The past decade has witnessed a rapid growth of graphene plasmonics and their applications in different fields. Compared with conventional plasmonic materials, graphene enables highly confined plasmons with much longer lifetimes. Moreover, graphene plasmons work in an extended wavelength range, i.e., mid-infrared and terahertz regime, overlapping with the fingerprints of most organic and biomolecules, and have broadened their applications towards plasmonic biological and chemical sensors. In this review, we discuss intrinsic plasmonic properties of graphene and strategies both for tuning graphene plasmons as well as achieving higher performance by integrating graphene with plasmonic nanostructures. Next, we survey applications of graphene and graphene-hybrid materials in biosensors, chemical sensors, optical sensors, and sensors in other fields. Lastly, we conclude this review by providing a brief outlook and challenges of the field. Through this review, we aim to provide an overall picture of graphene plasmonic sensing and to suggest future trends of development of graphene plasmonics. Surface plasmons are collective electron oscillations confined at surfaces due to strong interactions with photons. Although surface plasmons have been increasingly studied by scientists in recent decades, they were in use even centuries ago for making beautiful Gothic stained glass adorning medieval cathedrals , 2. ThesGraphene is a two-dimensional (2D) material with a 2D crystalline structure and atomically thin thickness. It exhibits extremely high mechanical strength and electrical mobility, is a semi-metal with zero bandgap, and can be easily sculpted into different structures such as nanoribbons or nanodisks with nanofabrication techniques. Due to its unique Dirac cone band structure with a linear dispersion relation, graphene shows extraordinary optical properties when used as a plasmonic material. In particular, graphene can localize electromagnetic fields within the small vicinity of graphene sheet, and its spectral features can be easily tuned by external electrostatic gating or chemical doping –9. For tGraphene plasmonics is a new and rapidly growing research area, with researchers only having started to fill the vacancy of experimental research on graphene plasmons in the past decade. In 2011, Ju et al. experimentally demonstrated that graphene plasmons can be induced by periodic graphene micro-ribbons, and the resonance can be tuned via ribbon width and electrostatic doping . Since tFollowing our introductory section, in Sect. Plasmons are the collective oscillations of electrons at the surface of a metal or metal-dielectric interface subjected to an external electromagnetic field. The plasmonic oscillations at the material surface or interface create confined electromagnetic fields, called surface plasmon polaritons (SPP). SPPs are common phenomena often found in metal or semi-metal surfaces such as graphene , 31. ComIn this section, we will discuss popular ways that have been applied for exciting graphene plasmons, such as introducing edges via micro/nano patterning, introducing and controlling defects, selective doping, and spatial modulations of electric potential.Introducing edges on graphene is an effective way for confining SPPs and inducing plasmon resonance –33. The Chen et al. experimentally demonstrated that incident light with a wavelength of 9.7 μm excited graphene plasmons when the width of patterned graphene was reduced to ~ 260 nm . The nea− 1 excitation frequency  direction was both downwards and upwards at either side of the ribbon. On the contrary, the Ez direction on the narrower ribbon was upwards on the right edge and downwards on the left edge, thereby inducing different SPR modes.Yan et al. demonstrated polarization dependent plasmonic resonance in graphene nanoribbons using a broadband excitation . LocalizGraphene plasmons can interact with defects, causing inhomogeneities in the density of states . For exaGraphene plasmons also interact with out-of-plane topographies of graphene, such as bubbles, blisters, or wrinkles –37. SuchMoreover, out-of-plane deformation can influence the propagation behavior of graphene plasmons. Slipchenko et al. reported computational studies on the behavior of plasmon propagation across a corrugation in graphene Fig. d 36]. F. F36]. F− 1   and changing the width of micro-ribbon structures . Plasmon}ωp∝n1/4 . Yan et }ωp∝n1/4 . In the − 1. However, the spectral peak became asymmetric with a much higher extinction intensity when it was coupled with the graphene plasmon peak. The transparency in the spectra between the broad plasmon peak and sharp phonon peak was prominent. The transparency window  became narrower as the graphene plasmon frequency was tuned by the nanoribbon width causing the frequency to approach the phonon frequency of AB stacked bilayer graphene  , 48. BraGraphene plasmon resonance can be tuned by introducing three-dimensionality to graphene . Kang etWhile graphene plasmons exhibited extraordinary intrinsic properties originating from high electron mobility, low loss of plasmons, high spatial confinement, and wide range of tunability, graphene has been combined with conventional plasmonic nanostructures for the enhanced sensitivities, sensing in visible wavelength, and more. In graphene-based hybrid plasmonic materials, graphene plays various roles owing to its smooth surface without dangling bonds, atomic thickness, chemical inertness, gas impermeability, and optical transparency. Moreover, graphene is a fluorescence (FL) quencher while contributing to surface enhanced Raman scattering (SERS) effects, which makes graphene an attractive option for sensing platforms –54. In tOne challenge in characterizing a target molecule using Raman spectroscopy is suppressing the target molecule’s FL emission when the target molecule exhibits high FL emission at a particular laser excitation. The high FL emission masks the Raman signals of the target molecule, making those signals difficult to detect. For example, rhodamine 6G (R6G) emits strong FL signals at 633 nm excitation wavelength, and the intensity of the FL signal is much larger than its Raman signal. However, graphene has shown its potential not only as an effective surface for Raman enhancement but also as a FL quencher.3 when the R6G and PPP molecules were adsorbed on graphene, allowing the Raman peaks to be observed. Similar effects have been reported elsewhere . . 2O3 FabFurthermore, hybrid structures of graphene with other plasmonic materials have been widely investigated for optimally utilizing not only the materials’ individual advantageous properties but also their synergistic functionalities. Specifically, combining plasmonic materials with graphene can compensate for the mismatch between the wave vectors of graphene plasmons and incident light, thereby expanding the detectable wavelength range. In addition, graphene can enhance SPR signals due to strong electromagnetic field confinement in hybrid materials .In hybrid structures, nanostructures of noble metals  are commonly combined with graphene. Graphene can serve as either a substrate or a protective layer for vulnerable materials, while metal nanostructures serve as nanoantennas . Nam et Alternative materials have been proposed as replacements for noble metals in these hybrid structures, as noble metals were found to sometimes induce defects in the graphene lattice due to chemical interactions , 113. PaGraphene has also been integrated with other types of plasmonic structures, such as optical metamaterials or metal gratings, for biosensing applications. Luxmoore et al. demonstrated a graphene nanoribbon sensor placed inside the gap of a complementary split ring resonator (CSRR) Fig. e 116]. . 116]. CAdditionally, graphene-on-metal SPR prisms have been widely applied for bio-applications, such as detecting DNA and antibodies –122. Whe2, NO2, N2O, and NO in real time. Due to the strong confinement of graphene plasmons and high adsorption of gas molecules on graphene, the graphene sensor’s detection limit was as low as 800 ppm. Bareza et al. further enhanced the adsorption of a target gas molecule, CO2, to lower the detection limit of their graphene-based plasmonic gas sensors by depositing an ultrathin layer of polyethylenimine (PEI) on top of graphene nanoribbons . . 132]. T− 9 M. These sensors were additionally applied for more practical chemical detection, such as detecting thiram from orange peels.Metal/graphene/metal hybrid structures can also serve as SERS substrates with high sensitivity. Nguyen et al. adopted graphene/Au film/Au nanorod hybrid structures for detecting pesticides including azinphos-methyl, carbaryl, and phosmet with detection limits of 5, 5, and 9 ppm, respectively . Dong et2 with enhanced SERS signals . . 136]. AVarious strategies have been proposed and demonstrated for achieving even higher photoresponsivity. Nanofabrication strategies such as e-beam lithography can be used for creating metal nanostructures with precise control of antenna size and shape as well as the gap between adjacent antennas for stable and uniform photosensing performance. For nanorod arrays with a 60 nm nanogap, the photoresponsivity towards mid-IR light was enhanced by more than 200 times to 0.4 V/W compared to the responsivity of photodetectors without nanostructures (< 2 mV/W) . By conto/Hz detected for mid-IR incident light. Lastly, theoretical studies have suggested that graphene plasmonic structures can be used for pressure and mass sensing [In addition to the aforementioned applications that have been widely investigated, plasmonic structures of graphene and graphene-integrated hybrid materials have additionally shown great potential for other sensing purposes. Dabidian et al. used graphene with a plasmonic metasurface layer to develop an interferometer for detecting nanoscale motions of reflecting objects . The dis sensing , 153.The past decade has witnessed a boost of fundamental studies of graphene plasmons as well as their applications in biosensors, chemical sensors, photodetectors, and other fields. Graphene enables high electromagnetic field confinements with low losses and long lifetimes. The plasmonic properties of graphene can be easily tuned by electrical gating or chemical doping. Moreover, graphene plasmons are in the wavelength range (THz and mid-IR) that other plasmonic materials cannot reach. These distinctive optical properties of graphene provide a foundation for various plasmonic sensing applications. However, in order to access graphene plasmons, the momentum mismatch between graphene plasmons and incident light must first be overcome. Researchers have employed different approaches for exciting plasmons in graphene, including patterning graphene into micro/nano patterned structures, introducing defects and curvatures, applying selective doping, or forming Moiré superlattices. Other plasmonic nanomaterials, including NPs, nano gratings, and metamaterials, have also been adopted and integrated with graphene for tuning plasmonic properties and enhancing optical absorption.5 cm2/Vs) and high Fermi velocity only two orders of magnitude lower than the speed of light [Graphene additionally shows potential for plasmonic applications with ultrahigh sensitivities towards very low concentrations of analytes and even label-free single molecule detection. Beyond detecting materials, surface plasmon of graphene can also be used for detecting property or structural changes within films, which may largely broaden its application in monitoring, diagnosing, and characterization. In addition, due to its high carrier mobility (theoretically > 2.5 × 10of light , graphenof light –157. It of light , 159. Adof light , 157. Inof light , 161 andof light , 163 allAlthough graphene plasmons have demonstrated great potential for various sensing applications, challenges still remain towards commercialization. Commercialization will not only require consistent device performance, but also demand scalable, reliable, and consistent fabrication processes with low costs. Currently, most graphene plasmonic sensor fabrication requires complex processes with expensive facilities. Moreover, reproducibility and quality-control in mass-production of graphene-based plasmonic sensors has not yet been fully addressed, as most lab-scale fabrications only yield one or a few devices at a time. In terms of device performance, synthesizing high quality graphene with single crystallinity and fewer defects will be the highest priority for graphene-based plasmonic sensors. Specifically, graphene with improved quality will have greater mobility and longer lifetime, thereby enhancing absorption, light-matter interactions, efficiency, spectral resolution, and sensitivity. From this point of view, batch production of high quality graphene via CVD –168, may


Tissue microarray analysis shows the positive correlation of TDP-43 and ABHD2 in HCC. Mechanistically, TDP-43 binds with the UG-rich sequence1 of ABHD2 3’UTR to enhance the mRNA stability of ABHD2, thereby upregulating ABHD2. Afterwards, TDP-43 promotes the production of free fatty acid and fatty acid oxidation-originated reactive oxygen species (ROS) in an ABHD2-dependent manner, so as to suppress apoptosis of HCC. Our findings provide insights into the mechanism of HCC progression and reveal TDP-43/ABHD2 as potential targets for the precise treatment of HCC.TAR DNA-Binding Protein 43 (TDP-43) has been well studied in neurodegenerative diseases, but its potential role in malignance is still unclear. Here, we demonstrate that TDP-43 contributes to the suppression of apoptosis by facilitating lipid metabolism in hepatocellular carcinoma (HCC). In HCC cells, TDP-43 is able to suppress apoptosis while deletion of it markedly induces apoptosis. RNA-sequencing identifies the lipid metabolism gene  TDP-43 acts as an RNA-binding protein that regulates the RNA stability of ABHD2 and affects the release of fatty acids and ROS, which in turn regulates apoptosis and affects the growth of liver tumors. The pathogenesis of HCC is complicated, it involves a variety of processes, such as chronic inflammation, liver fibrosis and oncogene dysregulation3. The dysregulation of oncogene expression always induces the disorder of the physiological state of cell, which results in excessive proliferation and suppressive apoptosis, accelerating the malignant progression of HCC4. Therefore, clarifying the mechanism of initiation and development is crucial for the targeted therapy in HCC.Hepatocellular carcinoma (HCC), one of the common malignant tumors around the world6, was originally identified as a DNA-binding protein capable of regulating human immunodeficiency virus 1 gene expression8. Later studies found that TDP-43, encoded by the tardbp gene, is a highly conserved DNA/RNA-binding protein. In the nucleus, TDP-43 can bind to RNA and play an important role in mRNA transcription, splicing, nuclear transport and stability9. In the cytoplasm, TDP-43 forms RNP ribonucleoprotein granules that regulate mRNA transport and promote the production of non-coding RNAs such as miRNA11. TDP-43 has been focused because of its vital role in neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer’s disease13. Recently, it has been discovered that TDP-43 is closely correlated with the progression of malignant tumors. Studies have shown that TDP-43 is dysregulated in breast cancer, lung cancer, melanoma and other malignant tumors16. In breast tumor, TDP-43 can interact with serine and arginine rich splicing factor 3 to modulate the selective splicing of downstream targets PAPR3 and NUMB mRNA, thereby promoting tumor proliferation and metastasis14. In lung cancer cells, TDP-43 enhances the mRNA stability of FasL by interacting with FasL mRNA, thereby mediating apoptosis and finally inhibiting the progression of lung cancer16.Transactivation (TAR) DNA-binding protein (TARDBP), also known as TDP-4317. This evidence indicates the potential role of TDP-43 in modulating cell metabolism in HCC. Abnormal metabolism is a common hallmark of cancer cells; especially glucose metabolism and lipid metabolism, cancer cells often reprogram these metabolic pathways to meet the energy needs of overgrowth and to resist apoptosis18. Liver is an important place for lipid metabolism, and plays a vital role in the absorption, synthesis, decomposition, and transport of lipids19. lipid metabolism disorder usually appears during the occurrence and development of HCC21. However, whether TDP-43 has an effect on lipid metabolism in the progression of HCC and the underlying mechanism are still unknown.Notably, a previous study shows that TDP-43 elevates the expression of the platelet isoform of phosphofructokinase, a rate-limiting enzyme of glycolysis, to promote cell growth in HCCABHD2. Molecular mechanism investigation reveals that TDP-43 can bind to the 3ʹUTR of ABHD2 mRNA and enhance the stability of ABHD2 mRNA, thereby upregulating the expression of ABHD2. TDP-43 was able to promote the production of free fatty acid (FFA) and fatty acid oxidation-originated reactive oxygen species (ROS) in an ABHD2-dependent manner, thereby promoting proliferation and inhibiting apoptosis in HCC cells. Our study provides a potential target for the precise treatment of HCC.In the present study, we uncover that the expression of TDP-43 is negatively related to the overall survival and relapse-free survival of HCC. TDP-43 suppresses the cell apoptosis of HCC through the lipid metabolism-related gene Bcl-2 and proliferation marker Ki67. Meanwhile, TDP-43 downregulated the proapoptotic genes p53 and Bax  was the most considerably down-regulated after TDP-43 knockdown  and obtained the transcriptome expression profiles of MHCC97H-KD-TDP-43 cells and control cells. After knocking down TDP-43, the expressions of 1392 genes were increased and 1418 genes were decreased .Next, we performed qRT-PCR and Western blot assay in different HCC cell lines to confirm the effect of TDP-43 on ABHD2 expression. Silence of TDP-43 efficiently decreased the expression of ABHD2, while TDP-43 overexpression obviously increased ABHD2 Fig. . Meanwhi5. Therefore, we wondered whether TDP-43 exerted a regulatory role by binding to ABHD2 mRNA. The RNA immunoprecipitation (RIP) assay in MHCC97H cells showed that TDP-43 could indeed bind to ABHD2 mRNA   Fig. . This ev24. After sequence analysis, we found there were two UG-rich sequences in the 3ʹUTR of ABHD2 mRNA. Therefore, we cloned the 3ʹUTR of ABHD2 mRNA and constructed it into a luciferase vector (pGL3-Control). The data showed that overexpression of TDP-43 could upregulate the luciferase activity of ABHD2 3ʹUTR, while TDP-43 silencing markedly inhibited the luciferase activity   Fig. , and detWT) Fig. . HoweverWT) Fig. . These a25. Under the action of triacylglycerol lipase, triacylglycerols (TG) are hydrolyzed to produce fatty acids27. Therefore, we examined the effects of ABHD2 on intracellular TG levels. The data displayed that deletion of ABHD2 obviously induced the accumulation of TG in SMMC-7721 and MHCC97H cells  family, has the lipase serine nucleophilic motif GXSXG and can participate in lipid metabolism by exerting triacylglycerol lipase activity28. Studies have shown that ROS is critical for cell survival, and small increases in ROS levels can lead to the activation of some proto-oncogenic pathways and facilitate cancer development30. We examined the cellular ROS level and found that ABHD2 could indeed induce the generation of ROS  were collected for subcutaneous tumor implantation in nude mice. After 42 days, the subcutaneous tumors of three groups were collected and the tumor weight was detected. The results showed that overexpression of ABHD2 effectively alleviated the inhibition of tumor growth caused by TDP-43 deletion Fig. . Examina31. Therefore, the exploration of strategies and targets for the treatment of HCC has never stopped. The poor prognosis of HCC is mainly related to the delay of diagnosis32. Study of biomarkers may provide more instructive information for the individualized diagnosis and/or prognosis of HCC. The role of TDP-43 in neurological diseases has been well studied, but its potential function in malignant tumors still needs to be explored. In our study, survival analysis manifested that the higher the expression level of TDP-43, the shorter the survival rate of HCC patients. Moreover, TDP-43 had been proved to inhibit apoptosis and promote proliferation in HCC cells, thus accelerating the malignant development of HCC. Our discovery is expected to provide a biomarker for the treatment of HCC, so as to be applied to the diagnosis and prognosis of patients.Over the years, with the development of medicine, we have made considerable progress in the diagnosis and treatment of HCC; However, statistics have showed that almost 70% of patients recur within 5 years after operation34. So, we supposed whether TDP-43 modulated lipid metabolism-related gene in HCC. In order to confirm our speculation, we detected the expression of all metabolism-related genes among the differential downregulated genes (Top50) and observed that the lipid metabolism-related gene ABHD2 was most dramatically downregulated after TDP-43 silencing. ABHD2, as a member of the mammalian ABHD family, is a triacylglycerol lipase with ester hydrolysis ability25. Conserved structural motifs shared by the members of ABHD family primarily play a role in lipid synthesis and degradation. In recent years, the ABHD family has emerged as a potential regulator of signal transduction and lipid metabolism34. The above findings led us to investigate whether TDP-43 could modulate lipid metabolism via ABHD2. TG and FFA content examination displayed that TDP-43 enhanced the TG-originated production of FFA in an ABHD2-dependent manner. Fatty acid oxidation (FAO)-originated ROS maybe a major source of ROS and plays a crucial role in oxidative stress during FFA overload in the liver36. By detecting intracellular ROS levels, we discovered TDP-43 was able to elevate the content of ROS through ABHD2. The production of ROS could activate multiple oncogenic signaling pathways and furthermore contribute to the pathogenesis of multiple diseases, including cancers38. Expectedly, we found that ABHD2 could robustly attenuate the promotion of apoptosis induced by TDP-43 deletion. Moreover, FFA treatment also efficiently alleviate TDP-43 deficiency-caused apoptosis. These evidences revealed that with the help of ABHD2, TDP-43 regulated lipid metabolism, which had an impressive effect on tumor cell apoptosis.To elucidate the mechanism of TDP-43 promoting HCC, we implemented high-throughput sequencing and found that TDP-43 was indeed closely related to the apoptotic pathway. Interestingly, KEGG analysis showed that metabolic pathways strongly responded to the changes in TDP-43 expression. Therefore, we suspected that TDP-43 was likely to suppress the apoptosis of HCC by affecting metabolic pathways. The further classification analysis of KEGG pathway discovered the close association between lipid metabolism and TDP-43. Lipids are critical to the cellular life as molecules for energy storage, membrane structure and signal transduction41, but whether it has molecular functions in these two cancers is still unknown. The Obinata team has reported that ABHD2 is an androgen-responsive gene, and it can promote the growth and migration of prostate cancer42. However, in HCC, the expression and underlying function of ABHD2 remains to be elucidated. In this study, we first put forward that ABHD2 played a crucial role in inhibiting apoptosis and promoting proliferation in HCC. A study in 2009 found that increased apoptosis could be observed in ABHD2 gene deficient mice43, which was consistent with our findings. In conclusion, our data suggested that ABHD2 had significance in tumor development and was expected to be a potential target for the precise treatment of HCC.Notably, few studies have addressed the role of ABHD2 in malignant tumors. It is reported that the expressions of ABHD2 in breast cancer and melanoma are obviously higher than those in normal tissuesABHD2, thus upregulating ABHD2. Interestingly, we also found that ABHD2 had a certain effect on the expression of TDP-43. Western blot and qRT-PCR data displayed that the level of TDP-43 expression was upregulated after ABHD2 overexpression  to enhance the mRNA stability of ion Fig. . EspeciaABHD2, enhance the stability of ABHD2 mRNA, and then upregulate ABHD2. Elevated-ABHD2 catalyzes the metabolic process of TG to FFAs, thus promoting the β-oxidation pathway and increasing the production of ROS, finally resulting in the suppression of apoptosis and facilitation of proliferation in HCC. Our study provides a theoretical basis for elucidating the role of RNA-binding proteins in the promotion of malignance. Meanwhile, TDP-43/ABHD2 can be used as potential target, which has important application value in the precise treatment of HCC.In summary, our study discloses a mechanism for TDP-43-promoted HCC progression Fig. . As an RTDP-43 was amplified by RT-PCR. During amplification, the enzyme digestion sites were introduced. The PCR products and vector pEGFP-C2 were double digested, respectively. Then, the PCR products were connected with pEGFP-C2 by T4 DNA ligase. Finally, the recombinant plasmid pEGFP-TDP-43 was used for exogenous overexpression of TDP-43. The pCMV3-ABHD2 expression plasmid was purchased form Sino Biological  and was used for ABHD2 overexpression. The 3ʹUTR of ABHD2 was amplified by PCR using specific primers and inserted into the pGL3-Control vector to generate the pGL3-ABHD2-3ʹUTR (WT). Then, two sites rich in UG sequence were separately deleted to construct two different ABHD2 3ʹUTR mutants (Del-1 and Del-2). All siRNAs used in this study were generated by RiboBio , and the sequences of siRNAs were listed in the Supplementary Table Using the mRNA extracted from SMMC-7721 cells as the template, the coding region sequence (CDS) of Total proteins were extracted by RIPA buffer  containing 1nmol/L PMSF (Beyotime). The proteins were resolved by SDS-PAGE and then transferred on a polyvinylidene difluoride membrane . After incubation with 5% skimmed milk at room temperature for 2 h, the blot was hybridized with the primary antibody at 4 °C overnight. After washing by TBST three times, the blot was incubated with the secondary antibody  at room temperature for 1 h. After adding the enhanced chemiluminescence , the immunoblots were obtained using Azure600 . The primary antibodies used in this study were GAPDH , TDP-43 , ABHD2 , Cleaved-caspase3 , p53 , Bax , Bcl-2, Ki67 .5 cells were plated into 6-well plates and were harvested after transfection for 48 h. After washing with precooled PBS, the binding buffer was added to resuspend cells. Subsequently, dye the cells with 5 μl Annexin V-FITC and 5 μl propidium . Within 1 h, the apoptotic cells were detected by flow cytometry. Three replicates were set in each group.In total, 1×10 To evaluate intracellular lipid metabolism, cells were seeded on a 6-well plate and collected after transfection for 48 h. According to the manufacturer’s operating instructions, the FFA levels were determined using a Free Fatty Acid Quantification Kit . For tumor metabolism analysis, the mice tumor tissues need to be ground in an ice bath first, and then assayed for FFA content according to the instructions.2O2 for 10 min. Sections were blocked in 10% normal goat serum for 30 min at room temperature, and then incubated overnight with indicated primary antibody at 4°C. After three washes with PBS, the slides were hybridized with the secondary antibody  for 50 min at room temperature. Finally, diaminobenzidine (DAB) (servicebio) was used for color development. After counterstaining with hematoxylin, dehydrate the slides and seal with neutral gum . Image J software was used for staining analysis.Paraffin-embedded xenograft sections were used for TDP-43 , ABHD2 , C-caspase3 , Bcl-2 , and p53  staining analysis. The antibody of Ki67 used in this experiment was purchased from Abcam . After deparaffinized and rehydrated, sections were laid in EDTA solution (pH9.0) for antigen retrieval by boiling in microwave oven for 8 min on medium heat. The slides were naturally cooled before treating with 3% HHCC tissue microarray (No. live c-1401) was purchased from Servicebio. It contains 32 cases of HCC tissues and paired non-tumor tissues, including hepatocellular carcinoma, cholangiocarcinoma, and mixed hepatocellular carcinoma-cholangiocarcinoma with different degrees of differentiation. See Supplementary table Foxn1nu mutation) for the xenograft experiment. The experiment followed the guidelines of the Ethics Committee of Xinxiang Medical University, and nude mice were fed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. 6- to 8-week-old male mice were grouped randomly (n = 5 or 6). After growing to the logarithmic growth phase, the cells were collected and implanted subcutaneously in the right shoulder of each nude mouse. One week after subcutaneous inoculation, the volume of tumor was measured every three days, the weight of mice was recorded as well. Six weeks later, the nude mice were euthanized and dissected, and the tumor was photographed and weighed. Then, the tumor tissues were analyzed by immunohistochemistry and free fatty acid content in order to analyze the related proliferation, apoptosis and metabolic indexes.Beijing Vital River Laboratory Animal company provided the BALB/c Nude mice  for RNA-seq. BGI is responsible for the construction of mRNA library and analysis of transcriptome data. For detailed protocols, please visit the official website of BGI. RNA-seq reads were quantified as Fragments Per kilobase Million.ABHD2 mRNA. Briefly, HCC cells were gathered by centrifugation and then lysed with RIP lysis buffer containing RNAase inhibitor. The protein A + G agarose beads  were re-suspended and incubated with TDP-43 antibody or control IgG for 1 h. Next, the cell lysate was incubated with the above-prepared agarose beads at 4°C overnight. After repeated cleaning with NT-2 buffer for 6–8 times, added protease K buffer into agarose beads and water bath at 55 °C for 30 min to remove protein. Finally, NT-2 buffer was added and immunoprecipitated RNA was extracted with Trizol reagent for qRT-PCR analysis.The RIP assay was conducted to examine the interaction of TDP-43 and t-test. Error bars represent ±s.d. A value of P < 0.05 was considered statistically significant. ***P < 0.001, **P < 0.01, *P < 0.05. The correlations between TDP-43 expression with the relapse-free survival and overall survival were separately analyzed via the online resource Kaplan-Meier Plotter  and GEPIA (http://gepia.cancer-pku.cn/). In the analysis of HCC tissue microarray, the correlation between TDP-43 and ABHD2 expression was determined according to Pearson’s chi-square independent test.All replicates displayed in this paper are biological replicates; technical replicates  were performed and used to generate the means for each biological replicate. The sample sizes and number of replicates were indicated in the figure legends. Graph pad prism 8.0  was used for statistical analysis. The statistical significance was evaluated by comparing the mean of three biologically independent experiments using a 2-tailed student’s Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


WCO verified the successful coating of WCO onto the sand surface and conversion to graphene, which possessed distinct functional groups and features of graphene materials. GSCWCO adsorbent effectiveness in removing Congo Red dye through batch adsorption was studied under the influence of different initial concentrations (20 to 100 mg/L), and the optimum pH (pH 2 to 10), contact time (5 to 240 min), and temperature (25 to 45 °C) were investigated. The GSCWCO showed removal rates of 91.5% achieved at an initial dye concentration of 20 mg L−1, 1.0 g of adsorbent dosage, a temperature of 25 °C, and 150 min of contact time. The GSCWCO exhibited a maximum capacity of 5.52 mg g−1, was well-fitted to the Freundlich isotherm model with an R2 value of 0.989 and had an adsorption mechanism that followed the pseudo-second-order kinetic model. Negative values of enthalpy (ΔH) and Gibbs free energy (ΔG) revealed that CR adsorption onto GSCWCO was a spontaneous and exothermic process. The presence of functional groups on the surface of GSCWCO with such interactions  was responsible for the anionic dye removal. Regeneration of GSCWCO adsorbent declined after four cycles, possibly due to the chemisorption of dyes with GSC that resulted in inefficient adsorption. Being a waste-to-wealth product, GSCWCO possessed great potential to be used for water treatment and simultaneously benefited the environment through the effort to reduce the excessive discharge of WCO.Waste cooking oil (WCO) appears to be a potential carbonaceous source for synthesizing graphene sand composite (GSC) adsorbent in removing pollutants. This study presents a green synthesis method of GSC using WCO as a sustainable carbon source for the synthesis of GSC through the thermal graphitization method. Characterization analysis conducted on GSC Hence, removing the pollutants and preventing them from being released into the environment have become more appealing and urgent to safeguard the environment. To date, a wide variety of wastewater treatment processes, including adsorption2, membrane separation3, anodic oxidation4, biodegradation5, coagulation and flocculation6, electrocoagulation7, and photocatalysis8 have been developed to remove the pollutants following the discharge regulations and to minimize the adverse impact on economic development on the water environment. Adsorption remains one of the preferred processes mentioned above, extensively deployed in the wastewater treatment system. Water utilities' acceptance of the adsorption process could be attributed to its simple, effective, and relatively low-cost operation advantages11. The success of an adsorption process mainly lies in the adsorbent, which should possess a large surface area or volume, and proper functionalities to capture the pollutants from wastewater12.Water is one of the most critical components supporting and nourishing the world's ecosystems. The growing urbanization and industrialization activities have significantly affected the ecosystem, releasing pollutants that deteriorate water quality. Hazardous pollutants, such as organic and inorganic materials, heavy metals, dyes, and pharmaceutical residues, are a significant concern to the environment and society due to their toxicity, bioaccumulation, persistence, and non-biodegradable natures13. One of the fascinating carbon nanomaterials, graphene, has captured the attention of many researchers. Graphene is a monolayer of sp2-hybridized carbon atoms arranged in a 2D honeycomb structure14. This material has garnered significant interest owing to its high available surface area, enhanced active sites, and better desorption property15. Furthermore, the relatively large and delocalized π-electron system of graphene could strongly influence the binding for target contaminants, making them very attractive and promising as an adsorbent in water treatment. Though graphene displays promising potential as an adsorbent in removing water pollutants, the immediate use of graphene in the actual application is hindered by economic feasibility  and technical difficulties  for chromium heavy metal pollutants. Such a high removal efficiency was attributed to the presence of charged and electron-donor groups of GSC that reduced the chromium ions and bound them through electrostatic interaction. These results confirmed that GSC effectively adsorbs and removes dissolved pollutants from wastewater. Despite all the advantages, GSC demonstrated some drawbacks in size and particle weight since GSC’s weight is much heavier than the other graphene nanomaterials. Furthermore, the majority of the carbon precursors used for the synthesis of GSC, or similar graphene-supported adsorbent, was sugar, which was coated onto the sand and graphitized to obtain the composite adsorbent. Using sugar as a carbon source is considered non-sustainable and impractical for large-scale synthesis of GSC due to cost factors and risk to food security24. The exploration of using more environmentally friendly carbonaceous sources for synthesizing GSC has been conducted. Alternative carbon sources such as asphalt25, date syrup26, palm oil mill effluent19, and oil palm frond juice27 have been successfully converted to graphene, where the GSC displayed satisfying removal efficiency for a wide range of pollutants.Several efforts have seen the immobilization or coating of graphene on sand  through a facile synthesis approach, where the composites were generally known as graphene sand composite (GSC)28. The improper disposal of WCO is a major environmental pollution problem, and its presence in sewers or drains would lead to blockages and operation issues in wastewater treatment plants29. To address these issues, WCO has been utilized as feedstock for various value-added products, including biofuels, plasticizers, binders, surfactants, biomaterials, and different building blocks32. The valorization of WCO has also been extended to the synthesis of graphene, where the WCO reportedly was successfully converted to graphene via a chemical vapour deposition approach, albeit the information on this topic remains scarce34. Nonetheless, this suggests that WCO possesses the potential as a carbon source for synthesizing graphene. However, the facile (in-situ) synthesis of GSC from WCO has yet to be investigated. Facile synthesis of GSC from WCO is essential as a complicated synthesis approach will render the economic feasibility of GSC in real applications impractical.Given this, waste cooking oil (WCO) appears as a potential low-cost and readily available carbon source for synthesizing GSC. Worldwide, it is estimated that more than 6 million tons of WCO are generated yearlyWCO adsorption mechanism. It is believed that the successful anchoring of graphene nanosheets onto superparticles realized via a simple chemical synthesis method can act as an inexpensive material for water pollutant removal. The valorization not only resolves the issues associated with graphene but also helps to minimize environmental pollution from improper disposal of WCO.In the presented study, we aimed to develop a novel multifunctional graphene composite from a cheap and locally available waste carbon source, WCO, which acts as a superior adsorbent for removing organic pollutants. WCO will become an excellent alternative to replace other expensive materials owing to its high carbon content and affordable source, which is more favourable for industrial-scale production. In the meantime, further investigation was executed to explore the feasibility of the as-synthesized adsorbent in the adsorptive removal of Congo red dye from an aqueous solution. Morphological and physicochemical analyses were conducted to verify the successful conversion of WCO to graphene. The adsorption kinetic, isotherm and thermodynamics were also determined to provide in-depth insight into the GSCRiver sand with an average particle size of 0.6–1.2 mm was used to support the immobilization and coating of graphene. The WCO was collected from the café operated on the campus. Sulfuric acid (purity 95–98%) was purchased from R&M Chemicals, whereas Congo red (CR) was obtained from Sigma-Aldrich, Malaysia. All the chemicals used were of analytical grade.i.From room temperature to 100 °C in 1 hii.From 100 to 200 °C in 1 hiii.From 200 to 400 °C in 1 hiv.From 400 to 750 °C in 3 hv.Held at 750 °C for 2 h (to ensure complete graphitization of WCO).The river sand was first washed thoroughly with ultra-pure water and dried in an oven. WCO was filtered using filter paper to remove solid objects and precipitate. Next, the WCO was added to sulfuric acid in a ratio of 25:1, continuously stirred, and heated at a temperature range between 120 to 150 °C. The mixture was then mixed with a known amount of sand and stirred for about six hours at a temperature ranging between 80 to 90 °C until the sand was wholly dried and coated with the WCO. Afterwards, the WCO-coated sand was placed in a crucible and heated in a furnace under a nitrogen atmosphere. The furnace was programmed at the temperature as follows:WCO was left to cool at room temperature overnight. The GSC was activated by immersing it in concentrated sulfuric acid and kept undisturbed overnight. Lastly, the activated GSCWCO was flushed with ultra-pure water and dried at 100 °C before being used in adsorption testing.The black sample produced – GSCWCO into 30 ml of synthetic CR solution and agitating in an incubator shaker operating at a constant speed of 150 rpm at 25 °C. The initial pH solution was altered by adding HCl or NaOH (0.1 M). The samples were taken out at a predetermined time, and the absorbance was analyzed with a UV–vis spectrophotometer  at a wavelength of 496 nm. The residual concentration was calculated from the calibration curve. The removal efficiency (R) and the adsorption uptake at the equilibrium stage (qe) were calculated through Eqs. (WCO (g) used in the study.Batch adsorption experiments were carried out to investigate the effect of various parameters such as initial concentration 20–100 mg/L), adsorbent dosage (0.2–1.2 g), contact time (5–240 min), pH (2–10), temperature (25–45 °C), and ionic strength (0–0.1 M) on the removal of CR. Each experiment was performed by adding 1 g of GSC00 mg/L, Additionally, detailed characterization procedures, including kinetic, isotherm, thermodynamic and regeneration studies, are provided in the Supporting Information –S6.−1 and 1594 cm−1, corresponding to the D and G bands, respectively. The presence of the G band indicates the stretching of C–C bonds between sp2-hybridized carbons in graphitic materials, whereas the D band represents defect and disordered graphene structure35. The intensity ratio (ID/IG) of the D and G peaks helps to measure the degree of disorder and crystallite size in the structure of carbon materials. The ID/IG ratio of GSCWCO was found to be 0.76, which shows a similar structural disorder as the graphene36. On the other hand, no 2D peak was observed between 2500 and 2700 cm−1, indicating the formation of amorphous carbon37. This postulation follows Gupta et al.38 and Ruiz-Hitzky et al.39, as it was a standard feature for graphene analogues from a chemically synthesized method. Hence, this result indicated the successful conversion of WCO to graphene composite.Raman spectroscopy was a primary technique used for determining the graphenic forms of carbon. Figure WCO is represented in Fig. 2. The sand particles were typically composed of different minerals, such as quartz, kaolinite, calcite, mica, etc. Based on the XRD spectra, the highest peaks of sand can be observed at 26.7° and 20.9° with a crystal plane of (101) and (100), respectively, explicitly referring to the structure of quartz and silica40. The XRD spectra of GSCWCO depict a strong peak at 26.8°, which exhibits the characteristic peak of the (002) plane with an interlayer spacing of 3.32 Å. This result reveals the formation of multilayer graphene structures obtained by the successful conversion of WCO on the surface of GSC. The existence of graphene was reflected by the display of strong peaks between the range of 20–27° (2θ), as reported in the literature16. In addition, the spectra were seen to have several small and medium diffraction peaks, indicating the presence of minerals from the sand25. Similar results were reported by Bajpai et al.41, where the trend of diffraction peaks for GSC was found to be lower as compared to sand. This may be attributed to the dispersion of graphene sheets over the sand surfaces that eventually lowered the intensity of the observed peak. The detailed analysis of the (002) plane of GSCWCO is shown in Table WCO (34.48 nm) was found to be in the range of crystallinity results obtained from the graphene synthesis using waste cooking palm oil (WCPO) via double thermal chemical vapour deposition (DTCVD) method42. This clearly showed that WCO has the potential to be used as a carbonaceous source for the formation of graphene-like carbon materials. The interlayer spacing, crystallized size, and graphene layer were estimated according to the method given in the supplementary information XRD pattern of sand and GSCplane of 01 and , which can be considered as impurities that occurs from the exposure of fresh oil to the atmosphere and sunlight.The functional groups of WCO and fresh oil were performed using FTIR analysis. Generally, WCO consists of triglycerides, glycerol molecules, a variable quantity of free fatty acids, and several polymerization compoundsWCO. The band around 3402 cm−1 was associated with the O–H stretching of the hydroxyl group. The peak at 1619 cm−1 was referred to as aromatic C=C vibrations, which suggests the basic structure of graphene. Meanwhile, the peak at 1794 cm−1 confirmed the presence of the carbonyl group in graphene as C=O stretching. It was reported that this observation confirmed the graphitization of WCO on the sand surface47. The wide peak at 1080 cm−1 was assigned to the stretching mode of the C–O bond20. The presence of oxygen functionality in this spectrum was said to help facilitate the adsorption process. The band intensity at 778 cm−1 reflects the Si–O stretching mode, indicating the presence of quartz in the sand particles.Figure WCO. Figure WCO. Four different peaks at 100, 152, 285 and 530 eV related to Si 2p, Si 2s, C 1s and O 1s, respectively, are observed in both patterns. However, after the graphitization of WCO on the sand surface, the silicon content slightly decreased, while there was a significant increase in the carbon content. The illustrated spectra of C 1s in Fig. WCO. The sp2 hybridized carbon has an essential peak at 282.2 eV and other peaks at 284.1, 285.4 and 286.5 attributed to C–C/C–H, C–O and O=C–O, respectively, which verified the successful coating of graphene and oxygen functionalized carbon atoms on the surface of GSCWCO. Based on the O 1s spectrum  compared to other reported BET surface areas of nano-sized adsorbents. This lower value indicates that the surface area of GSCWCO was affected mainly by the sand particles, which was in good agreement with the findings of Yang et al.50. The graphene sheets on the surface of sand tend to aggregate and re-stacking because of strong π–π bonds and Van der Waals interactions, resulting in a surface area lower than the theoretical value51. In addition, since the particle size and weight of sand were much more significant compared to nanomaterials, this could significantly reduce the BET surface area of GSCWCO as the enormous weight of sand led to a smaller surface area. Nevertheless, this finding suggested that the presence of graphene nanosheets having several active sites covering the sand surface has dominated the adsorption capabilities of GSCWCO and was sufficient to remove dye pollutants.The BET surface area is one factor influencing the adsorption performance. GSC53. Then, it slowly reached the plateau as the adsorption sites were fully occupied when the concentration of CR was increased. On the other hand, the greater collision between CR molecules and the adsorbent further enhances the adsorption process. This observation is consistent with previously reported findings54.The effect of initial concentration on CR adsorption was studied in the range of 20 to 100 mg/L with constant adsorbent dosage (1 g) at 25 °C. As indicated in Fig. WCO dosage increased from 0.2 to 1 g/ 30 ml. This indicates the larger amounts of vacant adsorption sites available for binding and greater surface area for adsoption55. The adsorption capacity would reduce its removal ability by increasing the adsorbent dosage beyond the optimum dosage values. The decrease in the adsorption capacity with increased adsorbent dosage was primarily attributed to the overlapping or occurrence of particle aggregation, which could lead to a decrease in surface area and adsorption sites57. A similar trend was reported by Hou et al.58 on CR removal from aqueous solution using chitin suspension. As a result, the optimum dosage of 1 g was chosen due to its high removal efficiency and fixed for the following experiments.To find the optimized dosage, the experiments were carried out using 0.2 to 1.2 g of adsorbent dosage at a fixed CR concentration of 20 mg/L at 25 °C. The results of adsorbent dosage on adsorption capacity and removal efficiency have been plotted in Fig. 59. Figure WCO was more favourable in an acidic medium (pH 2). The zeta potential measurements of the GSCWCO composite as a function of pH are displayed in Fig. WCO attained an isoelectric point at pH 6.5. The positive zeta potential in acidic pH was mainly due to the protonation of amine groups on GSCWCO. The predominant charge at the GSCWCO surface was positive at pH < pHZPC and negative at pH > pHZPC. In an acidic medium, a high amount of hydrogen ion concentration (H+) present in the solution protonates the surface of GSCWCO. Hence, a significantly strong electrostatic force of attraction occurred between the negatively charged CR molecules (–SO3−) and the positively charged adsorbent surface, resulting in a high percentage of dye removal61. Also, other interactions (π–π bonding) may be one of those factors contributing to the greater removal efficiency. In contrast, anionic CR dye adsorption was less efficient at elevated pH, as there was a competition between excess hydroxyl ion (OH−) and CR anions for adsorption sites and the repulsive forces exist between the negatively charged adsorbent, and anionic ions of CR could lead to a decrease in CR removal efficiency. Notably, the amount of CR could still be adsorbed at higher pH owing to the hydrophobic interaction mechanism in the adsorption process. A similar observation was reported for the sorption of CR on mesoporous-activated carbon62 and activated carbon-coffee waste63.pH solution is one of the crucial parameters influencing the interaction between the adsorbed molecules and adsorbent particles by affecting the charged nature of functional groups on the adsorbent and the ionization degree of dye pollutantsWCO. This was mainly due to the greater number of available vacant sites and the high driving force that eventually helped to speed up the removal rate of CR64. The adsorption slightly slows down in the later stages since the exterior surface of the adsorbent has been occupied and become saturated by the molecules of dye. Upon attaining the equilibrium condition, the removal rate could no longer increase as the remaining vacant sites had difficulty occupying the position. This may be ascribed to the repulsive forces between the molecules adsorbed on the surface and the bulk phase65. Conclusively, the equilibrium time for CR adsorption was attained within 150 min, where about 91.5% of CR had been removed.Contact time is essential as it significantly influences the favourability of the adsorption process. The effect of contact time on the adsorption of CR was carried out in the range of 5 to 240 min under optimized initial concentration (20 mg/L), dosage (1 g), and pH 2. As seen in Fig. 66. In this study, the influence of the temperature of GSCWCO on the elimination of CR was investigated at various temperatures, including 25, 35, and 45 °C with an optimum dosage of 1 g, pH 2, and an initial concentration of 20 mg/L. As shown in Fig. 67. Two plausible factors contribute to this phenomenon; the first indicates that the physical bonding between the dye molecules and the GSCWCO has weakened, resulting in reduced adsorption as the temperature rises. Another reason may be the increase in dye solubility, which creates stronger solute interaction with solvent68.Temperature is another critical factor influencing the mobility of pollutants and adsorbent propertiesWCO. Ionic strength was adjusted by adding different concentrations of NaCl solution, ranging from 0 to 0.3 mol/L. It was noticeable that there was a slight reduction in the CR uptake capacity from 5.58 to 4.64 mg/g as the NaCl concentration increased. This behaviour was mainly ascribed to the competition between CR and chloride anions (from NaCl) for the available active sites on the GSCWCO surface. The influence of ionic strength was weak, thus, suggesting that the electrostatic interaction was not the dominant mechanism for removing CR on GSCWCO. The such postulation was supported by the findings of Liu et al.69.Figure 2) was used to indicate the best-fitting model. Based on the results, it can be deduced that the adsorption of CR was well-fitted with the Freundlich isotherm model since the R2 value was found to be 0.989, which is greater than the other isotherm models. This can be explained by the fact that GSCWCO has a heterogeneous surface that can adsorb a multilayer of CR molecules. The heterogeneous adsorbent surface was known to have different types of adsorption sites for adsorbate to attach, with each site having different adsorption energy. According to the theory of Freundlich, the favorability of the adsorption process can be examined by indicating the adsorption intensity, n, which lies between 1 and 10, was said to have a higher intensity of adsorption70. As depicted in Table n) is closer to zero71.The experimental plots and data predicted by four isotherm models  in the form of the linearized equation are shown in Fig. 2 value of pseudo-second-order (R2 > 0.99) was closer to unity, indicating the adsorption of CR was operated through chemisorption. Chemisorption occurs when a chemical bonding is formed between the CR ions and GSCWCO. Besides, the calculated value of equilibrium adsorption capacity (qe) was closer to the experimental value  than the pseudo-first-order model. A similar result has been reported by Teow et al.27 using GSC from oil palm frond juice for the adsorptive removal of methylene blue.Four kinetic models, namely pseudo-first-order, pseudo-second-order, Elovich, and intraparticle diffusion kinetic models, were fitted to the experimental data to understand the adsorption process mechanism better. Figure 72. It was noted that the interaction behaviour between adsorbent and adsorbate solution could be well understood through 73. The results clearly showed the trend of The values of thermodynamic parameters, namely Gibb's free energy  interacted with various oxygen functionalities on GSCWCO (electron donor). At the same time, a hydrophobic effect exists between the GSCWCO surface's tails and CR's hydrophobic components at basic pH.It is crucial to understand the adsorption mechanism of organic dye on the novel adsorbent of GSCWCO also had an essential role in the adsorption process. In “WCO, which were responsible for adsorption. Moreover, an aggregated morphology and disappearance of pores were noticed from the post-adsorption characterization of GSCWCO using SEM, indicating the interaction of adsorbent with CR. In addition, the full-scale XPS spectrum  via a simple chemical approach was successfully reported in this study. WCO, as abundant and low-cost carbon precursors, was successfully converted to graphene sheets and coated on the sand surface. Raman and XRD analysis confirmed the formation of graphene-like structure materials. The SEM and EDX mapping showed that GSCWCO had a rough and porous surface that can contribute to the greater efficient removal of pollutants. The applicability of GSCWCO was tested for adsorptive removal of CR under optimal conditions . The sorption mechanism and rate of adsorption of GSCWCO were better to fit with Freundlich and pseudo-second-order model, respectively, giving a maximum adsorption capacity of 5.53 mg/g at the temperature of 25 °C. The negative value of Gibb's free energy, enthalpy, and entropy changes confirmed sorption's feasibility, spontaneity, and exothermic nature. A reusability study revealed that GSCWCO adsorbs CR up to four cycles with removal efficiency ranging from 75.5 to 59.4%. The π–π and hydrogen bonding interactions were postulated as the primary mechanism responsible for the adsorbent performance. Therefore, further development of graphene composite derived from carbon waste (WCO) would be an excellent potential for water pollution treatment and simultaneously help reduce the improper discharge of WCO.A novel approach of synthesized graphene sand composite from waste cooking oil (GSCSupplementary Information.


These findings indicate that warming strongly structures freshwater communities by reducing or increasing densities of different aquatic communities groups. It also enhances beta‐diversity, and many of these effects are modulated by habitat size or detrital resources.Freshwaters are among the most vulnerable ecosystems to climate warming, with projected temperature increases over the coming decades leading to significant losses of aquatic biodiversity. Experimental studies that directly warm entire natural ecosystems in the tropics are needed, for understanding the disturbances on aquatic communities. Therefore, we conducted an experiment to test the impacts of predicted future warming on density, alpha diversity, and beta diversity of freshwater aquatic communities, inhabiting natural microecosystems—Neotropical tank bromeliads. Aquatic communities within the tanks bromeliads were experimentally exposed to warming, with temperatures ranging from 23.58 to 31.72°C. Linear regression analysis was used to test the impacts of warming. Next, distance‐based redundancy analysis was performed to assess how warming might alter total beta diversity and its components. This experiment was conducted across a gradient of habitat size (bromeliad water volume) and availability of detrital basal resources. A combination of the highest detritus biomass and higher experimental temperatures resulted in the greatest density of flagellates. However, the density of flagellates declined in bromeliads with higher water volume and lower detritus biomass. Moreover, the combination of the highest water volume and high temperature reduced density of copepods. Finally, warming changed microfauna species composition, mostly through species substitution ( Effects of warming on structure of aquatic communities. Temperature strongly influences cell chemical composition, nutrient uptake, COEmpirical evidence of the impacts of warming on aquatic communities is often limited to observational studies with low replication or simplified, artificially assembled communities in laboratory experiments. However, freshwater communities occupying small water bodies trapped between bromeliad leaf‐axils, or ‘phytotelmata’ are becoming widely used to test the impacts of environmental change on entire communities and ecosystems ; (ii) warming would preferentially reduce the alpha diversity and density of some groups of the aquatic communities, especially larger organisms such as copepods.In this study, we experimentally warmed naturally assembled aquatic communities occurring within 50 tank bromeliads to quantify the impacts of warming on the structure of tropical freshwater aquatic communities. We tested how warming affects the density, local species richness, and beta diversity components of different groups of microflora and microfauna. We predict that warming would have stronger impact of microfauna communities than microflora communities because larger organisms are more sensitive to warming than smaller organisms , on the coast of São Paulo state Figure . We collNeoregelia johannis as the model system for our warming experiment as it is a large, dominant species in the region. Individual N. johannis hold almost 2 L of water on average, majorly contributing to freshwater habitat provision in an environment where standing water is rare  from the forest floor, to eliminate height stratification  and 2100 (+4°C) IPCC,  along wi2.2We performed two sample collections of the aquatic communities, at the beginning and at the end of the experiment. We collected a homogeneous mixture of 50 mL of water from all 50 microecosystems. Subsequently, 1.5 mL of this sample was conditioned in a 2‐mL cryogenic tube. The rest was fixed with the Lugol's acetic fixative (5%) totaling, at the end of the sampling, 100 samples (50 samples from each collection period). The samples were subjected to quantitative analyses of specific groups  and square root (for richness).We analyzed 50 community samples from the beginning and 50 community samples from the end of the experiment. The samples from the beginning of the experiment allowed us to establish the absence of baseline differences in aquatic communities at the start of the experiment across treatments. To assess how warming and volume affected the density and richness of microflora and microfauna (prediction i), and different taxonomic groups (prediction ii), multiple linear regressions were also fit to the data from the end of experiment. We included mean water temperature and volume as continuous predictor variables, final detrital mass as covariate, and their interaction, using the function “lm” from the stats package. We also performed the model comparison with the Akaike information criterion (AIC) to select the best model using the function “AIC” from the βrepl) and difference in richness (βrich), the Jaccard distance matrix was analyzed with the method proposed by Podani and Schmera . More specifically, the highest density of flagellates was found in bromeliad ecosystems with the highest temperature and detritus amount . Warming and bromeliad water volume did not alter the total density . Specifically, the highest copepod density was found in bromeliads with higher water volume and low temperatures  was deriving the changes in beta diversity with the experimental warming, that is, bromeliads with higher mean temperatures had more distinct composition, probably due to the occurrence of species more adapted to the warmer environment. Species replacement tends to be more common in the Neotropics, relative to temperate regions, due to relatively constant temperatures, limited acclimation potential, and narrower climate tolerances . This is because bacteria can support algal growth by recycling nutrients while also competing for essential nutrients and this can mediate remineralization and carbon sequestration  from upper trophic level groups . Such changes in trophic structure can alter important ecosystem processes such as decomposition and primary productivity. By limiting energy transfer to higher trophic levels, through negatively affecting microflora and microfauna, warming has both direct and indirect effects on the entire food webs. We argue that due to the controlled and replicated nature of this field experiment, our findings may be applicable to a wide range of aquatic environments in the tropics, most of which are experiencing changes in temperature regimes. Our findings suggest that large taxa that are strongly affected by climate warming should by prioritized for targeted conservation actions.This study provides new experimental evidence that warming can alter the diversity of aquatic communities in the tropics. However, to draw more general conclusions it would be fruitful to explore a long‐term effect of experimental manipulation, focusing on the process of species succession. The future study of both resistance and resilience of aquatic communities to warming would help to characterize more sensitive organismal groups and, consequently, possible functional losses. Future research should investigate these responses of aquatic communities across a wider range of experimental temperatures, including the heatwave periods ; data curation ; formal analysis ; methodology ; visualization ; writing – original draft ; writing – review and editing . Pablo A. P. Antiqueira: Conceptualization ; data curation ; writing – original draft ; writing – review and editing . Felipe R. Oliveira: Conceptualization ; methodology ; writing – original draft ; writing – review and editing . Bianca R. Meira: Conceptualization ; writing – original draft ; writing – review and editing . Fernando M. Lansac‐Toha: Data curation ; formal analysis ; writing – review and editing . Luzia C. Rodrigues: Methodology ; visualization ; writing – review and editing . Gustavo Q. Romero: Conceptualization ; methodology ; supervision ; writing – review and editing . Liam N. Nash: Conceptualization ; data curation ; formal analysis ; methodology ; writing – review and editing . Pavel Kratina: Conceptualization ; data curation ; methodology ; supervision ; visualization ; writing – review and editing . Luiz F. M. Machado‐Velho: Conceptualization ; methodology ; supervision ; writing – original draft ; writing – review and editing .The authors declare that they have no conflict of interest.Table S1Click here for additional data file.


Two cases of sarcoidosis referred to our clinic with neurological symptoms. They were diagnosed with multiple sclerosis using non‐invasive studies. The first patient refused treatment and died of myocardial infarction 6 months after visiting our clinic. The second received interferon‐beta and methotrexate with a favorable outcome after 3 years. Since the possible similar presentation of the two conditions could appear indistinct for certain diagnosis, accurate evaluation of symptoms and paraclinical data can provide the best approach to each condition. That is mainly because patients with sarcoidosis and MS are of similar ages and may present with overlapping clinical features such as damage to cranial nerves, myelopathy, and demyelinating lesions of the CNS.2A 53‐year‐old woman presented to our clinic in March 2019 for evaluation of lower limb weaknesses along with visual impairments starting from 2 months earlier. She was married with three children, had a history of a face lesion 5 years prior, for which she had visited a dermatologist. At that time, she was diagnosed with sarcoidosis based on a histopathological study of the lesion biopsy which revealed a pattern of non‐caseating granulomatous inflammation consistent with sarcoidosis, a chest CT study which found mild pleural thickening and hilar prominence on the right side, and serum angiotensin‐converting enzyme (ACE) level measurement, which was within normal range but close to the upper limit. Since then, she was put on oral prednisolone therapy. From the time she was diagnosed with sarcoidosis, she reported experiencing several episodes of ataxia, diplopia, and upper limb weakness, each lasting between 2 and 4 weeks. Those episodes were deemed due to NS and treated accordingly in other clinics. She also developed hypertension and diabetes mellitus, otherwise, her past medical, social, and familial histories were unremarkable. Neurological examination revealed glove and stocking sensory impairment and lower limb weakness with a distal force of 3/5 and proximal force of 5/5. Babinski sign was observed bilaterally and tendon reflexes were brisk. Considering NS, diabetic neuropathy, and MS as the primary differential diagnoses, nerve conduction velocity (NCV) studies, visual‐evoked potential test (VEP), and MRI were performed. NCV showed demyelinating polyneuropathy in the lower extremities, VEP results were 192 and 194 in the left and right eye, respectively, and showed prolongation of latency bilaterally. MRI study showed multiple demyelinating lesions in the periventricular, juxtacortical, and cervical spine areas , and juxtacortical demyelinating lesions suggestive of MS (Figure 4Sarcoidosis can produce several heterogenous clinical pictures, stemming from the multisystem nature of the disease. NS is uncommon, but also a potentially serious form of sarcoidosis. While NS mostly affects cranial nerves, it can also involve other parts of nervous system, including intracranial structures. The clinical manifestations in an individual depends on the location of the inflammatory process. The most commonly reported features of NS include cranial nerve neuropathy, headache, fatigue, nausea and vomiting, sensory abnormalities like visual disturbance, motor symptoms consisting of hemiparesis and paraparesis, meningitis, seizures, and spinal cord abnormalities.Furthermore, although cases of sarcoidosis in people with preexisting MS—particularly deemed, but not proven to be associated with certain DMTsFinally, no underlying mechanistic correlation between sarcoidosis and MS has been confirmed to date, although both are thought to involve genetic and immune‐mediated etiologies. Although not established, the HLA loci may be involved in both MS and sarcoidosis.5Differentiating MS from NS may be challenging in known cases of sarcoidosis. We reported our experience with two cases of sarcoidosis patients developing MS, who were diagnosed and managed without any major invasive procedures. This report, along with the previous ones, provides valuable insights on the diagnostic work‐up of neurological symptoms in sarcoidosis patients.PTN, ME, and AM have gathered relevant case information, searched the databases for relevant data, and drafted the initial manuscript. MS and NS were involved in validation of findings, review, and editing of the initial draft. All authors have read the final manuscript and approved it for publication.The authors have no conflicts of interest to declare.The patients or their next of kin provided written consent for publication of their anonymized cases.