Spatial and temporal patterns in microbial biodiversity across the Amazon river-ocean continuum were investigated along ∼675 km of the lower Amazon River mainstem, in the Tapajós River tributary, and in the plume and coastal ocean during low and high river discharge using amplicon sequencing of 16S rRNA genes in whole water and size-fractionated samples (0.2–2.0 μm and >2.0 μm). River communities varied among tributaries, but mainstem communities were spatially homogeneous and tracked seasonal changes in river discharge and co-varying factors. Co-occurrence network analysis identified strongly interconnected river assemblages during high (May) and low (December) discharge periods, and weakly interconnected transitional assemblages in September, suggesting that this system supports two seasonal microbial communities linked to river discharge. In contrast, plume communities showed little seasonal differences and instead varied spatially tracking salinity. However, salinity explained only a small fraction of community variability, and plume communities in blooms of diatom-diazotroph assemblages were strikingly different than those in other high salinity plume samples. This suggests that while salinity physically structures plumes through buoyancy and mixing, the composition of plume-specific communities is controlled by other factors including nutrients, phytoplankton community composition, and dissolved organic matter chemistry. Co-occurrence networks identified interconnected assemblages associated with the highly productive low salinity near-shore region, diatom-diazotroph blooms, and the plume edge region, and weakly interconnected assemblages in high salinity regions. This suggests that the plume supports a transitional community influenced by immigration of ocean bacteria from the plume edge, and by species sorting as these communities adapt to local environmental conditions. Few studies have explored patterns of microbial diversity in tropical rivers and coastal oceans. Comparison of Amazon continuum microbial communities to those from temperate and arctic systems suggest that river discharge and salinity are master variables structuring a range of environmental conditions that control bacterial communities across the river-ocean continuum. The phylogenetic composition of microbial communities in freshwater and marine water predicts, to some degree, the genomic potential of communities in each of these environments , and sha-1 of organic carbon to the Atlantic Ocean , early rising (December), and maximum (May) river discharge periods . AdditioFigure 1 and Supplemental Table Amazon River samples were collected on three cruises  from five stations: an upriver station (Óbidos), a major lower Amazon tributary river station (Tapajós), and near the mouth  using methods described in Figure 1 and Supplemental Table Water samples from the Amazon River plume were collected from the R/V Knorr on May 23 to June 21, 2010, and from the R/V Melville on September 5 to October 6, 2011  and un-screened (plume) water samples were collected on 0.2 μm Sterivex filters , and flooded with RNAlater preservative . Cells were also partitioned into two size fractions by sequential filtration through 2.0 μm pore-size, 142 mm diameter polycarbonate (PCTE) membrane filters  and 0.22 μm pore-size, 142 mm diameter Supor membrane filters . These filters were immediately submerged in RNAlater in sterile 50 mL tubes. Filtration and stabilization of all samples was completed within 30 min of water collection. Following RNAlater addition, all filters were incubated at room temperature overnight, frozen, dry-shipped in liquid nitrogen dewars, and stored at -80I°C until extraction. River samples were initially stored at -20°C before shipping to the United States.4+, NO3-, NO2-, PO4-, and Cl-) were determined by flow injection analysis using a Foss-Tecator FiaStar 5000A FIA Analyzer with samples that were filtered through Whatman cellulose acetate filters (0.45 μm pore-size) into acid-washed 60 mL high-density polyethylene bottles, preserved with thymol, and frozen at -20°C until analysis. Major ions  were measured in the same samples using a Horiba-Jobyn Ivon UltimaPro inductively coupled plasma-optical emission spectroscope. Dissolved inorganic carbon (DIC) concentrations were measured on a Shimadzu total carbon analyzer (Model TOC-VCPH) using water samples filtered through 0.47 μm cellulose acetate membrane filters (Millipore) into acid-washed 60 mL HDPE bottles with no headspace to avoid degassing and preserved with thymol (100 mg/1000 ml of solution). DIC samples were analyzed for total organic carbon before and after acidification with 6 N HCl and sparging to remove gas; DIC was calculated as the difference between non-acidified and acidified/sparged samples. Finally, river dissolved CO2 concentrations were calculated based on DIC, pH, and temperature measurements.River discharge, dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were determined as described in 3, NO3+NO2, and PO4) were described in Plume measurements of salinity, temperature, fluorescence, and nutrients , 1.5 M NaCl, 5% CTAB) (2O) in 50 mL conical tubes. Dislodged cells in RNAlater and rinse solutions were collected on Sterivex filters, triple-rinsed with sterile solution (PBS or H2O), processed as above, added to the 142 mm filter pieces, and submerged in 7–9 mL DEB. Thermus thermophilus strain HB8 (ATCC) genomic DNA was added to each sample once the filter was placed in DEB to serve as an internal standard to account for variable extraction efficiency  . Each 14ficiency . T. therome size . DNA wasTC AGRGTTTGATYMTGGCTCAG) and 338R with 454A linker and unique 8 base pair barcode denoted as ‘N’ (GCCTCCCTCGCGCCATCAG NNNNNNN CA TGCWGCCWCCCGTAGGWGT) , chloroplast, mitochondria, Archaea, and unclassified were removed and the remaining OTUs were rarified to 887 sequences per sample. Alpha-diversity was estimated using Catchall  shared computational cloud at the University of Maryland School of Medicine Institute for Genome Sciences (IGS) following Catchall , which cCatchall  and weigCatchall . Similar ANOSIM; . OTUs th ANOSIM;  and indv ANOSIM;  in R.Z-scores and analyzed using BV-STEP and BIO-ENV  within plume and river samples were computed using the CoNet (v1.1.1.beta) plugin within Cytoscape (v3.4.0)escribed . Two seps method  and falss method . Only 102 was highest in the mainstem, decreasing slightly from Óbidos to Macapá. Dissolved oxygen was lower in the turbid mainstem, with lowest values at Óbidos increasing toward the mouth at Macapá and Belém. The highest values were observed in the Tapajós where oxygen saturation approached 100% in September. DIN, nitrate, and ammonium concentrations were highly variable and followed no clear trends, but nitrite was positively related to river discharge. DOC and DON peaked in May in the Tapajós River and at Óbidos, but were otherwise similar across all stations and sampling dates.Amazon river biogeochemistry varied by season and by sampling site . Chlorophyll a, fluorescence, POC, and PN peaked in low salinity and DDA samples.Amazon River plume biogeochemistry varied as a function of river dilution, reflected in salinity, and was similar between the high river discharge in May 2010 and low discharge in September 2011 , and, among plume samples, was between size fraction (>2.0 μm and 0.2–2.0 μm)  , with the exception of low salinity station 10 in 2010  where the >2.0 μm fraction was similar to the 0.2–2.0 μm fraction. Communities in unfractionated “whole” water samples grouped with the 0.2–2.0 μm fractions . In contrast, there was no significant difference between the size fractions among river samples .The greatest variability in bacterial community composition was between river and plume  with overlapping salinity ranges: near-shore low salinity plume , off-shore high salinity plume , DDA blooms , and the plume edge  matching those in the 0.2–2.0 μm fraction . There was no clear seasonal change in plume communities except that the DDA assemblage was only observed in May/June 2010.Plume communities in the 0.2–2.0 μm fraction clustered into four groups  . Communities in the Tapajós River and at some Belém stations were distinct from the mainstem stations , and also differed between dates . We investigated cross-channel variability at mainstem stations by sampling the left, middle, and right side of the channel, and found no significant cross-channel variability at Óbidos and Macapá. At Belém, communities at the center and right stations were different than those on the left side of the channel in May 2011 , likely due to mixing with Tocantins River communities.River communities grouped primarily by sampling date  was significantly greater in the river than in the plume  among which conductivity (ρ = 0.553) and pH (ρ = 0.566) were the highest ranked. CCA analysis showed that conductivity and pH explain 34.2% of the variability. For mainstem river communities  the best model included six factors, with discharge (ρ = 0.906) and pH (ρ = 0.748) explaining 33.9% of variability. For tributary communities (Tapajós and Belém), the best model included 17 factors, with Cl- (ρ = 0.710) and Si (ρ = 0.678) explaining 66.2% of variability. For Tapajós communities alone, the best model included three factors, with discharge (ρ = 0.785) and Si (ρ = 0.777) explaining 60.4% of variability.The best model explaining variability in river communities included eight environmental factors (Table 2) likely because of the difference between the two size fractions. For the 0.2–2.0 μm size fraction and unfractionated samples the best model included four factors, with salinity (ρ = 0.749) and bacterial production (ρ = 0.790) explaining 13.6% of variability, based on CCA. For communities in the >2.0 μm size fraction the best model included two factors, with fluorescence (ρ = 0.494) and chlorophyll-a (ρ = 0.486) explaining 11.3% of the variability (Table 2). Variability in plume bacterial communities was better explained by environmental factors when major groupings of communities were analyzed separately (Table 2).The best model explaining variability in plume communities included five environmental factors, but the correlation was not strong  reflected this shift . Seasonal shifts were also observed for freshwater relatives of the Alphaproteobacteria SAR11, and Cyanobacteria (mainly Synechococcus and Merismopedia), both of which had highest proportions during low discharge . Cyanobacteria peaked in abundance in tributaries in the Tapajós and the Belém center and right bank stations  , and were among the indicator taxa for tributaries in September and December. In the mainstem river, indicator taxa accounted for large proportions of sequences in December (7%) and May (9%), but there were very few significant indicator taxa for September, representing only 1.9% of sequences , and Betaproteobacteria shifted seasonally, peaking during high discharge in May . Freshwater taxa were almost entirely absent from plume samples, averaging 0.001% of communities in samples with salinity <30, with the exception of one Actinobacteria OTU that was most abundant in both freshwater and high salinity plume samples and therefore likely represents closely related freshwater and marine taxa. The low salinity plume community was dominated by SAR11, Flavobacteriia, Gammaproteobacteria, and Synechococcus, and some samples included Sphingobacteriia and Firmicutes. High salinity plume communities were dominated by Synechococcus and SAR11, and many of the dominant low salinity taxa were rare or absent. As sample salinity increased toward the plume edge, the proportion of Synechococcus declined and the proportion of Prochlorococcus increased . Indicator taxa reflected this taxonomic shift between the low salinity plume and the plume edge . High salinity plume had few indicator taxa other than one very abundant Synechococcus in the 0.2–2.0 μm fraction and several Sphingobacteriia in the >2.0 μm size fraction. Plume edge indicator taxa were dominated by Prochlorococcus and Alphaproteobacteria  in the 0.2–2.0 μm fraction and an abundant Gammaproteobacteria in the >2.0 μm size fraction. In DDA communities, the proportion of Verrucomicrobia and Flavobacteriia was elevated and Synechococcus and Prochlorococcus were reduced . In the >2.0 μm fraction, DDA communities were very similar to high salinity communities, with the addition of the same Verrucomicrobia found in the 0.2–2.0 μm size fraction, and the Cyanobacteria Richelia, which is a diazotroph symbiont of DDA . In the river network, indicator taxa within each indicator group showed strong co-occurrence (266 positive edges and 0 negative edge) , and were much more highly connected for the May and December mainstem than the September mainstem. Indicator taxa in the May and December mainstem groups were also negatively correlated to each other, while September mainstem indicator taxa showed few correlations with themselves or with other indicator groups . Indicator taxa assigned in the May and September-December tributary groups showed mutual exclusion between groups (13 positive edges and 59 negative edges) and with mainstem groups (97 positive edges and 354 negative edges). This mutual exclusion from mainstem groups was stronger for indicator taxa in the September-December tributary group (250 positive edges and 53 negative edges) than the May tributary group (44 positive edges and 104 negative edges) .Indicator taxa for river and plume groups accounted for most of the taxa in co-occurrence networks , but these taxa varied greatly in the degree to which they showed co-occurrence (positive correlations) and mutual exclusion (negative correlations) . Indicator taxa in the low salinity group showed strong mutual exclusion from other indicator groups (85 positive edges and 652 negative edges), with the most mutual exclusion detected with plume edge indicator taxa (46 positive edges and 466 negative edges), and DDA indicator taxa (11 positive edges and 165 negative edges). In contrast, high salinity plume indicators were not strongly correlated with themselves or with taxa in any other group (67 positive edges and 58 negative edges). Indicator taxa in the DDA and plume edge groups showed the strongest co-occurrence between any of the plume indicator groups (141 positive edges and 8 negative edges) .Similar to the river network, indicator taxa in the plume network showed a high degree co-occurrence within the plume edge, low salinity, and, to a lesser extent, DDA groups (Table 2), and plume communities were strikingly different in DDA bloom samples than in other high salinity plume samples. These results suggest that although salinity provides physical structure to plume ecosystems, the composition of communities is driven by other environmental and biotic factors including nutrients, DOC chemistry, and phytoplankton community composition . Across all these systems, river discharge appears to be the master variable controlling seasonal changes in microbial communities. Discharge influences the environmental conditions that drive species sorting in microbial communities by controlling the flux of materials  and organisms from land and tributaries, and the water residence time available for species sorting to occur. Discharge also influences turbidity and the magnitude of solar insolation, and thus water temperature and phytoplankton production, both of which are potential controls on seasonal patterns in microbial community composition.Amazon River bacterial communities varied seasonally, tracking changes in river discharge and associated environmental conditions. Seasonality is typical of bacterioplankton communities , and hasa . In the The strongest signal of seasonality in river microbial communities was a shift from a high proportion of Betaproteobacteria during high discharge (May) to high proportions of Actinobacteria, Cyanobacteria, and freshwater SAR11 Alphaproteobacteria during low discharge (September and December). A similar pattern was seen in the Columbia River, with more Betaproteobacteria during the spring freshet, and more Actinobacteria and freshwater SAR11 during summer and fall . A studyIn-stream water residence time is negatively related to discharge in rivers , and in Figure 2). This contrasts with the Columbia River plume, where seasonality in microbial communities corresponded to seasonal changes in coastal upwelling conditions and to mixing of seasonally varying river communities , which show that chlorophyll fluorescence and several other factors contribute significantly to models of bacterial community composition within most regions of the plume.A number of studies show changing community composition along salinity gradients in estuarine and coastal environments . In the 43-), but also correlated with several other factors. This community had the highest proportions of Bacteroidetes (Flavobacteriia and Sphingobacteriia) and Gammaproteobacteria, and a recent study demonstrated high gene expression by these taxa in several of these samples , consistent with a parallel study of phytoplankton community composition , which are typically associated with oligotrophic conditions  mostly from the family Puniceicoccaceae. Verrucomicrobia are ubiquitous in the world’s oceans, averaging 1.8% of sequences in one global survey, and are more abundant in coastal waters . Relativ diatoms , suggest diatoms , particuParticle-attached bacteria are often different than free-living bacteria in the plankton of lakes , rivers Synechococcus, Prochlorococcus, and SAR11 Alphaproteobacteria, and elevated proportions of Gammaproteobacteria, Bacteroidetes, and Planctomycetes in the >0.2 μm size fractions. At low salinity station 10, sampled in June 2010, the gene expression patterns of particle-attached microbes were strikingly different than those of free-living bacteria, with elevated expression of genes for sulfur cycling, aromatic compound degradation and synthesis of vitamins , suggesting that these taxa are the strongest indicators of high discharge (May) and low discharge (December) conditions in the Amazon River. In contrast, September mainstem indicator taxa showed few correlations (positive or negative), suggesting that September indicator taxa represent a transitional community between the presumably stable communities that form during high and low discharge. Similarly, tributary indicator taxa showed strong co-occurrence within seasonal indicator groups and mutual exclusion between groups, demonstrating seasonality in tributary communities. Correlations between mainstem and tributary indicators were strongly negative during December but not during May, suggesting that tributary communities are more highly represented in the mainstem river during the high discharge season.We combined the two independent analysis techniques of co-occurrence networks and indicator analysis to differentiate between stable and transitional microbial communities, and to infer potential microbial interactions across the Amazon River continuum. Indicator taxa in the river network co-occurred strongly within the May and December mainstem indicator groups, and these groups were strongly negatively correlated to each other . In contrast, high salinity plume indicators showed few correlations, suggesting that the high salinity plume contains a transitional community between the low salinity plume and open ocean. DDA indicators were positively correlated with themselves and with plume edge indicators, and negatively correlated with low salinity indicators  suggesting that DDA communities develop from open ocean communities following DDA bloom development, and are very different than the communities that develop associated with non-DDA phytoplankton blooms at lower salinities. These results suggest that river plumes host transitional mixtures of organisms that grow into more stable communities under elevated growth conditions , and that the composition of these communities depends not on salinity but instead on factors that drive those high growth conditions.Plume indicators co-occurred strongly within the low salinity and plume edge groups, and these groups were strongly negatively correlated . Bacteroidetes were detected in the tropical Paraná River, but these taxa did not include Flavobacteriia  . Marine bacterial taxa exhibit latitudinal ranges (Amazon plume communities were different than Columbia plume and Bering Strait communities, and again one of the most important differences was low Flavobacteriia in the Amazon, along with a higher proportion of Cyanobacteria and Deltaproteobacteria (l ranges , and onel ranges . Howeverl ranges , and Flal ranges . In factl ranges , suggestThis cross-system comparison suggests globally consistent patterns and controls on the composition of microbial communities across river-ocean continuums. At the phylum/class level, river microbial communities were fairly similar and appeared to vary with river turbidity, supporting the idea that river microbial community composition depends on the ratio of allochthonous vs. autochthonous DOM. Plume communities tracked salinity in both the Amazon and Columbia plumes, and both systems were strongly influenced by mixing with coastal ocean communities, which differed greatly in the two regions. However, productive regions of both systems developed abundant populations of Bacteroidetes, including Flavobacteriia, suggesting that although plume communities are structured by salinity through buoyancy and mixing, the composition of plume-specific communities is controlled by factors unique to productive regions including nutrients, phytoplankton community composition, and DOM chemistry.MD for laboratory analysis, data analysis, intellectual contributions, and manuscript preparation. PY for project leadership, intellectual contributions, sample collection, and laboratory analysis. MM for project leadership, intellectual contributions, and manuscript preparation. VC for sample collection, laboratory analysis, and intellectual contributions. CF for sample collection, laboratory analysis, intellectual contributions, and manuscript preparation. AK for project leadership, sample collection, and intellectual contributions. PM for laboratory analysis, intellectual contributions, and manuscript preparation. JP for data analysis, intellectual contributions, and manuscript preparation. JR for project leadership, sample collection, and intellectual contributions. BS for sample collection, intellectual contributions, and manuscript preparation. HS for sample collection, laboratory analysis, and manuscript preparation. NW for sample collection, laboratory analysis, intellectual contributions, and manuscript preparation. BC for project leadership, intellectual contributions, data analysis, and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and > 95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation   •Insoluble phosphorylated TDP-43 from ALS patient brain and spinal cord can seed into cell culture to form TDP-43 inclusions.•These inclusions represent the full morphological spectrum of TDP-43 neuronal cytoplasmic inclusions observed in ALS.•TDP-43 inclusions can be serially propagated resulting in an increase in phosphorylated TDP-43 pathology.•TDP-43 aggregates can spread from cell to cell in a prion-like manner.•TDP-43 can be seeded to motor neuron-like cells producing both TDP-43 oligomers and reduced cell viability. This results in muscle spasticity, weakness, atrophy and eventual paralysis, where death is usually caused by degeneration of motor neurons innervating respiratory musculature . AccumulThe prion diseases are the prototypic protein misfolding disorders characterised by the accumulation of self propagating polymers of misfolded PrP. Cell to cell propagation of these misfolded protein polymers culminates in dissemination throughout the central nervous system (CNS). Different misfolded PrP subtypes, referred to as strains , can leaThe pathological hallmark of ALS is the deposition of ubiquitinated aggregated proteins which, in > 95% of all cases, contain TDP-43 . TDP-43 via a proposed templated seeding reaction in cell culture , the London Neurodegenerative Diseases Brain Bank  and the Queen Square Brain Bank for Neurological Disorders . All tissue samples were obtained with informed consent and under Research Ethics Commitee and Human Tissue Authority (HTA) regulations. ALS cases were clinically, electrophysiologically and pathologically definite cases. All brains were pathologically confirmed to have TDP-43 positive neuronal cytoplasmic inclusions. g, 30 min, 4 °C). The supernatant was discarded, and the pellet was briefly sonicated in the starting volume (500 μl) of 1 × BA containing 1% sarkosyl  with 1 × protease and phosphatase inhibitors, then incubated at 37 °C for 30 min. The sample produced was ultracentrifuged  and the supernatant was discarded. The pellet was briefly sonicated in 200 μl of 1 x BA containing 1% CHAPS  with the protease and phosphatase inhibitors, and ultracentrifuged for 20 min at 25 °C. The supernatant was removed and discarded and the pellet was resuspended 50 μl of 8 M urea (pH 7.5).To prepare phosphate-buffered saline (PBS) tissue extractions, each block of snap-frozen tissue was homogenised in 9 x tissue wet weight of PBS using a probe sonicator and 750ul aliquots were frozen at − 80 °C. For sarkosyl insoluble urea soluble fractions, CNS tissue was homogenised and extracted as described previously  but with g, 10 min). The supernatant was ultracentrifuged . The pellet was washed in sterile PBS, sonicated and re-ultracentrifuged . The final pellet was resuspended by sonication in 25 μl of sterile PBS per pellet. In order to achieve workable amounts of TDP-43 seeding, 4 to 6 aliquots of homogenate were extracted per CNS region.For the TDP-43 seeding the homogenate was extracted using the tissue homogenisation buffer (HB) made at 2 × concentration  containing a 2% sarkosyl detergent and protease and phosphatase inhibitors. Then 750 μl of this buffer was added to the homogenates to make a final of 1 × HB with 1% sarkosyl. These samples were incubated at 37 °C for 30 min and centrifuged  and 5 μl–10 μl of ligation reaction (i.e. 10% of the cell volume) were mixed and incubated on ice for 15 min then “heat shocked” for 30 s in a 42 °C heating block and returned to the ice for 10 min. After the addition of 500 μl–1 ml of LB-broth (for low or high copy number vectors respectively), the cells were incubated at 37 °C for 1 h with horizontal agitation at 150 rpm. Gentle centrifugation at 3000 × g formed a cell pellet which, after removal of the supernatant, was re-suspended in 100 μl of LB-broth. The full cell suspension was spread on an LB-agar plate containing an appropriate selection antibiotic and incubated overnight at 37 °C.The wild type FLAG tagged TDP-43 gene was previously cloned into the pFLAG-CMV2 vector which was a kind gift from Burratti and Barralle . The ful g. Cloned plasmid DNA was extracted from the bacterial cells using the QIAquick Spin Miniprep kit  according to the manufacturer's protocol. For larger quantities of DNA the bacteria were grown in 250 ml of LB broth and incubated overnight at 37 °C with agitation at 250 rpm. After this, the QIAGEN maxi prep kit  was used to extract and purify the DNA used for transfection according to the manufacturer's protocol. Final confirmation of cloned plasmid constructs was achieved by sequencing. All DNA sequencing was outsourced to a service provider, Source BioScience Ltd.Single, well-defined colonies were individually picked and cultured in 3 ml of L-broth containing the selection antibiotic. Following overnight incubation at 37 °C with vigorous horizontal agitation at 250 rpm, the cultured cells were harvested by centrifugation at 17,000 ×2.32 up to 80–90% confluency then were passaged at 1 in 10 and grown for up to a week where the medium was changed every 2–3 days.Human embryonic kidney (HEK) 293 and Neuroblastoma spinal cord 34 (NSC-34) cells were cultured in DMEM with high glucose and 5 mM Glutamine containing 10% FBS. Cells were grown at 37 °C and 5% CO2.4Cells were either grown in 24 well plates for IF staining or 6 well plates for western blotting. In each case transfection of plasmids was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. In brief, we used DMEM with no FBS as transfection media and added 0.75 μl of lipofectamine and 1 μl of P3000 reagent with 0.5 μg of plasmid DNA per 1 well of a 24 well plate, and 3.75 μl of lipofectamine with 5 μl of P3000 reagent and 2.5 μg of plasmid DNA per 1 well of a 6 well plate. g for 20 min at room temperature. The supernatant was used as the sarkosyl soluble (SS) fraction and the pellet was resuspended by sonication in either 25 μl of 8 M urea per 1 well of a 6 well plate for western blotting, or 25 μl of sterile PBS per 1 well of a 6 well plate for serial propagation. A BCA assay was performed on both fractions, and 10–20 μg of protein was loaded on a gel for blotting and 5 μg of protein from the PBS suspended pellet was transfected to the next culture.To inoculate the insoluble TDP-43 protein seed from the ALS CNS homogenates, the transfection process was the same as above but with 5 μg of protein in replacement of the DNA. Both plasmids and TDP-43 seeds were co-transfected and were incubated for a maximum of 3 days post transfection. For IF analysis the cells were fixed in 4% PFA. For western blotting and serial propagation the cells were harvested by trypsinising in 0.25% Trypsin with EDTA and centrifuged at 2000 rpm for 5 min. The media was discarded and the pellet was resuspended and sonicated in 50 μl of TS buffer  containing 1% sarkosyl and protease and phosphatase inhibitors. The samples were sonicated and incubated at 37 °C for 30 min. The samples were ultra-centrifuged at 290,000 ×2.5For serial propagation 5 μg of the sterile PBS fractions of the treated cells were co transfected with the FL WT TDP-43 plasmid and incubated for 3 days and harvested as before where half of the pellet was used for western blotting and the other half was resuspended in sterile PBS for further propagation.For aggregate spreading, HEK cells were grown to ~ 60–70% confluency in 24 well plates and either seeded with the FL WT and ALS inocula or transfected with GFP. After 3 days of incubation both wells were trypsinised in 0.25% trypsin for 5 min and spun down at 2000 rpm for 5 min. Both populations of cells were mixed together in a 1:1 ratio and plated onto a 22 mm diameter coverslip and incubated for a further 3 days. Cells were fixed in 4% paraformaldehye (PFA) for immunocytochemical analysis.2.6anti-TDP-43 (1:500) , monoclonal mouse anti-TDP-43 (1:500) , polyclonal anti-rabbit phospho-TDP-43 (pS409/410-1) (1:500) , polyclonal anti-rabbit TDP-O (1:500) (a gift from Dr. Yun Ru Chen), monoclonal anti-mouse FLAG M2  in 5% BSA for 2 h at room temperature. Cells were gently washed in PBS 3 times and incubated with fluorescent secondary anti-rabbit Alexa-Fluor 488 and anti-mouse or anti-rabbit Alexa-Fluor 568 antibodies (1:2000) (Invitrogen) in 5% BSA for 1 h and washed again as before. They were mounted with DAPI Fluoromount G onto slides, allowed to dry and visualised with a Zeiss 700 confocal microscope.Cells were gently washed with PBS and fixed for 15 min in 4% PFA then gently washed again in PBS and permeabilised with 0.5% triton X-100 in PBS for 10–15 min. The coverslips were blocked in 5% BSA for 30 min and stained with either polyclonal rabbit 2.7Samples were measured for protein concentration using a BCA assay (Bio—Rad) and either 10 or 20 μg of protein was loaded on to a gel. These samples were added to an equal amount of working SDS solution with β-mercaptoethanol and boiled at 100 °C for 5 min. The samples were vortexed and centrifuged at 14,800 rpm for 1 min. Equal amounts of protein from each sample were loaded on to a 15 well 16% tris glycine SDS PAGE gels  and run at 200 V for 70 min. Gels were transferred and blotted onto nitrocellulose membranes  for either 2 h at 35 V or 15 V at 4 °C overnight. Membranes were then blocked in 5% non-fat milk in PBS for 30 min and probed with either anti-rabbit polyclonal pTDP-43 (pS409/410)  (1:1000), monoclonal anti-mouse FLAG M2  or mouse monoclonal β-actin antibody Clone AC-15  , in 1% non-fat milk in PBS + 0.1% tween (PBST). Blots were washed 3 times for 5 min each in PBST and probed with secondary fluorescent antibodies: goat anti rabbit IgG antibody IRDye800 (green)  , Alexa Fluor 700 goat anti mouse IgG (red)   in 1% Non-fat milk in PBST. Blots were washed again 3 times for 5 min in PBST and washed once in PBS, then visualised with an Odyssey scanner.2.8For the assessment of cellular viability the 3--2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay was performed using MTT salt  resuspended at 1 mg/ml in sterile water. Cells were plated out in 96 well plates at 500 cells per well and co-transfected as previously described, then on day 3 20 μl of the 1 mg/ml MTT solution was added to each well and left to incubate at 37 °C for 2–4 h The media from each well was decanted and replaced with 100 μl of dimethyl sulfoxide (DMSO) until all the purple formazan product was dissolved and the plate was read at 570 nm. Absorbance readings were taken as a percentage of the mean value of non-treated cells.2.9t-test was used when comparing between two individual groups cases to generate a statistical p value. A one way ANOVA was used when comparing 2 or more groups with a post hoc Tukey test to compare all groups. Any p value below 0.05 was considered to be statistically significant .Densitometry analysis was performed using the raw data on the inbuilt Odyssey densitometry software and statistical analysis was carried out using Prism 4 software. An unpaired two tailed student's 33.1p < 0.001, n = 3) compared to the cells expressing the FL WT and FL WT seeded with control inocula. However, whilst the signal from the ALS spinal cord (SC) demonstrates an increase, this does not reach significance here  were able to seed TDP-43 aggregation. Therefore, samples exhibiting robust phosphorylated TDP-43 (pS409/410 or ‘pTDP-43’) pathology were selected for further experiments . In ordence here C. In thince here E. This ince here .de novo propagation, and not persistence of the pTDP-43 from the transfected ALS inocula, a time-course seeding experiment was conducted over 3 days  and day 2 and 3   E. This iregation C. Furtheregation C, and thp < 0.001).Noting that the TDP-43 seeding reaction is more efficient in cells expressing the full length TDP-43 wild type construct, we hypothesized that TDP-43 overexpression in these cells increases reaction efficiency (by increasing the substrate concentration). We measured the levels of sarkosyl soluble (SS) and insoluble (SI) TDP-43 on blots with densitometry, and confirmed a significantly increased levels of total TDP-43 in both SS and SI fractions in cells expressing the FL WT plasmid (‘+’) compared to the cells without the plasmid (‘–‘) , skein, dash, dot, and round inclusions  Fig. 2AAFig. 2A.onstruct N. These onstruct B and S1AAdditionally, the presence of diffuse cytoplasmic staining in the form of granular ‘pre-inclusions’ B suggest3.3in vitro and in vivo to maintain the strain specific conformational protein structures . These cells are immortalized motor neurons that express many physiological characteristics of primary motor neurons . In addip < 0.05) and spinal cord (**p < 0.01) compared to cells treated with TDP-43 plasmid alone and TDP-43 plasmid plus control -2,5-diphenyltetrazolium bromide (MTT) assay to assess cellular viability. We treated the NSC-34 cells with a control spinal cord sample (NC SC) and separate CNS regions that we had previously demonstrated significant seeding capacity, which were the ALS brain (TCX) and spinal cord (SC) samples. The MTT assay revealed no reduction in cell viability in cells treated with the ALS brain and spinal cord extracts alone in comparison the control brain extract B. Howeve control B. This wIncreasing evidence now implicates oligomeric species of aggregated proteins as the neurotoxic component in a number of neurodegenerative diseases . Indeed,4in vitro  suggesting that this seeding process is accelerated upon passaging. The increased accumulation of aggregated TDP-43 on serial passage may suggest more efficient propagation on passage and raises the possibility of the existence of strains of pathological TDP-43; a phenomena first demonstrated in the prion diseases, but more recently also demonstrated with alpha synuclein . We report the successful demonstration of TDP-43 pathology in the NSC-34 cell line expressing the TDP-43 plasmid and following exposure to ALS CNS extract. We also demonstrate that TDP-43 seeding can decrease the viability of these NSC-34 cells. As this decrease in cellular viability only occurs with the TDP-43 plasmid and ALS CNS extract exposure, it is highly probable that TDP-43 is the responsible species.It is still unclear if or how protein misfolding and aggregation is toxic to cells. One hypothesis is the formation of oligomeric aggregation intermediates. A number of neurodegenerative disease related proteins now demonstrate that the oligomeric species of misfolded aggregated proteins may be responsible for neurodegeneration in a number of disorders . Indeed,5in vitro directly from both ALS brain and spinal cord extracts. The morphology of the pathological TDP-43 inclusions is highly reminiscent of the TDP-43 pathology seen in the ALS patients, and all different morphologies of inclusions can be observed. We have also demonstrated that TDP-43 can propagate from cell to cell in co-culture experiments and can be serially passaged from cell extracts containing pTDP-43 aggregates. The increased accumulation of aggregated TDP-43 on serial passage may suggest more efficient propagation, and raises the possibility of the existence of strains of pathological TDP-43. Finally, we are able to induce this TDP-43 pathology in a pathologically relevant motor neuron-like cell line (NSC-34) and demonstrate decreased cellular viability and the formation of oligomeric species of TDP-43. These data further support the prion-like nature of TDP-43, confirming and extending accumulating evidence for the propagative nature of ALS. Indeed, our experimental paradigm could be used in in vitro assays to explore these prion-like mechanisms further and to screen for therapies to inhibit TDP-43 seeding, propagation and toxicity in order to potentially ameliorate this devastating condition.Our findings here demonstrate that TDP-43 can seed the formation of TDP-43 aggregates The following are the supplementary data related to this article.


Interlinking of two terpyridine ligands results in mononuclear chiral metal complexes (Fe and Ru) which were separated into their enantiomers. ii) bis(terpyridine) cage complexes 2-cFe(L1) and 2-cRu(L1) are described. The extraordinary design of the precursors 2Fe(L1) and 2Ru(L1) allows perfect preorganization for the final closing step. Due to the rigidity of the spacers between the two terpyridine moieties, the two isolated enantiomers barely racemize at room temperature in solution. The stable and axially chiral bis(terpyridine) Fe(ii) and Ru(ii) complexes were fully characterized by NMR-spectroscopy, UV-Vis spectroscopy, electrochemical measurements, high resolution mass spectrometry, circular dichroism measurements, and X-ray structural analysis.The syntheses of novel chiral M( To satisfy the octahedral coordination sphere of the central M(ii) ion, the three ligands wrap around the central metal in a helical arrangement, resulting in a spatial arrangement resembling a ship's propeller. The two enantiomers are distinguished by the helicity of the arrangement between the left-handed (Λ) and the right-handed (Δ) isomer (ii) ion. The two ligands are arranged perpendicular to each other and the helical chirality is lost. Attempts to maintain chirality in terpyridine complexes range from the attachment of chiral groupset al. which forms a dimetallic helical structure upon coordination of two Cu(ii) ionset al. which form chiral figure-of-8 shaped Fe(ii) complexes.Since the pioneering work of Louis Pasteur,) isomer .4 The heortho-ethynylphenyl moieties in 4 and 4′′ positions allowing for macrocyclization by Glaser-Hay type oxidative acetylene coupling chemistry. According to simple MM2 based molecular models, we hypothesized that there should exist rotamers of the metal complex having the ethynyl groups of neighboring ligands in close proximity favoring intramolecular coupling over intermolecular oligomerization.Here we present our new strategy to introduce axial chirality into metal terpyridine complexes, namely by twofold interlinking of the ligands surrounding the metal ion. The concept is sketched in While oxidative acetylene protocols have been used extensively for macrocyclization2-cFe(L1) and 2-cRu(L1) 2-c , and the2-cFe(L1) and 2-cRu(L1) are displayed in 1 was assembled following a published protocolSuzuki-type coupling reactions has already been reported.1 and boronic acid 2 gave the ligand L2 exposing two tris-isopropylsilyl (TIPS) protected ethynyl groups. Unfortunately, we were not able to find reaction conditions guaranteeing full conversion and thus, the target ligand L2 could only be separated from a monosubstituted side-product by automated size exclusion chromatography. For the assembly of the Fe(ii) complex the two ethynyl groups were first liberated by treatment with tetrabutylammoniumfluoride (TBAF) giving the bis-ethynyl functionalized ligand L1. In our first attempt the iron complex was synthesized with the TIPS masked ligand L2, but this strategy was revised quickly as the 2Fe(L2) complex was unstable when exposed to fluoride ions. The homoleptic 2Fe(L1) complex was obtained by the addition of FeCl2 to a solution of L1 in a mixture of methanol (MeOH) and dichloromethane (DCM). The instantaneous color change to deep purple indicated the complexation and after the removal of the solvents and the addition of water, the complex 2Fe(L1) was isolated in good yield (95%) upon precipitation with an excess of PF6– ions.The syntheses of the caged complexes 2Fe(L1) exposing four ethynyl groups in hand, its behavior under acetylene coupling conditions moved into the focus of interest. Thus the compound was exposed to commonly used Hay conditions.m/z = 459.2 was observed by ESI-MS corresponding to the signal of the doubly charged cation of the macrocyclic complex 2-cFe(L1) and the signal of the cation of the open precursor complex 2Fe(L1) at m/z = 461.2 was no longer present. The reaction mixture was worked up and the isolated purple solid was repeatedly washed with hexane. The completeness of the macrocyclization by oxidative acetylene coupling was displayed by the comparison of the crude 1H-NMR spectra of 2-cFe(L1) and its precursor 2Fe(L1). As displayed in 2Fe(L1) into diacetylenes in 2-cFe(L1).With the iron complex 2Ru(L1) failed, probably due to competing side reactions of the ethynyl groups at the elevated temperature required for the complexation. Using the more stable TIPS-protected ligand L2 the corresponding ruthenium complex 2Ru(L2) was obtained according to a reported procedure3 with AgBF4 in refluxing dimethylformamide (DMF) before L2 was added and the reaction mixture was refluxed for 3.5 days. Precipitation from aqueous NH4PF6 and excessive purification by column chromatography (CC) provided 2Ru(L2) in moderate yield (31%) as a red solid. The increased stability of the ruthenium complex allowed for quantitative deprotection of the ethynyl groups with TBAF in DCM at room temperature providing the complex 2Ru(L1). Applying similar macrocyclization conditions to those for 2Fe(L1) provided macrocyclized 2-cRu(L1) in an even better isolated yield of 96% as a red solid.A similar synthetic strategy for the ruthenium complex The importance of the preorganization of both ligands for the macrocyclization became obvious when high-dilution strategies to assemble the macrocycle failed  and 2-cRu(L1) were corroborated by their solid state structures. Single crystals of 2-cFe(L1) and 2-cRu(L1) suitable for X-ray analysis were obtained by vapor diffusion using diethyl ether/acetone and diethyl ether/acetonitrile, respectively. As a representative example the structure of 2-cFe(L1) is displayed in 2-cRu(L1) is displayed in the ESI.The target complexes and their precursors were fully characterized by 2-cFe(L1) displayed a compact packing with π-stacking interactions between alternating enantiomers, there is no similar intermolecular interaction observed for 2-cRu(L1). The solid state structures display the compact arrangement of the axially chiral macrocyclic ligand surrounding the central metal ion. In the macrocyclized complexes the octahedral coordination sphere is distorted as the planes defined by both terpyridines have an angle of 76.72° for 2-cFe(L1) and 75.11° for 2-cRu(L1) and are no longer perpendicular to each other. In spite of this distortion, the 2-cFe(L1) remains in its low spin state indicated by its purple color. The diacetylene bridges in both macrocyclized complexes are only slightly bent with angles between both triple bonds of 173.5° and 175.7° for 2-cFe(L1) and 2-cRu(L1), respectively. Their minor deviation from linearity points at both the perfect match of the ethynyl-preorganization in the precursors and the structural integrity and stability of the macrocyclized complexes.Both complexes crystallize as a racemate and in spite of their structural similarity, a surprisingly large difference in their solid state packing was observed  as the supporting electrolyte. The voltammograms were recorded against ferrocene as the internal standard and are displayed in the ESI  and Fe(ii) terpyridine complexes,iii) ion.Comparing both open precursors with their macrocyclized complexes, a 100 mV increase of the potential required to oxidize the macrocyclized species was observed in both cases, while only minor shifts  were recorded for the reduction potentials. In analogy to reported Ru(ii) and Ru(iii) polypyridyl complexesIn the case of the Ru complexes the bond lengths do not vary significantly between Ru(ii) and Fe(ii) terpyridine complexes.2g orbitals in an Oh symmetric environment can be found below the HOMO for all four complexes 2]2+ and the difference is even more substantial when considering its iron analogue.To further rationalize the results obtained by the cyclic voltammetry, DFT calculations were performed. Surprisingly, the highest occupied molecular orbital (HOMO) is according to these calculations not centered on the metal, as it would be expected for Ru and 2-cRu(L1) are non-emissive at room temperature, but photoluminescence is readily detectable in frozen matrices. In frozen butyronitrile at 77 K, the luminescence lifetimes are 10.5 μs for Ru(L1) and 8.6 μs for 2-cRu(L1). For Ru(tpy)22+ a luminescence lifetime of 8.0 μs was determined under similar conditions.3MLCT deactivation via energetically close-by metal centered (MC) excited states remains efficient in Ru(L1) and 2-cRu(L1).Of particular interest was the influence of the macrocyclization on the photophysical properties of the complexes. In analogy to the Ru(tpy)ii) and the Ru(ii) complexes could be separated into their enantiomers by high-performance liquid chromatography (HPLC) using a chiral stationary phase . The circular dichroism (CD) spectra of both enantiomers are displayed in 2-cFe(L1) and 2-cRu(L1), Cotton effects were observed in the entire detectable spectral range with opposite signs for both enantiomers. Only small samples of pure enantiomers were obtained due to the analytical character of the column. In spite of intense efforts, attempts to simulate the CD spectra have not been conclusive so far and thus, the assignment of the CD spectra to a particular enantiomer is currently not possible.The macrocyclization of the complexes provides racemic mixtures of both helical chiral enantiomers. Interestingly, the stiffening of the terpyridine ligand arrangement obtained by macrocyclization results in a considerable increase in the stability of the enantiomers. Racemates of both the Fe(2-cRu(L1) complex in various solvents at elevated temperature (e.g. butyronitrile at 200 °C oil bath temperature in a pressure tube or tetrachloroethane at 140 °C oil bath temperature) for prolonged periods did not show any racemization, pointing at the stability of the helical chiral species. Even more surprising was the stability of 2-cFe(L1) as to the best of our knowledge, Fe(ii) terpyridine complexes that do not racemize in solution have not been reported so far. And indeed, after screening of various solvents (see the ESI2-cFe(L1). To get a first idea of the thermodynamics of the racemization, the process was monitored by HPLC at temperatures between 60 °C and 90 °C  and Ru(ii) complexes are fully characterized by their solid-state structures. The obtained racemates of helical chiral complexes can be separated into the corresponding enantiomers by chromatography with chiral stationary phases. The reinforcement of the terpyridine ligand sphere upon macro-cyclization considerably increases the stability of the enantiomers and its influence on physical properties like redox behavior and optical features is discussed.In summary, a new type of compact helical chiral terpyridine metal complex is presented based on the metal templated macrocyclization of suitably functionalized terpyridine ligands. The macrocyclization based on oxidative acetylene coupling within the complex proceeds in excellent yields and the isolated Fe(We are currently investigating alternative macrocyclization reactions and are interested in approaches enabling an even tighter fixation of the surrounding ligand sphere.There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


Comparison of PD-L1+ and PD-L1− patients with matched staging showed increased cytoplasmic positivity in all four stages of PTC that correlated with a greater risk of recurrence and a poor prognosis, but increased membrane positivity significantly correlated with a greater risk of metastasis or death only in Stage IV patients. In conclusion, PD-L1 positive expression in PTC correlates with a greater risk of recurrence and shortened disease free survival supporting its potential application as a prognostic marker for PTC.Programmed death-ligand 1(PD-L1) expression on tumor cells is emerging as a potential predictive biomarker in anti-PD-L1 directed cancer immunotherapy. We analyzed PD-L1 expression in papillary thyroid carcinoma (PTC) and its variants and determined its prognostic potential to predict clinical outcome in these patients. This study was conducted at an academic oncology hospital which is a prime referral centre for thyroid diseases. Immunohistochemical subcellular localization (IHC) analyses of PD-L1 protein was retrospectively performed on 251 archived formalin fixed and paraffin embedded (FFPE) surgical tissues (66 benign thyroid nodules and 185 PTCs) using a rabbit monoclonal anti-PD-L1 antibody  and detected using VECTASTAIN rapid protocol with diaminobenzidine (DAB) as the chromogen. The clinical-pathological factors and disease outcome over 190 months were assessed; immunohistochemical subcellular localization of PD-L1 was correlated with disease free survival (DFS) using Kaplan Meier survival and Cox multivariate regression analysis. Increased PD-L1 immunostaining was predominantly localized in cytoplasm and occasionally in plasma membrane of tumor cells. Among all combined stages of PTC, patients with increased PD-L1 membrane or cytoplasmic positivity had significantly shorter median DFS (36 months and 49 months respectively) as compared to those with PD-L1 negative tumors (DFS, both 186 months with  Thyroid carcinoma (TC) is the most common malignant endocrine tumor with more than 500,000 diagnosed cases in the United States and one of the most rapidly increasing malignancies in both men and women . The estin-vitro experimental models indicate compromise of immune surveillance mechanism for cancer cells in the tumor microenvironment, by interaction with PD-1 . For the membrane staining cut off these respective values for sensitivity and specificity were 90% and 85% [with PPV = 88% and NPV = 87%]. From these analyses, the cut-off for PD-L1 increased immunopositivity in the cytoplasm was ≥ 4.5; for the plasma membrane ≥ 2.1 and for combined cytoplasm and membrane immunopositivity the cut-off was ≥ 5.25.Among 185 PTC tissues analyzed, 123 tissues (66.5%) showed cytoplasmic PD-L1 expression, whereas 74 tissues (40.0%) showed membrane localization Table , while iAmong the PTCs, no correlation was observed between PD-L1 expression and multifocality, capsule extension, microcarcinoma and perineural invasion. Importantly, we observed increased PD-L1 expression in benign chronic lymphocytic Figure  and Hashp < 0.001)    analysis was performed to determine the prognostic potential of PD-L1 expression in comparison to clinical and pathological parameters that were independently significant in Kaplan-Meier analysis. The parameters tested were PD-L1 staining in membrane, cytoplasm and combined membrane and cytoplasm staining in patients with different stages of PTC. Disease free survival (DFS) was calculated through Kaplan-Meier analysis for each of the stages. DFS is defined as time period between completion of primary treatment and detection of residual disease or recurrence of the disease or death. In this cohort during the follow up no death was reported in patients with stage I and II. While in stage III and IV, the reported death incidences were 2 and 5 respectively. Kaplan Meier analysis showed that among all the combined stages of PTCs, patients with PD-L1 cytoplasmic positive tumor cells showed significantly reduced median DFS (49 months) as compared to those with PD-L1 negative tumors  Figure . PD-L1 m) Figure . Further) Figure .+ and PD-L1− patients with matched staging and treatment history. Importantly, Stage I patients with PD-L1 cytoplasmic positive tumors had significantly shorter median DFS = 166 months as compared to those with PD-L1 negative tumors    Figure . Compari) Figure . Stage IImpaired immune surveillance has been shown to be an important factor in enhanced tumor cell aggressiveness that results in metastasis. The aim of our study was to investigate the subcellular expression of PD-L1 in different stages of PTC and to correlate its protein expression with disease aggressiveness and outcome. Our results showed that PD-L1 expression in tumor cells correlated with aggressive PTC and was associated with poor prognosis.Several other cancers have been reported to have increased PD-L1 expression in the plasma membrane alone or in both plasma membrane and cytoplasm –25. The In accord with previous reports –34, we a+ Tim-3+ CD8+ T lymphocytes showed varied degrees of functional exhaustion in patients with regionally metastatic PTC [In the current report a strong correlation between PD-L1 expression on tumor cells and long term prognosis has been documented in our cohort of PTCs . PD-L1 status has been proposed to be critical to promote tumor growth and metastasis , 36. An atic PTC .In summary, our data demonstrate that immunohistochemical analysis - based subcellular cytoplasm and membrane localization of PD-L1 expression and its association with reduced disease free survival in PTC patients, suggests its utility as a potential prognostic biomarker. This could assist in determining which PTC patients and its variants have a greater risk for a more aggressive clinical outcome in PTC management.The Research Ethics Board (REB) of Mount Sinai Hospital (MSH), Toronto, Canada, approved this study. Informed consent for scientific use of anonymized patients' data and tumor tissues was obtained from all patients (REB guideline #07-0212-E). All data were analyzed anonymously. Archived formalin-fixed paraffin-embedded (FFPE) tissue blocks from the MSH Tumor Bank were retrieved and reviewed by the pathologist. Clinical-pathological parameters were obtained from histopathological analyses and the clinical database and are summarized in Table FFPE tissue sections (5 μm thickness) from these patients were deparaffinized, hydrated with series of graded alcohol and antigen retrieval for PD-L1 was performed in Tris-EDTA buffer, pH 9.0 as described by us previously . After bImmunostaining scores were based on the percentage positivity and staining intensity as described previously . Sectiont-test as appropriate. The endpoint for this analysis was disease-free survival (DFS), which is defined as the length of time from the date of surgery on primary tumor to local or regional recurrence, distant metastasis or death. Survival curves based on PD-L1 expression were estimated using the Kaplan–Meier product-limit method with the log-rank test. Univariate Cox proportional hazards models were fit to identify factors significantly related to DFS. To assess whether the PD-L1 expression in tumor cells was an independent predictor of DFS, a multivariate Cox model was constructed to adjust for other patient/clinical characteristics that were significant in the univariate analyses. Two-way interaction terms between PD-L1 expression and other factors in the multivariate Cox model were also assessed. All analyses were two-sided, and significance was set at p value ≤ 0.05. Statistical analyses were performed using Sigma Plot 11 , SPSS statistics software version 23  and R software 3.2.2.The patient distribution and clinical features between PD-L1-positive and PD-L1-negative tumors were compared by chi-square test, or two-sample


Subsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas.PD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry.Pituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes.Human pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management. Pituitary tumors are the second most common intracranial neoplasms, comprising 10-15% of diagnosed brain tumors . Some piThe use of immune-based therapies has led to durable clinical responses in patients with a range of systemic cancers -7. In paThus, due to the dearth of adjuvant therapies for pituitary tumors refractory to conventional treatments and the recent success of checkpoint blockade immunotherapies in cancer, we investigated the expression of PD-L1 in a range of human pituitary tumors. We determined the level of expression first by RNAscope analysis, and then confirmed PD-L1 protein expression by immunohistochemistry. Lastly, we characterized the immune cell infiltrates in these tumors.in situ hybridization to detect PD-L1 mRNA and IHC to assess PD-L1 protein expression. Across all pituitary adenomas, variable expression of PD-L1 was observed  and non-functioning  adenomas revealed significantly higher mRNA levels in the functioning tumors  compared to recurrent (n = 14) pituitary adenomas  or atypical (n = 11) status  or MIB-1 > 3% (n = 28). No significant correlation was observed between PD-L1 mRNA or protein expression and MIB-1 index , T cell markers  and the immune regulatory receptor PD-1. An analysis of all tumors taken together shows variable CD3, CD4, CD8, CD45, and PD-1 expression Figure .p = 0.019 and p = 0.0001, respectively). All lymphocytic infiltrate markers appeared higher in adenomas with an elevated proliferative index, with a significantly increased expression in CD45+ cell infiltrates in adenomas with high (MIB-1 > 3%) compared to low (MIB-1 ≤ 3%) proliferative indices (p = 0.0093). Furthermore, CD3+, CD4+, and CD45+ cells trended to a higher expression in atypical adenomas compared to typical adenomas. PD-1 was significantly increased in non-functioning adenomas (p = 0.0018) and those with elevated proliferative indices (p = 0.003), while being comparable in primary and recurrent tumors.When analyzed by subclass of tumor Figure , both CDSubstantial work over the last fifteen years has begun to clarify the complex relationship between the immune system and cancer. Specifically, the “cancer immunoediting hypothesis” articulates that tumors dynamically evolve to evade immune responses in order to grow progressively . This shin situ RNA hybridization and immunohistochemistry, we assessed the expression of the immunoregulatory checkpoint molecule PD-L1 as well as the infiltrating lymphocyte population within a broad range of pituitary tumors. Together, our findings are the first to demonstrate expression of both PD-L1 mRNA and protein in pituitary tumors irrespective of tumor hormone secretion, proliferative index, aggressiveness level, or recurrence status. Previous studies have reported the presence of T-cell enriched populations of tumor infiltrating lymphocytes in pituitary adenomas [We investigated several immunological parameters in pituitary tumors with the goal of exploring new therapeutic avenues for treatment-refractory tumors. In particular, the location of the pituitary gland outside the blood brain barrier may render systemic therapies more effective. Using adenomas , 31. Funadenomas . In our PTEN loss in malignant brain tumors [EGFR mutations in non-small cell lung cancer [BRAF-mutant melanomas resistant to BRAF inhibition [The interaction between PD-L1 and the PD-1 receptor on T cells leads to an inhibitory signal that constrains the function of activated lymphocytes . Thus, Pn tumors , activatg cancer , and inchibition . Alternahibition . ConcomiPTEN and EGFR, that have been implicated in PD-L1 upregulation in other tumor types. However, we observed significant differences in broad copy number alterations in the functional subset compared to other pituitary tumors. Ongoing work is directed at exploring whether (a) other shared signaling pathway components downstream of PTEN and EGFR alterations may be more activated in functional tumors; (b) there are recurrent copy number alterations in pathway components that may be relevant to PD-L1 expression or regulation; and (c) a globally altered copy number state itself may be an activator of PD-L1 upregulation.The selective increase in PD-L1 expression observed in functional pituitary tumors raises a number of future areas of investigation. Our group has characterized the genomic landscape of a broad range of pituitary adenomas including functional and non-functional tumors, and there do not appear to be recurrent alterations in some of the genes, such as The clinical success of PD-1/PD-L1 blockade has generated enthusiasm in exploring the spectrum of tumor types that may respond to checkpoint inhibition. However, although durable responses to PD-1/PD-L1 therapies have been observed in a range of cancers including melanoma, non-small cell lung cancer, and renal cell carcinoma , reliabl+PD-L1+, TIL−PD-L1+, TIL+PD-L1−, and TIL−PD-L1− [+PD-L1+” tumors may have the best chance to respond to checkpoint immunotherapy. Thus, in our dataset, it is possible that functional and primary adenomas—in which there is high PD-L1 as well as increased T cells—may be particularly responsive to anti- PD-L1 therapy. In contrast, slowly proliferative pituitary tumors—in which there are decreased T cells that do not express PD-1, lower PD-L1 expression, and may be anergic or hypoactive—may be less responsive. In other CNS tumors, the presence of tumor infiltrating lymphocytes (TILs) has been correlated with improved survival in several malignancies, including gliomas, and is being examined as a potential prognostic biomarker. We anticipate that intracranial tumors may actually harbor distinct immunologic microenvironments depending on location—e.g., extradural or intradural, proximity to commissural organs, and such—and therefore additional work is needed to understand how distinct intracranial compartments differ. Ultimately, although tumor cell PD-L1 expression may predict clinical response to anti-PD-1/PD-L1 therapy, it is clear that additional work is needed to clarify the full scope of its predictive power as well as the contributions of other in situ parameters.Other groups have attempted to stratify tumors by the presence of tumor infiltrating lymphocytes and PD-L1 and have used these parameters to create 4 tumor types—i.e, TILL−PD-L1− , 46. TheFormalin-fixed, paraffin-embedded human pituitary tumor specimens were collected from the Department of Pathology, Brigham and Women's Hospital, with corresponding clinical records and pathology reports. Hematoxylin and eosin stained sections corresponding to each tumor were reviewed by two neuropathologists  for selection of specimens with greater than 70% estimated tumor purity. 48 tumors  were compiled in tissue microarray (TMA) format for subsequent analysis. The study was approved by the Institutional Review Boards of Brigham and Women's Hospital and Dana Farber Cancer Institute, Harvard Medical School.Growth hormone (GH) and prolactin (PRL) secreting tumors were classified as functioning. Only two ACTH-expressing adenomas were in the cohort and were excluded from analysis. Null cell adenomas and silent gonadotroph adenomas were classified as nonfunctioning.PD-L1 transcript levels were detected using RNAscope 2.0 HD brown detection kit . A custom-designed RNAscope probe (courtesy of Santagata laboratory) was used to stain the pituitary adenoma TMA. As described previously , 5 μm pahttp://www.cellprofiler.org/) [http://cellprofiler.org/examples/published_pipelines.html) [in situ hybridization signals (dots). The average number of dots per cell in each pituitary case was compared across groups. False positive recognition of stromal cells such as blood cells in regions of hemorrhage as well as dark staining of some nuclei by the counterstain were excluded by visual review. Cases with staining artifacts or poor tissue integrity following hybridization processing were excluded.RNAscope staining was analyzed using CellProfiler image analysis software (er.org/) . The pipes.html) . BrieflyPituitary TMA slides were baked at 60°C for one hour to melt excess paraffin. Immunohistochemistry (IHC) staining for CD3 , CD4 , CD8 , CD45 , PD-L1 . Single DAB stained images were obtained using color deconvolution. After adjustment of the color threshold, the intensity of the DAB positive staining was measured. A mean value (intensity/area) was used for statistical analysis. PD-L1 IHC staining intensity was also measured using color deconvolution and quantified by Image J. PD-1 and CDs IHC staining were quantified by blinded counting. Cases with staining artifacts or poor tissue integrity following immunohistochemical processing were excluded.Histochemical staining was quantified by color deconvolution as previously described . The TMAp value < 0.05 was considered significant.Results are expressed as median ± interquartile range, within minimum to maximal values. Statistical analysis was performed using GraphPad Prism , and comparisons were made using unpaired Mann-Whitney test. A


Ppd-D1, Vrn-A1 and Rht-B1) markers on quantitative trait loci (QTL) detection in a recombinant inbred lines (RILs) population derived from a cross between two spring wheat (Triticum aestivum L.) cultivars, ‘Attila’ and ‘CDC Go’, and evaluated for eight agronomic traits at three environments under organic management. The objectives of the present study were to investigate the effect of conventional management on QTL detection in the same mapping population using the same set of markers as the organic management and compare the results with organic management. Here, we evaluated 167 RILs for number of tillers (tillering), flowering time, maturity, plant height, test weight (grain volume weight), 1000 kernel weight, grain yield, and grain protein content at seven conventionally managed environments from 2008 to 2014. Using inclusive composite interval mapping (ICIM) on phenotypic data averaged across seven environments and a subset of 1203 informative markers (1200 SNPs and 3 gene specific markers), we identified a total of 14 QTLs associated with flowering time (1), maturity (2), plant height (1), grain yield (1), test weight (2), kernel weight (4), tillering (1) and grain protein content (2). Each QTL individually explained from 6.1 to 18.4% of the phenotypic variance. Overall, the QTLs associated with each trait explained from 9.7 to 35.4% of the phenotypic and from 22.1 to 90.8% of the genetic variance. Three chromosomal regions on chromosomes 2D (61–66 cM), 4B (80–82 cM) and 5A (296–297 cM) harbored clusters of QTLs associated with two to three traits. The coincidental region on chromosome 5A harbored QTL clusters for both flowering and maturity time, and mapped about 2 cM proximal to the Vrn-A1 gene, which was in high linkage disequilibrium (0.70 ≤ r2 ≤ 0.75) with SNP markers that mapped within the QTL confidence interval. Six of the 14 QTLs  were common between the conventional and organic management systems, which suggests issues in directly utilizing gene discovery results based on conventional management to make in detail selection (decision) for organic management.Recently, we investigated the effect of the wheat 90K single nucleotide polymorphic (SNP) array and three gene-specific ( Puccinia triticina Eriks. f. sp. tritici), stripe rust (P. striiformis f. sp. tritici), stem rust (P. graminis f. sp. tritici), common bunt (caused both by Tilletia tritici and Tilletia laevis) and Fusarium head blight (caused mainly by Fusarium graminearum) (http://www.pgdc.ca). The availability of various improved wheat cultivars not only for good agronomic characteristics and quality traits, but also with better resistance to wheat diseases, have increased average wheat productivity in Canada by approximately four-fold from 0.8 Mg ha-1 in 1961 to 3.1 Mg ha-1 in 2014 (http://faostat3.fao.org). Wheat is grown in the region both under conventional and organic management systems, with the demand for organic production intensified in the last decade for different reasons, including concerns on human health, food quality and environment [www.intechopen.com). Most wheat breeders in western Canada develop semi-dwarf cultivars, which require high inputs  to produce high grain yield and attain satisfactory protein content, but such cultivars types often produce lower grain yield in organic management due to weaker weed competitiveness [More than 85% of wheat in Canada is produced in the western Canadian prairie provinces of Alberta, Saskatchewan and Manitoba, with a small proportion in British Columbia and eastern Canada . Wheat bRht-B1 gene specific markers. The soil at both organically and conventionally managed sites was an Udic Boroll (Orthic Black Chernozem in Canadian system) with loam or clay loam texture, neutral pH (6.7 to 7.4), and high soil organic matter (6 to 11%). The crop rotation on the conventionally managed site was wheat-pea-canola, while it was wheat followed by a green manure rye plow-down in the organic management site. The conventionally managed sites received 36–40 kg ha-1 fertilizer (11–52–0 N–P2O5–K2O) banded with seed at the time of planting, while the organically managed sites received neither chemical fertilizer nor compost [Ppd-D1, Vrn-A1 and Rht-B1) [The Wheat Breeding group at the University of Alberta has been evaluating the performance of wheat cultivars and breeding lines under both conventional and organic management systems in Alberta, Canada , 7, 9–17 compost , of whic compost –23. In o Rht-B1) . That stThe present study was conducted on a mapping population of 167 RILs developed from a cross between two spring wheat cultivars—‘Attila’ (CM85836-50Y-0M-0Y-3M-0Y) and ‘CDC Go’. As described in our previous studies , 20, ‘AtPpd-D1 [Vrn-A1 [Rht-B1 [DNA extraction and genotyping was done as described in our previous studies , 21. BriPpd-D1 , Vrn-A1  [Vrn-A1  and Rht- [Rht-B1  gene spehttp://wheat.pw.usda.gov/ggpages/wgc/98/Intro.htm), which consisted of three letters for trait acronym, lab designation (dms = Dean Michael Spaner) and chromosome. Genetic maps and QTL graphs were drawn using MapChart v2.1 [Ppd-D1, Rht-B1 and Vrn-A1 gene specific markers and all SNPs that mapped on chromosomes 2D, 4B and 5A, respectively, was evaluated by computing the r2 values using TASSEL version 5.2.30 [Linkage analysis was performed as described in the ‘Cutler’ × ‘AC Barrie’ mapping population , while aart v2.1 . The ext-2, with kernels 2 g heavier and 0.6% higher grain protein content, but was 2 cm taller and yielded 277 kg ha-1 less grain than ‘Attila’. In our previous study conducted from 2008 to 2010, ‘CDC Go’ yielded approximately 420 kg ha-1 more grain than ‘Attila’ [-1 grain. The phenotypic distribution of least square means averaged across seven environments was normal (P > 0.050) for all traits, except test weight  were used for QTL mapping. The number of markers retained for QTL mapping varied from 4 on chromosome 1D to 150 on 2B (Detailed results on the SNP and gene specific markers and linkage maps used in the present study were presented in our previous study . After e50 on 2B . There w2 (QTil.dms-6A.1), flowering time (QFlt.dms-5A), plant height (QPht.dms-4B) and grain yield (QYld.dms-2D.2); two QTLs each for maturity (QMat.dms-4B and QMat.dms-5A.2), grain protein content (QGpc.dms-2D.2 and QGpc.dms-4B) and test weight (QTwt.dms-5A and QTwt.dms-5B.3); and four QTLs for kernel weight . All QTLs associated with each trait exhibited mainly additive effects and QTL by QTL interactions were negligible (R2 < 2%). The QTL for tillering mapped at 70 cM on chromosome 6A (QTil.dms-6A.1) and accounted for 11.2% of the phenotypic variance across seven environments. RILs that had ‘Attila’ alleles at the two flanking markers for QTil.dms-6A.1, on average, had 3.4 more tillers than those RILs homozygous for ‘CDC Go’ alleles. However, this QTL was not detected in any of the individual environments; instead, we found 6 other environment specific QTLs on 4A (QTil.dms-4A), 5A (QTil.dms-5A), 6A (QTil.dms-6A.2) and 7A  that were associated with tillering in 2009, 2013 and/or 2014 environments , flanked by the Vrn-A1 gene, and explained 16.8% of the phenotypic variance across the seven environments , and explained from 12.6 to 13.0% of phenotypic variance of the individual environments (QFlt.dms-4A), 4B (QFlt.dms-4B) and 6B (QFlt.dms-6B), which individually explained from 7.6 to 9.0% of the phenotypic variance. The two QTLs for maturity mapped at 80 cM on chromosome 4B (QMat.dms-4B) and at 297 cM on 5A (QMat.dms-5A.2), which individually explained 15.9 and 14.0%, respectively, and altogether accounted for 29.9% of the phenotypic variance across the seven environments. The favorable alleles for QMat.dms-4B and QMat.dms-5A.2 originated from ‘Attila’ and ‘CDC Go’, respectively. RILs that were homozygous for the favorable alleles at the two flanking markers of each QTL matured about two days earlier than those RILs that were homozygous for the unfavorable alleles. When individual environments were considered, QMat.dms-4B and QMat.dms-5A.2 were detected at the same confidence interval in five (2009 to 2014 except 2011) and four  out of the seven environments, respectively  and explained 18.4% of the phenotypic variance. Rht-B1 gene mapped 34.5 cM distal to QPht.dms-4B and 27 cM distal to one of the flanking SNP markers, wsnp_Ra_c1146_2307483. RILs that had the ‘CDC Go’ alleles at the two flanking markers for QPht.dms-4B were 7.7 cm shorter than those RILs that were homozygous for ‘Attila’ alleles (QPht.dms-4B was consistently detected at the same position in five (2010 to 2014) of the seven environments, but the proportion of phenotypic variance explained by QPht.dms-4B was variable, ranging from 11.9 to 23.9% (QPht.dms-2D.2), 5A (QPht.dms-5A) and 6B (QPht.dms-6B.1 and QPht.dms-6B.2), which individually explained from 3.2 to 13.3% of the phenotypic variance in the 2009, 2010 and 2013 environments, respectively (QYld.dms-2D.2) that explained 9.3% of the phenotypic variance across the seven environments. The photoperiod response Ppd-D1 gene mapped 66 cM distal to QYld.dms-2D.2. RILs with ‘Attila’ alleles at the two flanking markers for QYld.dms-2D.2 produced 375.7 kg ha-1 more grain yield than those RILs homozygous for ‘CDC Go’ alleles. When individual environments were considered, QYld.dms-2D.2 was detected at the same confidence interval in four  of the seven environments, explaining from 6.0 to 11.1% of the phenotypic variance at individual environments. In addition, five environment-specific QTLs associated with grain yield were also identified on chromosomes 3A, 6B, 7A and 7D explaining from 6.3 to 10.9% of the phenotypic variance  and at 239 cM on 5B (QTwt.dms-5B.3), and they explained 6.1 and 10.1% of the phenotypic variance, respectively , at 79 cM on 6A (QTkw.dms-6A.1), at 4 cM on 6D (QTkw.dms-6D.2) and at 158 cM on 7B (QTkw.dms-7B.1). Each QTL individually explained from 6.7 to 12.1% and altogether accounted for 35.4% of the phenotypic variance across seven environments  and at 80 cM on 4B (QGpc.dms-4B). QGpc.dms-2D and QGpc.dms-4B explained 13.4 and 6.3%, respectively, and together accounted for 19.7% of the phenotypic variance across seven environments  environments, while QGpc.dms-4B was detected only in 2010. In addition, three environment-specific QTLs on chromosomes 2B, 3A and 5B were detected that individually explained from 6.8 to 8.3% of the phenotypic variance at a single environment (The two QTLs associated with grain protein content across seven environments mapped at 62 cM on 2D (ronments . The favironment .QFlt.dms-5A) and maturity (QMat.dms-5A.2) in the combined data across seven environments plus plant height (QPht.dms-5A) in 2013 environment , plant height (QPht.dms-4B) and grain protein content (QGpc.dms-4B). RILs with ‘Attila’ alleles at the two flanking markers for this QTL on 4B were different (p ≤ 0.008) than those with ‘CDC Go’ alleles for maturity, plant height and grain protein content, but not for the other five traits (QYld.dms-2D) and grain protein content (QGpc.dms-2D). RILs carrying ‘CDC Go’ alleles at the two flanking markers for the coincidental QTL on 2D were different (p ≤ 0.001) from those possessing ‘Attila’ alleles for both grain yield and grain protein content, but not for the other six traits , two for maturity on 4B and 5A (QMat.dms-4B and QMat.dms-5A.2), one for plant height on 4B (QPht.dms-4B) and two for kernel weight on 4A and 6A (QTkw.dms-4A and QTkw.dms-6A.1). The percentage of phenotypic variance explained by QFlt.dms-5A and QPht.dms-4B was the same under organic and conventional management. In the conventional management system, both QMat.dms-5A.2 and QTkw.dms-6A.1 showed reduction in phenotypic variance by 3.2–3.7%, while those of QMat.dms-4B and QTkw.dms-4A showed an increase by 5.3–10.0% as compared with organic management. In a previous study conducted using the DArT-based low density markers [QHt.dms-4B) associated with plant height was common between the two management systems. Although we identified five more QTLs that were common between the organic and conventional management systems using the SNPs than the DArTs, most QTLs still remained management specific.Using the averaged phenotypic data across three organically and seven conventionally managed environments, we uncovered a total of 24 QTLs, of which 6 QTLs were common between the two managements, while the remaining 18 QTLs were detected either under conventional (8) or organic (10) environments. The number of QTLs detected for flowering time, maturity, pant height and tillering remained the same irrespective of the management system and number of environments. For the other traits, we found one or two fewer QTLs for test weight, kernel weight and grain yield, and two additional QTLs for grain protein content under conventional than organic management system. Each QTL identified under organic management individually explained from 5.5 to 18.8% of phenotypic variance, which is similar to the 6.1 to 18.4% phenotypic variation obtained under conventional management. Overall, the total phenotypic variance explained by all QTLs associated with each trait under conventional and organic management system differed from 0.4% for flowering time and plant height to 19.7% for grain protein content. The six common QTLs between the conventional and organic management included one for flowering time on chromosome 5A (QFlt.dms-5A) and maturity (QMat.dms-5A.2) between 294 and 298 cM interval on chromosome 5A, which accounted for 14.0–16.8% of the phenotypic variance for both traits, which is equivalent to a reduction in both flowering and maturity time up to 3 days  between the Vrn-A1 gene specific marker and the SNPs that mapped within the QTL confidence interval on chromosome 5A; however, the LD values were not high enough to confidently suggest that the QTL is the same as the Vrn-A1 gene. In addition, the QTL only accounted for 14–17% of the phenotypic variance, which is not a typical effect for single genes. The alternative scenario is that the Vrn-A1 gene may be tightly linked with the coincident QTL region, but the statistical methods used for linkage analysis and QTL mapping failed to discriminate them due to small number of recombinants in our RIL mapping population to break up the linkage, which has been discussed in detail in our previous study [In western Canada, where the growing season is short and days are long, the development of early maturing wheat cultivars is important to avoid frost damage, which can affect both yield and grain quality , 32. In o 3 days . One of osome 5A , 33 and osome 5A , 35. In us study .QPht.dms-4B) mapped at 82 cM on chromosome 4B  and grain protein content (QGpc.dms-4B) , which is too low to suggest any association between the coincidental QTL and the Rht-B1 gene.One of the QTLs for plant height  . This co.dms-4B) . This QT markers  and 33.5QTil.dms-6A.1) on chromosome 6A that accounted for 11.2% and 44.9% of the phenotypic and genetic variance, respectively. In spring wheat, QTLs associated with tillering have been reported near Gli-A2 (Xpsr10) on the short arm of chromosome 6A and several other chromosomes [QYld.dms-2D.2) that explained 9.3% and 22.1% of the phenotypic and genetic variance across seven environments, which is equivalent to an increase in grain yield by 376 kg ha-1 (Ppd-D1) on chromosome 2D has been the focus in breeding for early maturing wheat cultivars to better adapt to their environments [Ppd-D1 gene, which resulted in a reduction in maturity up to 5 days, but showed a yield penalty of 436 kg ha-1 [Ppd-D1 gene, which is genetically far. Pairwise LD values between the Ppd-D1 gene and all SNP markers that mapped around QYld.dms-2D.2varied from 0.002 to 0.035 (data not shown), which is too small to suggests any association between QYld.dms-2D.2 and the Ppd-D1 gene.We identified a single QTL from ‘Attila’ for tillering  that explained 8.3% of the phenotypic variance across three environments; and (ii) two QTLs associated with kernel weight on chromosome 4A and 6A that together explained 18.7% of the phenotypic variance across three environments [QTwt.dms-5A and QTwt.dms-5B.3) associated with test weight that individually explained 6.1 and 10.1%, respectively  that individually explained 6.7–12.1% of the phenotypic variance (QTkw.dms-4A) and 6A (QTkw.dms-6A.1) were common between the present and previous [QTkw.sdau-6A) that explained from 6.1 to 13.2% of the phenotypic variance across three environments [In our previous study using DArT markers, we reported (i) a single QTL associated with test weight on chromosome 1B  and 4B (QGpc.dms-4B) that individually explained 13.4% and 6.3% of the phenotypic variance, respectively, and altogether accounted for 18.7% of the phenotypic variance  mapped 5 cM distal to the QTLs for grain protein content (QGpc.dms-2D), but the genetic confidence interval between QYld.dms-2D.2 (62–70 cM) and QGpc.dms-2D (59–63 cM) showed an overlap; both QTLs mapped 61–66 cM distal to the Ppd-D1 gene, so neither QYld.dms-2D.2 nor QGpc.dms-2D are associated with the Ppd-D1 gene. RILs with ‘Attila’ alleles at the two flanking markers of QYld.dms-2D.2 produced 375.7 kg ha-1 more grain yield but 0.3% lower grain protein content than those with ‘CDC Go’ alleles  still remained unexplained even after doubling marker density, which may be due to gaps on some chromosomes and/or lack of SNP polymorphism on some chromosomes, such as 3D and 4D. Only 6 of the 14 QTLs identified under conventional management were common with those identified under organic management, which clearly suggest the management specificity of the majority of QTLs. Our results from the different studies conducted in the ‘Attila’ × ‘CD Go’ population using both low and high density markers suggest the need in conducting management specific gene discovery studies and provides insight on difficulty in directly utilizing genes or major effect QTLs identified based on phenotypic data from conventional management to make selection under organic management.S1 FigThe arrows indicate values of the two parents. See (DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The understanding of these seemingly connected phenomena is lacking. Here we demonstrate, using Raman spectroscopy and in situ electrostatic doping techniques, that the intensity is directly modulated by an additional contribution from photoluminescent π-conjugated domains surrounded by sp3 carbon regions in graphene monolayers. The findings are further underpinned by a model which correlates the individual Raman mode intensities to the degree of functionalisation. We also show that the position shift in the spectra of solvent-based and powdered functionalised graphene derivatives originates predominantly from the presence of edge-to-edge and edge-to-basal plane interactions and is by large functionalisation independent.Covalent functionalisation of graphene is a continuously progressing field of research. The optical properties of such derivatives attract particular attention. In virtually all optical responses, however, an enhancement in peak intensity with increase of sp We also propose a model which allows to quantify the degree of functionalisation by using the individual Raman mode intensities. Furthermore, we demonstrate that while the coupling of the photoluminescence affects the intensity of the optical response in few-layered restacked homogeneous systems, the shift in peak position can be attributed to edge-to-edge and edge-to-basal plane interactions in (disordered) solvent-based systems and powders.We selected Raman spectroscopy, including a combined cf. Methods) to cover a wide range of θ. 2525We refer to covalently modified monolayer graphene obtained by different reaction routes (cf. Methods) and in the central areas of the flakes. For the purpose of this study, we focused on the dependence on the degree of functionalisation of the individual Raman D- and G-mode intensities, ID and IG, respectively  centre content was observed , m an exponent associated with the density of states of the π-conjugated domains and fB(E) the Boltzmann distribution function. Following our initial assumption that the modulation of the optical response is a general effect, a specific coupling of the Raman signal to the PL can be expected, and thus the D-mode intensity to the lowest order can be written aswhere and the G-mode intensityI0 corresponding to the G-peak present in non-functionalised graphene. D0 and G0 account for the coupling strength of the D and G mode to the PL, respectively. It should be noted that all the parameters  in cf. with cf.2 domains.The experimental data in DI and GI on the π-state occupation for the case of oxo-G (θ ~ 50%) at room temperature. A single oxo-G flake2 (300 nm) substrate  atoms illuminated by the laser beamπ-conjugated domain size in oxo-G of approximately 1 nm2252) domains, since electrostatic doping only changes the π-state occupancy.In θ of a monolayer graphene can be unambiguously determined by solely relying on the actual intensities, DI and GI. Our model is also commensurate with the existing approaches which refer to the ratio of the Raman D- and G-modes, ID/IG, as a typical measure for the number of defects and disorder introduced1433DI/GI ratios with θ-dependence as reported in literature1433θ the uncertainty in the fitted ID/IG ratios tends to increase, which suggests that in this regime higher-order terms in The consistency between our experimental data and our model shows that the functionalisation degree DI/GI combined with the geometrical models is well known to be a limited description: First, no material-specific parameters are accounted for. Second, considering the addend related formation of π-conjugated domainsLD14333) centres). In other words, the DI/GI ratio is only suitable in the limit of a low θ (below ca. 1%). Third, no unique solution for LD is provided. And fourth, ID/IG leads to a loss of information which is contained in the individual ID and IG values and thus gives an incomplete measure regarding θ. This is illustrated in ID and IG values decreased by 35 to 40%. In contrast, the ID/IG ratio was reduced by less than 8%  transferred on top of highly oriented pyrolytic graphite (HOPG) (cf. Methods). The Raman response was measured in the central areas (off-edge) of the flakes for different numbers of layers which were identified by AFM profile imaging . θ = 50%). Analogous data for r-oxo-G (θ = 0.07%) are shown in ID and IG positions in the central area of the r-oxo-G flakes remain constant irrespective of the layer number, a distinct shift can be observed at and close to the flake edges. Considering that the oxo-G at θ = 50% has on average significantly smaller π-conjugated domains with associated stronger PL contribution at our laser lines than r-oxo-G at θ = 0.07%, the reported shift in position of peaks in the optical response of solvent-based and powdered functionalised graphene is primarily not associated with the sp2 domains. Therefore, it can be attributed to edge-to-edge and edge-to-layer  interactions within such polydisperse systems.Importantly, the fixed position of the Raman peaks suggests the shift observed in non-aligned systems11617ID/IG ratio approaches, therefore making analysis of graphene derivatives less ambiguous. Furthermore, we show that a similar intensity-based approach should be feasible to quantify the defect density in the case of few-layered, well-ordered systems. However, the presence of edges, interlayer interactions and different stacking geometries in a (disordered) bulk or solvent-based system can lead to a position shift in the optical response. Our results also provide insight into the optical response of graphene quantum dots and assemblies of those where edge-to-edge interactions can play a role. Finally, the coupling of PL to Raman excitations can be expected to occur in other covalently functionalised layered 2D materials, too, and is probably even enhanced if they are inherently PL active unlike graphene.Our combined study using Raman and electrostatic doping provides evidence that the generally observed modulation of the optical response in graphene derivatives is associated with photoluminescent sptert-butylphenyl) iodonium hexafluorophosphate was deposited from solution (CH2Cl2) on a monolayer flake. The reaction of 4-tert-butylphenyl (tBP) cations was subsequently activated by a laser pulse  within the Raman spectrometer. Generated phenyl cations reacted with the carbon lattice of graphene by covalent bond formationcf. https://graphene-supermarket.com/CVD-grown-graphene).Oxo-graphene (oxo-G) of 50% was synthesized from natural graphite in sulfuric acid with potassium permanganate as oxidantRaman spectroscopic characterization was carried out on a Horiba Jobin Yvon LabRAM Aramis confocal Raman microscope (excitation wavelength: 532 nm) with a laser spot size of ~1 μm . Raman spectra were taken with a 532 nm laser line, 1 s acquisition time, 1 mW power, at room temperature. Statistical Raman measurements were obtained through a motorized x-y table in a continuous linescan mode (SWIFT-module).2 were prepared using Langmuir-Blodgett Minitrough from KSV NIMA. Oxo-G was dissolved in methanol/water mixtures. The subphase was water. Oxo-G transfer was carried out at the pressure of 10 mN m−1. Oxo-G was subsequently reduced by vapor of a 1:1 volume mixture of hydriodic acid and trifluoroacetic acid at 80 °C.Langmuir-Blodgett Films of oxo-G on HOPG and SiOAll relevant data are available from the authors on request.How to cite this article: Vecera, P. et al. Degree of functionalisation dependence of individual Raman intensities in covalent graphene derivatives. Sci. Rep.7, 45165; doi: 10.1038/srep45165 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.