of dTDP-43 . In ordet day 10 , the elat day 10 , a time-In the past decade several lines of evidence have shown that TDP-43 plays a key role in the pathogenesis of several neurodegenerative disorders, including ALS and FTLD . NotwithDrosophila eye, a tissue that does not need TDP-43 for its development  with different levels of the AggIn transgene expression. This characteristic was useful to model differential levels of the TDP-43 aggregation process. In fact, only the elav-Gal4>UAS_5A line  showed motility impairment at the larval stage, whereas both the elav-Gal4>UAS_5A and elav-Gal4>UAS_2B lines resulted in a reduced lifespan and impaired climbing ability during adulthood. These latter effects were more severe when the aggregation was more efficient. In fact, lifespan was reduced to about a quarter  and a half  with respect to the median survival of control elav-Gal4>UAS_Egfp flies. Importantly, these effects were observed at a physiological growth temperature (25°C).Taking all the above points into consideration, we have now optimized an aggregation inducer with minimal TDP-43 sequences and tested it both in HEK293 cells and in 93 cells B and 2. Δ23 larvae  plasmid was generated by site-directed mutagenesis using the pcDNA5/FRT/TO-Flag-TDP-12×-Q/N plasmid  as a temFor generation of stable cell lines, HEK293 flip-in cells were grown in DMEM-Glutamax-I  supplemented with 10% fetal bovine serum (Gibco-BRL) and antibiotic and antimycotic stabilized suspension . Cells were transfected using Effectene Transfection reagent  following the manufacturer's instructions. Co-transfection of 0.5 μg of plasmid together with 0.5 μg of pOG44  vector allowed recombination in the genome of the cells. After co-transfection, cells were grown in DMEM-Glutamax-I supplemented with 10% fetal bovine serum with antibiotic and antimycotic until they reached 80% of confluence. The stable integration of the plasmid was then gradually selected using 100 μg/ml Hygromicin B (Gibco-BRL) and 10 μg/ml of Blasticidin (Gibco-BRL). Once cells were selected, expression of the protein was achieved by adding 1 μg/ml of tetracycline (Sigma-Aldrich) to the culture medium.5 Flag-TDP-43-WT, Flag-TDP-43-12×Q/N and AggIn cells were seeded in 6-well plates and induced with tetracycline for 72 h. Uninduced cells were used as a control. After induction, cells were collected and RNA was extracted using Trifast reagent  according to the manufacturer's instruction. Reverse transcription was performed using M-MLV Reverse Transcriptase (Gibco-BRL) following the manufacturer's protocol. A PCR with TAQ DNA Polymerase  was performed for 35 amplification cycles  to amplify POLDIP3, BIM and MADD cDNAs. The primers used to test the splicing pattern of POLDIP3, BIM and MADD endogenous genes were: POLDIP3 forward (5′-GCTTAATGCCAGACCGGGAGTTGGA-3′); POLDIP3 reverse (5′-TCATCTTCATCCAGGTCATATAAATT-3′); BIM forward (5′-TCTGAGTGTGACCGAGAAGG-3′); BIM reverse (5′-TCTTGGGCGATCCATATCTC-3′); MADD forward (5′-GACCTGAATTGGGTGGCGAGTTCCCT-3′); MADD reverse (5′-CATTGGTGTCTTGTACTTGTGGCTC-3′).For the splicing assay, 5×105 cells seeded in 6-well plates and induced for 72 h. Uninduced cells were also seeded as control. After induction, cells were collected and lysed with 100 μl of RIPA lysis buffer  supplemented with Complete protease inhibitor cocktail . Cell lysates were incubated at +4°C for 30 min, then lysed by sonication and centrifuged at 500 g at +4°C for 5 min. Total protein amount in cell lysates was then quantified by the Bradford method, and 20 μg was loaded in a 10% SDS-PAGE. An anti-Flag  primary antibody and a HRP-labeled anti-mouse-IgG  secondary antibody were used for protein detection. Western blotting using a primary anti-POLDIP3  antibody and a secondary HRP-labeled anti-rabbit-IgG  antibody was also performed to detect POLDIP3 isoforms.Protein expression of the AggIn construct was analyzed in 5×105 HEK-AggIn cells were seeded on slides and induced with tetracycline for 72 h. Non-induced cells were also seeded as a control. Immunofluorescence was performed as previously described , and were maintained and crossed in a humidified incubator at 25°C with a 12-h-light–12-h-dark cycle.AggIn and EGFP constructs were cloned in the pUASTattB vector and subsequently sequenced. The constructs were used to create transgenic flies by standard embryo injections . Transgenes were subsequently balanced on the required chromosome to obtain fly stocks. wAdult flies were collected for 2 days from eclosion and transferred to fresh food tubes in a 1:1 male:female ratio . At the third day, death events were scored and viable flies were transferred to fresh tubes. The same was done every 3 days. Survival proportions were plotted as percentage of living flies against days. More than 120 flies were tested for each genotype.The assay was performed as previously described . BrieflyAge-synchronized cohorts of flies were transferred without anesthesia to a 50-ml glass cylinder, and tapped to the bottom with cotton. After a period of adaptation of 30 s, the climbing ability of flies was quantified as number of animals that reached the top of the cylinder (10 cm) in 15 s. Flies were assayed in batches of 20  and the test was repeated three times for each batch of animals. More than 120 flies were tested for each genotype. The number of flies that reached the top was converted into a percentage value, and the mean±s.e.m. percentage was calculated for at least six experiments.Wandering third-instar larvae were selected, gently washed and transferred to a Petri dish (0.7% agarose in distilled water). After a period of adaptation (30 s), the peristaltic waves were counted within 2 min. At least 20 larvae were assayed for each genotype. The median number of peristaltic waves performed in 2 min by each genotype was plotted on a graph (mean±s.e.m.).Drosophila heads were homogenized in lysis buffer . Proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes , blocked overnight in a 5% non-fat dried milk solution and probed with the following primary antibodies: rabbit anti-Drosophila TDP-43 , mouse anti-SYX 8C3s , anti-CSP2c , mouse anti-tubulin CP06  and mouse anti-FLAG M2  antibodies. The membranes were incubated with the secondary antibodies HRP-labeled anti-mouse-IgG  or HRP-labeled anti-rabbit-IgG . Finally, protein detection was performed with Femto Super Signal substrate (Thermo Scientific) for anti- TDP-43 and anti-CSP2c immunoblotting and with ECL western blotting substrate (Thermo Scientific) for anti-syntaxin and anti-tubulin antibodies.Protein expression was quantified using the NIH ImageJ software  and normg for 10 min at 4°C. An aliquot was taken at this point as the input, and after a further centrifugation step at 100,000 g for 30 min at 4°C, the supernatant was collected as the soluble fraction. The resulting pellet was re-suspended in urea buffer  and collected as the insoluble fraction. Proteins were separated by 10% SDS-PAGE. The different samples were loaded in a 1:1:1 ratio for the input, soluble and insoluble fractions. Proteins were immunoblotted as already described for fly head samples and probed with the following reagents. Primary antibodies: mouse anti-FLAG M2 , rabbit anti-GFP  and mouse anti-tubulin CP06  antibodies. Secondary antibodies: HRP-labeled anti-mouse-IgG  or HRP-labeled anti-rabbit-IgG .The solubility assay was performed as previously described . Briefly2, MgCl2 4 mM, KCl 2 mM, NaCl 128 mM, sucrose 35.5 mM and Hepes 5 mM pH 7.2), fixed for 20 min in 4% paraformaldehyde, washed in PBS with 0.1% Tween 20 and blocked with 5% normal goat serum  in PBS with 0.1% Tween. Primary antibodies [anti-HRP  and anti-SYX 8C3s  antibodies] were incubated overnight at 4°C and then the secondary antibodies, Alexa-Fluor-488-conjugated anti-rabbit-IgG and Alexa-Fluor-555-conjugated anti-mouse-IgG , were incubated for 2 h at room temperature. SlowFade Gold (Gibco-BRL) was used for the mounting. Images were acquired on a Zeiss LSM 510 Meta Confocal Microscope with a 63× oil lens and 40× lens, then analyzed using NIH ImageJ software (Third-instar larvae were selected, briefly washed in water and dissected in saline solution (0.1 mM CaClsoftware .The larvae analyzed for these experiments were processed simultaneously and the same microscope settings were employed to acquire all images. The presynaptic terminals of second abdominal segment on muscle 6 and 7 were analyzed. The samples were double labeled with anti-HRP and anti-SYX antibodies: the mean intensity of both was quantified and a ratio calculated (adapted from


Graphene is a two-dimensional material that offers a unique combination of low density, exceptional mechanical properties, large surface area and excellent electrical conductivity. Recent progress has produced bulk 3D assemblies of graphene, such as graphene aerogels, but they possess purely stochastic porous networks, which limit their performance compared with the potential of an engineered architecture. Here we report the fabrication of periodic graphene aerogel microlattices, possessing an engineered architecture via a 3D printing technique known as direct ink writing. The 3D printed graphene aerogels are lightweight, highly conductive and exhibit supercompressibility (up to 90% compressive strain). Moreover, the Young's moduli of the 3D printed graphene aerogels show an order of magnitude improvement over bulk graphene materials with comparable geometric density and possess large surface areas. Adapting the 3D printing technique to graphene aerogels realizes the possibility of fabricating a myriad of complex aerogel architectures for a broad range of applications. Aerogels are ultra-lightweight porous materials that possess some remarkable properties. Here, the authors use a 3D printing technique to fabricate just such a material out of graphene, exhibiting large surface area, high conductivity and supercompressibility while maintaining good structural integrity. Graphene is an emerging class of ultrathin carbon membrane material126810121416202122232425262728293031323334352021272829303335The functional characteristics of cellular materials are mostly determined by the intrinsic properties of their chemical composition, porosity and cell morphologies444346In this work, we demonstrate a 3D printing strategy for the fabrication of 3D graphene aerogels with designed macroscopic architectures. Our approach is based on the precise deposition of GO ink filaments on a pre-defined tool path to create architected 3D structures. Two key challenges in this process are developing a printable graphene-based ink and maintaining the intrinsic properties of single graphene sheets  in the 3D printed structures. To this end, we have developed a new GO-based ink and printing scheme that allows the manufacture of porosity-tunable hierarchical graphene aerogels with high surface area, excellent electrical conductivity, mechanical stiffness and supercompressibility.−1 GO) that do not possess the required rheological behaviour for a 3D printable ink as they are low-viscosity (η) Newtonian fluids20−1 GO) that can also make high-quality graphene aerogels30−1, the GO suspension shows orders of magnitude higher apparent viscosity than reported at lower concentrations−1 is a shear-thinning non-Newtonian fluid, which is necessary for a printable ink. Further increasing the GO concentration to 40 mg ml−1 results in another order-of-magnitude increase in apparent viscosity, which further improves printability. Finally, addition of hydrophilic fumed silica powders to the GO suspensions imparts additional increases in viscosity. Silica filler serves as a removable viscosifier by imparting both shear thinning behaviour and a shear yield stress to the GO suspension to further enhance the printability of the GO inks. −1 GO suspensions without fillers exhibit a plateau value of its elastic modulus (G′) ∼1,000 Pa and a yield stress (τy) ∼40 Pa, respectively. By adding 20 wt% silica powders into pure 20 mg ml−1 GO suspensions, both elastic modulus and yield stress increase by approximately an order of magnitude. Meanwhile, the addition of 10 wt% silica filler increases the elastic modulus and yield stress of 40 mg ml−1 GO suspensions by over an order magnitude. The magnitudes of these key rheological parameters are in good agreement with those reported for other colloidal inks designed for this 3D filamentary printing technique−1 GO suspension ink is printable, the silica-filled GO inks were preferred due to their superior rheological properties and facile removal of the silica during post-processing. In addition to these, GO inks exhibit the desired viscoelasticity and they have a long pot life.The first challenge for this fabrication strategy is to develop printable GO inks, by tailoring the composition and rheology required for reliable flow through fine nozzles, and self-supporting shape integrity after deposition . Printable GO ink development is challenging because most GO-based graphene aerogels begin with fairly dilute precursor GO suspensions  of 1 mm and a filament diameter (d) of 0.25 mm, resulting in a spacing-to-diameter ratio (L/d) of 4 (4)2CO3) to directly crosslink graphene sheets via the functional groups  or resorcinol (R) and formaldehyde (F) with sodium carbonate as a catalyst to ‘glue' the sheets together. The use of organic sol–gel chemistry (R–F solution) to build the GO network led to a more open, less crosslinked network  of 4 . By simp/d) of 4 , which i/d) of 4 . After t/d) of 4 , there a/d) of 4 . Figure  network  compared network . The abi network .−1) and G  bands with weak, broad D′ and G′ features identical to those previously reported for bulk aerogels2929Modifying the GO suspensions to make suitable inks has the potential to alter the properties of the final aerogel; however, most properties of the 3D printed graphene aerogels were found to meet or exceed those of the bulk material. For example, techniques such as Raman spectroscopy, X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDS) were applied to see how microstructure, graphene layering and degree of GO reduction compare with bulk graphene aerogels. Raman spectra of the 3D printed graphene aerogels  all show2 g−1) and large mesopore volumes (2–4 cm3 g−1), consistent with the SEM images and comparable to bulk graphene aerogels in the literature2929Standard graphene aerogels are also notable for their large surface areas, low densities and high electrical conductivities. These characteristics are also evaluated for the modified formulations that we used to create the inks and are presented in σ) as a function of strain (ɛ) for all bulk and printed structures. The compressibility of these graphene aerogels is displayed in Graphene aerogels are also known to be remarkably stiff and flexible. To quantify the mechanical properties of these aerogels, we conducted in-plane compression tests to measure the compressive stress  and density (ρ)29303150515253E∝ρ2.5, consistent with other studiesE for the graphene microlattices was about one order of magnitude larger than that of most bulk graphene aerogels with same densities. In other words, the printed graphene aerogels can maintain the stiffness values of higher-density bulk aerogels to much lower densities. This phenomenon is also commonly observed in traditional cellular materials, such as honeycombs, which exhibit stiffness values in certain loading directions that rival higher-density bulk solidsFinally, the effect of macroscopic architectural design on the mechanical properties is also reflected in superior rigidity of the graphene aerogel microlattices compared with bulk aerogels at the same overall geometric density. It has been shown that the stiffness of many conventional cellular solids is significantly diminished as their densities decrease due to quadratic or higher power scaling relationships between Young's modulus . After sonication, the lateral dimensions were in the range of 150–400 nm. For GO inks gelled using the native functionality on the GO sheets, ammonium carbonate ((NH4)2CO3) solution (0.3 wt%) was used. In a typical synthesis, GO inks are prepared by mixing 6 g of 40 mg ml−1 GO suspension, 0.343 g (NH4)2CO3 solution and 0.7 g fumed silica , respectively. For GO inks gelled using organic sol–gel chemistry, the sol–gel mixture consisted of an aqueous solution of resorcinol (R), formaldehyde (F) and sodium carbonate catalyst (C). The R:F mole ratio was 1:2, the R:C mole ratio was 200:1 and the resultant R–F concentration was 11 wt% R–F solids. In a typical synthesis, 3.6 g of 40 mg ml−1 GO suspensions, 2 g of R–F solution and 0.7 g of fumed silica are mixed. A planetary centrifugal mixer (Thinky) was used for mixing these samples for 2 min in a 35-ml container using a custom adaptor.Raw single-layer GO powder (Cheaptubes) was produced by the Hummer method−1, followed by a 10-min rest period to allow the ink structure to reform. A stress sweep from 10−2 to 103 Pa at a constant frequency of 1 Hz was conducted to record the storage modulus and loss modulus variations as a function of sweep stress. The yield stress (τy) was defined as the stress where storage modulus falls to 90% of the plateau value. A strain sweep from 10−1 to 102 s−1 was also performed to record the apparent viscosity at varying shear rates.Rheological properties of the inks were characterized using a stress-controlled Rheometer  with a 40-mm-flat plate geometry and a gap of 500 μm. All measurements were preceded by a 1-min conditioning step at a constant shear rate of 1 sd). An air-powered fluid dispenser  provided the appropriate pressure to extrude the ink through the nozzle. The target patterns were printed using a three-axis positioning stage  bath, with an initial nozzle height of 0.7d to ensure adhesion to the substrate. Three-dimensional periodic microlattices were assembled by patterning an array of parallel (rod-like) filaments in a meanderline-like pattern in the horizontal plane such that the orientation of each successive layer was orthogonal to the previous layer. Three-dimensional honeycomb structures were fabricated by stacking hexagonal unit-cell arrays into a lattice and then printed directly upon previous layers. Printed parts were cured in sealed glass vials at 85 °C. After gelation, the wet GO gels were removed from the glass vials and washed in acetone to remove water from the pores. Supercritical CO2 was used to dry the GO gels, and they were thermally reduced at 1,050 °C under nitrogen. Finally, etching in hydrofluoric acid solution was used to remove the silica nanoparticle filler.The GO ink was housed in a 3 ml syringe barrel (EFD) attached by a luer-lok to a smooth-flow tapered nozzle  for at least 24 h to remove all adsorbed species. XRD measurements were performed on a Bruker AXS D8 ADVANCE X-ray diffractometer equipped with a LynxEye 1-dimensional linear Si strip detector. The samples were scanned from 5 to 75° 2θ. The step scan parameters were 0.02° steps and 2 s counting time per step with a 0.499° divergence slit and a 0.499° antiscatter slit. The X-ray source was Ni-filtered Cu radiation from a sealed tube operated at 40 kV and 40 mA. Phases in the samples were identified by comparison of observed peaks to those in the International Centre for Diffraction Data (ICDD PDF2009) powder diffraction database, and also peaks listed in reference articles. Goniometer alignment was ensured using a Bruker-supplied Al2O3 standard.The dimension and weight of the samples were determined with a caliper with an accuracy of 0.01 mm and an ultra-micro balance  with an accuracy of 0.001 mg. The relative density was calculated from the measured mass and volume of each specimen. The compressive characteristics of printed specimens were measured using a universal testing machine (Instron 5943) equipped with a 1,000 N load cell at a nominal rate of 5 μm sC.Z., E.B.D., J.D.K., C.M.S. and M.A.W. conceived and designed experiments. C.Z., M.A.W., T.Y.-J.H. and A.M.G. prepared samples. M.A.W. and C.Z. were involved in electrical analysis. M.A.W. performed SEM, Raman, XRD, EDS and surface area analysis. C.Z. performed rheological and mechanical experiments. M.A.W., C.Z., E.B.D. and C.M.S. were mainly responsible for preparing the manuscript with further inputs from other authors. All authors discussed the results and commented on the manuscript.How to cite this article: Zhu, C. et al. Highly compressible 3D periodic graphene aerogel microlattices. Nat. Commun. 6:6962 doi: 10.1038/ncomms7962 (2015).Supplementary Figures 1-5.An animation showing the fabrication scheme used to print the graphene aerogel microlatticesReal-time video showing the 3D printing of GO ink in 2,2,4- trimethylpentane (isooctane) bath.


Graphene plasmons have been found to be an exciting plasmonic platform, thanks to their high field confinement and low phase velocity, motivating contemporary research to revisit established concepts in light–matter interaction. In a conceptual breakthrough over 80 years old, Čerenkov showed how charged particles emit shockwaves of light when moving faster than the phase velocity of light in a medium. To modern eyes, the Čerenkov effect offers a direct and ultrafast energy conversion scheme from charge particles to photons. The requirement for relativistic particles, however, makes Čerenkov emission inaccessible to most nanoscale electronic and photonic devices. Here we show that graphene plasmons provide the means to overcome this limitation through their low phase velocity and high field confinement. The interaction between the charge carriers flowing inside graphene and the plasmons enables a highly efficient two-dimensional Čerenkov emission, giving a versatile, tunable and ultrafast conversion mechanism from electrical signal to plasmonic excitation. Graphene plasmons have gained significant interest thanks to their high field confinement and low phase velocity. Here the authors show theoretically that charge carriers propagating in graphene can excite plasmons through a quantum Čerenkov emission process in two dimensions, in the form of plasmonic shock waves. However, the realization of plasmonic sources that are electrically pumped, power efficient and compatible with current device fabrication processes ), is a formidable challenge. In recent years, several groups have demonstrated the potential of surface plasmons as a platform for strong and ultrafast light–matter interaction345789711121516Achieving ultrafast conversion of electrical to optical signals at the nanoscale using plasmonics1Here we show that under proper conditions charge carriers propagating inside graphene can efficiently excite GPs, through a two-dimensional (2D) Čerenkov emission process. Graphene provides a platform in which the flow of charge alone is sufficient for Čerenkov radiation, eliminating the need for accelerated charge particles in vacuum chambers and opening up an opportunity for the study of the Čerenkov effect (ČE) and its applications, especially as a novel plasmonic source. Unlike other types of plasmon excitations, the 2D ČE manifests as a plasmonic shock wave, analogous to the conventional ČE that creates shockwaves in a three-dimensional (3D) medium. On a quantum mechanical level, this shockwave is reflected in the wavefunction of a single GP emitted from a single hot carrier.νF≈106 m s−1) are considered possible, even in relatively large sheets of graphene  of enhanced emission. This tunability also reveals regimes of backward GP emission and regimes of forward GP emission with low angular spread, emphasizing the uniqueness of ČE from hot carriers flowing in graphene. Surprisingly, we find that GP emission can also result from intraband transitions that are made possible by plasmonic losses. These kinds of transitions can become significant and might help explain several phenomena observed in graphene devices, such as current saturation17The mechanism of 2D ČE is enabled by two fundamental characteristics of graphene. On one hand, hot charge carriers moving with high velocities (up to the Fermi velocity Recent studies2526v>vp) may seem unattainable for charge carriers in graphene, because they are limited by the Fermi velocity v≤vF, which is smaller than the GP phase velocity vF<vp, as shown by the random phase approximation calculations1929The conventional threshold of the ČE in either 2D or 3D . This approach has been shown to give good agreement with experimental results8121333yz plane and the charge carrier is moving in the z direction . q= satisfies vp=vp(ω) or vp(q) obtained from the plasmon dispersion relation as vp=ω/q. The momenta of the incoming (outgoing) charge carrier ki= ) correspond to energies v=Ek/|ℏk|, which equals a constant (vF). The only approximation in where c is the speed of light and ϕ for the outgoing charge and θ for the GP, both relative to the z axis, which is the direction of the incoming charge. This notation allows us to simplify the [SP] for charge carriers inside graphene to |SP|2=cos2(θ−ϕ/2) or |SP|2=sin2(θ−ϕ/2) for intraband or interband transitions, respectively. The emission is restricted to two angles θ=±θČ , as reflected in the delta function in where ℏ→0 in the above expressions we recover the classical Čerenkov angle cos (θČ)=νp/ν and the classical 2D ČE, which can also be obtained from a purely classical electromagnetic calculation. However, although charge particles outside of graphene satisfy ℏω<<Ei, making the classical approximation almost always exactv>vp), v=vF) to reside between θČ=0 in ℏωcutoff=2Ei/(1±vF/vp), exactly matching the points where the red curves in ℏω=Ei+EF due to the interband transition being limited by the Fermi sea of excited states. Since Ei can be larger than EF, this implies that GP emission from electrical excitation can be more energetic than photon emission from a similar process . Finite temperature will broaden all cutoffs by the expected Fermi–Dirac distribution. However, for most frequencies, the GP losses are a more significant source of broadening.We note in passing that by setting qI=qI(ω), derived independently for each point of the GP dispersion curveγ width, defining γ(ω)=qR(ω)/qI(ω). The calculation can be done partly analytically , we modify the matrix elements calculation by including the imaginary part of the GP wavevector ytically  yieldingℏω≤Ei−EF . The dip in the spectrum at the boundary between interband and intraband transitions  tip, ballistic transport in graphene with high drain–source voltage and photoexcitation). GPs at near-infrared frequencies have already been shown to exist through direct and indirect experimental evidence383940Ei as high as (approximately) 3 eVvp(ω) and γ(ω) as parameters; thus, the basic predictions of the equations and the ČE features we describe will continue to hold regardless of the precise plasmon dispersion. For example, a recent paperThe interband ČE in There exist several possible avenues for observing the quantum ČE in GPs, having to do with schemes for exciting hot carriers. For example, apart from photoexcitation, hot carriers have been excited from tunnelling current in a heterostructurep–n junction474850To generate hot carriers that are also directional, a possible method would be to apply a strong drain–source voltage across a graphene Ei), for instance, may be generated from a tunnelling current or p–n junction. The ČE spectrum corresponding to an arbitrary hot carrier excitation energy distribution is readily computed by integrating over a weighted distribution of ČE spectra for monoenergetic hot carriers. An example is presented in EF and 1.1EF, respectively. Dividing by max(Ei)+EF (upper limit on the energy invested into a hot carrier), we get 77% and 78% (respectively) energy conversion efficiency. Here we assume that not more than one plasmon is emitted from each hot carrier—the efficiency is higher when GP emission from intraband trasitions is significant, as then multiple emissions from a single hot carrier are probable . Emission rates on the order of Γω∼1 are two to three orders of magnitude higher than those found in the conventional ČE in a 3D homogeneous dielectric, Γω=0.0013 for v=0.6c to Γω=0.0055 for v→c. In graphene, the high ČE emission rate over a broad range of Ei owes itself to the low phase velocity and high confinement of GPs over a wide frequency range, which are properties unique to 2D plasmons.Future studies could directly generalize our approach to regimes of higher rates of excitation of hot carriers, by solving for the steady-state occupation probability. Here we assume the hot carriers are sparse enough so that they relax to the Fermi sea after their GP emission without creating a significant occupation that would exclude the transitions of other hot carriers. Altogether, higher excitation rates are therefore expected to cause gradual saturation of the GP emission and thus reduce the conversion efficiency. Importantly, the conversion efficiency remains high even when the carriers' energy distribution is broad, as implied by the high ČE rate of emission for the representative values of Notably, the ČE emission of GPs can be coupled out as free-space photons by creating a grating or nanoribbons—fabricated in the graphene, in the substrate, or in a layer above it . For regular plasmons, this mismatch is typically overcome using a grating ; however, this results in relatively low coupling efficiency. In general, the efficiency of out-coupling is similar to the efficiency of in-coupling, for example, 2% has been shown in the best cases of ref. Furthermore, electrical excitation is interesting in itself due to its direct generation of radiation, without the need to use another radiation source (as in coupling lasers from free space). The unavoidable disadvantage of this scheme lies in the broad spectral range of the generated plasmons, compared with the highly monochromatic spectrum possible when using an external laser.Ei≈2EF and above) are involved. In this latter case, one expects single-particle excitations to prevail over the contribution of the plasmonic resonances7The hot carrier lifetime due to GP emission is defined by the inverse of the total rate of GP emission , while also supporting meV plasmons that can have slow phase velocities, partly due to the higher refractive indices possible in such low frequencies6676869Ei and EF, the ČE could form part of the explanation for the observed frequency up-conversionWe conclude with some very intriguing yet at this stage admittedly very speculative comments. Effects associated with the highly efficient emission of interband GPs and the unexpected emission of intraband GPs predicted by our quantum ČE theory may have already manifested themselves in current graphene experiments, even in ones that do not involve any optical measurement, such as transistor-based graphene devices221723The data supporting the findings of this study are available within the article and its How to cite this article: Kaminer, I. et al. Efficient plasmonic emission by the quantum Čerenkov effect from hot carriers in graphene. Nat. Commun. 7:11880 doi: 10.1038/ncomms11880 (2016).Supplementary Figure 1, Supplementary Notes 1-4 and Supplementary References


Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 , ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 , ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate bocaparvovirus 2 , ungulate bocaparvovirus 3 (porcine bocavirus 5), ungulate bocaparvovirus 4 (porcine bocavirus 7) and ungulate bocaparvovirus 5 . PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure.Porcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples.Seven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6–99.5 % identity with the North American strains) to be the North American strains nearest relative.These results are the first to report the presence of PPV6 in North America and demonstrate that the virus is found in multiple geographic areas in the United States and in Mexico. The overall prevalence of PPV6 in PRRSv viremic animals is relatively low. Further, all of the PPV6 genomes found in North America are most closely related to a PPV6 strain first identified in 2014 in healthy pigs from the Tianjin province of China.The online version of this article (doi:10.1186/s12985-015-0401-6) contains supplementary material, which is available to authorized users. Parvoviridae consists of many small, non-enveloped, single-stranded DNA viruses that infect a wide variety of species [Parvoviridae is further divided into two subfamilies: the Parvovirinae that infect vertebrates and the Densovirinae that infect invertebrates. Parvovirinae is comprised of eight genera; Copiparvovirus, Tetraparvovirus, Erythroparvovirus, Bocaparvovirus, Dependoparvovirus, Amdoparvovirus, Aveparvovirus and Protoparvovirus [The family  species . Parvovirvovirus . Parvovirvovirus . The geni.e. PCV-2, PRRS) were more likely to be positive for ungulate protoparvovirus 1, ungulate tetraparvovirus 2,ungulate tetraparvovirus 3, ungulate copiparvovirus 2, or ungulate bocaparvovirus 2 as well.Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 , ungulate tetraparvovirus 2 PPV3), ungulate tetraparvovirus 3 , ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate bocaparvovirus 2 , ungulate bocaparvovirus 3 (porcine bocavirus 5), ungulate bocaparvovirus 4 (porcine bocavirus 7) and ungulate bocaparvovirus 5 . Prevalence studies conducted in Europe and the United States have shown that ungulate tetraparvovirus 2, ungulate tetraparvovirus 3 and ungulate copiparvovirus 2 occur in both areas with varying percentages of infected animals depending upon animal age, sample type, clinical status, presence of additional viral agents and time frame. Prevalence of parvoviruses in European pigs ranged from 6.4 % for ungulate tetraparvovirus 3, 9.7 % for ungulate tetraparvovirus 2, 6.4 % for ungulate copiparvovirus 2, and 13.44 % for ungulate bocaparvovirus 2 in samples collected between 2006 and 2011 , ungulat. In 2009i.e. pseudorabies, PRRSv, PCV2) [PPV6 is the most recent parvovirus of swine to be described . PPV6 wav, PCV2) . Using av, PCV2) .Of the eight described porcine parvoviruses, ungulate protoparvovirus 1 and ungulate bocaparvovirus 5 have been isolated in cell culture. However, only ungulate protoparvovirus 1 has been demonstrated to be a causal agent of porcine reproductive failure –14. In cHere we report the first identification of PPV6 from swine in the United States and Mexico. Seven nearly full-length genomes with high similarity to Chinese PPV6 were found during next generation sequencing of PRRSv positive serum samples. Prevalence of PPV6 within the PRRSv positive dataset was then determined by screening all samples by quantitative PCR (qPCR).E < 1×10−5) by BLASTn search to PPV6 sequences described in China in late 2014 [Serum samples positive for PRRSv by qPCR submitted to the Kansas State Veterinary Diagnostic Laboratory, the Iowa State University Veterinary Diagnostic Laboratory, and the South Dakota State University Animal Disease Research and Diagnostic Laboratory were further analyzed by viral metagenomic sequencing. The Illumina  MiSeq data yielded seven samples which produced multiple contigs that showed significant expectation scores  (amino acids HDXXY) and the calcium binding loop (amino acids YXGXG), features found in many other parvoviruses, was identified in ORF2 by a motif search in CLC Genomics Workbench 7.0 . The Chinese PPV6 PLA2 domain of amino acids HDIRY was conserved in the NA-derived genomes. This domain is variable in ungulate copiparvovirus 2 viral variants as it is present in PPV5 but absent in PPV4. The calcium loop motif of YXGXR was additionally conserved in the Chinese PPV6 and the NA-derived genomes. This sequence differed from the other shared motifs of YXGXF found in ungulate bocaparvovirus 1, ungulate bocaparvovirus 5, adeno-associated dependoparvovirus A and avian adeno-associated dependoparvovirus 1 and YXGXG of the remaining parvoviruses.These North American-derived (NA) PPV6 genomes contained two non-overlapping open reading frames (ORF) whose amino acid sequences showed homology to the Parvo non-structural 1 (NS1) superfamily and Parvo capsid by BLASTp search. This search also identified homology between ORF1 of PPV6 and the replicase proteins of other members of Parvovirinae, as well as the amino acid alignments of ORF1 (NS1) and ORF2 (capsid) were evaluated for the best-fit model of sequence evolution in MEGA6 [Alignments of the nucleotide sequences of the seven new nearly full-length genomes and 31 complete or nearly full-length genomes encompassing the diversity of genera in the in MEGA6 . The besin MEGA6 , 16. In The reconstructed phylogenies for each alignment are shown in Figs. Pairwise comparisons of the seven new genomes showed that the NA-derived sequences shared 98.3 – 100 % nucleotide identity with 0 – 106 nucleotide differences. ORF1 and ORF2 showed 99.6 – 100 % (0 – 8 differences) and 99.3 – 99.6 % (15 – 56 differences) nucleotide identity respectively between the NA-derived sequences. Amino acid identities of ORF1 and ORF2 were 99.9 – 100 % (0 – 1 differences) and 99.2 – 99.7 % (4 – 13 differences).Variation within the Chinese sequences range from 96.8 – 99.9 % at the nucleotide level and 97.4 – 99.9 % in the amino acid sequence of VP1 which is the more variable of the PPV6 ORFs . The NA Comparisons of the genome sequences of the PPV6 clade with its sister clade containing PPV4 and PPV5 found 44.6 – 44.9 % identity with PPV4 and 43.9 – 44.6 % identity with PPV5.Alignment of the VP1 amino acid sequence identifies only two regions of greater than 100 amino acids in length that are conserved, 415 – 787 and 1090 – C-terminus, there is also a slightly smaller conserved stretch from 164 – 256 . These positive samples were confirmed with overlapping PCR and sequencing of the 441 bp fragment. The prevalence of PPV6 in the serum samples tested was 13.2 % (22/167). PPV6 was detected in PRRSv positive samples from every state tested .Copiparvovirus as discussed by Ni et al. [Phylogenetic analysis of seven newly described parvovirus genomes recovered by viral metagenomic sequencing of PRRSv positive porcine serum samples and 31 parvovirus genomes downloaded from GenBank show that these new genomes cluster with high support with the PPV6 genomes described in late 2014 from China and should be considered new strains of PPV6 as indicated by Cotmore et al. [i et al. .Although, the samples in this study have no age data associated with them, the prevalence of PPV6 in North America is similar to the prevalence found in finishing pigs in China . PPV6 viDespite the noncontiguous geographic dispersion of PPV6 in the United States and Mexico the strains found in North America are very closely related to each other with homology between strains, from different geographical locations, ranging from 98.3 to 100 % at the nucleotide level and 99.2 – 100 % at the amino acid level of ORFs1 and 2. These strains are more closely related to each other than the Chinese strains are to each other at both the nucleotide sequence and amino acid sequence of the predicted proteins, 97.1 – 99.6 % and 97.3 – 99.6 % respectively . The NorAmong all of the strains identified both in North America and China the sequence diversity is highest in ORF2, the capsid protein. This conforms to the predicted behavior of the virus in response to the host immune system, in that the capsid protein would be under the greatest direct pressure from the host immune system. Since ORF2 mutates at a faster rate than ORF1, mapping the rate of change in ORF2 might provide a method for identifying the timeframe in which PPV6 was introduced into North America.These results are the first to report the presence of PPV6 in North America and indicate that the virus is found in multiple states in the United States and found in approximately 13 % of pigs with PRRSv viremia. Further, all of the PPV6 genomes found in North America appear to share a single source virus very similar to the strain first identified in China from healthy pigs in Tianjin in 2014. Additional sampling of PPV6 genomes with good spatial and temporal data in North America and China may elucidate ways in which viruses such as these are transported across the globe and provide insights into improved biosecurity.The samples used in this study originated from swine submissions to the Kansas State Veterinary Diagnostic Laboratory, the Iowa State University Veterinary Diagnostic Laboratory, and the South Dakota State University Animal Disease Research and Diagnostic Laboratory. The protocol for this study was approved by the Kansas State University Institutional Biosafety Committee.Serum samples were centrifuged for five minutes (min) at 7500 rpm to remove any large particulates. One hundred and eighty microliters (μL) of clarified serum was treated with nucleases and incubated at 37 °C for 90 min as described by Allander et al. . NucleicPaired end reads for each sample were imported into CLC Genomics Workbench 7.0 software . For each sample, reads were assembled into contigs using the De Novo Assembly function with default parameters. Contig consensus sequences were identified by BLASTn against the non-redundant nucleotide database at NCBI. The GenBank accession number with the best E-value was used for reference-based assembly using the entire set of paired reads. Consensus sequences were extracted from the reference-based assemblies and used in phylogenetic analysis and sequence comparisons.Parvovirinae, thirty-one genomes representing all eight genera were downloaded from GenBank  using default parameters. Nucleotide alignments of the genomes, NS1 and capsid and amino acid alignments of NS1 and capsid were evaluated for the best model of evolution in MEGA6 under the Bayesian Information criterion [Galleria mellonella densovirus in MEGA6.To determine the relationship of the seven PPV6-like genomes within the in MEGA6  or on thriterion . PhylogeNucleotide and amino acid sequences of the seven new genomes and the five Chinese PPV6 genomes were aligned in ClustalW and then imported into CLC Genomics Workbench 7.0  for pairwise comparison. Comparisons were made of percent identity and number of nucleotide or amino acid differences. Comparisons were also made with alignments of the new genomes and ungulate copiparvovirus 2.Based on the consensus sequences obtained through viral metagenomic sequencing and the PPV6 sequences in GenBank two pairs of primers were designed, using CLC Main Workbench 7.0 . One pair of primers, PPV6-F44/PPV6-R53, amplified a 976 bp segment of ORF 2 and the second primer pair, PPV6-F/PPV6-R, amplified a 618 bp fragment. These two pairs of primers when used together form an overlapping pair of primers which amplify two additional fragments of 618 and 441 bp in length. The reaction mixture was composed of 1 μL of sample, 1 μL of 10 μM of each of the primers, 10.5 μL distilled water, and 12.5 μL GoTaq Green Master Mix . The thermal cycling conditions for single product amplification and overlapping amplification were 95 °C for 5 min, 35 cycles of 94 °C 30 s, 51 °C 30s, and 72 °C 1 min, followed by a 7 min elongation step at 72 °C. The presence of amplicons of the appropriate size was determined by electrophoresis and staining with ethidium bromide. A real-time PCR assay was also developed using a pair of detection primers (PPV6 qPCR F/PPV6-R53) and probe designed to bind and amplify a portion of the conserved region of ORF2. The real-time PCR reaction mixture was composed of 1 μL of sample, 0.5 μL of 10 μM probe, 0.5 μL of 10 μM of each primer, 10 μL distilled water, and 12.5 μL of GoTaq Colorless Master Mix . The thermal cycling conditions were 95 °C for 5 min, 40 cycles of 95 °C for 15 s, 50 °C for 15 s, and 72 °C for 15 s. Amplification and quantification was performed on a Bio-Rad CFX96 . Both the overlapping PCR and real-time PCR specificity was confirmed through testing of samples containing ungulate tetraparvovirus 3, ungulate protoparvovirus 1, porcine circovirus type 2 (PCV2), and Torque teno sus virus 1 (TTSuV1). Primer and probe sequences are shown in Table The NA-derived PPV6 genomes are available in GenBank accession numbers KR709262-KR709268.


Testosterone attenuates postnatal hippocampal neurogenesis in adolescent male rhesus macaques through altering neuronal survival. While brain-derived neurotropic factor (BDNF)/ tyrosine kinase receptor B (TrkB) are critical in regulating neuronal survival, it is not known if the molecular mechanism underlying testosterone’s action on postnatal neurogenesis involves changes in BDNF/TrkB levels. First, (1) we sought to localize the site of synthesis of the full length and truncated TrkB receptor in the neurogenic regions of the adolescent rhesus macaque hippocampus. Next, (2) we asked if gonadectomy or sex hormone replacement altered hippocampal BDNF and TrkB expression level in mammalian hippocampus (rhesus macaque and Sprague Dawley rat), and (3) if the relationship between BDNF/TrkB expression was altered depending on the sex steroid environment.We find that truncated TrkB mRNA+ cells are highly abundant in the proliferative subgranular zone (SGZ) of the primate hippocampus; in addition, there are scant and scattered full length TrkB mRNA+ cells in this region. Gonadectomy or sex steroid replacement did not alter BDNF or TrkB mRNA levels in young adult male rat or rhesus macaque hippocampus. In the monkey and rat, we find a positive correlation with cell proliferation and TrkB-TK+ mRNA expression, and this positive relationship was found only when sex steroids were present.We suggest that testosterone does not down-regulate neurogenesis at adolescence via overall changes in BDNF or TrkB expression. However, BDNF/TrkB mRNA appears to have a greater link to cell proliferation in the presence of circulating testosterone. Hippocampal neurogenesis may modulate hippocampal function and plasticity across life. Previous studies have suggested that estrogen and testosterone enhance neurogenesis in adult animals (reviewed in ). Long tThe BDNF gene contains multiple 5′ untranslated exons that are spliced onto a common 3′ exon that encodes the mature BDNF protein -9. AdditDue to the differing action of TrkB receptors on neurogenesis, we sought to characterise the anatomical distribution of cells expressing TrkB-TK+ and TrkB-TK- within the neurogenic regions of the rhesus macaque hippocampus. We hypothesised that the degree of cell proliferation (Ki67) would be positively correlated with TrkB-TK- expression and that the degree of cell survival and/or early neuronal markers (BrdU and DCX) would be positively correlated with TrkB-TK+ expression. We also hypothesised that gonadectomy may result in increased TrkB-TK+ and BDNF expression as a mechanism to support nacent cell survival during adolescence. Increased cell survival previously identified in gonadectomised monkeys  may be mWe found that gonadectomy or sex steroid replacement did not alter BDNF or TrkB mRNA levels in young adult male rat or rhesus macaque hippocampus. In the monkey, TrkB-TK+ mRNA showed limited expression in the neurogenic subgranular zone but was positively correlated with cell proliferation (Ki67+ cells). In the rat, Ki67 mRNA expression was positively correlated with TrkB-TK+ and BDNF mRNA expression only in the presence of sex steroids. Adolescent gonadectomy did not appear to potently influence neurogenesis in the rat.Macaca mulatta) from the NIH Animal Center’s primate field station were recruited into this study. Subjects, housing conditions, surgery and endocrine measurements for this cohort have been described in detail in Richards et al. . Samples were run alongside a seven point standard curve using hippocampal cDNA derived from a subset of 20 cases, including samples from all groups. In monkeys, TBP and SDHA were used as housekeeping genes, on the basis that their geometric mean was unchanged by gonadectomy . Samples were run alongside a seven point standard curve using serial dilutions of cDNA derived from hippocampus RNA pooled from all 12 monkeys. Table RT-PCR was performed in triplicate, by TaqMan Gene Expression Assays  using an ABI Prism 7900HT Fast Real-Time PCR System and a 384-well format. In rodents, the geometric mean of the expression of two housekeeping genes (YWHAZ and GUSB) was used to calculate the normalizing factor for gene expression, and were selected on the basis that they did not differ between treatment conditions [in vitro transcription kit as recommended by the manufacturer . [35S] antisense and sense riboprobes were labelled to a specific activity of ~2 × 109 cpm/μg by addition of radiolabelled UTP and were purified by ethanol precipitation. In situ hybridisation histochemistry was performed on 14-μm coronal sections containing the hippocampus as previously described , or in 2) was calculated and averaged across ~16 slides/monkey spaced evenly across the extent of the dentate gyrus.Immunohistochemistry for Ki67 and BrdU and cell density measurements were previously performed in Allen et al. . We have2, BrdU+ cells/mm2 and DCX mRNA expression). In the rat, planned comparisons were between TrkB variant mRNA expressions and measures of neurogenesis  in all treatment groups combined. Pearson’s correlations were also used to analyse the relationship between Ki67 mRNA expression and BDNF or TrkB variant mRNA expression in each rat treatment group separately. All statistical tests were performed with STATISTICA 12  and all graphs were produced with GraphPad Prism 6 . Significance was set at p < 0.05 for all tests.Outliers were removed by Grubb’s test . This resulted in the removal of 2 data points from the rhesus macaque data  and 5 data points from the Sprague Dawley data . Two-tailed paired sample t-test were performed to analyse regional differences in TrkB mRNA+ cell localisation in monkeys. Two-tailed independent sample t-tests were performed to analyse the difference in mRNA expression between intact and gonadectomised monkeys, data are presented as percent change in mRNA levels relative to the intact group ± SEM. One-way ANOVA was performed to determine differences in mRNA expression between intact, gonadectomised and sex hormone replaced rat groups, data are presented as percent change in mRNA levels relative to the intact group. Pearson’s correlations were performed to analyse the relationship between neurogenic markers  and neurotrophin expression. In rhesus macaque, planned comparisons were between TrkB variant mRNA expression and measures of neurogenesis (Ki67+ cells/mm2 in the subgranular zone (first 40 μm from granule cell layer) and an average of 122 TrkB-TK+ mRNA+ cells/mm2 in the outer 40 μm. There was an average of 1102 TrkB-TK- mRNA+ cells/mm2 in the subgranular zone, and an average of 344 TrkB-TK- mRNA+ cells/mm2 in the hilus. There was a significantly higher density of TrkB-TK- mRNA+ cells in the subgranular zone compared to TrkB-TK+ mRNA+ cells .As distinct TrkB isoforms appear to regulate different stages of neurogenesis , we invep > 0.05). Further, we did not detect a change between intact and gonadectomised animals in TrkB-TK+ or TrkB-TK- mRNA in the dentate gyrus or hilus by in situ hybridisation (film-based)  (data not shown).In the monkey, there were no significant group differences in mRNA expression of any BDNF or TrkB transcript examined (see Table 3) in this cohort [in situ hybridisation) and Ki67+ cells/mm3 in the dentate gyrus  ]. Neither form of the TrkB receptor mRNA was correlated with cell survival (BrdU+ cells/mm3) in the dentate gyrus .We hypothesised that TrkB-TK- mRNA expression in the dentate gyrus would be related to our previously calculated measure of cell division (Ki67+ cells/mms cohort . Contrar) Figure . Conversey Table . FurtherF4.64 = 1.8, p = 0.13)], doublecortin [(DCX) ] or TUC4 [(DPYSL3) ]  compared to Gdx rats that received no hormone replacement.In Sprague Dawley rats there was no overall significant treatment effect on hippocampal mRNA expression of Ki67 , and wiTestosterone can be aromatised to estradiol and thus can signal via both AR and ER pathways. Testosterone may have direct effects on neurogenesis via ERα or ERβ, which are expressed in proliferating cells (Ki67+) and immature neurons (Dcx+) in male rats . In maleEstradiol or DHT replacement did not influence expression of any neurogenic marker. Our results are consistent with previous studies suggesting that while estradiol influences neurogenesis in female rats, it does not influence cell proliferation or neuronal differentiation, at least to the same extent, in adult male rats ,56. Our It is unknown if there are adolescent-specific changes in the influence of estradiol on neurogenesis in female rats, as there have been no studies to our knowledge on the effect of sex hormone manipulation in adolescent female rats. Similarly, DHT treatment has been previously shown to impact cell survival (not assessed in this study), but does not impact cell proliferation or differentiation in adult male rats ,52. As wSupraphysiolocial testosterone may also influence cell proliferation via sex steroid receptor independent mechanisms. High levels of testosterone antagonise the glucocorticoid receptor in muscle tissue , and decAlthough manipulation of sex steroid levels did not alter total hippocampal BDNF/TrkB mRNA levels, but as testosterone may alter cell proliferation in the rat and this was related to TrkB-TK+ expression, we sought to determine if the relationship between Ki67 and BDNF/TrkB mRNA expression would be altered in different sex steroid environments. Sex steroid removal via gonadectomy uncoupled the relationship between TrkB-TK+ and cell proliferation. Similarly, levels of several BDNF transcripts  were only related to cell proliferation in the presence of sex steroids. The strongest and most numerous correlations were in the intact and in the testosterone replaced groups, suggesting this “coupling” effect may require both androgenic and estrogenic mechanisms. It is possible that sex steroids control the expression of shared factors that influence both proliferation and TrkB-TK+/BDNF expression Figure .Figure 9While changes in the overall levels of hippocampal BDNF/TrkB mRNA do may not mediate the influence of testosterone on neurogenesis at adolescence in the mammalian hippocampus, BDNF and TrkB-TK+ mRNA levels do appear to be indirectly related to cell proliferation Figure . Our res


Transactive response DNA binding protein 43 (TDP-43) is detected in pathological inclusions in many cases of Alzheimer’s disease (AD) and mild cognitive impairment (MCI), but its pathological role in AD and MCI remains unknown. Recently, TDP-43 was reported to contribute to pathogenesis in amyotrophic lateral sclerosis through its interaction with p65 nuclear factor κB (NF-κB) resulting in abnormal hyperactivation of this signaling pathway in motor neurons. Hence, we investigated the interaction of TDP-43 with p65 in the temporal cortex of subjects with a clinical diagnosis of MCI (n = 12) or AD (n = 12) as well as of age-matched controls with no cognitive impairment .Immunoprecipitation and immunofluorescence approaches revealed a robust interaction of TDP-43 with p65 in the nucleus of temporal lobe neurons in four individuals with MCI (named MCI-p). These MCI-p cases exhibited high expression levels of soluble TDP-43, p65, phosphorylated p65 and low expression levels of β-amyloid 40 when compared to AD or NCI cases. The analysis of cognitive performance tests showed that MCI-p individuals presented intermediate deficits of global cognition and episodic memory between those of AD cases and of NCI cases and MCI cases with no interaction of TDP-43 with p65.From these results, we propose that enhanced NF-κB activation due to TDP-43 and p65 interaction may contribute to neuronal dysfunction in MCI individuals with episodic memory deficits. Accordingly, treatment with inhibitors of NF-κB activation may be considered for MCI individuals with episodic memory deficits. Transactive response DNA binding protein 43 (TDP-43) has been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusion (FTLD-U) -4. TDP-4Mild cognitive impairment (MCI) refers to a transitional state between normal cognition and early dementia, especially AD . MCI is Recent lines of evidence suggest that in ALS, soluble fractions of TDP-43 interacts with p65 subunit of nuclear factor κB (NF-κB) in the nucleus of neurons and glial cells, and that an upregulation of TDP-43 may contribute to pathogenesis by causing abnormal hyperactivation of p65 NF-κB . These fThe following antibodies were used in this study: anti-actin , anti-neuronal nuclear antigen (NeuN) , anti-p65 , anti-p65 , anti-phospho-p65 , anti-tau 13 , anti-tau CP-13 , anti-TDP-43 2E2-D3 , and anti-C-TDP-43 .Samples from the brain cortex were obtained from participants in the Religious Order Study, a longitudinal clinicopathology study of aging and dementia from which an extensive amount of clinical and neuropathology data were available . The stuAt death, each case was assigned a Braak score  based onFor quantification of TDP-43 and p65, postmortem frozen samples (~ 100 mg) from the temporal cortex from 36 study participants were homogenized in a Tris-buffered saline (TBS) consisting of 50 mM Tris-base, 138 mM NaCl, 2.7 mM KCl, protease inhibitor cocktail  and phosphatase inhibitor cocktail , and centrifuged at 20,800 g for 20 min at 4°C. After protein determination was performed by Bradford method , the supernatant was used as a sample (TBS-soluble fraction).For quantification of total tau and Aβ, postmortem samples from the temporal and inferior parietal cortex from the same 36 study volunteers were homogenized and centrifuged sequentially to generate a TBS-soluble protein fraction and a detergent-insoluble protein fraction (formic acid extract) as described . Tissue Aβ40 and β-Amyloid 42 (Aβ42) concentrations were measured using specific human Aβ ELISA kits  according to the manufacturer’s recommendations.TDP-43, p65 and tau were quantified using Western immunoblot. Proteins (15 μg/sample) were heated at 95°C for 5 minutes in SDS sample buffer. For immunoprecipitation study, anti-TDP-43 polyclonal  or anti-p65 polyclonal antibody  was bound to protein G-coated magnetic beads  and was incubated with 50 μg of lysate overnight at 4°C. After washing, immunoprecipitates were eluted with SDS sample buffer. Samples were resolved by 10% SDS-PAGE and transferred to a PVDF membrane . The membrane was incubated with anti-p65  or anti-TDP-43 2E2-D3 antibody , and western blot image was obtained using a chemiluminescence detection kit . Each protein was estimated by standardization with actin.Immunofluorescence labeling was performed on 6-μm-thick sections of paraffin-embedded temporal cortex samples from the brains. Before immunostaining, the sections were microwaved 2 × for 2 minutes each in 0.01 mol/L citrate buffer, pH 6.0, for antigen retrieval. Sections were incubated with anti-TDP-43 2E2-D3  and anti-p65 antibodies  and subsequently with corresponding Alexa 488 and 633 antibodies . The nuclei were counterstained with Dapi . Sections were observed by confocal laser microscopy . Autofluorescence, such as lipofuscin pigments, were detected using 575–630 nm bandpass emission filter.χ2 test or Kruskal-Wallis test, followed by Dunn multiple comparisons test. For comparisons between 2 groups, Mann–Whitney test was performed. Statistical analyses were done using GraphPad Prism 5 . Statistical significance was set at p < 0.05.Data are expressed as means ± SD. Statistical comparisons of data between groups were performed using the All subjects signed an informed consent and Anatomical Gift Act donating their brain for studies of aging and dementia. The study was approved by the Institutional Review Board of Rush University Medical Center.2O2[2test, the frequency of positive phospho-p65 were increased in MCI .TBS-soluble fraction of temporal cortex of subjects with NCI, MCI and AD was used for immunoblotting and coimmunoprecipitation experiments. Immunoblotting experiments revealed that the levels of both TDP-43 and p65 were elevated in 5 individuals with MCI , 2 individuals with AD (Subjects 2 and 13) and 4 individuals with NCI   displaying nuclear interaction between TDP-43 and p65 NF-κB in the neurons from the temporal lobe Figures  but not TDP-43 cytoplasmic inclusions, which consist mainly of TDP-43 C-terminal fragments of ~25kD, were first described as pathological hallmark of ALS and FTLD-U cases ,3. The CNeuropathology analysis in Table Our results showed the accumulation of p65 and the presence of phosphorylated p65 at ser536 in a subgroup of persons with MCI (MCI-p) Figure , Table 1We propose that enhanced NF-κB activation due to TDP-43 and p65 interaction may contribute to neuronal dysfunction in MCI individuals with episodic memory deficits. Accordingly, treatment with inhibitors of NF-κB activation may be considered for MCI individuals with episodic memory deficits before their progression to AD.The authors declare they have no competing interest.YO, JAS, DAB, FC and JPJ designed the study. YO and CT made the assessments. YO, CT, JAS, DAB, FC and JPJ drafted the manuscript. All authors contributed to the manuscript and approved the final manuscript.a-c Sections from the temporal cortex of MCI-p , AD without the interaction of TDP-43 with p65 in coimmunoprecipitation experiments  or NCI-p  were incubated with anti-TDP-43 and anti-p65 antibodies and subsequently with corresponding Alexa 488 and 633 antibodies, and imaged by confocal laser microscopy. Autofluorescence was detected using 575–630 nm bandpass emission filter. Arrows indicate the nuclear TDP-43 positive cells colocalized with p65 (a). Arrowheads indicate the nuclear TDP-43 positive cells without colocalization with p65 (a-c). Scale bars, 50 μm.TDP-43 colocalizes with p65 in the neuronal cells from the temporal cortex of individuals with MCI-p. Click here for fileSelected characteristics of subjects from the religious order study with a clinical diagnosis of Alzheimer’s disease.Click here for file


Spontaneous proliferation, a hallmark of Human-T Lymphocyte Virus Type 1 (HTLV-1) infection, is particularly higher in HAM/TSP patients compared to asymptomatic carriers. However the role of the proliferation in the pathogenesis of HAM/TSP is still unknown. The identification of drugs that modulate the spontaneous proliferation may be important for the treatment of HAM/TSP disease. We have evaluated the effect of three different quinoline compounds  on the modulation of spontaneous proliferation of peripheral blood mononuclear cells from HTLV-1 patients with HAM/TSP. The cells from 8 HAM/TSP patients were cultivated in the presence of serial concentrations of quinolone compounds. Cell proliferation was assessed by 3[H]thymidine incorporation. Cell viability was measured by optical density in the presence of MTT. The ultrastructure analysis was done using a transmission electron microscope. Quinoline compounds were not toxic at the concentrations evaluated. The IC50 was 18.5 µM for compound A, 30.5 µM for compound B and 3.4 µM for compound C. The three compounds inhibited more than 90% of spontaneous proliferation. Cultured cells in the presence of quinolone compounds showed vacuoles presented with myelin-like membranes, probably autophagic vacuole-like compartments, observed by electronic microscopy. In conclusion, the quinoline compounds showed no toxicity and were able to inhibit the spontaneous proliferation of T cells from HTLV-1-infected individuals. New assessments are being applied to understand how the quinoline compounds act on cells by decreasing cell proliferation.


A human IgG1 anti-PD-L1 antibody, MSB0010718C, induced ADCC activity in vitro against all three human bladder cancer cell lines following treatment with IFN-γ. In initial in vivo murine studies, the growth of s.c. MB49 tumors in syngeneic mice was significantly delayed following three i.p. injections of 400 µg of the MSB0010718C anti-PD-L1 antibody. An orthotopic bladder model consisting of the MB49 cells tagged with luciferase (MB49-luc) was also used to evaluate the antitumor efficacy of the anti-PD-L1 antibody. MB49-luc cells were instilled intravesically (bladder) in B6 mice. Beginning at 7 or 10 days post-instillation, three i.p. injections of the anti-PD-L1 antibody substantially reduced tumor volumes, as determined by intravital imaging, leading to long-term tumor-free survival for 40-60% of the treated mice. While initial immune cell subset depletion studies implicated both CD4+ and CD8+ T cells, continuing efforts will further define the cellular mechanisms responsible for the antitumor effects of the anti-PD-L1 antibody. Taken together, these results suggest that MSB0010718C therapy might be used to activate both innate and adaptive immune mechanisms to treat PD-L1-expressing bladder tumors. Furthermore, the MB49 tumor model can be used to evaluate the combined effects of anti-PD-L1 and other therapeutic agents, particularly ones that induce IFN-γ production and tumor PD-L1 up-regulation. MSB0010718C is currently being evaluated in a Phase I clinical trial (NCT01772004).Antibodies that block PD-1/PD-L1 interactions have shown efficacy against both lung and skin cancers in early-stage clinical trials, and may also be effective in other tumor types, particularly bladder tumors. PD-L1 expression has been previously reported to correlate with high-grade tumors, a high recurrence rate, and reduced survival rate in patients with bladder cancer. These findings and the high frequency of somatic mutations found in bladder tumors indicate that bladder cancer patients may respond well to anti-PD-L1 therapy. Both murine  and human  bladder cancer cell lines constitutively express PD-L1 as determined by flow cytometry. As expected,