Forkhead box C2 (FOXC2) is a crucial factor involving in various cancers. However, its functions in hepatocellular carcinoma (HCC) is unknown. Here, we explored the role of FOXC2 in the progression of HCC and its potential mechanisms.FOXC2 expression in HCC tissue and cells were detected by immunohistochemistry or western blot and real-time PCR. CCK8, wound healing and transwell assay were used to measure cell growth and invasion. Tumor formation experiment was carried out to assess the tumorigenicity of HCC cells. Regulation of FOXC2 on Ang-2 was validated by luciferase assay and complementary experiments.Increased FOXC2 expression was found to be associated positively with more aggressive clinicopathologic features. HCC patients with higher FOXC2 expression had significantly shorter overall survival. FOXC2 expression was indentified as an independent risk factor for resectable HCC. Increased FOXC2 expression accelerated the migration and invasion of HCC cells, accompanied by enhanced Ang-2 expression. Likewise, FOXC2 knockdown yielded opposite results. Moreover, FOXC2 stimulated the activation of the Ang-2 promoter. Suppression of Ang-2 expression hindered the FOXC2-mediated EMT processs, cell migration and invasion of HCC.FOXC2 is a novel prognostic predictor for HCC and may facilitate the growth and invasion through Ang-2. As a fatal cancer with poor prognosis, hepatocellular carcinoma (HCC) causes the third-highest number of cancer-related mortality in sub-Saharan Africa, East Asia and the second-highest number deaths for males in China, with an increasing number of cases in Europe and the United States –3. In thA range of processes occurring in the cell, like metabolism, stress resistance, cell cycle arrest, apoptosis, and aging are regulated by the FOX (Forkhead box) family of proteins, which characteristically possess a DNA binding domain, that is evolutionarily conserved –7. The FAbnormal angiogenesis is one of the hallmarks of malignancy and a research hotspot for the development of novel targeted regimen . The A nTherefore, the expression levels and prognostic significance of FOXC2 in HCC tissues was determined in the present study. Additionally,we explored potential FOXC2 target genes involved in cell invasion and migration. Luciferase assays and western blot analysis were used to identify Ang-2 as a target gene of FOXC2. Ultimately, we knocked down endogenous Ang-2, using its shRNA, to elucidate the role of FOXC2 in tumor development.Paraffin-embedded HCC samples were obtained from the Nanfang hospital biobank. 280 HCC patients treated at Nanfang hospital between January 2009 and December 2014 were included. The inclusion criteria were (1) histologically confirmed the diagnosis and (2) no previous treatment. The exclusion criteria were (1) serious complications, (2) presence of other malignant diseases, or (3) incomplete follow-up data. For tumor staging, the Cancer staging system defined by the 7th Edition of the American Joint Committee was used. Besides, 40 pairs of HCC and respective adjacent non-tumorous samples were collected to analyze the relationship between FOXC2 and Ang-2.Nude mice were purchased from Southern Medical University, Laboratory Animal Center. Normal hepatic cell line L02 and HCC cell lines including sk-hep-1, hep3B, SMMC-7721, Huh-7 and MHCC-97H were purchased from the Chinese Academy of Sciences, Shanghai Cell Bank (China). 293T cells were purchased from ATCC (American Tissue culture collection). All cells were maintained in RPMI1640  containing 10% fetal bovine serum at 37 °C.For the gene knockdown (KD) studies, shRNA containing lentiviral particles for either FOXC2 or Ang-2 were purchased from Santa Cruz Biotechnology (Cat. No: sc-43767-V and sc-39305-V). The virus was collected after transfection for 60 h into 293T cells with all vectors and then infected target cells along with 8 mg/mL polybrene (Sigma-Aldrich). Western blotting was performed for selection of stable and independent clones.Lentiviral expression clones LV-pGV208-FOXC2, lentiviral packaging plasmids (pHelper 1.0 and 2.0) and control vector LV-pGV208 were purchased from Genechem . The supernatant containing lentivirus particles were harvested after transfection for 48 h in 293T cells and then were used to infect Huh-7 and MHCC-97H cells. Based on EGFP assay, lentiviral-infected cells were sorted with flow cytometry to achieve 100% cell infection. Overexpression of FOXC2 was confirmed through western blot.The protein of each group was collected after cell lysis with RIPA buffer and quantified using a BCA assay kit from Abcam . 20 µg of protein was separated on 10% SDS-PAGE gel, electrotransferred to nitrocellulose membranes , blocked with 1% BSA for 1 h and incubated with rabbit primary antibodies in 1:1000 dilution, including anti-Ang-2(#2948S), anti-FOXC2(#12974S),anti-E-cadherin(#14472S), anti-vimentin(#5741S), anti-fibronectin(#4705S) and anti-β-actin(#4970S) . Subsequently, the membranes were washed extensively with PBST  followed by incubation of secondary antibody, HRP-conjugated anti-rabbit IgG incubation for 1 h at room temperature. The ECL method was then used to visualize protein bands.® III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed with a CFX96 Real-Time System (Bio-Rad) in the presence of 2X SYBR green master mixture (Invitrogen) in a final reaction volume of 10 μL. The primer sequences were: FOXC2 sense 5′-CCTACCTGAGCGAGCAGAAT-3′, antisense 5′-ACCTTGACGAAGCACTCGTT-3′, GAPDH sense 5′-TGTTGCCATCAATGACCCCTT-3′, and antisense 5′-CTCCACGACGTACTCAGCG-3′. The internal control was GAPDH. All experiments were run in triplicates in three independent experiments.Total RNA was extracted from HCC cells according to manufacturer’s instructions using Trizol (Invitrogen). 1 μg of RNA was used to synthesize cDNA with the SuperScriptCCK-8  was used to assess the cell proliferation as manufacturer’s instructions recommend. Briefly, cells were inoculated in 96-well plates at the density of 1.5 × 103 cell/well, and cultured in incubator for 24 h before evaluated at day 1, 2, 3, 4, 5, respectively. CCK-8 solution was then dripped into each well, and the plate was transferred to the incubator for 4 h. Finally, OD value at 450 nm was detected by HBS-1096B microplate reader .4 cells were inoculated in each well of a 6-well plate and maintained for 24 h in an incubator. Next day, a 1 mL pipette tip was used to scratch the cells at a width of 500 μm and then washed with RPMI-1640. At 24 and 48 h after scratch, cells were observed with an inverted microscope and photograph. Data summarize three independent experiments.The migration of cells was determined through wound healing tests following a standardized protocol. Briefly, 2 × 10Cells were incubated in PBS  and the upper chambers coating with 50% Matrigel (BD Biosciences) of 24-well transwell plates . After incubating for 24 h, invaded cells were examined and photographed through bright field microscopy  after staining with 0.5% crystal violet. The invasion rate was determined by counting the invaded cells in five randomly selected fields of each chamber under a fluorescence microscope . Data summarize three independent experiments.Tissue sections were boiled in citrate-hydrochloric acid (pH 6.0) for 30 min for epitope retrieval and incubated at 4 °C overnight with anti-FOXC2  and anti-Ang-2  primary antibodies. Immunostaining was performed using the Envision System with diaminobenzidine .The stained sections were scored independently by two pathologists who were blinded to the clinical parameters. FOXC2 expression was evaluated using the extent and intensity of staining. The staining intensity was scored as 0-no staining, 1-weak staining(seen as light yellow), 2-moderate staining(seen as yellow–brown),or 3-strong staining(seen as brown).The extent of staining was scored as 0(0%), 1(1 to 25%), 3(51 to 75%) and 4(76 to 100%), based on the percentage of positive stain relative to the entire cancerous area or the entire section for normal samples. The final staining score (0 to 7) was defined through the sums of the intensity and extent scores. Tumors with a final staining score < 3 were considered as low expression, while those with high expression had scores > 3 to 7.The binding regions of FOXC2 on Ang-2 promoter were predicted on the high-quality transcription factor binding profile database JASPAR . Three s6 HCC cells in 100 µl 1:1 of PBS and matrigel on both sides of each mouse. The growth rate of the tumor was monitored using bioluminescent imaging. Mice were anesthetized with 0.7% pentobarbital sodium (10 μl/g) and injected intraperitoneally with 150 mg/kg luciferin . Tumors were imaged through the IVIS Lumina II platform and analyzed with Live Image software . Each group had six mice.FOXC2-OE Huh-7 and FOXC2-OE-Ang-2 KD Huh-7 cells were transfected to express luciferase. 6 to 7-week-old male NOD/SCID mice  were injected were with 2*10t test or ANOVA; The differences were assessed using SPSS 19.0 software . P ≤ 0.05 was considered as statistically significant.All data are presented as mean ± SEM unless stated otherwise. Statistical significance (P < 0.05) was determined by two-side 280 paraffin-embedded HCC samples and 40 normal (non-cancer) samples were immunohistochemically analyzed for FOXC2 expression. According to the intensity of IHC staining, no or weak staining of FOXC2 protein was seen in 115 of 280 (41.1%) paraffin-embedded HCC tissues, while moderate staining (in the membrane and cytoplasm of cancer cells) was observed in 104 of 280 (37.1%) samples and strong staining was observed in 61 of 280 (21.8%) samples. In 40 non-cancerous control tissues, no FOXC2 staining was seen in 32 cases and weak expression was seen in two cases  and late stage (stage III-IV). In the early stage group, 69 patients showed high FOXC2 expression in tumor cells. Patients with high FOXC2 expression suffered from worse OS as compared to those with low FOXC2 expression  and overexpression (OE) HCC cell lines using MTT assay. As shown in Fig. To analyze the impact of FOXC2 on the migration of both HCC cell lines, we performed wound-healing assays. As illustrated in Fig. 2 = 0.709, p < 0.0001). Thus, FOXC2 may impact the production of Ang-2 to induce metastasis and invasion in HCC.According to the above results, we further analyzed FOXC2 and Ang-2 expression in 40 HCC tissues, of which 60.2% showed high FOXC2 expression. The result showed that tissues with high FOXC2 expression also exhibited high Ang-2 expression. As shown in Fig. Since Ang-2 also plays a crucial role in metastasis and invasion of malignancy, we further investigated whether FOXC2 modulates Ang-2 expression in MHCC-97H and Huh-7 cells via western blot. As shown in Fig. To determine whether Ang-2 is a direct target of FOXC2, three Ang-2 promoter fragments  were cloned into a pGL3-control vector for dual luciferase reporter assay. As observed in Fig. As expected, Ang-2 knockdown (KD) abated the growth of HCC cells with FOXC2 overexpression (OE) Fig. a, b. As The function of Ang-2 knockdown on the FOXC2-induced EMT process in HCC was assessed by measuring the expression of E-cadherin, vimentin, and fibronectin in FOXC2 OE and FOXC2 OE Ang-2 KD cells through western blot. Reduced Ang-2 expression in FOXC2 OE cells led to the enhanced expression of epithelial marker E-cadherin and weakened the expression of mesenchymal markers fibronectin and vimentin Fig. g. These The aforementioned in vitro findings strongly indicated that FOXC2 promoted the proliferation and invasion of HCC cells by modulating Ang-2 expression. We next evaluated the effect of FOXC2 on tumor growth by in vivo optical imaging in NOD/SCID mice.FOXC2 OE and FOXC2 OE Ang-2 KD Huh-7 cells with luciferase activity were injected into the flanks and shoulders of NOD/SCID mice Fig. a and e. By far, recurrence and metastasis remain the major cause of HCC-related mortality, and the underlying molecular mechanisms are still to be clarified.As a focus of current research field, FOXC2 has been reported as an oncogene in a range of cancers. It was well accepted that FOXC2 was a metastasis-related gene and might consequently serve as a predictor for the treatment response or prognosis of cancer patients in clinic use . In lineFurther analysis revealed a positive correlation between the expression of FOXC2 and Ang-2, led to our assumption that FOXC2 promotes Ang-2 expression for the invasion and metastasis of HCC. Luciferase assay showed that FOXC2 noticeably augmented the transcriptional activity of Ang-2, together with the result that Ang-2 expression was decreased in FOXC2-knockdown MHCC-97H and Huh-7 cell lines, suggesting that Ang-2 was a direct target of FOXC2.Hypervascularity is one of the remarkable features of HCC. In the past decades, the development of neovascular targeting therapy has dawned on HCC patients. The current therapeutic target of the angiogenic pathway includes VEGF signaling, angiopoietin 1(Ang-1), Ang-2 and tyrosine-protein kinase receptor Tie2. Agents targeting VEGF axis like bevacizumab, axitinib, lenvatinib, pazopanib, ramucirumab, sunitinib and vandetanib has been approved for the treatment of various cancers including colorectal cancer, non-small cell lung cancer, cervical cancer,ovarian cancer. Angiogenesis inhibitor targeting both VEGFR and Tie2 like cabozantinib, regorafenib and sorafenib have also shown efficacy in the treatment of renal cancer, thyroid cancer, gastrointestinal stromal tumor and liver cancer . Among tTo sum up, this study indicated that FOXC2 promote HCC migration and invasion, and the regulation of Ang-2 exacerbates these effects. Thus, FOXC2 and Ang-2 might be potent and promising targets for the prevention and treatment of metastatic HCC.Additional file 1: Figure S1. Fibronectin, vimentin and E-cadherin expression in FOXC2 OE and FOXC2 OE Ang-2 KD tumors were analyzed through western blot.


As a derivative material of graphene, graphene oxide films hold great promise in thermal management devices. Based on the theory of Fourier formula, we deduce the analytical formula of the thermal conductivity of graphene oxide films. The interlaminar thermal property of graphene oxide films is studied using molecular dynamics simulation. The effect of vacancy defect on the thermal conductance of the interface is considered. The interfacial heat transfer efficiency of graphene oxide films strengthens with the increasing ratio of the vacancy defect. Based on the theoretical model and simulation results, we put forward an optimization model of the graphene oxide film. The optimal structure has the minimum overlap length and the maximum thermal conductivity. An estimated optimal overlap length for the GO (graphene-oxide) films with degree of oxidation 10% and density of vacancy defect 2% is 0.33 μm. Our results can provide effective guidance to the rationally designed defective microstructures on engineering thermal transport processes. Graphene-oxide (GO) films show fascinating performance and have been widely applied in various fields ,3,4,5,6.The peculiarity and complexity of the flux transfer are a bottleneck for the thermal conductivity thin-film material at atomic scale. Efforts can be made to research the influence factors and thereby improve the quality of such heat transfer efficiency. A multilayered film can be fabricated by a single sheet in a layer-by-layer assembly approach See in a. This no ~ 100) . The vaco ~ 100) ,32. Meano ~ 100) ,33. Someo ~ 100) ,35 and to ~ 100) , could bDue to the limitation of experimental methods and techniques, the structure with a continuous large individual GO sheet is difficult to synthesize. The GO film structure with gaps is more easily formed See in b 37]. I. I37]. IIn this article, we highlighted a theoretic formula framework based on Fourier law to decouple the effects of in-plane and out-of-plane on thermal transfer in overlap GO films model. The in-plane thermal property of GO has been studied in our previous work . In thisy-axis is set to 1, and the value of In this work, a novel effective theoretical formula of GO films thermal conductivity is deduced based on the Fourier law, systematically considering the heat transfer both in-plane and out-of-plane directions. The derivation of the formula is similar to the previous work on the thermal properties of graphene-based layered materials . Herein,Bring Equation (2) into Equation  and eliminate A general solution of Equation  isAs shown in Using Equation (7) and a few mathematical techniques to substitute for From the above relations, the effective thermal conductivity From Equation (9) it can be found that uctivity ,42. λ caThe existence of the constant 2 is due to the periodic conditions of RVE.In this work, we perform classical molecular dynamics (MD) simulations to study the t groups . The hydt groups  simulatit groups ,44 impleMD simulations are performed using the large-scale atomic/molecular massively parallel simulator (LAMMPS) ,46. The i-th slab and The system is equally divided into 100 thin slabs along the heat transfer direction. The heat source and sink slabs are located at the middle and the two ends of the model, respectively. The time step of 0.1 fs is selected for integration on the equations of atomic motion in the simulations. The system reaches the equilibrium state at 300 K in the Nosé–Hoover thermal bath for 0.2 ns. Then, the system exchanges the kinetic energies every 100 time steps between the coldest atom in the heated slab and the hottest atom in the sink source slab for 0.8 ns. The total heat flux 8 Wm−2 K−1), the corresponding model is the graphene overlap structure. The effect of thermal transfer between GO strengthened gradually with an increasing degree of oxidation. When 8 Wm−2 K−1. 8 Wm−2 K−1, whereas both the degree of oxidation and ratio of the vacancy defect are the maximum  to the out-of-plane direction, which results in the increment of the thermal conductance. The vector arrows among the overlap structure are intensive as the increasing ratio of single vacancy and a hydroxyl group with heat flux see a. Especi6 GWm−3 K−1, 6 GWm−3 K−1, −3 K−1, 6 GWm−3 K−1 by fitting the curves in To obtain the Another key point in this work is to find the optimal overlap length From the above discussion, us works , and the−1 K−1, 6 GWm−3 K−1, When the ratio of vacancy defect is constant, the overlap length corresponding to In summary, an analytical model is developed to investigate the thermal properties of overlap GO films. The influence of coupled hydroxyl group (the ratio


In the current research, the biodiesel was prepared from feedstocks of Neem oil and Karanja oil employing a single step direct transesterification method using acid-base catalysts simultaneously. The fuel properties of both Neem and Karanja biodiesel along with different biodiesel-diesel blends were studied and compared. Biodiesel produced from Neem oil was found better in terms of kinematic viscosity, calorific value and cloud point for all its blends with diesel compared to Karanja biodiesel-diesel blends. Experiments were conducted to study the effects of addition of graphene nano particles on fuel properties of biodiesel-diesel blends. The B20 biodiesel-diesel blend was selected, which was blended with graphene nano particles in different proportions  to get different stable and symmetric B20-nano blends. The fuel properties except kinematic viscosity were further improved with higher dosages of nano particles with the biodiesel-diesel blend. The performance and emissions tests were conducted on 4-stroke variable compression ratio diesel engine. Higher concentrated B20-nano blends of Neem (NOME20GO105) and Karanja (KOME20GO105) resulted in 31 and 30.9% of brake thermal efficiency, respectively, compared with diesel of 32.5%. The brake-specific fuel consumption (BSFC) was reduced by 10 and 11% for NOME20GO105 and KOME20GO105, respectively, compared to their respective B20 blends. Similarly, carbon monoxide (CO) was reduced significantly by 27 and 29% for NOME20GO105 and KOME20GO105, respectively.  The mixture was shaken well for proper mixing. Later boron triflouride (BF3), which acts as an acid catalyst, was added to the reactor containing methanol and sodium methoxide mixture. Finally, crude oil was added to the reaction mixture. The crude oil, methanol, sodium methoxide and boron triflouride were taken in the ratio 1:4:0.1:0.1, respectively. The reaction was carried out for just two hours at 50 °C. After 2 hours of direct transesterification, the reaction was stopped. The author used petroleum ether to separate biodiesel from the reaction mixture in his previous published research. The method described was not economical and was consuming more time when performing evaporation and condensation of petroleum ether . TherefoIn this study, Graphene oxide (GO) has been used as a fuel additive synthesized using chemical vapor deposition (CVD). GO possess excellent thermal and electrical conductivities, higher magnetism besides high surface area to volume ratio. It improves the convective heat transfer coefficient, calorific value, cetane number of fuel if it is blended with the fuel. Furthermore, it reduces ignition delay and enhances micro-explosion. 2 v−1 s−1, surface area 2630 m2g−1, thermal conductivity 3300 Wm−1K−1 and purity of 95–98.5%The size of GO NPs is 23–40 nm, Young’s modulus 1.0 TPa, an inherent mobility of 200,000 cmThe physico-chemical properties of Neem and Karanja and their respective methyl esters along with their blends have been found in accordance with the ASTM D6751 standards. Experiments were conducted on single cylinder four stroke variable compression ratio engine with electric starter connected to eddy current type dynamometer for loading as shown in Throughout the experiments, the compression ratio, fuel injection pressure and ignition timing were kept constant at 17.5: 1, 205 bar and 23° before top dead center, respectively. To record different emissions such as HC and CO, an AVL DiGas444G gas analyzer  was used, which works on a non-dispersive infra-red technology (NDIR). Smoke in the exhaust gas was measured by the Smoke Meter AVL . All the readings were noted after the engine is reached steady state. The engine was loaded with the help of an eddy current dynamometer. The uncertainties and accuracies of the measured parameters in the current investigations are given in R in Equation (3) reveals the function of X1, X2,……nX, and X1, X2,……nX indicates the no. of obtained readings. Therefore, The variance of the measured factors estimated utilizing Gaussian distribution as described in Equation (1), under the of limits confidence of ±2σ. The limit 2σ reflects the average bound of 95% of the calculated values.e limits .By utilizing Equation (4), the uncertainties in various quantified and specific variables were analyzed. The errors bars were by averaging 3 readings and included in all the engine characteristics using NOME, KOME and graphene oxide fuel blends.2/s) biodiesel is less by 4.97% compared to Karanja (5.0901 mm2/s) biodiesel at 40 °C as shown in The calorific values of diesel, Neem biodiesel and Karanja biodiesel are 45,369, 40,050 and 39,538 kJ/kg, respectively, and are shown in The nano-additives are used to improve some of the biodiesel fuel properties, which further improve the performance of the engine besides reduced emissions with no or little engine modifications. Nano particles (NPs), which possess enhanced surface area/volume ratio, also act as a catalyst. The higher surface area/volume ratio improves the quality of air and fuel mixture and, hence, increases the rate of chemical reaction during combustion . Higher In the current investigation, an ultrasonication technique is used to stably blend the nanoparticles in the fuel blends. Initially, different concentrations of nanofluids are prepared using bath and probe sonication method. The nanofluids are blended with a diesel–biodiesel (B20) blend, initially the nanofluids  are transferred to diesel–B20 fuel blend, to remove excess moisture, the blends are heated at 60 °C using magnetic stirrer; later, it is sonicated for one hour using bath sonication and, finally, the nano fuel blends are stably dispersed using probe sonication at a frequency of 15–20 Hz for 20 min for each fuel blend. These steps enable stable dispersion of nanoparticles in fuel blends, thus preventing the nanoparticles from agglomerating. Ultrasonication technique avoids the agglomeration to the larger extent and, hence, is used in the present research for the preparation of NPs dispersed in base biodiesel fuel. Metal based nano additives results in agglomeration, clustering and settling due to their short-term stability. Hence, in the present research, the author has used graphene oxide, a carbon-based NP, to make the Biodiesel-Diesel-Nano blends. Nano additives are blended with a B20 biodiesel-diesel blend in the three different dosage levels of 35, 70 and 105 ppm. They are denoted as NOME20GO35, NOME20GO70 and NOME20GO105 for Neem Biodiesel-Diesel-Nano blends. Similarly, KOME20GO35, KOME20GO70 and KOME20GO105 are for Karanja biodiesel-diesel–graphene oxide nano blends.Higher kinematic viscosity of the fuel affects the fuel supply system in the engine. The fuel injection system and charge preparation are reliant on density, volatility and viscosity of fuel, which are often interdependent. The kinematic viscosity of biodiesel-diesel blends increase with the addition of nano additives as shown in The variation of brake thermal efficiency (BTE) with load for diesel, NOME20, KOME20, along with different dosage levels  of graphene oxide NPs has shown in The brake-specific fuel consumption (BSFC) is an important performance parameter of an IC engine. Hydrocarbons are the result of incomplete combustion of the fuel. Diesel engines are the major source of formation of smoke and are more spontaneous at maximum engine load conditions, which can be observed from Unavailability of sufficient quantity of oxygen for combustion inside the combustion chamber is the main reason for incomplete combustion and, hence, formation of CO. The formation of CO with brake power for different fuel blends has been shown in The current research is a comparison of fuel properties biodiesel and their blends prepared by using the non-edible feedstocks of Neem and Karanja by employing direct transesterification process. Furthermore, B20 biodiesel-diesel blends were blended with graphene oxide NPs in different proportions. The following points are drawn from the results.Biodiesel prepared by direct transesterification reduced the reaction timing.Neem biodiesel is slightly better compared to Karanja in terms of calorific value, kinematic viscosity. There is a marginal difference in cloud point and flash point.The fuel properties of both blends improved with addition of higher dosages of graphene oxide nano particles.Maximum BTE of diesel, NOME20GO105 and KOMEGO105 is 32.5, 31 and 30.9%, respectively, at full load.BSFC for NOME20GO105 and KOME20GO105 was reduced by 10 and 11%, respectively, compared to NOME20 and KOME20.CO for NOME20GO105 and KOME20GO105 was reduced by 27 and 29%, respectively, compared to NOME20 and KOME20.BTE, BSFC, CO, HC and smoke were slightly better for the diesel-fueled engine compared to NOME20GO105 and KOME20GO10 run engines.


Lactate is an important organic molecule that is produced in excess during anaerobic metabolism when oxygen is absent in the human organism. The concentration of this substance in the body can be related to several medical conditions, such as hemorrhage, respiratory failure, and ischemia. Herein, we describe a graphene-based lactate biosensor to detect the concentrations of L-lactic acid in different fluids (buffer solution and plasma). The active surface (graphene) of the device was functionalized with lactate dehydrogenase enzyme using different substances  to guarantee stability and increase selectivity. The devices presented linear responses for the concentration ranges tested in the different fluids. An interference study was performed using ascorbic acid, uric acid, and glucose, and there was a minimum variation in the Dirac point voltage during detection of lactate in any of the samples. The stability of the devices was verified at up to 50 days while kept in a dry box at room temperature, and device operation was stable until 12 days. This study demonstrated graphene performance to monitor L-lactic acid production in human samples, indicating that this material can be implemented in more simple and low-cost devices, such as flexible sensors, for point-of-care applications. Lactate is present in various biological processes and medical conditions, and it has an important role in the anaerobic metabolic pathway ,2. UsualMost of the reported biosensors used to detect lactate concentration depend on a specific enzyme to catalyze the reaction and enhance the selectivity; the product of the reaction is usually proportional to the analyte concentration ,9,10,11.In the clinical environment, lactate concentration is commonly measured using a blood gas analyzer that is large bench-top point-of-care (POC) equipment and it has certain limitations regarding processing time and sample preparation ,8,24. ThGraphene is a single-layer material with a large surface area and paramount electronic and chemical properties that can increase the sensitivity and specificity of a biosensor when applied as an active layer ,35,36,37In this study, we present a multiplexed solution-gated graphene-based field-effect transistor sensor to determine the concentration of lactate in different fluids. The lactate was detected using the LDH enzyme to catalyze the chemical reaction in the biological fluid. The enzyme was immobilized using different substances to guarantee stability and selectivity over the active layer. The transfer characteristics of the common-gate GFET were measured for each concentration in different electrolytes (buffer solution and blood plasma). The selectivity was verified using interference substances , and the stability of the GFET devices was monitored for up to 50 days.AZ-1512, SU-8 2075, hexamethyldisilazane (HMDS), and (1-methoxy-2-propyl) acetate (SU-8 developer) were purchased from MicroChem Corp. . Polydimethylsiloxane (PDMS) (Sylgard 184 A/B) was obtained from Dow Corning . Graphene layers grown by chemical vapor deposition (CVD) were acquired from Graphenea . Human plasma, uric acid, ascorbic acid, iron (III) chloride powder, and phosphate-buffered saline were supplied by Sigma-Aldrich Corp. . Illustrations were created with BioRender.com.The fabrication of the coplanar electrodes on a glass substrate was reported in our previous works . Briefly, the glass substrate was spin-coated with the adhesion promoter (HMDS) and with the positive photoresist (AZ-1512). The electrodes were patterned using a photomask and ultraviolet radiation over the glass by a mask aligner . The titanium and the gold layers were deposited over the glass using a vacuum thermal evaporator system (Evaporation System SHE-6T-350D). The CVD-grown graphene coated with a PDMS layer was transferred onto the region between the eight pairs of electrodes (source and drain) after the copper layer was etched away, and the supportive layer (PDMS) was detached from the graphene during an acetone bath. The PDMS microfluidic channels containing eight channels were bonded onto the substrate by UV–ozone treatment. In The electrode array was assembled with PDMS channels and the active layer surface was treated with different substances to guarantee stability and selectivity of the device. The enzymatic matrix was fabricated using Nafion, chitosan, lactate dehydrogenase enzyme, and glutaraldehyde. First, 3 µL of Nafion (NA) was drop-casted over the graphene for 1 h, after which the channels were washed with phosphate-buffered saline (PBS). Nafion is a sulfonate fluoropolymer–copolymer and is used as a membrane to block anionic interference species, such as ascorbic acid ,44,45,46Dirac) of the bare graphene was determined considering the lowest drain current (ID) measured while applying a constant bias (VD) on the drain terminal of 0.4 V and varying the bias (VG) applied to the gate. The microchannels were cleaned with deionized water and dried in an oven after the characterization of the bare devices. Then, the enzyme was immobilized following the steps mentioned in the previous subsection to produce the immobilization matrix. The transfer characteristics of the graphene layer were verified using an HP Agilent 4145B Semiconductor Parameter Analyzer  while using the buffer solution (PBS) as an analyte. The Dirac point voltage  to focus on clinical applications. A schematic of the experiment is illustrated in Dirac of 1 V (standard deviation (SD) of 0.03 V). The same was reproduced for the individual gate structure, with a mean VDirac of 1.03 V (SD of 0.06 V). The Dirac point voltage values of each structure are presented in The measured transfer characteristics of the graphene-based devices were used to define reference points before the immobilization matrix formation and the lactate assays. In our previous work, a solution-gated GFET with a coplanar electrode array was developed for cancer diagnostics . The intAfter electrical characterization, all the microchannels were cleaned with deionized water and the lactate detection assay was performed. First, the enzyme was injected over the graphene surface into the eight microchannels. The device was placed in the probe station, and different concentrations of L-lactic acid (0.25 to 10 mM) were mixed with the LDH enzyme, considering that the reference line for bare graphene was measured with the buffer solution only. The concentration range was defined based on the reference values used to detect sepsis shock in blood samples, since lactate is a prognosis marker among septic shock patients ,7. Dirac are shown in Dirac values demonstrate a linear relationship with lactate concentration in 2 = 0.8549). Adding high concentrations of L-lactic acid in the channel with the presence of the LDH enzyme causes the production of more positive and neutral charges (NADH and H+), then the neutrality point is higher when the concentration increases [The Dirac point voltages were measured after the LDH enzyme catalyzed L-lactic acid, resulting in a product that doped the graphene surface with negative charges, as shown in Dirac obtained with bare graphene. Based on the results presented in Dirac only shifted without the presence of chitosan. However, the matrix was still too thick for the microchannel; some devices experienced issues regarding the flow of analyte in the channel, even after drying them for more than 60 min. To overcome this clogging issue, a cleaning procedure with deionized water was repeated more than three times after the deposition of each component in the matrix. New devices were fabricated to evaluate the proposed immobilization matrix. It is necessary to reduce the number of layers over the active layer to avoid clogging the microchannels. Since Nafion and glutaraldehyde are essential to blocking the determined substances, we verified the performance of the enzyme with and without chitosan, since this material is an electron mediator that can improve the LDH enzyme immobilization capacity ,19,48. ADirac values of devices measured during the 50 days are shown in Dirac increased greatly after 12 days of measurement, as did the number of failed devices. The instability of the enzyme can be considered a drawback of the method presented in this study, and it is necessary to evaluate a new storage approach to guarantee the enzyme function for at least 6 months, since the device will be adapted as a point-of-care sensor in future study.Considering the final immobilization matrix composed of Nafion, chitosan, LDH, and glutaraldehyde, stabilization of the enzyme was verified for up to 50 days while the devices were kept in a dry box at room temperature. In this study, we tested the storage of the devices in a refrigerator and a dry box, although the devices maintained in a refrigerator had no electrical response even after 2 days of storage. We decided to proceed only with the devices stored in the dry box. For each day measured, 5 mM of L-lactic acid were injected into the microchannel, and the signal response was recorded. The VDirac increased from the lowest to the highest concentration in blood plasma samples. For each concentration, three channels were used to obtain the error bars. The variation of the VDirac for the selected biological fluid is presented in 2 = 0.9787, n = 3). Compared with the works in the literature that also used human plasma samples, the linear response observed with the graphene-based device was similar or superior, and we also investigated a wider range of concentrations [Previously, the performance of common-gate GFET devices was verified using the proposed immobilization in buffer solutions with different concentrations of L-lactic acid. Then, blood plasma was used as an electrolyte to confirm the capability of the device to detect lactate in real human samples. First, blood plasma was injected (10 µL) into the channels after being mixed with different concentrations of lactate, from 0.25 to 7.5 mM, based on the reference values in the literature considering clinical emergencies where the level of lactate is abnormal . As obsetrations ,51. BesiDirac of the buffer solution was used as a reference point. The plasma sample containing 7.5 mM of L-lactic acid was injected into the same microchannel as when the transfer characteristics were measured; this procedure was repeated for each interference species with concentrations that were considered for a normal blood sample from a healthy human. New experiments were performed to evaluate the interference from electroactive species in the human samples ,21. The Dirac values are presented in The GFET devices were measured, and the output signals were analyzed, and the VDirac, indicating a limitation of this enzyme with the respective immobilization matrix. Additionally, different interference substances  were injected into the GFET devices during the lactate assays to verify the selectivity of the sensor. The variation of VDirac was significant after injection of the plasma sample with 7.5 mM of lactate considering the reference point (PBS solution), and the maximum shift observed was 0.09 V among interference substances. Considering the results obtained in this study, graphene demonstrated the potential to detect L-lactic acid in human fluids without the interference of other biochemicals. To the best of our knowledge, a GFET device to detect the concentrations of lactate in human samples had not been reported, also considering the combination of elements in the immobilization matrix. With the implementation of low-cost materials to our proposed method, it is possible to detect lactate in a fast and precise diagnostic method for clinical applications taking advantage of the properties of the graphene-related materials, e.g., graphene oxide and reduced graphene oxide. The next step of this study is to include two new analytes to increase the prediction of a sepsis shock by developing a flexible, low-cost, and disposable chip for a point-of-care device with a rapid and accurate response.We demonstrated in this study a graphene-based biosensor to provide rapid quantitative measurement of lactate in the buffer solution and blood plasma. Determination of L-lactic acid was performed by measuring the transfer characteristics of graphene in a field-effect transistor structure with different concentrations of the analyte. Different materials were tested for the immobilization matrix to guarantee the stabilization and selectivity of the device; the best combination includes Nafion, chitosan, lactate dehydrogenase, and glutaraldehyde. The proposed immobilization matrix was structured considering the role of each biochemical to improve the performance of the graphene-based sensor. After the measurements, a linear response was observed for all the samples, including the blood plasma, with different concentrations (from 0 mM to 7.5 mM). The capability of detection of lactate at high concentrations (up to 7.5 mM) indicates that the proposed device can be implemented for POC applications for different disorders, such as septic shock and left ventricular failure. The presence of chitosan was essential to maintain the stability of the LDH enzyme, which was evaluated through the electrical measurements after storing the GFET devices at room temperature in a dry box for up to 50 days. After 12 days, there was a significant shift of V


Finding prey is essential to survival, with marine predators hypothesised to track chemicals such as dimethyl sulfide (DMS) while foraging. Many predators are attracted to artificially released DMS, and laboratory experiments have shown that zooplankton grazing on phytoplankton accelerates DMS release. However, whether natural DMS concentrations are useful for predators and correlated to areas of high prey biomass remains a fundamental knowledge gap. Here, we used concurrent hydroacoustic surveys and in situ DMS measurements to present evidence that zooplankton biomass is spatially correlated to natural DMS concentration in air and seawater. Using agent simulations, we also show that following gradients of DMS would lead zooplankton predators to areas of higher prey biomass than swimming randomly. Further understanding of the conditions and scales over which these gradients occur, and how they are used by predators, is essential to predicting the impact of future changes in the ocean on predator foraging success. Kylie Owen et al. sample concurrent prey biomass and natural dimethyl sulfide (DMS) concentration, and show that these variables are correlated in air and seawater. Agent simulations show that following fine-scale gradients of DMS would lead zooplankton predators to higher prey biomass, shedding light on how marine predators may use these cues for foraging. The chemical compound that has received the most attention as a potential foraging cue is dimethyl sulfide (DMS). DMS in the ocean (DMSaq) and atmosphere (DMSg) are produced by the breakdown of dimethylsulfoniopropionate (DMSP), which is synthesised by phytoplankton4. DMS is the largest natural source of atmospheric sulfur and it plays an important role in cloud formation and likely in climate regulation5. Data collected in the context of climate science has shown that high concentrations of DMS occur in regions characterised by high productivity, being correlated to multiple factors such as chlorophyll-a concentration and mixed layer depth7. However, no previous research has determined whether natural DMS concentrations could accurately convey information on prey patch quality to a predator.Marine predators face the challenge of finding prey resources that are patchy and ephemeral in nature. Odour landscapes have been proposed as a strategy that animals may use to locate prey in a vast and largely featureless ocean7, with changes shown in response to meteorology, solar radiation, and phytoplankton photophysiology8. Many biotic  and abiotic (e.g. temperature changes) factors are known to influence DMS production10. In addition, grazing by microzooplankton has also been shown to play a large role in DMS concentration in surface waters in some regions11. These species are too small to be prey for many large predators such as marine mammals, seabirds, and turtles that have been the centre of most behavioural research on the role of DMS in foraging. The high number of factors that influence DMS production, and large spatial and temporal variation in DMS concentration, may result in DMS being an uninformative foraging cue for predators. Despite all of the biological and oceanographic drivers of variability in DMS, for zooplankton predators to rely on DMS as an accurate foraging cue, an association between DMS concentration and prey biomass is essential.DMS concentration has been shown to be highly variable over both space and time14. In addition, many marine predators, such as fish15, turtles16, marine mammals17 and seabirds18 have been shown to be attracted to artificially released DMS. This has led to the theory that zooplankton predators use this chemical to locate prey1 and the hypothesis that the attraction of predators to areas of DMS concentration may be the link between the sulfur, carbon and iron cycles in high latitude oceans19. However, the majority of studies on the behavioural response of predators to DMS have been completed in captivity or involved artificially introducing DMS into the environment19. To the best of our knowledge, the relationship between prey biomass and natural DMS concentration has never been compared in the field.Zooplankton grazing on phytoplankton has been shown to accelerate the rate of DMS release in laboratory experimentsg and DMSaq) and prey biomass (zooplankton and fish), at a scale that is likely to be relevant to a foraging predator. Our aim was to determine whether there was a correlation between prey biomass and DMS and whether gradients of DMS existed at fine-scales, that could be utilised by predators to increase the prey biomass encountered while foraging. We found that zooplankton biomass and DMS concentrations in air and seawater are correlated and that smooth gradients of DMS exist at fine spatial scales that would lead zooplankton predators to areas of higher prey biomass in the ocean.Here, we present concurrent high-resolution measurements of multiple phases of DMS (DMSaq and DMSg) and prey (zooplankton and fish) biomass during surveys conducted during day time hours from Chatham Harbor, Cape Cod, MA, USA. These surveys covered a total of 220.2 km over 5 days in June 2019 ), and the DMSaq concentration ranged from 1.3 to 19.5 nM (mean 8.0 ± 5.6 nM)   for zooplankton and 55–910 m2 nm−2 (mean 153 ± 141 m2 nm−2) for fish. The concentration of both DMSaq  and DMSg  were found to be positively correlated to the biomass of zooplankton , there were rarely times when zooplankton biomass was low (close to zero)  and DMSg   ranged from 0.0 to 464.3 mton Fig. . Althougro) Fig. . In contro) Fig. . The bio05) Fig. .Fig. 4ThIn order to test whether DMS gradients could be used by predators to locate areas of higher zooplankton biomass, despite the high spread in the data, we utilised agent simulations, where agents acted as pseudo-predators. There were two scenarios tested: (1) a tracking DMS experiment, where each agent was instructed to follow the DMS gradient, moving to the neighbouring cell with the highest DMS until a local maximum was reached, and (2) a random movement experiment, where agents were instructed to move randomly for the same length distribution as agents in the tracking DMS experiment. The distribution of the track lengths for the random movement experiment were limited to the same distribution as the tracking experiment for two reasons: (1) to ensure that the search effort was the same for both the random movement and tracking DMS experiments, and (2) as the maximum prey biomass encountered along each track was selected, having no limits on the length of the random movement tracks would allow all random agents to ultimately find the cell with the highest prey biomass in the entire area. The highest prey biomass encountered along all of the tracks was selected, as this is more likely to reflect true predator behaviour, with animals ending their search and beginning to feed as soon as prey is located. For the random tracking experiment, 1000 iterations were completed, and the mean of the maximum prey encountered across the 1000 tracks was compared to the tracking DMS experiment. Only one iteration was needed for the tracking DMS experiment as this is deterministic.aq concentrations lead agents to areas that had significantly higher prey biomass compared to when the agents moved randomly   .Tracking DMS16) Fig. . However74) Fig. . When tr74) Fig. . Across 10, and that DMS was sampled at a single depth while prey biomass was integrated over a larger depth range. Nonetheless, the occurrence of higher zooplankton biomass in regions of higher DMS suggests that natural concentrations of this chemical would allow DMS to act as a valuable foraging cue for zooplankton predators. Many marine predators, such as fish20, marine mammals21 and sea birds22 forage primarily on zooplankton, ranging from copepods to krill. Previous research has shown that attraction towards areas of DMS release is stronger in zooplankton predators compared to higher trophic level predators23. The lack of a positive correlation between fish biomass and DMS concentration may explain why higher trophic level predators show a weaker or no response to the chemical23. It is possible that time lags between DMS production and the build-up of fish biomass, or the diffusion of the chemical both spatially and temporally, may result in DMS being a valuable foraging cue only to zooplankton predators that will find higher prey biomass in areas of higher DMS concentration.We show that a correlation between zooplankton biomass and the concentration of DMS in air and seawater occurs in the ocean. Although it has been suggested that DMS is likely to be a useful foraging cue for marine predators, to the best of our knowledge, no previous research has demonstrated that higher concentrations of DMS are linked to higher prey biomass. The observed correlation between prey biomass and DMS is notable, especially given the many factors that influence the production and concentration of DMS24, likely relying on a combination of senses to assist with finding the highest density prey patches in the localised area25. It is unlikely that DMS is used by predators in isolation, with predators using information from a number of sensory cues at different scales to reduce foraging effort. Examining how animals respond to the gradients of this chemical could provide much-needed information on the sensory capabilities of many marine predators. In addition, understanding the scales over which these gradients provide useful information on prey quality could also increase understanding of the scales at which different senses are used for foraging.Higher prey biomass is likely to be a high source of DMS, which would then cause DMS to diffuse into the surrounding area and make the area of elevated DMS larger than the prey biomass area. We observed with the agent simulations that tracking gradients of DMS lead predators to areas that were characterised by higher DMS, but not necessarily the highest prey biomass in the area. Once in an area of high DMS and elevated prey biomass relative to the surrounding area, zooplankton predators may then initiate area-restricted search behaviourg in the air as they forage over the ocean surface26. Here we have shown that tracking DMSaq concentration would lead predators to areas of higher zooplankton biomass, but no significant benefit of tracking DMSg was found. We also observed that DMSaq lead agents to significantly higher prey biomass than DMSg, which was unexpected given the similarity in the strength of the correlation observed between prey biomass and both phases of DMS. We speculate that the difference in the success of DMS as a foraging cue in seawater vs. air is the result of the higher mobility of DMSg in wind, relative to the movement of DMSaq in seawater, making the spatial overlap between DMSg and prey biomass less reliable. In addition, the data used for creating the DMS maps took 8 h to collect, thus, especially for DMSg, it cannot be considered an instantaneous measurement. On the day of sampling, the wind direction was from west to east, which is evident in the eastward shift in the distribution of DMSg relative to DMSaq  tropical open oceans30. However, DMSg concentration has been reported to be higher at night32, likely due to photochemical decomposition of DMSg during the daytime. Our findings of a correlation between zooplankton biomass and DMS concentration suggest that variation in zooplankton grazing pressure in surface waters during the day and night may also influence DMS concentration throughout the day. Higher DMS concentrations at night could be a benefit to predators that may rely on vision to forage during the day and then switch to a stronger reliance on chemical cues in the dark. If animals can sense DMSaq, then the deeper exploratory dives completed by marine predators may be a mechanism to assist predators to locate zooplankton at depth. Understanding whether the exploratory diving behaviour of marine predators is also linked to DMS concentration may then provide insight into the energetics of marine predator foraging, particularly for air-breathing predators that may choose to continue or stop an exploratory dive based on the chemical information obtained during the dive.In this study, we focused on surface prey (in the upper 10 m) based on an assumption that prey in the upper water column would be most strongly associated with surface DMS25. It is worth noting that the results of this study were based on 5 days of field research in one location. Therefore, a priority for further research should be collecting additional data on concurrent DMS concentration and prey biomass in order to validate that the results are consistent in other conditions and locations. Much further research is required to fully understand the conditions under which these gradients occur, and the full range of oceanographic and biological factors that influence the strength of the correlation. For example, phytoplankton species composition is known to influence DMS production, and this factor may explain why in some areas prey biomass was high, while DMS concentration remained low. Alternatively, time lag effects could also play a role in explaining this pattern, with zooplankton (being longer-lived) biomass often remaining high long after phytoplankton has been consumed, and DMS has presumably dispersed. The variation in DMS concentrations observed in this study was relatively high despite the small observation area. This highlights the need for further fine-scale measurement of these spatially and temporally dynamic chemicals in order to fully understand their role in foraging ecology. Measuring the movement and distribution of marine predators in relation to natural DMS concentrations would provide some insight into whether predators respond to these gradients. Given the assumed link between DMS and global climate, understanding the role that DMS plays in predator foraging behaviour is essential to achieving an understanding of how the foraging success of predators may vary in changing oceans.We show that the link between DMS concentration and prey biomass that is necessary for predators to use DMS as an accurate foraging cue occurs in the ocean. This is a necessary condition to investigate which predators use these gradients to find prey and the scales over which DMS may be important to predators. How the use of these gradients interplays with other sensory modalities also needs further investigation, as it is likely that different senses are used at different scalesMV Noah departing from Chatham Harbor, Cape Cod, MA, USA, during daylight hours from the 23 June to 28 June 2019. This is a known area of high primary productivity and a hotspot for baleen whale foraging activity33.Data collection occurred over 5 days on board the aq) and air (DMSg) were conducted using a sequential vapour generation chemiluminescence instrument34. Water was collected from 1 m depth and approximately 1 m beside the vessel into an overflow tank with an exchange rate of 12 L min−1. We used a high flow-through pump and water in the overflow tank was replaced every couple of seconds. Air samples were collected via an inlet filter and a Teflon tube, attached to the roof of the pilothouse, well in front of the exhaust at the stern, in order to avoid contamination. Water samples were analysed for DMSaq and gas samples were analysed for DMSg automatically as distinct samples at a 7.5 min interval following a previously developed protocol34.Measurements of DMS in seawater  six transects in a lawn mowing pattern to obtain a 2D distribution of DMS concentration in order to conduct agent simulations, (2) targeting high and low productivity patches (based on echosounder data and observations of predators foraging in the area) in an attempt to cover a full range of possible prey biomass values in the area, (3) a drifting experiment using a sea anchor to stay in the same water mass and (4) a long-transect offshore and back. All data were used to assess the overall correlation between DMS and prey biomass.35.Concurrent prey measurements were conducted using a multi-frequency down-looking echosounder . The EK80 was operated in narrowband mode and both the EK80 and ES60 echosounders operated with ping rates of 0.5–2.0 Hz (depending on bathymetry), pulse lengths of 256 microseconds, and output power of 500, 250 and 100 W  during the surveys. Echosounders were mounted on the port side of the vessel with the transducer faces at a depth of 0.5 m. All frequencies were calibrated during the study using a 38.1 mm diameter Tungsten carbide sphere37. To ensure that our acoustic measure of small zooplankton biomass was not including backscatter from individual or aggregations of fish, we identified regions of backscatter consistent with fish (volume backscattering strength > −70 dB) using the 38 and 200 kHz echograms, and then removed those same regions from the 710 kHz echogram before additional analysis. This procedure may lead to underestimates of zooplankton biomass as we may exclude some regions that had both copepods and fish present.Because different organisms, such as small zooplankton and fish, scatter sound with different efficiencies depending on the acoustic frequency used, we used data from all three frequencies to measure the backscatter from small (<5 mm length) crustacean zooplankton such as copepods. In general, 38 kHz systems can detect fish with swim bladders, 200 kHz systems can detect fish without swim bladders and large mesozooplankton (such as krill > 20 mm), but neither can detect small copepods. However, small zooplankton produces measurable backscatter at 710 kHz2 nm−2, a parameter widely used in fisheries acoustics as a proxy for zooplankton or fish biomass38), for each 8 m vertical by 100 m horizontal bin. Fish biomass was similarly estimated using the 38 kHz vertically integrated NASC data. We conducted three net tows using a 50 cm diameter, 2.5 m long ring net with 200-micron mesh towed 1–2 m below the surface for 2–5 min duration to collect ground-truth data on the fish and mesozooplankton present. Small copepods (lengths < 3 mm) were the most abundant taxa, although cladocerans, fish eggs and other small crustaceans were also caught. Pelagic fish aggregations were mostly mackerel which were caught by hook and line during breaks in the vessel survey operations.Because of this approach, we were limited in analysing data from regions where we had both 38 and 710 kHz data. The 38 kHz system has a larger nearfield region (roughly 2 m from the transducer) so we were limited to analysing backscatter data from 3 m and deeper. Since DMS measurements were made in the upper water column, we analysed acoustic data from 3 to 10 m water depths to try to best match the sampling region while also measuring sub-surface zooplankton aggregations. Backscatter at 710 kHz was binned  to roughly match the DMS sampling resolution and then vertically integrated (depths from 3 to 10 m) and converted to a zooplankton biomass value as measured by the Nautical Area Scattering Coefficient . As the prey biomass data were not normally distributed, correlations were computed using a two-sided Spearman’s rank-order correlation within the corr function in Matlab (version R2019a). Significance was determined at 2 area was sampled as a series of six transects and interpolated to a grid cell size of 0.0025°, equal to 208 m. Agent simulations were also computed in Matlab using the DMS maps and were used to represent pseudo-predator movement through the area39. We completed two experiments: (1) a tracking DMS experiment, and (2) a random movement experiment. As a starting point, one agent was placed in every grid cell (n = 783) across the whole area. In the tracking DMS experiment, each agent was instructed to move from their initial position to an adjacent grid cell with the highest DMS concentration (DMSaq or DMSg) out of the eight grid cells adjacent to its current position. This behaviour was repeated until they reached a local maximum, where all of the adjacent grid cells had a lower DMS concentration than the current grid cell. We expect real predators would stop their search behaviour, with their movements no longer following the DMS signal, when prey is located, as opposed to the agents that continued searching until a local maximum in DMS was reached. As a result of this, the grid cell along the path that had the highest prey biomass was labelled and used to derive a frequency distribution of maximum prey biomass encountered when tracking DMS. In addition, a random movement experiment was completed, where an agent started in each grid cell but was instructed to move randomly. The length of the tracks for the random movement experiment was pulled from a distribution that was equal to the distribution of track lengths in the tracking DMS experiment. This was to prevent random agents from searching the entire area and always encountering the highest prey biomass in the area and to ensure that the search effort by agents was consistent in both experiments. The tracking DMS experiment was only run once, as it is deterministic, however, the random experiment was completed 1000 times from each grid cell. The maximum prey biomass encountered was recorded for each of the 1000 random tracks from each cell. The mean of the maximum prey encountered over these 1000 iterations was then compared to the maximum prey encountered in the tracking DMS experiment.DMS and prey biomass maps (Supplementary Data g and DMSaq (n = 783). In addition, the same test was used to compare the mean of the maximum prey biomass encountered when the agents followed DMSg to the mean of the maximum prey biomass encountered when following DMSaq.Since prey biomass measurements are likely to be spatially correlated and thus individual grid cells are not necessarily independent, and agents beginning in the same grid cell (random movement and tracking DMS) could be considered paired samples, non-parametric Wilcoxon signed-rank tests were completed in R Statistical Software (version 3.6.1) to compare the mean of the maximum prey biomass encountered by agents moving randomly, to the mean of the maximum prey biomass encountered when the agents followed either DMSSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


The “distal axonopathy” hypothesis in amyotrophic lateral sclerosis (ALS) proposes that pathological changes occur at the neuromuscular junction (NMJ) early in the disease. While acetylcholinesterase (AChE) plays an important role in the functionality of the NMJ, its potential role in ALS remains unexplored. Here, we identified AChE as a limiting factor regulating muscle/motor neuron connection in a vertebrate model of ALS. Knockdown of the TAR DNA-binding protein 43 (TDP-43) orthologue in zebrafish resulted in early defects of motor functions coupled with NMJ disassembly. We found that a partially depleted tdp-43 caused a decrease of ache expression. Importantly, human AChE overexpression reduced the phenotypic defects in the tdp-43 loss of function model, with amelioration of post- and pre-synaptic deficits at the NMJ. In conclusion, our results provide a better understanding of the role of TDP-43 in the NMJ organization and indicate AChE as a contributing factor in the pathology of ALS. In particular, it may be implicated in the early defects that characterize NMJs in this major neurodegenerative disorder. TARDBP [TARDBP mutations in 2008 [Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem disease caused by progressive loss of both upper and lower motor neurons . The patTARDBP , in the TARDBP ,4. The d in 2008 ,6 brough in 2008 ,8 able t in 2008 . Togethe in 2008 ), TDP-43G93A mouse, which replicates several key features of ALS, reduced NMJ complexity at presymptomatic stages has been described, corresponding to an augmented number of disassembled acetylcholine (ACh) receptor clusters on muscle from the mutant SOD1 transgenic mice [Increasing evidence shows early morphological and biochemical changes at the neuromuscular junction (NMJ) before complete muscle fiber denervation and functional symptom onset in ALS patients ,12. In pnic mice .Structurally, the NMJ is a highly complex cholinergic synapse where the majority of the molecules responsible for its formation are also crucial for its stability and function at the adult stage .In this context, the enzyme acetylcholinesterase (AChE) is a master regulator of the signal transduction. In mammals, AChE exists as three distinct variants coming from alternative exon splicing  ,16,17. TIn addition, the enzyme has several non-classical roles in neuronal regeneration and development, as demonstrated in dopaminergic neurons ,25; in rDanio rerio) permit the overcoming of these issues since this vertebrate does not appear to express a functional bche and has not been reported to undergo alternative splicing at the respective 5′ or 3′ ends [In mammals, our understanding of AChE cellular mechanisms is complicated due to the presence of butyrylcholinesterase (BChE), another enzyme able to hydrolyze ACh, although less effectively , and thr 3′ ends . Thus, i 3′ ends ,36, consSeveral studies on pathological tissues ,38,39 anData summary: In this study, we describe for the first time a functional link between tdp-43 and ache in zebrafish. For this purpose, we employed the zf model previously described by our team , where kDanio rerio) were maintained at the ICM  fish facilities and bred according to the National and European Guidelines for Animal Welfare. Experiments were performed on wild-type embryos from AB and TL genetic strains. Zebrafish stored in incubators at 28.5 degrees remain at embryonic stages until approximately the end of the third day post-fertilization, before they hatch out of the chorion [Adult and larval zebrafish  Nonidet P−40/0.5% (w/v) Triton X-100 supplemented with a cocktail of protease inhibitors . Fish were then sonicated and centrifuged at 14,000× g at 4 °C for 20 min. Supernatants were collected and frozen at −80 °C until biochemical analysis.A morpholino, binding to the ATG sequence, responsible for translational initiation, was designed to knockdown (KD) tardbp  and AChE gene . A control morpholino (mis-std), containing mismatch nucleotides and not binding anywhere in the zebrafish genome, was used to assess the specificity of the observed phenotype (5′-CCTCTTACCTCAgTTACAATTTATA-3′). Human TARDBP wild-type (WT) mRNA (WT-TDP-43) was transcribed from NotI-linearized pCS2þ using SP6 polymerase with the mMESSAGE Machine Kit . AChE-T plasmid (WT-AChET) under the cytomegalovirus (CMV) promoter–enhancer was a generous gift from Dr. J. Saez Valero  . MicroinZebrafish embryos at 48 hpf were analyzed to identify any major morphological abnormalities . Subsequently, embryos that did not display any developmental abnormalities were touched lightly at the level of the tail with a pipette tip in order to evaluate their locomotor behavior. TEER episodes were performed only in zebrafish that appeared morphologically normal and were recorded with a Grasshopper 2 Camera  at 30 Hz. The videos were then analyzed using the manual tracking plugin of ImageJ 1.45r software as previously described .Animals were fixed in 4% paraformaldehyde for 3 h at room temperature. After fixation, the embryos were rinsed several times with PBS and then incubated in PBS containing 1 mg/mL collagenase  to remove skin. The collagenase was washed off with PBS Triton X-100  and heads were cut away. After an incubation of 30 min in blocking solution , the embryos were incubated overnight at 4 °C in synaptic vesicle 2  antibody diluted in blocking solution. The embryos were then washed and incubated for 30 min in PBST containing α-bungarotoxin conjugated to Alexa 488 . The embryos were rinsed several times with PBST and then incubated in freshly prepared block solution containing a secondary antibody  for 3h at RT before mounting on glass slide in 50% glycerol. The NMJs were visualized using a Leica SP8 Inverted scanning confocal microscope. All captured images of stained embryos were processed using Imaris Image Analysis software and ImageJ.4, and 5 mM potassium ferricyanide (pH 6.0). The reaction was stopped with washes in PBS. Then, the sections were dehydrated, cleared with xylene, coverslipped, and imaged with the Nanozoomer scanner .The Karnovsky and Roots method adapted for zebrafish was used . OptimalA modified micro assay version of the colorimetric Ellman’s method was used to measure AChE . A totalTotal RNA was isolated from injected fish using TRIzol Reagent (Sigma) according to the manufacturer’s protocol. First-strand cDNAs were obtained by reverse transcription of 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Roche), according to the manufacturer’s instructions. Quantitative PCR amplification was performed with SyBer2X Gene Expression Assays using the primers listed in p < 0.05.All data values for the zebrafish and cells experiments are expressed as mean with error bars representing standard errors of mean (SEM) with significance determined using one-way ANOVAs. Differences between groups were identified via Bonferroni post hoc comparisons. All analyses were performed using Prism 5.0 . Significance level was set at n = 12 for tdp-43 KD, vs. 15.82 ± 3.786, n = 10 for mis-CTRL, p < 0.05; sv2 orphans: 47.67 ± 4.206, n = 9 for tdp-43 KD, vs. 31.41 ± 4.311, n = 9 for mis-CTRL, p < 0.05; Several genes mutated in ALS patients and implicated in motor neuron degeneration have been studied in zebrafish, including superoxide dismutase (SOD1) , alsin . Then, tSigns of endplate denervation in ALS have been observed in patients ,54 as weachRα) and γ (achR γ), and for the muscle histone deacetylase 4 (hdac4), already described as molecular markers of denervation intensity [achRα was increased  that can be rescued by the expression of the corresponding human gene (hTDP-43) . Because of the impossibility to extract proteins specifically from muscle, we used Karnovsky and Roots reaction on sagittal embryo sections to detect ache activity in muscles .n = 11 for tdp-43 KD, vs. 99.99 ± 23.44, n = 11 for mis-CTRL, p < 0.05; Furthermore, we performed qPCR analysis in order to quantify ache expression at the transcript level. After KD of tdp-43, endogenous ache transcript was found to be reduced by 40%  or TARDBP (hTDP-43) genes after KD of either of these genes in zebrafish.To determine whether a functional interaction occurs between ache and tdp-43, we performed rescue experiments by co-injecting the human n = 27 for tdp-43 KD + hAChE-T, vs. 7 ± 3.613, n = 12 for tdp-43 KD vs. 100 ± 10.19, n = 26 for mis-CTRL; velocity: 91.65 ± 6.572, n = 27 for tdp-43 KD + hAChE-T, vs. 23.27 ± 4.229, n = 12 for tdp-43 KD vs. 100 ± 3.924, n = 26 for mis-CTRL; total time: 103.4 ± 12.09, n = 27 for tdp-43 KD + hAChE-T, vs. 21.43 ± 5.14, n = 12 for tdp-43 KD vs. 100 ± 10.62, n = 26 for mis-CTRL; Expression of hAChE-T in tdp-43 knockdown fish (orange) significantly improved the swimming behavior at 48hpf  gradually lost motility up to 48 hpf . The oveSince tdp-43 knockdown in zebrafish induces ach receptors reorganization, reminiscent of the diffuse achRs distribution previously seen in mutant zebrafish (achesb55) lacking of ache activity , we evalIn both double injections (KD tdp-43 + hAChE-T and KD ache + hTDP-43), we did not detect any pre/post synaptic orphans as compared to tdp-43 or ache KD conditions B,C. FurtThese data suggest the existence of a functional relationship where ache and tardbp operate and confirm the important role that ache has during development and NMJ stability.In this study, we extended our understanding of a major pathological factor in ALS, TDP-43, and its potential contribution in this neuromuscular disorder. Here, we established that tdp-43 plays an important action in regulating ache expression and levels in zebrafish. We demonstrated that ache levels that remain membrane-bound are decreased upon tdp-43 knockdown. Importantly, motor phenotype and the NMJ’s defects due to the tdp-43 depletion were rescued upon restoration of ache levels and activity.Drosophila [In the overwhelming majority of ALS cases, TDP-43-mediated neurodegeneration is primarily caused by the loss of normal function of TDP-43 in nuclei without being mutated with a consequent accumulation in the cytosol resulting in pathological aggregation ,59,60. Hosophila  as well osophila . Our finThe relevance of these findings is evident when taking into consideration the role that AChE displays during neuronal development and NMJ synapse stabilization independently of its activity in cholinergic neurotransmission . Thus, pDespite AChE being considered an “old actor”  in scienIn the mature NMJ instead, the AChE classical role involves the hydrolysis of ACh , and hasachR α subunits are increased and hdac4 expression augmented. The increase of these factors is particularly interesting since they are clearly upregulated in ALS-denervated muscle [Similarly, the zebrafish model lacking ache catalytic activity presents smaller and fragmented ach receptors clusters, reminiscent of the anatomical abnormalities observed in muscle/NMJ biopsies from ALS patients ,78. Mored muscle ,56,79,80AChE mRNA or rather indirectly TDP-43 and AChE can exert a role in NMJ maintenance through trans-acting factors [In our tdp-43 KD model, NMJs clearly lose their integrity, and ache down-expression can be one of the major causes of NMJ dysfunctions. The discovery that tdp-43 LoF decreases ache expression in zebrafish could provide a novel mechanistic insight for nerve-muscle integrity disruption in ALS pathogenesis. Since TDP-43 downregulation appears to be an early defect in ALS, AChE expression could also be altered at early pre-symptomatic stages of the disease, leading to NMJ remodeling. Indeed, to reinforce these findings, a recent study that performed spatial transcriptomic analysis in ALS pathological tissue observed reduced AChE levels . Since N factors .Certain limitations arise from the usage of zebrafish models in this current study. Importantly, compared with other vertebrates, zebrafish embryos appear to be devoid of ache splicing . For thi


Purpose: Diffuse large B cell lymphoma (DLBCL) is one of the most common B cell lymphomas, which displays heterogeneous pathologies. Programmed cell death 1/ programmed cell death ligand 1 (PD-1/PD-L1) plays an essential role in immunosuppression in multiple malignancies. Signal transducer and activator of transcription 3 (STAT3)-positive patients also have an independently inferior clinical outcome. However, there are no reports on the effect of plasma soluble PD-L1 (sPD-L1) combined with plasma STAT3 on the prognosis of DLBCL. In this study, we investigate the relationships between plasma sPD-L1 combined with STAT3 and clinical prognosis of DLBCL.Methods: Levels of plasma sPD-L1 and STAT3 were quantified using ELISA in eighty-seven DLBCL patients. Multiplexed immunofluorescence staining was performed to visualize the expression of PD-L1 in twenty-nine matched FFPE specimens from all patients.Results: The survival analysis revealed that the progression-free survival (PFS) and overall survival (OS) in high sPD-L1 level group were poorer than that in low sPD-L1 level group . Similarly, the PFS and OS in high STAT3 level group were also poorer than that in low STAT3 level group. Multivariate cox regression analysis showed that both high sPD-L1 and high STAT3 levels were the independent prognostic factors negatively affecting survival. In addition, patients with DLBCL having high levels of both sPD-L1 and STAT3 had a worse outcome than those patients having any one high or low levels of both (P < 0.001).Conclusions: We therefore revealed that high levels of plasma sPD-L1 and STAT3 are associated with inferior outcome for DLBCL patients, suggesting that combined measurement of their levels in plasma may be a promising prognostic strategy for DLBCL patients. Diffuse large B cell lymphoma (DLBCL) is one of the most common B-cell lymphomas, accounting for approximately 30-40% of non-Hodgkin's lymphoma (NHL) PD-L1, also known as B7-H1/CD274, is an immune inhibitory receptor expressed on the surface of T cells, B cells, and monocytes upon activation. It is a member of the B7 family and an important ligand of PD-1  Three oncogenic pathways have been observed in DLBCL including the constitutively activated NF-κB, JAK/signal transducer and activator of transcription (STAT), and PI3K/AKT/mTOR pathways Although high level of plasma sPD-L1 is associated with poor prognosis in DLBCL patients Our study included eighty-seven patients with de novo DLBCL whose peripheral blood was available and received R-CHOP  or R-CHOP-like regimen. Plasma samples from the peripheral blood of all patients were collected before systematic treatment and frozen at -80 °C until further analyses. Meanwhile, 29 matched formalin-fixed paraffin-embedded (FFPE) tissue from patients before treatment were collected. This study was approved by the Clinical Research Ethics Board of Tianjin Medical University Cancer Institute and Hospital . All patients provided written informed consent, and this study was conducted in accordance with the principles of Declaration of Helsinki and Good Clinical Practice guidelines.Levels of plasma sPD‐L1 and plasma total STAT3 were measured using enzyme linked immunosorbent assay (ELISA) kits namely, Human PD-L1 ELISA kit  and Human Signal Transducer and Activator of Transcription 3 (STAT3) ELISA Kit  according to the manufacturer's instructions. Each sample was analyzed in duplicates. The intra-assay and inter-assay coefficients of variation were below 20%.+. The mean value of PD-L1 positivity across images acquired from 20 fields of view was calculated. Phenotyping and quantification were carried out by a researcher who was blinded to the sample identities and clinical outcomes.In order to precisely detect the PD-L1 and PAX-5 (paired box 5) expression, multiplexed immunofluorescence staining was performed in twenty-nine matched formalin-fixed paraffin-embedded (FFPE) tissues from patients before treatment according to the Opal immunostaining protocol, as described in our previous study P < 0.05 was considered statistically significant.We used SPSS 25.0 statistical software  and Graphpad Prism 8.0  to perform all statistical analyses. Pearson's chi-squared test was used to analyze the correlations between plasma sPD-L1 and plasma STAT3 levels. The relationships between clinicopathologic parameters and sPD-L1 or STAT3 levels were analyzed via chi-square test. The association between plasma sPD-L1 and PD-L1 expression in tumor tissue was assessed by linear regression analysis. Progression-free survival (PFS) was calculated from the date of diagnosis to that of disease progression, relapse, or death from any cause. Overall survival (OS) was calculated from the date of diagnosis to that of last follow-up or death. PFS and OS were estimated using the Kaplan-Meier method and log-rank tests were performed. Table P = 0.013), elevated LDH (P = 0.012) and β2-M levels (P = 0.001), international prognostic index (IPI) score > 2 (P = 0.003), non-GCB subtype (P = 0.042), B symptom (P = 0.012), DEL (P = 0.021), and poor clinical response (P = 0.002). High STAT3 levels were related to the advanced stage (P = 0.041) and IPI score > 2 (P = 0.044). Furthermore, we also found that there were no correlations between plasma sPD-L1 level and plasma STAT3 level .Using a cut-off value of median sPD-L1 level, 1.21 ng/ml, we separated the patients into two groups, which is similar to the previous study P < 0.001, Figure P < 0.001, Figure P = 0.005, Figure P = 0.013, Figure P = 0.046, Figure P = 0.002, Figure P = 0.013, Figure P = 0.053, Figure Median follow-up duration was sixty range: 2-106 months) months. All eighty-seven patients were available for the 3-year PFS and OS analysis. We found that 3-year PFS and 3-year OS rates were 74.7% and 79.3%, respectively. The survival analysis revealed that patients with high sPD-L1 level in the entire cohort , β2-M , IPI scores , B symptom , DEL , high sPD-L1 level , high STAT3 level  were associated with poor prognosis in patients with DLBCL. Multivariate cox regression models were further performed to determine the prognostic value of sPD-L1 and STAT3 level. We found that IPI score , high sPD-L1 level , and high STAT3 level  were the independent prognostic factors negatively affecting survival.Subgroup analyses were performed according to gender, age, clinical stages, LDH level, β2-M level, IPI grades, B symptoms, sPD-L1 level, and STAT3 level. Table 2χ = 26.289, P < 0.001; Figure P = 0.107). However, on comparing groups 1 and 2, we found that in patients with high sPD-L1 level, those with high STAT3 level had poorer survival rates than patients with low STAT3 level (P = 0.013). These findings suggested that if high STAT3 level led to the upregulation of sPD-L1 level, patients had the worst prognosis. If high STAT3 level did not lead to the upregulation of sPD-L1 level, there was no significant difference in patient survival (P = 0.107). According to present reports, overexpression of STAT3 may upregulate the expression of PD-L1 and relate to poor overall survival Among all patients, twenty-five displayed high sPD-L1 and high STAT3 levels (group 1), nineteen showed high sPD-L1 and low STAT3 levels (group 2), nineteen exhibited low sPD-L1 and high STAT3 levels (group 3), and twenty-four showed low sPD-L1 and low STAT3 levels (group 4). We performed survival analyses to compare these four groups according to their combination of sPD-L1 and STAT3 levels. Kaplan-Meier survival curves showed that group 1 patients had the worst OS compared with other groups ] and high STAT3 levels  were the independent prognostic factors negatively affecting survival. We also found that the PFS and OS for high sPD-L1 level group was poorer than that for low sPD-L1 level group in all patients as well as in the non-GCB and GCB-DLBCL subgroups. As demonstrated previously P = 0.041) and IPI score (P = 0.044). These findings were consistent with previous reports P < 0.001) compared with all other patients. Interestingly, we also found that there was no significant difference in OS in patients with DLBCL having low sPD-L1 levels and high or low STAT3 levels (P = 0.107). However, the patients with high sPD-L1 and STAT3 levels had poorer OS compared with those having high sPD-L1 levels and low STAT3 levels (P = 0.013). These findings also suggest that we can stratify DLBCL patients according to plasma sPD-L1 and STAT3 levels into different risk groups.In our study, we showed that high sPD-L1 levels [HR (95% CI) = 6.284 (1.390-28.397),  4.158 1.2-14.627,2R = 0.3787, P = 0.0004, Figure The correlation between PD-L1 expression in the tumor tissue and plasma sPD-L1 is controversial In summary, the levels of plasma sPD-L1 and plasma STAT3 were independent prognostic factors negatively affecting survival, and high levels of both showed a significantly worse survival rate compared with that by any one high or low level of both. In the era of personalized diagnosis and treatment, combined measurement of the levels of plasma sPD-L1 and STAT3 may be a promising prognostic strategy for DLBCL patients.


They demonstrate cell type-specific vulnerability to both seeded aggregation (induced by post-mortem tissue extracts) and upon treatment with highly purified recombinant TDP-43 oligomers.Smethurst  Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by motor neuron loss, resulting in muscle wasting, paralysis and eventual death. A key pathological feature of ALS is cytoplasmically mislocalized and aggregated TDP-43 protein in >95% of cases, which is considered to have prion-like properties. Historical studies have predominantly focused on genetic forms of ALS, which represent ∼10% of cases, leaving the remaining 90% of sporadic ALS relatively understudied. Additionally, the role of astrocytes in ALS and their relationship with TDP-43 pathology is also not currently well understood. We have therefore used highly enriched human induced pluripotent stem cell (iPSC)-derived motor neurons and astrocytes to model early cell type-specific features of sporadic ALS. We first demonstrate seeded aggregation of TDP-43 by exposing human iPSC-derived motor neurons to serially passaged sporadic ALS post-mortem tissue (spALS) extracts. Next, we show that human iPSC-derived motor neurons are more vulnerable to TDP-43 aggregation and toxicity compared with their astrocyte counterparts. We demonstrate that these TDP-43 aggregates can more readily propagate from motor neurons into astrocytes in co-culture paradigms. We next found that astrocytes are neuroprotective to seeded aggregation within motor neurons by reducing  cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell-type specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS. In summary, we have developed a human, clinically relevant, and cell-type specific modelling platform that recapitulates key aspects of sporadic ALS and uncovers both an initial neuroprotective role for astrocytes and the cell type-specific toxic effect of TDP-43 oligomers. Amyotrophic lateral sclerosis (ALS) is caused by the selective degeneration of motor neurons in the brain and spinal cord, which results in rapidly progressive paralysis and invariable death, commonly by respiratory failure. ALS has a lifetime risk of ∼1 in 400 in individuals with European ancestry . Ten perAn increasing number of neurodegenerative diseases are considered to have ‘prion-like’ characteristics , which iAgainst this background, we and others have shown that TDP-43 pathology can be directly and reliably reproduced in HEK293 and NSC-34 cells via a templated seeding reaction from human sporadic ALS post-mortem brain and spinal cord tissue .Here we have induced a pathological TDP-43 seeded aggregation reaction in control human iPSC-derived motor neurons and astrocytes on an endogenous background level of TDP-43 expression using sporadic ALS spinal cord tissue extract serially passaged in cell culture (spALS). These TDP-43 aggregates were phosphorylated and morphologically representative of ALS post-mortem tissue. We found that astrocytes could also be seeded with TDP-43 aggregates but at lower frequency than motor neurons, implying cell-type specific differences in vulnerability. We found that proteasomal inhibition could both exacerbate seeded TDP-43 aggregation and significantly increase cell death in motor neurons when compared to astrocytes. We were also able to demonstrate that TDP-43 pathology spreads from motor neurons to astrocytes preferentially, but could also be observed spreading from astrocytes to motor neurons. We next found that astrocytes—both by physical co-culture and conditioned medium experiments—are neuroprotective to seeded aggregation within motor neurons by reducing  cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell type-specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS.Detailed methods are provided in the Initial sarkosyl-insoluble extracts from frozen CNS tissue for seeding were prepared as previously described  and from Days 7 to 14   C. Howeve < 0.01) D.P < 0.001) compared to treatment with the proteasome inhibition alone (∼4%) (P < 0.01) compared to proteasome inhibition alone (∼6%) (P < 0.001) (P < 0.01) than treatment with MG132 alone  E and F. ne (∼6%) G, suggesne (∼6%) H. Treatm< 0.001) I and J w< 0.001) I. After < 0.001) J. The fr32 alone J. Howeve32 alone K and L.P < 0.001). After this time point the percentage of astrocytes containing TDP-43-positive aggregates significantly decreased from 27.3% to 11.3% (P < 0.001) indicating a clearance or dilution of TDP-43 aggregates over time [To determine if TDP-43 aggregates could spread between motor neurons and astrocytes, we cultured control untreated astrocytes together with motor neurons that had been treated with our optimized seeded aggregation paradigm described above for 3, 7 and 14 days [P < 0.05) and again to 1% at Day 14 (P < 0.01) [Next, we investigated the propagation of TDP-43 aggregates from astrocytes to motor neurons by performing seeded aggregation in astrocytes and then co-culturing with control untreated motor neurons for 3, 7 and 14 days [P < 0.01) and 20.4% (ACM) (P < 0.01) (P < 0.01) (P < 0.01) with astrocyte co-culture and 32.2% to 19.3% (P < 0.01) with ACM treatment  treatments significantly reduced the percentage of cells containing TDP-43-positive aggregates from 37.8% to 23.4% (astrocyte co-culture) ( < 0.01) A and B.  < 0.01) C. Finallreatment D. These reatment .There is now accumulating evidence to support the notion that soluble oligomeric species of misfolded proteins are the cause of toxicity in several neurodegenerative diseases with prominent protein misfolding . We nextP < 0.001) compared to the control  after 24 h, detected via an increase in activated Casp3 immunolabelling  for 24 h . We have previously demonstrated that serial passage of TDP-43 pathology further enhances its potency and the increased abundance of seeded aggregation demonstrated in the experiments performed in The number of TDP-43 aggregates reproducibly and significantly increased in frequency with longer incubations, which is consistent with either The lack of toxicity in human iPSC-derived motor neurons treated with seeded aggregation alone in this study likely reflects low frequency and abundance of TDP-43 inclusions observed. However, upon proteasomal inhibition and subsequent seeded aggregation, the number of TDP-43 aggregates and activated Casp3-positive cells significantly increased compared to proteasome inhibition alone suggesting that such TDP-43 seeded aggregation is indeed toxic under defined cellular states. The sequential proteasome inhibition and inoculation of the spALS spinal cord extract resulted in the increased formation of TDP-43 aggregates in both human iPSC motor neurons and astrocytes. Indeed, a mouse motor neuron-driven expression of proteasome dysfunction, but not autophagy, resulted in a robust model of TDP-43, FUS, ubiquilin-2 and optineurin mislocalization and aggregation with a resultant ALS phenotype  in primary neuron and astrocytes co-culture happens in a cell contact dependent manner via tunnelling nanotubes , the UK Medical Research Council (FC010110), and the Wellcome Trust (FC010110). R.P. holds an MRC/MND Association Lady Edith Wolfson Senior Clinical Fellowship (MR/S006591/1) and is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre.The authors report no competing financial interests.awz419_Supplementary_InformationClick here for additional data file.