Fusarium crown rot (FCR) is a chronic disease of cereals worldwide. Compared with tetraploid wheat, hexaploid wheat is more resistant to FCR infection. The underlying reasons for the differences are still not clear. In this study, we compared FCR responses of 10 synthetic hexaploid wheats (SHWs) and their tetraploid and diploid parents. We then performed transcriptome analysis to uncover the molecular mechanism of FCR on these SHWs and their parents.We observed higher levels of FCR resistance in the SHWs compared with their tetraploid parents. The transcriptome analysis suggested that multiple defense pathways responsive to FCR infection were upregulated in the SHWs. Notably, phenylalanine ammonia lyase (PAL) genes, involved in lignin and salicylic acid (SA) biosynthesis, exhibited a higher level of expression to FCR infection in the SHWs. Physiological and biochemical analysis validated that PAL activity and SA and lignin contents of the stem bases were higher in SHWs than in their tetraploid parents.Overall, these findings imply that improved FCR resistance in SHWs compared with their tetraploid parents is probably related to higher levels of response on PAL-mediated lignin and SA biosynthesis pathways.The online version contains supplementary material available at 10.1186/s12864-023-09268-7. Triticum aestivum, AABBDD), is the youngest allohexaploid formed nearly 10,000 years ago as a hybrid between an early-cultivated tetraploid wheat  and diploid wild goatgrass  [Hexaploid wheat, or common wheat  . Comparehii, DD) . Hexaplohii, DD) . Owing thii, DD) , 5.Fusarium pseudograminearum, is a soil-borne disease that seriously impacts wheat and barley production. This disease was first reported in Australia and now, it has become a prevalent disease, threating cereal production worldwide [Fusarium crown rot (FCR), primarily caused by orldwide , 7. Signorldwide , 9. Accoorldwide . FCR hasorldwide  reportedorldwide , currentF. pseudograminearum can provide new strategies to develop varieties with better resistance to FCR. In wheat, several transcriptome analyses have been performed to investigate defense responses in wheat after FCR infection [F. pseudograminearum infection are very similar to those induced by F. graminearum and F. culmorum [A better understanding of the mechanisms of the host defenses against nfection –15. For nfection . In a confection . More reculmorum .An interesting phenomenon is that hexaploid wheat is generally more resistant to FCR infection than tetraploid wheat. Differences in FCR resistance between tetraploid and hexaploid wheat have been uncovered by several studies. For instance, Daniel et al. (2008) reported that FCR inoculation reduced the yield of durum wheat by 58%, whereas the average yield loss in bread wheat was only 25% across different soil types . Liu et T. turgidum (AABB) and Ae. tauschii (DD). We performed transcriptome analyses of two SHWs and their tetraploid and diploid parents following FCR infection to identify genes and pathways associated with improved FCR resistance of SHWs. We found evidence that defense pathways regulated by phenylalanine ammonia lyase (PAL) genes are altered in SHWs compared with their tetraploid and diploid parents. We also measured six physiological parameters related to FCR resistance. Our findings revealed the genetic basis of improved FCR resistance in SHWs, which may help to develop highly FCR-resistant varieties in future.Recent studies have shown that reprogramming of transcriptomes, which occurs in newly formed hexaploid wheat, has distinct effects on growth vigor and growth adaptation following allohexaploidization , 19–21. T. turgidum ssp. dicoccoides, T. turgidum ssp. turgidum and T. turgidum ssp. dicoccon) and diploid parents (including Ae. tauschii ssp. tauschii and Ae. tauschii ssp. strangulata) were used (Table T. turgidum (AABB) and diploid goat grass Ae. tauschii (DD) were used as parental lines to generate SHWs [T. turgidum was used as a maternal parent and pollinated with Ae.tauschii anthers. S1 seeds were obtained from triploid F1 hybrids (ABD) after spontaneous chromosome doubling and were grown as the first generation of allohexaploid plants (AABBDD) [T. turgidum and 14 from Ae. tauschii) were used for further experimentation. Spontaneous chromosome doubling resulted from the union of un-reduced gametes [1 hybrid. All materials were preserved and provided by Triticeae Research Institute of Sichuan Agricultural University.In the study, 10 sets of SHWs and their tetraploid parents (including ate SHWs . T. turg(AABBDD) . Only pl gametes . No embrFusarium pseudograminearum isolate (NLYY), collected from Henan Province of China, was provided by Henan Institute of Science and Technology. The inoculum preparation, inoculation and FCR assessment were performed as described previously [6 spores/mL and then used directly for inoculation or stored at − 20 °C until needed. Tween 20 was added (0.1% v/v) to the spore suspension prior to use.A eviously . Briefy,Fusarium pseudograminearum isolate (Fp inoculation) or distilled water (mock inoculation) following the protocol described previously [Fp inoculation) or in distilled water (mock inoculation) for 1 min and seedlings were planted into the 50-well plastic trays containing sterilised nutrient soil. The plastic trays were arranged in the incubator and the settings for the incubator were: 25/16 (± 1) °C day/night temperature and 65%/85% day/night relative humidity.Seedlings at 4 days post-germination, were inoculated with eviously . Ten seeX/5 N)×100, where X is the scale value of each plant, n is the number of plants in the category, and N is the total number of plants assessed for each material.FCR severity was assessed on a 0–5 scale at fourth weeks after inoculation, where “0” represents no symptom and “5” represents whole seedling completely necrotic . A diseaT. turgidum ssp. turgidum and Ae. tauschii ssp. tauschii, and SHW-2 from the cross of T. turgidum ssp. dicoccoides and Ae. tauschii ssp. strangulata) and their tetraploid and diploid parents were selected for RNA-seq. The experimental design for each material contained two treatments (mock and Fp inoculation), one time point (3 days post inoculation) and three biological replicates. And a total of ten samples for each biological replicate were used for RNA extraction. Samples were harvested by cutting the stem bases (1 cm) at 3 days post inoculation and snap-frozen in liquid nitrogen and kept at − 80 °C until processed. Total RNA was isolated using a HiPure HP Plant RNA Mini Kit  according to the manufacturer’s instructions.Two SHWs (SHW-1 from the cross of https://ngdc.cncb.ac.cn/search/). Sequence quality assessment, trimming and RNA-seq analysis was performed using CLC Genomics 12.0 software with default parameters. The processed RNA-seq data in SHWs were aligned to complete mRNA (cDNA) sequences of Chinese Spring Reference genome IWGSCv1.1 . While RNA-seq data in tetraploid parents and diploid parents were respectively aligned to mRNA sequences of AABB sub-genomes  and DD sub-genome  from Chinese Spring Reference genome IWGSCv1.1. The expression level of each gene was calculated using TPM (Transcripts Per Kilobase of exon model per Million mapped reads ). Differentially Expressed Genes (DEGs) were restricted with the absolute value of log2 (fold Change) ≥ 1 and False Discovery Rate (FDR) < 0.05 as the threshold by performing pairwise comparisons of Fp treated and corresponding mock samples. DEGs were separated into up-regulated and down-regulated genes and used as individual test sets for GO enrichment analysis. GO term enrichment analysis was done using Triticeae-GeneTribe (http://wheat.cau.edu.cn/TGT/) [A total of 36 libraries were sequenced in the 150 bp paired-end mode using Illumina NovaSeq6000 platform at Berry Genomics Corporation . Sequence files were deposited at the National Genomics Data Center (NGDC) Genome Sequence Archive under BioProject ID PRJCA011781 (cn/TGT/) .http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and listed in Table −∆∆CT). Genes with Ct values > 40 cycles were regarded as having no expression value.Thirteen DEGs were randomly selected and assessed using real-time quantitative PCR (RT-qPCR). Primers were designed using Primer-BLAST tool (Fp inoculation), one time point (3 days post inoculation) and three biological replicates. And a total of ten samples for each biological replicate were used. Samples were harvested by cutting the stem bases and snap-frozen in liquid nitrogen and kept at − 80 °C until processed.PAL activity, chitinase activity, salicylic acid (SA) content, jasmonic acid (JA) content and abscisic acid (ABA) content of 5 SHWs  and their tetraploid and diploid parents were determined using the ELISA method at FANKEL Industrial Corporation . The experimental design for each line contained one treatment . To measure basal lignin levels in stem bases, the experimental design for each line contained two time points (seedling and heading stage) and three biological replicates. And a total of ten samples for each biological replicate were used. Samples were harvested by cutting the stem bases and dried to constant weight.p < 0.05.Paired samples T-test was performed in Excel 2019 and significant differences was claimed at p < 0.01) infection than their parents  values than their diploid parents, whereas all SHWs had lower DI values than their tetraploid parents  randomly selected from the DEGs of two SHWs  enrichment analysis was performed to classify the functions of DEGs. This analysis revealed that 90, 48, and 26 terms related to biological processes were enriched (FDR < 0.05) in up-regulated DEGs of SHW-1 and its tetraploid and diploid parents, respectively , whereas 24, 36, and 17 functional biological process categories were enriched in down-regulated DEGs . In regard to SHW-2 and its tetraploid and diploid parents, 62, 45, and 20 functional categories under biological process were respectively enriched in up-regulated DEGs , whereas 33, 31, and 14 categories were enriched in down-regulated DEGs . GO terms enriched in up-regulated DEGs of SHWs were more abundant than those of their parents. Several defense-related pathways were commonly enriched in up-regulated DEGs of SHWs and their tetraploid and diploid parents, including cinnamic acid biosynthetic process (GO:0009800), chitin catabolic process (GO:0006032), L-phenylalanine catabolic process (GO:0006559), regulation of defense response (GO:0031347), glutathione metabolic process (GO:0006749), response to oxidative stress (GO:0006979)  SA contents of SHWs compared with their tetraploid parents  and heading (p < 0.01) stages  were significantly higher in SHWs than in their tetraploid parents (0.0992 U/g FW) and diploid parents (0.0933 U/g FW) , thereby exhibiting transgressive performance over both diploid and tetraploid parents.FCR has dramatically increased worldwide as a result of drier seasonal conditions and the adoption of moisture-preserving cultural practices, such as minimum tillage and stubble retention , 7. TetrTranscriptome reprogramming due to the introduction of the D genome occurred in SHWs, leading in turn to global changes in gene expression . These gAs an entry-point enzyme in the phenylpropanoid biosynthesis pathway, PAL converts phenylalanine to trans-cinnamic acid and free ammonia, thereby playing an important role in lignin and salicylic acid biosynthesis. Many studies have shown that PAL acts as a positive regulator of resistance to various plant biotic stresses –36. In tTaDIR-B1 improves resistance to FCR in wheat by increasing the accumulation of lignin [Lignin, an important component of the plant cell wall, provides an effective barrier for plants to resist pathogen infections and plays a crucial role in plant disease resistance . A recenf lignin , thus inThe importance of SA in plant defense against pathogen attack has been thoroughly documented , 39–42. Higher levels of response on PAL-mediated lignin and SA biosynthesis pathways may contribute to improved FCR resistance in SHWs compared with their tetraploid parents Fig. . HoweverPrevious transcriptome analyses have revealed various genes and pathways associated with FCR resistance in wheat, such as anti-microbial proteins, oxidative stress-related proteins, signaling molecules, primary and secondary metabolism, pathogen sensing and signaling, cellular transport and detoxification, and benzoxazinoid biosynthesis –15. HoweT. turgidum and Ae. tauschii. We confirmed improved FCR resistance in the SHWs compared with their parental genotypes. The transcriptome analysis of two SHWs and their tetraploid and diploid parents suggested that multiple defense pathways responsive to FCR infection were stronger in the SHWs than in the parents. Notably, dozens of PAL genes, involved in lignin and SA biosynthesis, exhibited a higher level of expression to FCR infection in the SHWs. Physiological and biochemical analysis validated that PAL activity and SA and lignin contents of the stem bases were higher in SHWs than their tetraploid parents. In summary, this study suggested that improved FCR resistance in SHWs compared with their tetraploid parents is probably related to higher levels of response on PAL-mediated lignin and SA biosynthesis pathways.In this study, we compared the FCR responses of 10 SHWs and their parental genotypes, Below is the link to the electronic supplementary material.Table S1. Primers for real-time quantitative PCR.Additional file 1: Table S2. RNA-seq read information and mapping statistics of 2 SHWs and their tetraploid and diploid parents.Table S3. Differentially expressed gene (DEGs) of 2 SHWs and their tetraploid and diploid parents.Table S4. GO terms enriched by up-regulated DEGs of 2 SHWs and their tetraploid and diploid parents.Table S5. GO terms enriched by down-regulated DEGs of 2 SHWs and their tetraploid and diploid parents.Table S6. PAL activity, chitinase activity, SA content, JA content and ABA content of the stem base in 5 SHWs and their parents following FCR infection.Table S7. Total lignin content of the stem base of 10 SHWs and their tetraploid parents at the seedling and heading stage.Figure S1. Validation of data of RNA-seq by RT-qPCRAdditional file 2:


Sporadic inclusion body myositis (sIBM) is an idiopathic inflammatory myopathy with invasion of CD8 T cells in muscle and aggregation of proteins in the sarcoplasm. TDP-43 and p62 are two proteins that aggregate in affected muscle, and have been suggested as specific markers for sIBM over other inflammatory myopathies. TDP-43 is also mislocalised from the nucleus to the sarcoplasm in sIBM. It is not clear if inflammation precedes protein aggregation in sIBM. This study investigated if exposure to cytotoxic inflammatory cells caused TDP-43 and p62 aggregation or TDP-43 mislocalisation in cultured myotubes. TALL-104 coculture was highly cytotoxic to myotubes after 24 h. Secretion of IFNγ and TNFα were higher in cocultures compared to monocultured TALL-104 cells, indicating activation. TALL-104 cells attached to and infiltrated myotubes. There was no effect of TALL-104 coculture on TDP-43 or p62 sarcoplasmic aggregate size or frequency. However, there was decreased localisation of TDP-43 to the nucleus with TALL-104 coculture compared to control. In an in vitro setting, cytotoxic immune cells did not cause TDP-43 or p62 sarcoplasmic aggregation, suggesting cellular cytotoxicity may not trigger aggregation of these proteins. However TALL-104 coculture influenced TDP-43 localisation, suggesting cytotoxic immune cells may contribute to TDP-43 localisation shifts which is observed in sIBM. The disease is characterised by muscle weakness especially in the quadriceps and finger flexors, with presentation usually asymmetrical affecting the non-dominant limb more6. It is slowly progressive and many patients requiring mobility aids at later stages, with the average time to requiring mobility aids of 10 years for a walking stick7 and 10–15 years for a wheelchair7. A common progressive symptom is dysphagia, which occurs in up to 40–86% of cases and can be a cause for mortality9.Sporadic inclusion body myositis (sIBM) is the most common form of myositis in people over the age of 5010. These CD8 + T cells are minimally proliferative and differentiated towards an effector cell phenotype with high cytotoxicity11. The inhibitory receptor and marker of differentiated CD8 cells, killer cell lectin-like receptor G1 (KLRG1), is highly expressed on muscle-invading and circulating CD8 T cells in sIBM patients, and is co-expressed with cytotoxic genes11. Other T cell differentiation markers have been identified in sIBM cytotoxic T cells, including the senescent-associated CD5713 and the differentiation marker T-bet1. As well as inflammation, sIBM is also characterised by degenerative non-inflammatory features. This includes the presence of rimmed vacuoles in affected fibres14, inclusion bodies and protein aggregates within the sarcoplasm and rimmed vacuoles15. Two proteins that are aggregated and potentially dysregulated in sIBM muscle are TDP-43 and p62.sIBM remains an idiopathic disease with no singular distinct cause and many mechanisms implicated in this disease. Broadly, pathological features of sIBM can be split into inflammatory and non-inflammatory/degenerative. Within affected skeletal muscle fibres of sIBM patients there is infiltration of inflammatory cells, mostly CD8 + cytotoxic T cells, into the endomysium and the muscle fibres themselves16, and stress sensing17. It is also involved in the removal of cryptic exons from some mRNAs18. Cryptic exons are usually excluded from mature mRNAs molecules19, and their inclusion in mature mRNAs can lead to splice variants causing premature stop codons or frameshift mutations.TDP-43 is an RNA/DNA binding protein involved in mRNA transport, stability, splicing regulation21. TDP-43 was found accumulated in all biopsies of definite sIBM cases and 31% of possible sIBM cases20, whilst a separate study found 67% of sIBM cases were positive for TDP-43 aggregates22. sIBM patients also show higher levels of 25 and 35 kDa truncated fragments of TDP-43 compared to healthy controls23. The nuclear localisation of TDP-43 is decreased in sIBM compared to healthy controls21. The presence of TDP-43 within the sarcoplasm with a lack of nuclear TDP-43 has been suggested as a marker of sIBM, with its presence detected in 25% of sIBM myofibres, whereas rimmed vacuoles were detected in 2.8% of sIBM myofibres24. It is not clear how TDP-43 aggregation and cytoplasmic mislocalisation may contribute to sIBM pathology. Potentially, cytosolic sequestration of TDP-43 in aggregates causes TDP-43 to lose its homeostatic functions. Indeed, sIBM patient muscle has widespread alterations in RNA metabolism pathways compared to controls25. sIBM muscle has altered TDP-43-controlled cryptic exon inclusions compared to control patients in ASAP2 26. Furthermore, detection of cryptic exons in sIBM muscle was 84% sensitive and 99% specific for sIBM compared to healthy control and other muscle disease patients27.TDP-43 is aggregated in the sarcoplasm in sIBM28. As with TDP-43, p62 is aggregated within the sarcoplasm of sIBM patients20, and p62 aggregates are often associated with vacuolated areas in affected sIBM fibres30. Myofibres with high p62 levels also show high levels of TDP-43 and mitochondrial staining23. p62 aggregates in sIBM muscle can be large, and approximately half of the p62 aggregates colocalised with LC3, suggesting a potential role for defective selective autophagy in sIBM31. The combined presence of TDP-43 and p62 in sIBM muscle has been suggested as a specific marker for sIBM over other inflammatory myopathies22.p62 is involved in selective autophagy and the ubiquitin proteasome system (UPS)33. This study aims to investigate in an in vitro system whether exposure to cytotoxic immune cells in the form of the TALL-104 can trigger non-inflammatory features observed in sIBM, focussing on TDP-43 and p62 sarcoplasmic aggregation and TDP-43 mislocalisation.Currently it is not known whether degenerative or inflammatory features of sIBM arise first, and if there is an interaction between these features. Knowing if inflammation or protein aggregation precedes other characteristics would help the understanding of this disease and could help in the development of treatments targeted towards the initiating events in sIBM pathology. Here, coculture of myotubes and the human cytotoxic cell line TALL-104 was used, which triggers cytotoxicity via a major histocompatibility complex (MHC)-independent pathway through perforin, granzyme, Fas ligand, and TRAIL (TNF-related apoptosis-inducing ligand)All materials were purchased from ThermoFisher Scientific, UK unless otherwise stated.Skeletal muscle-derived cells from Lonza Clonetics™  or Cook MyoSite®  from healthy donors were grown in Ham’s F10 nutrient mix (11550043) supplemented with 20% FBS (10500064), 1 µM dexamethasone , 10 ng/mL basic fibroblast growth factor , and 1% penicillin–streptomycin. Skeletal muscle-derived cells were differentiated in N2 differentiation medium: DMEM/F12 1:1 (11320074), 1% N2 supplement (17502048), 1% L-glutamine, 1% insulin-transferrin-selenium (41400045), and 0.2% penicillin–streptomycin. TALL-104 cells were purchased from LGC standards, UK. TALL-104 cells were cultured in ATCC-modified RPMI with 20% FBS and 20 ng/mL IL-2 premium grade .4 cells per 24-well with two days proliferation or 5.5 × 104 cells per well with one day proliferation followed by 5 days differentiation. TALL-104 cells were centrifuged at 170× g and resuspended in N2 myotube differentiation medium with 20 ng/mL IL-2 for addition to myotube cultures. Different effector to target (E:T) ratios of 0.5:1, 1:1, 2.5:1, and 5:1 of TALL-104 cells were added based on the initial seeding density of myogenic cells for 4, 24, or 48 h. For ELISA (enzyme linked immunosorbent assay) and immunofluorescent experiments analysing TDP-43 and p62, 1:1 E:T ratio for 24 h was used.Myotubes were seeded at either 4 × 10Cytotoxicity was measured using CytoTox 96® non-radioactive cytotoxicity assay  following manufacturer’s instructions. Cytotoxicity was measured in medium samples from the cultured myotubes after TALL-104 coculture or IL-2 control. Substrate solution was added to samples before addition of stop solution, and optical density read at 492 nm using a Multiskan™ FC plate reader. Results were normalised to a medium-only control and a positive cytotoxicity control, where cells were incubated in lysis solution for 45 min before commencing the assay. Cytotoxicity was expressed as a percentage of the positive lysis control.Skeletal muscle-derived cells were seeded on Gibco Geltrex (A1569601)-coated 13 mm round coverslips in 24 well plates. The cells were allowed to proliferate for 48 h before differentiation for 5 days before coculture with TALL-104 cells for 24 h. Cells were washed and fixed with 4% formaldehyde solution  for 10 min at room temperature. Cells were permeabilised with 0.1% v/v triton X100 in PBS for 15 min before blocking with 5% goat serum in PBS for 30 min to 1 h. Primary antibodies TDP-43 1:150 (10782–2-AP) and p62 1:100  diluted in blocking buffer were added by inverting the coverslip onto a 30 µL drop of antibody solution on Parafilm®, and incubating in a humidified chamber overnight at 4 °C. Coverslips were incubated in appropriate Alexa Fluor™-conjugated secondary antibodies  for 1 h at room temperature. Cells were washed and HCS CellMask™ deep red (H32721) was added at 1:5000 in PBS for 30 min to visualise all cell contents. Afterwards, DAPI  at 5 μg/mL in PBS was added for 5 min. After washing, cells were mounted onto glass slides with DAKO fluorescence mounting medium .To analyse TDP-43 localisation, images were captured using an Olympus IX81-ZDC inverted widefield fluorescent microscope at 10× magnification. Four images were analysed per condition per myogenic donor. DAPI and HCS CellMask™ staining was used to observe subcellular compartments of the nucleus and sarcoplasm respectively. Based on the observed staining, TDP-43 was classified as being within the nucleus, sarcoplasm, nucleus and sarcoplasm, or neither (not expressed). Localisation was quantified for both single nucleated and multinucleated cells. Localisation was expressed as a percentage of total number of cells observed with HCS CellMask™ for each localisation compartment.For p62 and TDP-43 aggregation analysis, cells were imaged using a Zeiss LSM 800 confocal microscope. Eight z-stacks each composed of two images approximately 0.9 µm apart from one experiment were captured. Images were analysed using NIH Image J (USA). Obtained images were split into individual colour channels and converted to 8-bit. p62 and HCS CellMask™ channels were manually thresholded.p62 particle frequency and size was determined in the p62 channel using NIH Image J using the “analyse particles” function with pixel size over 3. p62 frequency was the total number of particles obtained per image with the “analyse particles” function and p62 size was the average particle size per image. In the thresholded cell mask channel, percentage area of image coverage was obtained. p62 particle frequencies were normalised to percentage area coverage per image using HCS CellMask™ staining, to account for differences in myotube size.2. TDP-43 aggregate frequencies were normalised to percentage area coverage per image of cell mask staining using the cell mask coverage. TDP-43 colocalisation with p62 was assessed for each identified TDP-43 aggregate using thresholded p62 and TDP-43 channels. Any area of overlap between TDP-43 and p62 objects over two pixels was considered colocalised. Percentage colocalisation is the percentage of TDP-43 aggregates that colocalised with p62 puncta for all aggregates observed for one donor under one condition. p62 puncta and TDP-43 aggregate analysis was conducted blinded.The same images for p62 analysis were used for TDP-43 sarcoplasmic aggregate analysis. Sarcoplasmic aggregates were identified manually as punctate areas of staining. Using Image J, freehand regions were drawn around aggregates to measure their area in µmDuoSet® Enzyme-linked immunosorbent assays (ELISA) were purchased from R&D systems, UK. Human IFN gamma (DY285B), and human TNF alpha (DY210) were used. ELISA were performed following manufacturer’s instructions. Nunc MaxiSorp™ immunoplates were coated with recommended dilutions of capture antibody, blocked with 0.2 µm filtered 1% w/v BSA  (reagent diluent) for 1 h, before incubation of samples and standards diluted in reagent diluent for 2 h. Recommended concentrations of detection antibody diluted in reagent diluent were added for 2 h in sealed plates. Streptavidin horse radish peroxidase (HRP) diluted in reagent diluent was incubated for 20 min before washing and addition of substrate solution  for 20 min. Colour development was halted with 2N sulphuric acid. Optical density was determined using a Multiskan™ FC plate reader by subtracting readings at 540 nm from 450 nm to account for any imperfections in the plate.Statistical analysis and graph plotting was performed with GraphPad Prism 7. Normal distribution of data was tested with Shapiro Wilk test before choosing appropriate statistical tests. All statistical tests performed were two-tailed. Data are displayed as mean ± standard error of mean for normally distributed data or median ± interquartile range for non-normally distributed data.34, therefore it was not expected that the inclusion of this cytokine in controls would have any detrimental effect on myogenic cells, and IL-2-containing medium showed low myotube cytotoxicity. These myogenic cultures show limited continuous differentiation after day 4  and TNFα (r2 = 0.9335) secretion with increasing E:T ratio when considering only the median concentration value for each condition, showing increased cytokine secretion was due to higher TALL-104 cell number.To examine whether coculturing TALL-104 cells with myotubes affected activation of TALL-104 cells, ELISA was used to measure secreted levels of IFNγ and TNFα in the culture medium. IFNγ and TNFα levels were measured after 24 h TALL-104-myotube coculture. This was compared to TALL-104 cells cultured in myotube medium without myotubes as a negative control, where IFNγ and TNFα were not detected above 1 pg/mL. There was higher IFNγ and TNFα secretion at 2.5:1 and 5:1 compared to monocultured TALL-104 .Immunofluorescent microscopy was also used to assess TDP-43 sarcoplasmic aggregation with TALL-104 exposure. Figure p < 0.0001). The localisation with the largest percentage of TDP-43 regardless of treatment condition was nucleus and sarcoplasm with mean 83 ± 3.71% (Tukey’s post-hoc test).The localisation of TDP-43 within myogenic cells was assessed when cultured with 1:1 E:T ratio of TALL-104 for 24 h compared to skeletal muscle derived cells treated with IL-2-containing media for the same length of time. There was a significant difference in the interaction between TDP-43 localisation and treatment condition, showing TDP-43 localisation was altered with TALL-104 coculture (two-way ANOVA) Fig. . The onl37. However, the importance of amyloid-β in sIBM has been called into question38. The presence of amyloid β aggregates in sIBM myofibres may be low, with some studies showing as low as between 039 and 0.4%40 of myofibres containing amyloid deposits. On the other hand, amyloidβ was found over-represented in rimmed vacuoles compared to other areas of the sarcoplasm15. It was decided not to focus on this protein here. Whilst TDP-43 and p62 are suggested as specific sIBM markers, aggregates of these proteins have been observed in other muscular diseases. TDP-43 aggregation is seen in oculopharyngeal muscular dystrophy, desminopathy, myotilinopathy, and hereditary IBM42, however, these diseases are not immune-mediated. With no distinctive TDP-43 aggregate pattern in sIBM compared to other diseases42, the combined presence of TDP-43 and CD8 + T cells may serve as selective markers of sIBM. p62 is identified in other inflammatory myopathies, particularly immune-mediated necrotising myositis (IMNM) but undetectable in normal muscle44.This study aimed to investigate the effects of cell-mediated cytotoxicity on TDP-43 and p62 degenerative features in skeletal muscle. Other proteins are also implicated in sIBM, most notably amyloid-β45, similar to what was observed here. The effect of coculturing isolated sIBM CD8 + T cells with autologous myotubes has been tested, showing 1 out of 4 sIBM patient’s CTLs showed cytotoxicity against autologous myotubes46. This suggests some sIBM patient CTLs may be primed against skeletal muscle antigens.This study builds upon previous work examining cytotoxic immune cell interactions with skeletal muscle cells. Coculture of antigen-specific CD8 + T cells and transgenic antigen presenting myotubes was used to model polymyositis. These myotubes died with increasing lysis at increasing densities of CTLs and the CD8 T cells infiltrated into the myotubes49. In rat myotubes, treatment with IL-1β triggered TDP-43 mislocalisation to the sarcoplasm, without triggering inclusion body-like protein aggregates50. These results show inflammation can precede non-inflammatory sIBM features in myotubes. However, sIBM-derived myotubes cultured under electrical stimulation showed TDP-43 and p62 aggregation51, highlighting a potential muscle-intrinsic mechanism of aggregation in sIBM. The effects of cell-mediated cytotoxic immunity on TDP-43 and p62 in myotubes has not previously been investigated. The work presented here contributes to understanding the interplay of inflammation on degenerative sIBM-like features.Previous in vitro models of sIBM have been created. IL-1β and IFNγ treatment of healthy human myotubes was used to investigate the effects of these cytokines on a range of degenerative characteristics altered in sIBM33. Blocking NK cell receptors NKp46, NKG2D, and 2B4 prevented TALL-104-mediated lysis, therefore cytotoxicity is mediated through NK cell receptors, independent of MHC recognition32. Instead, they resemble a mixed lineage sharing features of NK and CD8 T cells56.Here, TALL-104 cells were used as a proxy for CD8 T cells due to their continued growth in culture and easy availability. TALL-104 is a lymphoblastic leukaemic cells line expressing CD8, CD3, and T cell receptor proteins as well as natural killer (NK) cell markers including CD56 and CD16160. NK cells can target autologous skeletal muscle cultures from healthy patients, whilst autologous myotubes were not targeted or killed by CD8 cytotoxic T cells61. This suggests culturing myogenic cells may reveal epitopes capable of stimulating NK cells that are usually hidden or absent in vivo. The lack of reactivity of TALL-104 cells to normal brain cells57 indicates cytotoxicity against myotubes is unlikely to be due to HLA-mismatching or anti-self recognition.It was expected TALL-104 coculture would not be cytotoxic as myotubes should lack epitopes that trigger cytotoxicity. Furthermore, TALL-104 spare normal cells whilst targeting tumourigenic cells62 where TDP-43 forms puncta with muscle-specific mRNAs. In cells undergoing apoptosis there is a rapid degradation of mRNA63, therefore TDP-43’s homeostatic roles could cease with TALL-104 coculture. However, selection bias where only cells with a higher propensity to survive TALL-104 cytotoxicity may have influenced these results. Whilst a decrease in nuclear TDP-43 was observed with TALL-104 coculture, the localisation did not shift to the sarcoplasm only, showing a “mislocalisation” of TDP-43 was not recapitulated. However, this suggests cytotoxic inflammatory cell exposure can contribute to TDP-43 subcellular shifts, which could contribute to the overall phenotype of TDP-43 mislocalisation as seen in sIBM.The results presented here suggest exposure to cytotoxic immune cells does not precede TDP-43 and p62 sarcoplasmic aggregation. The decreased likelihood of images containing TDP-43 aggregates may reflect a change in homeostatic functions of TDP-43, such as RNA processing in myo-granulesThere are several limitations to this study. Firstly, TALL-104 cells mediate cytotoxicity through MHC-independent mechanisms, as well as being a neoplastic cell line. sIBM CD8 + T cells are highly differentiated, therefore the TALL-104 cell line is unlikely to share similar activation features to native sIBM CTLs. TALL-104 were identified based on size and high nuclear to cytoplasmic ratio, but co-staining with CD8 or other T cell markers would have been preferrable. The inflammatory state of the myotubes themselves was not tested, which could have been investigated by examining major histocompatibility complex I and other inflammatory markers on myotubes. To further characterise the cytotoxic reactivity of TALL-104 to myotubes, granzyme and perforin analysis could be conducted. Finally, in comparison to the slowly progressive nature of sIBM, the cytotoxicity of TALL-104 cells here was acute, limiting the relevance of these experiments.Overall, the exploratory results in this study show exposure to cytotoxic immune cells did not trigger TDP-43 or p62 sarcoplasmic aggregation as seen in sIBM, suggesting in vitro aggregation of these proteins were not preceded by inflammatory cytotoxicity. Conversely, TALL-104 cells triggered a shift in TDP-43 subcellular localisation, showing cytotoxic cells may contribute to the sIBM-like characteristic of TDP-43 mislocalisation to the sarcoplasm. This study helps towards understanding whether inflammation or protein aggregation arises first in sIBM.Supplementary Information.


The current review aims to provide an overview of the most recent research in the last 10 years on the potentials of graphene in the dental surgery field, focusing on the potential of graphene oxide (GO) applied to implant surfaces and prosthetic abutment surfaces, as well as to the membranes and scaffolds used in Guided Bone Regeneration (GBR) procedures. “Graphene oxide” and “dental surgery” and “dentistry” were the search terms utilized on the databases Scopus, Web of Science, and Pubmed, with the Boolean operator “AND” and “OR”. Reviewers worked in pairs to select studies based on specific inclusion and exclusion criteria. They included animal studies, clinical studies, or case reports, and in vitro and in vivo studies. However, they excluded systematic reviews, narrative reviews, and meta-analyses. Results: Of these 293 studies, 19 publications were included in this review. The field of graphene-based engineered nanomaterials in dentistry is expanding. Aside from its superior mechanical properties, electrical conductivity, and thermal stability, graphene and its derivatives may be functionalized with a variety of bioactive compounds, allowing them to be introduced into and improved upon various scaffolds used in regenerative dentistry. This review presents state-of-the-art graphene-based dental surgery applications. Even if further studies and investigations are still needed, the GO coating could improve clinical results in the examined dental surgery fields. Better osseointegration, as well as increased antibacterial and cytocompatible qualities, can benefit GO-coated implant surgery. On bacterially contaminated implant abutment surfaces, the CO coating may provide the optimum prospects for soft tissue sealing to occur. GBR proves to be a safe and stable material, improving both bone regeneration when using GO-enhanced graft materials as well as biocompatibility and mechanical properties of GO-incorporated membranes. The landscape of modern healthcare has been continuously shaped by groundbreaking advancements in materials science and technology . One sucPromising uses for graphene include lots of biomedical fields . GraphenOral surgery encompasses a spectrum of procedures, ranging from routine tooth extractions to complex maxillofacial reconstructions ,15,16,17At the heart of graphene’s allure lies its remarkable physical properties. Structurally, graphene consists of a single layer of carbon atoms arranged in a hexagonal lattice . This arGraphene’s electrical conductivity is equally intriguing. Its high electron mobility opens the door to applications involving electrical stimulation and biosensing. In the context of oral surgery, this property could lead to the development of implantable devices capable of monitoring healing processes in real time, thereby enabling timely interventions in case of complications . AdditioA cornerstone of successful oral surgery is the interaction between surgical materials and the complex biological environment . In-deptMoreover, graphene’s interactions with immune cells have raised intriguing possibilities for modulating immune responses during surgical interventions ,29. ThisThe prevention of post-operative infections remains a critical challenge in oral surgery . GraphenWhile the potential applications of graphene in oral surgery are undeniably exciting, several challenges must be addressed to ensure safe and effective clinical implementation. The scalable synthesis of graphene materials suitable for surgical use, long-term biocompatibility assessments, and regulatory approvals are among the foremost challenges. Additionally, the development of standardized surgical protocols and techniques for incorporating graphene into existing procedures is essential to ensure seamless integration and optimal outcomes ,38.As the frontiers of materials science and oral surgery intersect, graphene emerges as a transformative force poised to reshape the landscape of surgical practices . Its extThis systematic review was conducted by the standards of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA), and it was submitted to PROSPERO with number ID 453609.Graphene oxide, dental surgery, and dentistry were the search terms utilized on the databases  to select the papers under evaluation, with the Boolean operators “AND” and “OR”.The search was restricted to just items released in English during the previous ten years (July 2013–July 2023).The reviewers, who worked in pairs, chose works that satisfied the following criteria for inclusion: (1) animal studies; (2) clinical studies or case reports; and (3) in vitro and in vivo studies.Exclusion criteria were systematic reviews, narrative reviews, and meta-analyses.The screening procedure, which was carried out by reading the article titles and abstracts chosen in the earlier identification step, allowed for the exclusion of any publications that varied from the themes looked at.The complete text of publications that had been determined to match the predetermined inclusion criteria was then read.Reviewer disagreements on the choice of the article were discussed and settled.The quality of the included papers was assessed by two reviewers, RF and EI, using the reputable Cochrane risk-of-bias assessment for randomized trials (RoB 2). The following six areas of possible bias are evaluated by this tool: random sequence generation, allocation concealment, participant and staff blinding, outcome assessment blinding, inadequate outcome data, and selective reporting. A third reviewer (FI) was consulted in the event of a disagreement until an agreement was reached.Keyword searches of the Web of Science (55), Scopus (38), and Pubmed (200) databases yielded a total of 293 articles.The subsequent elimination of duplicates (61) resulted in the inclusion of 232 articles.Of these 232 studies, 213 were excluded because they deviated from the previously defined inclusion criteria.The screening phase ended with selecting 19 publications for this work.The results of each study are reported in The study data was selected by analyzing the study design, number of patients, intervention, and outcomes .This review discusses the potential of graphene oxide (GO) as a promising nanomaterial with exceptional physical and chemical properties. Recent research has focused on its applications in biomedical fields such as tissue engineering, antimicrobial materials, and implants . The revIn order to manage infection and stop bone loss, peri-implantitis therapy must remove polymicrobial biofilms from the implant site and lessen tissue invasion. Brushing and GO at high concentrations effectively decontaminate biofilms from exposed titanium surfaces, as shown by Qin et al. .The study by Ren et al. utilized GO as a coating on titanium foils to deliver drugs and enhance the growth and differentiation of rat bone mesenchymal stem cells (rBMSCs) into osteoblasts  46]. Th. Th46]. This study focuses on enhancing the antibacterial and cytocompatible attributes of titanium alloy implants by employing electrophoretic deposition to create bioglass (BG)-GO composites . The resThe physiochemical properties of GO-carbon fibers (CF)-PEEK on titanium implants were analyzed by Qin et al., who revealed that these coatings might greatly reduce the coefficient of friction of alloy and improve wear resistance . In addiS. mutans and P. gingivalis adhesion compared to the non-irradiated group [Photothermal therapy (PTT), an alternative antibacterial treatment, has a substantial impact on deactivating oral microbiota. The study by Park et al. involved applying graphene possessing photothermal characteristics onto a zirconia surface using atmospheric pressure plasma, followed by an assessment of its antibacterial effects against oral bacteria . To coated group . This reed group .Cheng et al. analyzed a new type of coating using a combination of GO and the antimicrobial peptide (AMP) Nal-P-113 on a smooth titanium surface . The stuS. mutans bacterial attachment and growth were significantly reduced on the Zr-GO surface, while the attachment of MC3T3-1 cells remained similar, but their growth and specialization improved on Zr-GO compared to Zr [The study by Jang et al. aimed to investigate the impact of applying GO onto a zirconia (Zr) surface on bacterial bonding and osteoblast activation . Two groed to Zr .S. mutans bacterial attachment and promoted osteoblast growth and specialization, suggesting a potential prevention of peri-implantitis by deterring bacterial adhesion and enhancing implant success by improving bone attachment [Conclusively, GO-coated zirconia hindered tachment .Recently, scientific research on graphene has focused on regenerative techniques in oral surgery, going on to investigate the efficacy of graphene oxide added to membranes or scaffolds compared with conventional methods with the hope that the results, combined with the potential of stem cells, will lead to a new class of nanomaterials with unique properties and a significant impact in the field of nanotechnology and oral health.For example, the evaluation of a bone graft material consisting of biphasic calcium phosphate (BCP) coated with rGO was the focus of the investigation by Jeong-Woo Kim et al. Osteoblast viability decreased as the concentration of rGO nanoplates increased in terms of cytotoxicity, with significant decreases at higher concentrations, while new bone production dramatically increased compared with the control group in in vivo tests using rat calvarial lesions. In fact, according to micro-CT and histomorphometric evaluations, rGO-coated BCP groups had higher volumes and percentages of new bone. The rGO4 group (with a 4:1000 ratio of rGO to BCP) showed the highest bone volume, demonstrating that the concentration of rGO in the composite material is important for bone regeneration .In contrast, Erika Nishida et al. investigated the effects of adding a GO monolayer solution to a three-dimensional collagen scaffold for possible use in bone tissue engineering . A speciThe research results of Jong Ho Lee et al. suggest that composite nanoparticles of hydroxyapatite (HAp) and rGO have extraordinary potential in enhancing the proliferation and osteogenic differentiation of pre-osteoblastic MC3T3-E1 cells.In particular, extracellular calcium deposition in MC3T3-E1 cells was significantly enhanced by rGO/HAp composite nanoparticles, and clearly, calcium accumulation is a key sign of bone tissue creation and extracellular mineralization, two crucial steps of bone regeneration. This implies that composite nanoparticles could create a favorable environment for bone tissue development, thereby promoting osteogenesis. In addition, the enzymatic activity of alkaline phosphatase (ALP), an early indicator of osteogenic differentiation, was significantly elevated in the presence of composite nanoparticles. This shows that composite nanoparticles can accelerate the differentiation process of pre-osteoblastic cells.The presence of composite nanoparticles also had a favorable impact on the deposition of osteogenic proteins, including osteopontin (OPN) and osteocalcin (OCN), which are essential markers of osteogenic cell development, suggesting a favorable impact on the expression of proteins important for bone growth. Therefore, these results also appear to be on the same wavelength as the other studies mentioned .The work of Izumi Kanayama et al.  investigS. aureus) and Gram-negative (E. coli) bacteria were analyzed; the antibacterial action of the composite was significant, and a larger clean zone was observed for higher concentrations of GO in the composite. The biocompatibility of the GO/HAp/PCL composite was also evaluated using MC3T3-E1 preosteoblast cells. Cell viability studies showed that the cytotoxic effects of the composite were minimal at lower concentrations: the cytotoxicity caused by HAp seemed to be attenuated by the addition of GO to the composite structure. It is also possible that GO has the ability to promote cell development, as it increases cell attachment and proliferation when added to the composite. Ultimately, the work showed that it was possible to successfully synthesize a GO/HAp/PCL composite with promising antibacterial qualities and biocompatibility [In the work of Chingis Daulbayev et al. , a compotibility ,71,72.In recently published work by Milena Radunovic et al. , the effThe study concluded that GO-coated collagen membranes limit inflammatory processes and promote attachment, proliferation, and osteogenic differentiation of DPSCs, emphasizing that efficacy is dose-dependent (the 10 g/mL concentration of GO produces the best results) .The study presented by Letizia Ferroni et al.  investigIn the very recent study by Elham-Sadat Motiee et al.  in 2023,The objective of the study by Alana P C Souza et al.  was to dThe risk of bias in the included studies is reported in The field of graphene-based engineered nanomaterials in dentistry is expanding due to their superior mechanical properties, electrical conductivity, and thermal stability. Graphene and its derivatives can be functionalized with bioactive compounds and added to dental materials, enhancing their properties. These materials stimulate tissue regeneration, cell differentiation, and proliferation while being biocompatible and low in toxicity. Dental implants and abutments with graphene coatings exhibit improved cytocompatibility, antibacterial properties, and osteoblast growth.Graphene-enhanced scaffolds and membranes for guided bone regeneration (GBR) also have improved physical properties, leading to enhanced bone formation. Laboratory tests indicate increased secretion of osteogenic markers and reduced inflammatory markers on graphene-coated materials. However, there are concerns about the safety of graphene and its derivatives, necessitating further research to understand their long-term effects. Overall, graphene materials hold great potential for improving oral surgery procedures in the future.


Conductive Ag electrodes were formed on PET substrates by inkjet printing. A thin film of GO-MoTe2 was deposited on the Ag electrode used for adsorbing humidity. The experiment’s results demonstrate that MoTe2 are attached to GO nanosheets uniformly and tightly. The capacitive output of the sensors with various ratios of GO/MoTe2 has been tested for different levels of humidity (11.3–97.3%RH) at room temperature (25 °C). As a consequence, the obtained hybrid film exhibits superior sensitivity (94.12 pF/%RH). The structural integrity and interaction of different components were discussed to afford the prominent humidity sensitivity performance. Under the bending condition, the output curve of the sensor has no obvious fluctuation. This work provides a low-cost way to build flexible humidity sensors with high-performance in environmental monitoring and healthcare.This study focuses on a novel humidity sensor composed of graphene-oxide (GO)-supported MoTe Humidity monitoring plays an essential role in the fields of aerospace, industry, agriculture, and even our daily lives. Resonance ,2, optic2 (molybdenum ditelluride) has attracted considerable attention in numerous fields, such as energy storage and optoelectronics, due to its high surface-to-volume ratio and favorable surface energy level [2 is used as a new semiconductor material for gas and pressure sensors. Wu et al. [2 gas sensor for NO2 detection with greatly enhanced sensitivity and recovery rates under ultraviolet (UV) illumination. Seunghyun et al. [2 engineered by strain. Meanwhile, graphene oxide (GO), one of the most popular materials around the world, is an important kind of material for preparing flexible sensors, especially humidity sensors, due to its dispersibility [2/mSiO2) via screen printing. The fabric humidity sensor exhibited high sensitivity (14.8 MΩ/% relative humidity (RH)) and low hysteresis (2.71 %RH) at a humidity interval from 23% to 97%RH. Chi et al. [Recently, transition metal dichalcogenides (TMDs) have excited many researchers owing to their excellent physicochemical properties and wide electronic applications ,22,23. Agy level ,25. Curru et al.  reportedn et al.  demonstrsibility , hydrophsibility , and larsibility . Zhu et sibility  designedi et al.  preparedi et al.  fabricat2 nanosheets for humidity detection was firstly demonstrated. Herein, we fabricated Ag electrodes by inkjet printing technology on a PET substrate. Then, a GO/MoTe2 sensing material was deposited on the electrodes by a drop-casting method. The morphology and nanostructure of the GO/MoTe2 nanofilm were confirmed by means of FTIR, Raman, SEM, and TEM. As a result, the sensor exhibited high response, good repeatability, and stability. At length, the underlying sensing mechanism of GO/MoTe2 toward humidity is also discussed.In this work, a printable and flexible capacitance sensor based on GO-supported MoTe2 powder was offered by Muke Nanotechnology Co., Ltd. . All other chemicals were at least of analytical grade and utilized without further purification.Graphene oxide  was supplied by XFNANO Co., Ltd. . The MoTe2 dispersion solution was synthesized by mixing different ratios of MoTe2 powder into 10 mL GO solution (2 mg/mL), and then the mixture was treated with ultrasound for 1 h at 100 w. Finally, three kinds of MoTe2/GO nanocomposite solutions with mass ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 were obtained. The obtained dispersion solution was utilized for modifying as-prepared electrodes by means of a drop casting approach. In total, 2 droplets (1 μL single droplet volume) of the mixture solution were deposited by microinjector on the working electrode area of as-prepared flexible substrate. As control groups, pure GO and MoTe2 were, respectively, deposited on as-prepared electrodes by the same method.The GO/MoTeSEM images were examined by FEI Inspect F50 (SEM), and TEM images were inspected by FEI Talos F200S (TEM). The Fourier transform infrared spectroscopy (FTIR) was analyzed by Nicolet IS 10. Raman measurements were performed by Horiba Scientific LabRAM HR Evolution.2 sensor in various saturated salt solutions with different relative humidity (RH) levels [The humidity sensing experiments were carried out by placing the GO/MoTe) levels . The cap2/GO nano-hybrid are shown in 2 are confirmed. The wide absorption peak at 3458 cm−1 is attributed to the O-H stretching vibrations between the C-OH groups and water molecules [−1 is assigned to C=O stretching of carbonyl moiety functional groups. The sharp, tiny absorption peak at 1410 cm−1 is attributed to CO- carboxylic. The absorption peak at 663 cm−1 in nanocomposite can be assigned to MoTe2 [FT-IR spectrums of the graphene oxide and MoTeolecules . The shato MoTe2 .2 film. The measurements were conducted using a 532 nm laser. Combined with the spectra of MoTe2, the spectra of MoTe2/GO illustrated the four peaks at 120 (Bu), 145 (Bu), 197 (Bg), and 276 (Ag) cm−1 for the film blended with MoTe2, affirming the crystalline layered structure of our film. The positions and relative intensities of the four peaks match well with the previous literature reports [−1 and ~1583 cm−1 are defined as the D-band and G-band of graphene, respectively. The intensity ratio (ID/IG) of the two bands equals 1.5 and 1.19 for GO and MoTe2/GO, respectively. The ID/IG ratio can be seen as the crystallinity of graphene and a measure of defects in the skeleton structure of graphene. When the hydroxyl content is larger, the regularity of the material is worse. The GO used in this work may contain a lot of hydroxyl groups, which makes the material more hydrophilic. reports ,39,40. T2, and GO/MoTe2. From 2 exfoliated from bulk ones observed by SEM and TEM. 2 was well-dispersed in GO film. By comparing SEM and TEM images, it reveals that MoTe2 is doped between the GO sheets. In 2 (1 1 2) plane are observed with an 0.275 nm interfacial spacing.2/GO composites with different ratios were prepared. The humidity properties of the capacitive MoTe2/GO sensors at 500 Hz were measured and investigated as follows, as shown in 2 as the sensing material had only a 323 pF capacitance change from 11.3 %RH to 97.3 %RH. The response of the sensor with pure MoTe2 was remarkably lower than that of the sensor based on pure GO at different humidity conditions; however, the response of the sensor coated with MoTe2/GO (1:5) toward relative humidity reached 4386 pF in the whole RH range, which is about 13.5 times that of the sensor with pure MoTe2 and 1.3 times that of the sensor based on pure GO, respectively. These results show that the humidity sensitivity of the sensing film could be improved by doping MoTe2 in GO film.Humidity sensors based on MoTe2—too much is as bad as too little. At the outset, with the increase in the amounts of MoTe2 in GO film, the humidity response of sensor increases. When the mass ratio of both materials in the MoTe2/GO composite is 1:2, the humidity response of the sensor obtains the maximum value (capacitance change: 8093 pF), which is 25 times and 2.2 times those of the sensor with pure MoTe2 and the sensor with pure GO, respectively. Therefore, in the following further research, the sensor films are all in this ratio. Unfortunately, when the mass ratio of the two materials is further magnified (MoTe2/GO (1:1), MoTe2/GO (2:1), and MoTe2/GO (5:1)), the humidity response of sensor would actually decrease. It might be that excess MoTe2 could cover up not only the hydrophobic region of GO, but also part of the hydrophilic groups of GO, thus inhibiting the sensing performance improvement of the humidity sensor. It can be found that the addition of MoTe2 in GO thin film has little effect on its hydrophilicity under low humidity. However, under moderate and high humidity conditions, doping an appropriate amount of MoTe2 into GO thin films can significantly improve its hydrophilicity.The degree of improvement, however, depends on different addition amounts of MoTeIn To evaluate the accuracy and efficiency of the humidity sensor, the hysteresis characteristic is also a significant parameter. The dynamic hysteresis loop curve is shown in The repeatability of the as-fabricated sensor under both flat and bending state was studied between 11.3%RH and 97%RH. The response and recovery time is one of the important performance indicators to evaluate the sensor. 2, CH4, ethanol (C2H5OH), NH3, and NO2, have been tested to evaluate the cross-sensitivity of the sensor. The capacitance of the sensor to these gases (at 5000 ppm) was measured, as shown in Various gases, such as CO2/GO film is considered as a potential humidity sensing material. The combination of MoTe2 nanoparticles and GO nanosheets greatly enhances the ability of humidity sensing. MoTe2 with a sphere nanostructure can be embedded between nanosheets to play a supporting role, resulting in a great favor to the absorption and diffusion of water molecules [2/GO film can offer a higher proportion of available active sites for water molecule absorption and can accelerate the process of adsorption and desorption on the film surface owing to the larger surface specific area compared with GO [The above experimental results show that the sensor possesses excellent humidity sensing performance. Therefore, the MoTeolecules . Moreove with GO .−1, and the cycle of one breath is approximately 3–5 s [−1) and bradypnea (respiratory rate < 12 times min−1). As shown in −1, and the mouth breathing rate is 15 times min−1. Compared with a normal breathing (by nose) response, the capacitance variation range is larger due to the wetter and stronger airflow from the tester’s mouth. We simulated two kinds of abnormal breathing (tachypnea and bradypnea) and carried out a breathing experiment, as shown in In the field of health monitoring, with the development of wearable devices, new requirements are put forward for flexible sensors, especially in respiratory monitoring. Taking into account the response and recovery characteristics of the prepared sensor, it has ability to perform real-time respiratory monitoring in a non-contact manner. The normal respiratory rate of adults in a calm state is about 12–20 times minly 3–5 s . AbnormaWe also tested the non-contact skin humidity sensing performance, and the test method is shown in 2 nanosheets were deposited on the conductive Ag electrodes formed on PET substrates by inkjet printing to detect humidity. The SEM and TEM graphs demonstrate that MoTe2 are well-dispersed to GO nanosheets uniformly and tightly. By comparing the response of each sensor, the optimal composite ratio of materials is found out. The capacitive performance of the sensor has been tested at different states . The obtained hybrid film exhibits excellent sensitivity. Moreover, after bending, the response of the sensor does not change significantly, which is beneficial to its application in wearable devices. Due to its good response and recovery characteristics, the sensor also has a good performance in respiratory monitoring and non-contact measurement. The structural integrity and interaction of different components were discussed to afford the prominent humidity sensitive performance. This work proposed a low-cost and easy-to-operate preparation method of a flexible humidity sensor with high performance in environmental monitoring and medical care.In this paper, graphene-oxide (GO)-supported GO-MoTe


FUNDC1 is co-expressed with TDP-43, and both TDP-43 and FUNDC1 expression is correlated with genes associated with mitochondrial protein import pathway in brain samples of patients diagnosed with TDP-43 proteinopathy. FUNDC1 promotes mitochondrial translocation of TDP-43 possibly by promoting TDP-43-TOM70 and DNAJA2-TOM70 interactions, which is independent of the LC3 interacting region of FUNDC1 in cellular experiments. In the transgenic fly model of TDP-43 proteinopathy, overexpressing FUNDC1 enhances TDP-43 induced mitochondrial damage, whereas down-regulating FUNDC1 reverses TDP-43 induced mitochondrial damage. FUNDC1 regulates mitochondria-mediated TDP-43 degradation not only by regulating mitochondrial TDP-43 import, but also by increasing LONP1 level and by activating mitophagy, which plays important roles in cytosolic TDP-43 clearance. Together, this study not only uncovers the mechanism of mitochondrial TDP-43 import, but also unravels the active role played by mitochondria in regulating TDP-43 homeostasis.Though TDP-43 protein can be translocated into mitochondria and causes mitochondrial damage in TDP-43 proteinopathy, little is known about how TDP-43 is imported into mitochondria. In addition, whether mitochondrial damage is caused by mitochondrial mislocalization of TDP-43 or a side effect of mitochondria-mediated TDP-43 degradation remains to be investigated. Here, our bioinformatical analyses reveal that mitophagy receptor gene  TDP-43 gene have been identified only in a fraction of familial ALS-TDP and FLTD-TDP patients, but not in the majority of TDP-43 proteinopathy cases. Dysregulation of TDP-43 expression and protein homeostasis may contribute to the pathogenesis in most cases of TDP-43 proteinopathy.TDP-43 proteinopathy is a group of neurodegenerative diseases characterized neuropathologically by the presence of protein inclusions containing phosphorylated TDP-43 protein . This isMitochondrial damage is an early event and an important aspect of the pathogenesis of TDP-43 proteinopathy . A rangemt), and TDP-43 can be degraded by mitochondrial protease LONP1   for 30 min at 37 °C. To stop the reaction, 20 mM Tris-HCl, pH 7.8 was added at room temperature for 15 min. Mitochondria fractions prepared from DSP-treated cells were then used for Co-IP as described above.E. coli Rosetta strain . Bacteria were grown at 37 °C in LB medium with 100 μg/ml ampicillin and induced with 0.5 mM isopropyl-D-thiogalactopyranoside (IPTG) for 18 h at 16 °C. The cells were harvested and suspended in buffer A  and French pressed on ice at 1200 bar for 4 times. The lysate was centrifuged, and the pellet was resuspended with NTA buffer  containing 8 M Urea and loaded onto a Ni-NTA column . The column was washed with NTA buffer containing 8 M Urea and then TDP-43 bound to the column was eluted with NTA buffer containing 0.25 M imidazole and 8 M Urea. The collected fraction was either flash-freeze or dialyzed against 20 mM Tris, pH 8.0, 0.2 M NaCl for the following experiment. Human TDP-43 protein with a myc-his-tag was purified using Ni-Sepharose  following Tet-induction (1 μg/mL tetracycline) of the inducible HEK293 cells for 36 h. Purified human TDP-43 was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining or by immunoblotting with anti-TDP-43 or anti-myc antibody.A cDNA encoding human full-length TDP-43 protein was cloned into vector pET32M3C, expressed as a fusion protein carrying amino-terminal thioredoxin and 6×his-tag and purified from the 2, 3% BSA, 4 mM ATP, 4 mM NADH), and 10 μg purified TDP-43 was then added to start the import assay. Unless specified otherwise, the solution was incubated for 30 min at room temperature. The mitochondria were collected following centrifugation at 8000 × g for 5 min at 4 °C. The mitochondria pellets were washed twice with 1 ml wash buffer at 8000 × g for 5 min at 4 °C, followed by “Proteinase K protection assay”.Mitochondria were purified according to “Biochemical fractionation experiments”. Mitochondria were suspended in 500 μL import buffer , the cells were harvested and mitochondria were purified, followed by “Proteinase K protection assay” and immunoblotting analysis. In different groups, the dosage and transfection time for the negative control siRNA (NC) were the same as the siRNAs targeting corresponding genes. The target sequences of siRNAs were listed as follows: DNAJA2: 5’-GGAAGAAGGTGATTAAAGA-3’; HSPA8: 5’-GCTGGTCTCAATGTACTTA-3’; TOM70: 5’-GAGGCAGCATGTACATGCA-3’.The siRNA was from RIBOBIO . Corresponding siRNA  was incubated with 30 μL lipofectamineRNA-seq datasets of human frontal cortex samples were downloaded from GEO database, including those from control subjects , or Two-way ANOVA followed by a post hoc test. For enumeration data, differences between different groups were analyzed using the Chi-square test or Fisher’s exact test, and Bonferroni correction was used for multiple comparisons. Measurement data are presented as mean ± standard error of the mean (SEM). Significance is indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001. The correlation coefficient was calculated using the Spearman method. Graphs were prepared using the R package “ggplot2” (v2.0.0) [Quantification of immunoblotting, TEM, and fluorescence data was performed using ImageJ2 (v2.3.0). Data were collected in Excel (Microsoft) and analyzed using the R program (v3.3.3). Differences between different groups were analyzed using a one-sided Student’s (v2.0.0) .Reproducibility checklistSupplementary informationOriginal Data File


N = 76), and whether it varied depending on maternal involvement in childcare and the perceived quality of past experiences of care.Attentional bias toward infant faces is associated with parental sensitivity and supports the infant-caregiver attachment relationship, ultimately fostering child health outcomes. However, experience-related determinants of parents' attentional bias to infant faces have been poorly investigated. We examined attentional bias to infant versus adult faces in a sample of same-sex mothers (A Go/no-Go attentional task was used to compare the effects of infant and adult faces in retaining attention. Maternal involvement in childcare was measured using items addressing nurturing behaviors. Memories of past experiences of care were collected using the short-form version of the Parental Acceptance-Rejection scale.Results confirmed that infant faces induced greater attentional bias compared to adult faces. More involved mothers were more biased, in terms of attention, to infant versus adult faces. Attentional bias to infant versus adult faces increased as mothers felt more rejected by their own fathers during childhood.Our findings suggested that attentional bias to infant faces might be associated with past experiences of care and direct commitment in childcare in same-sex mothers. Robust and accurate empirical findings on same-sex parent families are essential to inform social policies supporting these families’ well being. Parent–infant interactions rely on the ability to express signals through facial expressions . In partBehavioral tasks assessing attentional bias in parents typically present infant cues as distractors during conflict tasks . The attentional bias index refers to the difference in attention captured by distinct stimuli , which suggests a greater cognitive engagement to a particular stimulus. Using an attentional Go/no-Go task, Psychological characteristics of mothers have been found to modulate their attentional bias to infant faces . PearsonThe attentional bias toward infant faces might be also linked to the perceived quality of care received during childhood. According to Bowlby's theoretical work (1969/1982), repeated interactions with attachment figures are schematized in the form of Internal Working Models (IWM) that synthesize the main self-other interactive dynamics and are automatically activated by individuals when processing new situations. Similarly, in the theoretical framework of the Interpersonal Acceptance-Rejection Theory  confirm that infant faces retain more attention compared to adult faces; ii) investigate whether the attentional bias to infant versus adult faces is associated with mothers’ involvement with childcare; iii) investigate whether individual variations in past experiences of care with mothers' own caregivers are associated with the attentional bias to infant versus adult faces. First (i), we expect that infant faces interfere with the task performance more than adult faces, slowing RTs to peripheral stimuli in Go conditions. Given that an attentional bias to infant distressed faces has been specifically found in previous research ; 2013, wN = 76 mothers being in a same-sex couple participated in the study. Contact with most mothers was made through the Italian Association “Famiglie Arcobaleno” , which sent an invitation to participate in the study to all its members through the mailing list. A snowball sampling method was also used, such that mothers who participated in the study were asked to forward the study invitation to other same-sex mother families who might be interested in joining the study. To be included in the study sample, i) the age of mothers’ only or youngest child should range between 2 and 36 months; ii) mothers raised their child since birth. Both members of each same-sex couple were first invited to participate in the study together. However, whilst 70 mothers (92 %) participated in the experiment with their partner, 6 mothers (8 %) participated alone. Only participants who completed the whole experimental procedure (n = 73 mothers) were included in the final analyses; whilst two mothers did not complete the task, one mother was excluded from the final sample for technical issues while performing the task. The majority of participants were Italian (95.8 %), but 3 of them (4.2 %) reported to be of a different nationality . The Socio-Economic Status (SES) was calculated according to A group of Sociodemographic data was collected. Mothers were asked to answer all the questions by referring to the youngest child in case of multiple children.not at all; 2 = rarely; 3 = a few times a week; 4 = About once a day; 5 = more than once a day) were summed, with a higher score reflecting a higher degree of involvement. Cronbach's alpha was satisfactory (α = 0.81). The McDonald's ω total was 0.87.To assess parental involvement in childcare, mothers completed 10 items  adapted from the Parental primary caregiving role Structured Interview . The orialmost always true to 1 = almost never true). In this study, the two total scores had a good reliability .Mothers completed the Italian validated short-form version  of the PMothers completed a modified Go/no-Go task derived from an established paradigm  to measuTo overcome geographical barriers in recruiting the sample, all the experimental procedures were conducted online. Self-reports were administered through Qualtrics . The experimental task was run on JATOS . The tast-tests were run to check for potential differences within the sample. Correlation analyses were implemented. Missing data in self-reports was not replaced. Analyses were conducted for evaluating the number of correct answers for different blocks. The overall accuracy for Go trials was 96.6 %, which confirmed the ability of participants to complete the task as instructed. The percentage of false alarms  were 3.9 %. Response accuracy was analyzed using a generalized mixed-effects model. RTs were analyzed via linear mixed-effects models . Pearson correlation analyses did not show any significant associations between maternal involvement and PARQmother or PARQfather. PARQmother and PARQfather showed a low positive correlation .β = −0.015, SE = 0.002, t = −9.070, p < .001), as infants slowed RTs to a greater extent compared to adult faces. This effect remained statistically significant after increasing the complexity of the random effect structure by varying all the slopes  and only the slope of the main effects . Model 1 also evidenced a main effect of emotional valence , as mothers allocated greater attention to sad faces compared to happy and neutral faces. However, this effect was not statistically significant in all the subsequent models. The interaction between face age and emotional valence was not significant. In Model 2 . It highlighted a two-way interaction effect between face age and maternal involvement . Interestingly, more involved mothers were more biased, in terms of attention, toward infant versus adult faces . The interaction effect between face age and PARQmother was not significant. Model 4 confirmed the main effect of face age  and highlighted a two-way interaction between face age and PARQfather . That is, those mothers who felt more rejected by their own father were more biased, in terms of attention, by infant versus adult faces  has been considered an optimum point of reference for assessing caregiving involvement , we deciM = 46) was greater than the ones previously reported in other studies  = 35.81, M(wave2) = 34.00 and M(wave3) = 33.52; From the attachment theory perspective , the intContrary to our hypothesis, we did not find that attentional prioritization of infant over adult faces varied in relation to early care experiences with one's own mother. Compared to previous studies , it shouSome limitations of our study should point toward future directions. Future research might clarify the role of individual mental health as a potential covariate in relation to the behavioral findings e.g., . In non-In a considerable sample of same-sex mothers, we examined the contribution of past and current experiences of care on the attentional bias toward infant faces. Taken together, mental representations of care built during childhood and direct commitment in childcare were associated with same-sex mothers’ attentional bias toward infant faces. Apart from having a value on its own, inclusive research of different family contexts is needed conceptually and methodologically, as this might allow researchers to move beyond sex-related differences in families and consider different roles and arrangements . OverallThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Interactions between the programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) lead to immune evasion in various tumors and are associated with poor prognosis in patients with pancreatic cancer; however, the roles of PD-L1-containing exosomes in pancreatic cancer is poorly understood. Here, we investigated the correlation between circulating exosomal PD-L1 (exoPD-L1) and PD-L1 expression in tumor tissue, and survival outcomes in patients with advanced PDAC.Exosomes were derived from pre-treatment serum samples isolated using ExoQuick kit from 77 patients with advanced pancreatic cancer. Exosomal PD-L1 (exoPD-L1) was detected by enzyme-linked immunosorbent assay, and matched tumor tissues PD-L1 expression were evaluated by PD-L1 immunohistochemistry (22C3) assay, described with combined positive score. Cutoff value of exoPD-L1 for survival was assessed with receiver operating characteristic curve analysis. Kaplan-Meier analysis was performed to obtain median overall survival (OS), and hazard ratio was estimated using a stratified Cox regression model.p = 0.023). There was a significantly higher proportion of elevated exoPD-L1 levels in patients with positive PD-L1 expression compared to patients with negative PD-L1 expression (p = 0.001). Patients were classified into groups with low and high exoPD-L1 levels using ROC curve-derived cutoffs . At a median follow-up of 8.39 months, the median OS was 13.2  and 6.36 months  in the low and high exoPD-L1 groups, respectively . ExoPD-L1 levels did not affect the proportion of CD8+CD69+ effector cytotoxic T cells in either of the groups (p = 0.166).The median exoPD-L1 serum concentration was 0.16 pg/mg, with undetected levels in seven patients. ExoPD-L1 levels were significantly higher in patients with systemic disease than in those with locally advanced disease (The serum-derived exoPD-L1 levels were higher in metastatic pancreatic cancer than locally advanced disease. Collectively, higher serum exoPD-L1 levels in patients with advanced pancreatic cancer suggested worse survival outcomes and may have clinical implications.The online version contains supplementary material available at 10.1186/s12885-023-10811-8. Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a dismal 5-year overall survival (OS) rate of only 9% . AlthougIn many solid tumors, T-cell activation is inhibited by the interaction between the immune checkpoint programmed cell death ligand 1 (PD-L1) and programmed cell death receptor 1 (PD-1), which is an inhibitory receptor of activated T-cells. Suppression of T cell activation facilitates immune evasion by cancer cells and is associated with poor prognosis in patients with cancer . Thus, tThe TME of PDAC comprises regulatory T cells (Tregs), myeloid-derived suppressive cells (MDSCs), and tumor-associated macrophages (TAMs) that inhibit effector CD8 + T cells . PD-L1 oIn the present study, we investigated the correlation between PD-L1 expression in tumor tissue and circulating exoPD-L1 in patients with advanced PDAC. Furthermore, we evaluated whether circulating exoPD-L1 is associated with survival outcomes in patients with advanced PDAC. We also examined the correlation between CD8 + T cell counts and circulating exoPD-L1 levels.We enrolled 77 patients with advanced PDAC diagnosed at the Catholic University of Korea, Seoul St. Mary’s Hospital, between 2017 and 2021. Patients with available serum samples at the time of diagnosis for advanced disease were eligible according to the following criteria: (1) histologically confirmed PDAC; (2) locally advanced or metastatic disease; (3) survival confirmed at the time of data collection; (4) Eastern Cooperative Oncology Group (ECOG) performance status (PS) from 0 to 2. Peripheral blood samples were collected from patients before initiating systemic chemotherapy in a first-line palliative setting. Blood samples were delivered to the laboratory, immediately centrifuged at 2,500 × g for 10 min and stored in 2–4 mL aliquots at − 80 °C until use. Tumor tissue specimens were obtained from patients with PDAC who had undergone surgery or core needle biopsy. The study protocol was approved by the Institutional Review Board (IRB) of The Catholic University of Korea, Seoul St. Mary’s Hospital . Informed consent was obtained from all the patients. All procedures were conducted in accordance with Korean regulations and the Declaration of Helsinki.Serum exosomes were isolated using the ExoQuick exosome precipitation solution  as per manufacturer’s instructions. Thawed serum samples (500 µL) were centrifuged at 12,000 × g for 15 min to remove cells and cell debris. The supernatants were transferred to a sterile tube, mixed with ExoQuick reagent 120 µL) and incubated for 1 h at 4 °C, and then centrifuged at 1,500 × g for 30 min. The supernatant was removed, and the precipitate was centrifuged at 1,500 × g for 5 min to remove the residual ExoQuick solution and obtain precipitated exosome in the pellet. After discarding the supernatant, the pellets were resuspended in 250 µL sterile phosphate-buffered saline (PBS) and stored at − 80 °C until required. The NanoSight NS300 instrument with nanoparticle tracking analysis 2.3 software  was used to analyze exosomal size distribution . Exosome µL and iBriefly, 50 µg exosomes were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes  for western blot analysis. The membranes were incubated with primary antibodies against TSG101  and CD63 . Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies , and the bands were visualized using a chemiluminescent western blot reagent .The expression of PD-L1 in serum-derived exosomes was detected using human PD-L1 DuoSet ELISA kits  according to the manufacturer’s protocols. The optical density was measured at 450 nm using a VersaMax Microplate Reader , and protein levels were calculated according to standard curves. The protein concentration of the isolated exosomes was determined using a BCA protein assay kit  according to the manufacturer’s instructions. The concentration of exoPD-L1 was standardized per mg of exosomal protein. For the detection of cytokines in the plasma, commercially available ELISA kits for transforming growth factor beta 1 (TGF-β1) (#DY240-05), TGF-β2 (#DY302), granzyme B (#DY2906-05), interferon-gamma (IFN-γ) (#DY285B-05), and interleukin (IL)-1β  were used according to the manufacturer’s protocol.Higher levels of sPD-L1 are associated with a poor prognosis in patients with various solid tumors . HoweverFormalin-fixed, paraffin-embedded tissues (4-µm-thick sections) from patients with advanced PDAC were tested for PD-L1 expression using the PD-L1 immunohistochemistry (IHC) 22C3 pharmDx assay  on an Autostainer Link 48 system with an EnVision DAB Detection System  following the manufacturer’s instructions. PD-L1 immunoreactivity was evaluated by two pathologists using a multihead microscope. PD-L1 expression in the specimen was described using a combined positive score (CPS), which is defined as the number of PD-L1 stained cells  divided by the total number of viable tumor cells and multiplied by 100 Peripheral blood mononuclear cells were isolated using Ficoll-Paque PLUS  density-gradient centrifugation and stored in liquid nitrogen until further analysis. Thawed peripheral blood mononuclear cells were incubated with anti-CD3 , anti-CD45 , anti-CD8 , and anti-CD69  antibodies for 20 min at 4 °C in the dark. The stained cells were analyzed using flow cytometry , and data were analyzed using FlowJo software .Descriptive statistics are reported as proportions and medians with ranges or interquartile ranges (IQR). A time-dependent ROC curve was used to assess the predictive performance of the clinical factors associated with survival outcomes. The cutoff values of the clinical factors were calculated based on Youden’s index. Differences between groups and associations between clinicopathological features and exoPD-L1 were determined using chi-squared and Fisher’s exact tests for categorical data and an unpaired t-test with Welch’s correction for continuous data. Pearson’s correlation coefficient was used to evaluate the correlation between exoPD-L1 and tumor PD-L1 expression. OS was estimated from the date of diagnosis of advanced disease to the time of last follow-up or death. Survival curves were estimated using the Kaplan–Meier method, and survival differences were compared using a two-tailed log-rank test. Cox proportional hazards regression models were used to identify the effects of the clinical factors on survival. The hazard ratio (HR) and 95% confidence intervals (CIs) were estimated for each factor. Results with two-sided p-values < 0.05 were considered statistically significant. Statistical analyses were performed using SPSS for Windows  and GraphPad Prism version 8.0 .The clinicopathologic characteristics of the 77 patients with advanced PDAC are summarized in Table Rs = 0.788; p < 0.0001; Fig. p = 0.023; Table ExoPD-L1 was undetectable or below the lower detection limit in seven of the 77 patients (9.0%). The median value of exoPD-L1 was 5.87 , and the median value of exoPD-L1 normalized with exosomal protein was 0.16 . Standardized exoPD-L1 concentrations were associated with corresponding exoPD-L1 levels .Among the 77 patients, PD-L1 expression in the tumor tissue was evaluated in 68 patients, and the correlation between tissue PD-L1 and serum exoPD-L1 levels was assessed. The median CPS of tumor PD-L1 was 3.0 IQR, 0–20), and 19 patients (27.9%) had negative tumor PD-L1 expression, defined as CPS < 1% . No linear correlation was observed between the tissue PD-L1 CPS and serum exoPD-L1 values in patients with advanced PDAC  with 52.6% sensitivity and 69.2% specificity. According to the cutoff value, the entire cohort was classified into low exoPD-L1 (45 patients) and high exoPD-L1 (32 patients) groups. The median OS was 13.2 months  in the low exoPD-L1 group, as compared with 6.36 months  in the high exoPD-L1 group , 56 patients (72.7%) had died. Among these deaths, 51 (92.7%) were related to disease progression, and five (8.9%) were attributed to treatment-related adverse events. The median follow-up was 8.39 months . In the entire cohort, the median OS was 9.28 months , and OS rates at 6 months, 9 months, and 1 year were 62.3%, 50.1%, and 40.6%, respectively. The optimal cutoff value that discriminated survivors at nine months was determined using the ROC curve. The optimal cutoff value of exoPD-L1 was 0.165 pg/mg, and the area under the curve was 0.617  and higher baseline neutrophil-to-lymphocyte ratio (NLR)  were significantly associated with worse OS. In addition, patients who received systemic chemotherapy had longer OS  than those who did not  and effector CD8 + T cell (p = 0.166) proportions were comparable between the two groups according to the exoPD-L1 level (Fig. p = 0.861; Fig. p = 0.031; Fig. The proportion of CD3 + CD45 + CD8 + T cells was measured in each patient, and the fraction of CD69 + CD8 + T cells was assessed to investigate cytotoxic T cell activity. The total cytotoxic T cell (Activation of the PD-1/PD-L1 signaling pathway can suppress T cell responses in various cancers, thereby leading to immune evasion . To the In a recent meta-analysis, elevated tumor PD-L1 expression was correlated with advanced stage and poor OS in patients with PDAC. However, the IHC methods for PD-L1 expression were not validated . High plIn this study, exoPD-L1 levels were not significantly correlated with clinicopathological features, except for the disease status. Patients with systemic disease had elevated exoPD-L1 levels compared to those with locally advanced disease, which was consistent with previous reports wherein exoPD-L1 levels correlated with advanced disease stage in patients with head and neck and lung cancer , 26. TheWe assessed the correlation between tumor PD-L1 IHC status and serum exoPD-L1 levels in patients with pancreatic cancer for the first time. Serum exoPD-L1 levels were higher in the positive than in the negative PD-L1 expression group. Consistently, in a previous study, tumor PD-L1 expression was associated with a higher percentage of PD-L1-expressing exosomes in patients with non-small cell lung cancer . In contSimilar to sPD-L1, serum-derived exoPD-L1 may be elevated in patients with increased systemic inflammation provoked by the inflammatory tumor type of pancreatic cancer . PancreaCirculating exosomes can interact with immune cells through various mechanisms. PD-L1-containing exosomes can suppress CD8 + T-cell activity by downregulating CD69 expression on the surface of activated T cells . AdditioSeveral limitations of our study need to be considered. First, the association between treatment resistance and exoPD-L1 was not evaluated because exoPD-L1 was not serially measured after treatment. Second, none of the patients in this study received anti-PD-1/PD-L1 blockade immunotherapy; therefore, we could not investigate the impact of exoPD-L1 on the response to anti-PD-1/PD-L1 blocking therapy. Third, tissue evaluation in relapsed patients was based on surgically resected specimens rather than relapsed tissues. Finally, the relationship between exoPD-L1 and tumor-infiltrating CD8 + T cells in the TME could not be investigated, as only serum-derived exosomes were evaluated and not exosomes from the tumor tissue.In conclusion, we found that exoPD-L1 was higher in PDAC patients with systemic disease than in those with locally advanced disease. Serum-derived exoPD-L1 levels correlated with PD-L1 expression in tumor tissue. Additionally, patients with PDAC and high exoPD-L1 levels had worse survival outcomes than those with low exoPD-L1 levels. Further studies exploring the association between PD-L1 expressing exosomes derived from tumor tissue and cytotoxic or immunosuppressive T cells in the TME are required to validate the clinical importance of exoPD-L1 in patients with pancreatic cancer.Below is the link to the electronic supplementary material.Supplementary Material 1


Magnaporthe oryzae is the causal agent of rice blast disease and can infect over fifty grass species. We demonstrate that rice blast fungi encode two diterpene synthases that produce normal pimara-8,15-diene and manoyl oxide scaffolds. Phylogenetic analysis of diterpene synthases among rice blast pathotypes showed functional conservation of these two core diterpene synthases amongst all pathotypes and suggests further expansion in those infecting select grass species. These insights into the blast fungal terpenome may inform efforts to counteract deleterious phytopathogens in crucial food crops.Plant-pathogenic fungi harbor various specialized metabolites including diterpenoids that function as hormones and virulence factors. The fungus  Ascomycota produce a diverse array of specialized metabolites with versatile roles that confer a competitive advantage in nature regarding virulence, interspecies communication and competition, and biotic and abiotic stress protection -geranylgeranyl diphosphate (GGPP) , also known as rice blast fungus, destroys 10–30% of total rice yield annually worldwide. Other than rice, the fungus infects over fifty grass species, the most alarming being its recent discovery in wheat  to shortlist bifunctional diterpene synthases. Class II cyclases are characterized by the DxDD motif involved in protonation-initiated cyclization. Class I active site has the characteristic Dxx motif that facilitates ionization initiated cyclization of the class II intermediate  and primers in Synthetic codon-optimized diterpene synthase genes for heterologous expression in E. coli cells pre-transformed with pIRS (to increase yield) for recombinant expression of each diterpene synthase. The resulting recombinant strains were grown at 37°C overnight in 5 ml of TB media (supplemented with 100 mM phosphate buffer pH 7.5 and appropriate antibiotics). One ml of overnight culture was used to inoculate 50 ml of TB media and the cultures were incubated at 37°C and 160 rpm till mid-log phase. At this point, the temperature was dropped to 16°C for 1 h and then 1 mM isopropylthiogalactoside (IPTG) was added for induction and 50 mM sodium pyruvate was added as a carbon supplement. The induced culture was further incubated for 3 days at 16°C and 160 rpm to allow product formation. The resulting products were extracted adding equal volume of hexane and the organic layer was separated, dried and re-dissolved in 1 ml of n-hexane for product analysis.The diterpene synthase products were determined using a modular metabolic engineering system  into yeast ZXB competent cells using previously described  values in the range from 60 to 600 starting from 9 min after sample injection until the end of the run. For identification of enzymatic products, the retention time and mass spectra of products were compared to authentic standards  on Agilent 7890/7000C GC-QQQMS using an Agilent HP-5MS column with 1.2 ml/min helium flow rate. Samples (1 ul) were injected in splitless mode using an Agilent 7693 autosampler. The following temperature program was used: the oven temperature started at 70°C, which was maintained for 1 min, and then increased at a rate of 20°C/min to 300°C, where it was held for another 2 min. The mass spectrum was recorded by mass-to-charge ratio  in M. oryzae reference genome (Mo70_15) to produce diterpenes. Using BLAST analysis, and presence of characteristic protein domains for published genome analysis we identified two putative diterpene synthases  among M. oryzae pathotypes that infect different host plants. Utilizing the diterpene synthase mining approach described in the Methods section and depicted in M. oryzae proteomes covering 14 pathotypes , brome grass (Bromus), and fingermillet (Eleusine) compared to rice (Oryza) pathotype. MoDiTPS6 is uniquely present in the pathotypes from two grasses –Triticum and Bromus. The observed expansion of DiTPS enzyme family aligns with several studies that have highlighted the fluidity of fungal genomes and expansion of various gene families  common ancestral origin of ent-kaurene synthases and MoDiTPS1, 4 and 6 clade. MoDiTPS2 and 5 have same node of divergence and do not group with any characterized DiTPS. However, DiTPS3 associates with a known phyllocladanol synthase , these were biochemically characterized using a previously described modular metabolic engineering system  or normal specific abietadiene synthase (AgAS) ,15-diene, indicating that this is relevant stereoisomer of CPP produced by its class II active site. The DiTPS2 class I active site also reacts with syn-CPP resulting in unidentified products but does not form any products with ent-CPP . Subsequent co-expression of the class I mutants with GGPP resulted in production of the expected intermediate 8α-hydroxy-CPP by DiTPS1 and CPP for DiTPS2 . To probs of CPP . To asseroxy-CPP ; MoDiTPS ent-CPP . These r ent-CPP .B71 and MZ5-1-6 may have variable regulator genes. The putative DiTPS2 cluster shows the presence of a complete and conserved biosynthetic gene cluster among M. oryzae pathotypes with precursor GGPP synthase (MGG_00026), oxidases and/or cytochromes P450 , dehydrogenase (MGG_00017); transcription factor (MGG_00032), and transporter (MGG_00040) , oxidases/cytochromes P450 , ligase (MGG_01951), and could possibly include a UDP glycosyl transferase (MGG_01961) . The synG_00040) . The biothotypes . These rMagnaporthe pathotypes infecting rice and other grass species. From the literature, the two DiTPSs were transcriptionally upregulated under various conditions in rice and fingermillet  are phylogenetically conserved across Magnaporthe pathotypes demonstrates an expansion of diterpene synthases that could be involved in adaptation of pathotypes for infection of other grasses. In particular, via intra-species duplication generated from the two core diterpene synthases (Fusarium species where terpene synthases were conserved across species, independent of host variety (Wiemann et al., F. fujikuroi. Nevertheless, this research provides a strong basis for further studies to investigate if the observed phylogenetic expansion is limited to duplication, but generally results in pseudogenes rather than neofunctionalization or whether the observed inactivity is an artifact of the experimental process. Our results also highlight the need for production of both pimara-8,15-diene and ent-manoyl oxide activity, as well as hinting at a relationship to pathogenicity. Thus, our study defines the diterpene biosynthetic capability in Magnaporthe pathotypes and provides impetus to further investigate the importance of chemical diversification in evolution and adaptation of plant pathogenic fungi. As terpenes mediate diverse functional roles, the conserved production of at least these two core diterpenes suggests a biological role for these diterpenes in the rice blast fungus.Interestingly, the phylogenetic analysis across ynthases . Terpeneynthases . Our resM. oryzae (Peters, ent-manoyl oxide, which are distinct from the diterpene scaffolds produced by rice (M. oryzae pathotypes. The BGC for MoDiTPS1 is more variable amongst the isolates which is consistent to the demonstrated variability in our biochemical analysis. The conservation of backbone formation and BGC suggest a conserved role of MoDiTPS2 in M. oryzae pathotypes. It would be worthwhile to confirm the BGC through experimental expression in heterologous hosts such as Aspergillus and yeast to not only identify the bioactive diterpenoids but understand the variability in the clusters.From the host plant perspective there is a strong diterpene-based chemical defense response in rice after infection by  Peters, . We show by rice . These dM. oryzae, the biochemical characterization described here provides information to enable experimental differentiation at the host-pathogen interface.As diterpenoids are essential for defense and resistance of rice to M. oryzae contains two diterpenes synthases that are evolutionarily conserved across different pathotypes, suggesting important roles for the resulting diterpenes. These labdane-related diterpenes produced by Magnaporthe are functionally distinct from those produced by rice. This research provides a strong basis for further studies of in vivo confirmation of gene function including testing whether the observed phylogenetic diversification confers an evolutionary advantage. Our study defines the diterpene biosynthetic capability in Magnaporthe pathotypes and provides insight to the importance of chemical diversification in evolution and adaptation.In conclusion, it is now evident that The original contributions presented in the study are included in the article/AS performed all the experiments. AS and HY performed the computational analysis. AS and SM analyzed the data and wrote the article. SM designed the research and supervised the project. All authors commented on the results and manuscript. All authors contributed to the article and approved the submitted version.Financial support was provided by the Department of Biochemistry and Molecular Biology, UMass Amherst and Richard and Susan Smith Family Foundation, Newton MA to SM. AS was supported by Lotta M. Crabtree Fellowship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.