CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools. The identification of events occurring at target sites is critical to determine the safety of CRISPRbased DNA editing tools. Here, the authors show that LINE-1 retrotranspositions can occur frequently at canonical CRISPR/Cas9 editing sites, but are rare with prime editors and base editors. Canonical CRISPR/Cas9 DNA editing induces double-strand breaks (DSBs) that are resolved by the classical non-homologous end joining pathway (c-NHEJ) and typically result in insertions or deletions (indels) of relatively small DNA sequences at both on-target and off-target sites, leading to disruption of the target sequence4. While c-NHEJ is the main cellular DNA repair pathway of CRISPR/Cas9-induced DSBs, homology-directed repair (HDR) can compete by inserting homologous DNA sequences that result in precise gene editing5. More rarely, DSBs induced by CRISPR/Cas9 can lead to larger genomic rearrangements such as large chromosomal deletions, inversions, or translocations10 or even more catastrophic events such as chromothripsis11 and chromosome loss14. Additional outcomes of the canonical CRISPR/Cas9 editing include integrations of exogenous sequences including lentivirus16, adeno-associated virus (AAV)17, plasmids19, and small DNA fragments21. These events exploit the availability of frequent DSBs generated by CRISPR/Cas9 to favor the insertion of exogenous DNA fragments using microhomology-mediated end joining (MMEJ)22.Genome editing with CRISPR/Cas9 holds great promise for the treatment of human genetic diseases28, little is known on the frequency and mechanisms of retrotransposon integration into CRISPR/Cas9 editing sites because few such events have been described so far only in mouse zygotes28. Retrotransposons are self-propagating sequences that generate de novo insertions through reverse transcription of RNA intermediates. In humans, the only autonomously active family is the long interspersed element-1 (LINE-1 or L1)29. L1-derived sequences are prevalent in the genome (~17% of the human genome30), and ~100 loci are full-length, retrotransposition competent L1s35. These competent sequences code for two proteins: open reading frame 1 protein (ORF1p) and ORF2p. ORF1p associates with L1 RNA to serve as an RNA chaperone36, while ORF2p contains endonuclease (EN)37 and reverse transcriptase (RT)29 activities that are critical for retrotransposition of L1 RNA38. Recently, the ORF0 was identified on the antisense strand of primate LINE-1 5′UTRs. ORF0 can induce the generation of fusion proteins of ORF0 with proximal exons and is translated as a short peptide that enhances LINE-1 mobility39.While it is known that mobile genetic elements can be captured at DSB sites41. The mechanism includes generation of a DNA nick by the EN activity of ORF2p at the consensus target sequence (5′-TT/AAAA-3′) to expose a 3′-OH followed by the formation of a primer-template structure by base pairing between thymines at the cleavage site and the PolyA stretch at the 3′ end of L1 mRNA44. The RT activity of ORF2p extends the DNA 3′-OH using the L1 RNA template to generate L1 cDNA, which is process into de novo L1 insertions through poorly understood mechanisms38. These L1 insertions are commonly incomplete because the majority of L1 insertions is 5′-truncated. In contrast, EN-independent L1 retrotransposition is hypothesized to occur at sites of damaged DNA and on the lagging strand of DNA replication forks, and does not require to start from intact L1 3′ ends with the constraint of a canonical consensus motif45.Canonical L1 retrotransposition takes place by target-primed reverse transcription (TPRT)46. A native L1 reporter (L1RP) uses native L1RP sequences and the native L1 5′UTR promoter to closely mimic endogenous L1 retrotransposition, while a synthetic L1 reporter (L1-ORFeus) contains a codon-optimized human L1 sequence that allows to distinguish de novo L1 insertions from insertions of inactive endogenous L1 fragments. In this work, we describe the occurrence of de novo L1RP and L1-ORFeus insertions into multiple canonical CRISPR/Cas9 editing sites in three human cell lines by PolyA-seq and an improved amplicon sequencing, and use structures of more than 2500 de novo L1 insertion events to infer underlying mechanisms. Moreover, we show that de novo L1-ORFeus insertions are rare in CRISPR-based genome editing tools (PE and BE) that do not require the formation of DSB intermediates.Two different types of reporters are commonly used to study L1 retrotransposition events in cellular models46 coding for ORF1p, ORF2p, and a GFP reporter for retrotransposition47  activity (L1-ORFeus-ENm (H230A), hereafter termed as L1-ENm) or reverse transcriptase activity (L1-ORFeus-RTm (D702Y), hereafter termed as L1-RTm) of ORF2p severely limited L1 integrations as compared to wild type  and one inactive gene (RAG1) for inducing DSBs by CRISPR/Cas9, two loci differing in their transcriptional activity and chromatin accessibility. Human RAG1 gene is also a proposed target for gene correction therapy52. We transduced HEK293T cells with the CRISPR/Cas9-containing lentivirus at day 5 of L1 expression and retrotransposition, then we analyzed repair outcomes of CRISPR/Cas9-mediated DSBs at day 11 on the bulk cell population  where CRISPR/Cas9 has been applied for therapeutic editing to prevent HIV entry into cells56  tail typical of canonical TPRT products, similar to a previously described approach43  tracts (range 15–75 bp)  techniquebp) Fig. . In contBy PolyA-seq, we identified de novo L1-ORFeus insertions from L1-ORFeus expressing cells that peaked in occurrence at the CRISPR/Cas9 target sites and, strikingly, extended for about 5 kilobases 5′ or 3′ of the DSB Fig. . These LMYC CRISPR off-target #1 site (MYC OT1)59 further validated these findings, in which RT-dependent retrotransposition of the spliced L1-ORFeus transcript were observed  (L1RP-ENm) or the RT mut (D702Y) (L1RP-RTm) reporters Immunoblot analysis showed an expression of the ORF1p protein consistent across L1RP, L1RP-ENm, and L1RP-RTm reporters, and slightly lower than ORF1p protein expression observed with the L1-ORFeus reporter  enable precise genome editing, including targeted insertions, deletions, all 12 possible base-to-base conversions and their combinations, using Cas9 nickase (H840A) fused to an engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) that is programmed with a pegRNA to edit DNA sites64. PE2 nicks the target site to expose a 3′-hydroxyl group that could be used to prime the reverse transcription of edit-containing pegRNA directly into the target site, while PE3 makes a second nick on the non-edited strand to induce its replacement and increase editing efficiency. Importantly, PE3b was then developed as an improved strategy that nicks the non-edited strand only after edited strand flap resolution to minimize the presence of concurrent nicks and potential DSB in PE364  control, or nick only for PE3b served as a negative control since this nicking sgRNA binds only to the edited sequence64  or PE3 (MYC + 5GtoC) editing, though were at a much lower level compared to canonical CRISPR/Cas9 64 , the APOBEC1 enzyme is fused with a nickase Cas9 (D10A) for targeting via sgRNA and with UGI to increase the accuracy and efficacy of base editing, converting C•G base pair to T•A base pair. In adenine base editors (ABEs), a E. coli-derived tRNA adenine deaminase (TadA) is fused with nickase Cas9 to convert an A•T base pair to a G•C base pair. Briefly, in contrast to dCas9-fused BE2, BE3 uses nickase Cas9 to specifically nick the non-edited strand to increase the editing efficiency69. Fusing BE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing70, while optimization of codon usage and nuclear location sequences (NLS) resulted in the most advanced and efficient AncBE4max or ABE8e72. Similar to prime editors, BE systems do not require the formation of a DSB intermediate for editing67. Thus, we also assayed for de novo L1-ORFeus insertions in multiple editing sites targeted by several CBEs  and one ABE (ABE8e)  Fig. .Fig. 8DeMYC and FANCF loci, the base editing efficiency was high in all five BEs, with highest in AncBE4max and ABE8e, and comparable in cells expressing L1-ORFeus or L1-RTm  currently being evaluated for gene therapy of sickle cell disease and β-thalassemia74  editing sites were consistent with their low levels of indels formation as reported72 17 vectors encoding CRISPR/Cas9, as well as DNA fragments from freely available DNA20. In contrast to these known events that rely on double-stranded DNA (dsDNA) sequences that are inserted during DSB repair, the mechanism of de novo L1 insertions is quite distinct, and appears to rely on the ability of RNA to template break repair in an RT-dependent manner at CRISPR/Cas9-mediated DSBs. Since large insertions are difficult to detect and can be easily missed by PCR and DNA sequencing that normally used to evaluate the safety and outcomes of CRISPR/Cas9 editing19, we developed PolyA-seq together with improved amplicon sequencing to fully appreciate these L1 insertions. Together, these approaches captured more than 2500 de novo L1 retrotransposition events at CRISPR/Cas9 editing sites, as well as 141,490 insertions of the lentiviral sequences that encodes for the CRISPR/Cas9 and 179,264 integrations of genomic DNA sequences that mostly originated from regions flanking the DSB.In this work, we describe LINE-1 retrotransposon insertions into CRISPR/Cas9-mediated DSBs as an outcome of canonical CRISPR/Cas9 genome editing as well as PE3 prime editing. Retrotransposed sequences are thus added to a growing list of insertions known to occur at DSBs generated by CRISPR/Cas9, including various vectors such as plasmids used for HR-mediated editing45. De novo L1 insertions were initiated by CRISPR/Cas9-induced DSB formation and extended for about 5 kilobases 5′ and 3′ of the DSB, consistent with extensive resection of the DSB9 before L1 integration. In contrast, canonical retrotransposition events, which were EN-dependent and showed TT/AAAA insertion site preference predominated in more distant regions from the CRISPR/Cas9-mediated DSBs, including on the same chromosome or in the rest of the genome.Mechanistically, de novo L1 insertions at sites of genome editing are similar to canonical retrotransposition events in some ways, but distinct in others. Like canonical retrotransposition, de novo L1 insertions at genome editing sites are dependent on RT activity but they are distinct in that they are largely independent of EN activity and show a random sequence motif pattern instead of the typical preference for TT/AAAA target sites Figs.  and 7h w19. Although we induced CRISPR/Cas9 DSBs 5 days after L1 plasmid transfection, still few copies of plasmid are present in cells at the time of CRISPR/Cas9 cutting. Consistently, the rare sequences found in GFP control samples aligned to the GFP portion of the vector and are likely plasmid integrations  and induced pluripotent stem cells (iPSCs)84 that are frequently used for CRISPR/Cas9 editing86. Although HEK293T express lower L1 ORF1p than iPSCs  is the most active autonomous L1 element in the genome87. We detected L1Hs sequences with bias toward the 3′ of the L1Hs both in MYC and RAG1 editing sites  (L1-ENm), and pCEP4 L1-ORFeus-RTm (D702Y) (L1-RTm) can be found in Supplementary Sequence 88. The GFP control vector was generated by cloning an intron-less GFP reporter in the same pCEP4 vector backbone. pCEP4 L1RP (L1RP) is modified from the Boeke Lab46 and published previously60 (Addgene plasmid #131392). pCEP4 L1RP uses native L1RP sequences89 and the native L1 5′UTR promoter, sharing the same pCEP4 vector backbone and GFP-Artificial Intron cassette with pCEP4 L1-ORFeus vector; likewise, two mutants pCEP4 L1RP-ENm (H230A) and pCEP4 L1RP-RTm (D702Y) were generated , pCMV-BE3 (Addgene plasmid #73021), pCMV-BE4-Gam (Addgene plasmid #100806), pCMV-AncBE4max (Addgene plasmid #112094), and pCMV-ABE8e (Addgene plasmid #138489) were gifts from David Liu. Oligonucleotides were purchased from Integrated DNA Technology (IDT), and sequences of sgRNAs and pegRNAs used in this work were listed in Supplementary Table 59, sequences were listed in Supplementary Table The human codon-optimized, synthetic L1-ORFeus was modified from the human codon-optimized, synthetic L1-ORFeus constructs published by the Boeke Labeus-ENm H30A , penicillin-streptomycin (100 units per ml), and L-glutamine (2 mM). Cells were cultured at 37 °C with 5% CO5 HEK293T cells, 2 × 105 HeLa/U2OS cells were seeded on 6-well plates. The following day, for L1-ORFeus in HEK293T/HeLa cells, cells were transfected at ~60–70% confluency with 1.5 μL X-fect polymer  and 5 μg indicated pCEP4 L1-ORFeus plasmids, as well as pCEP4 GFP control vector according to the manufacturer’s protocols. For L1-ORFeus in U2OS cells and L1RP in HEK293T cells, cells were transfected at ~60–70% confluency by Fugene HD  with 1 μg indicated plasmids following the manufacturer’s instructions. The media was changed 8–12 h after transfection. Two days post-transfection, medium was supplemented with puromycin (Sigma) at 1 μg/mL (for HEK293T/U2OS cells) or 2 μg/mL (for HeLa cells) to enrich the transfection positive cells (thus to enrich L1 retrotransposition events). The media was changed every 2 days. Cells were cultured for indicated days post-transfection, followed by flow cytometry and intron assay analysis on the bulk population of cells without GFP sorting. For flow cytometry, data acquisition was performed using a FACSVerse flow cytometer (BD Biosciences). For intron assay, genomic DNA was extracted using rapid lysis buffer containing 20 μg/ml Proteinase K and incubation at 56 °C overnight, followed by standard isopropanol extraction, wash in 70% ethanol, and resuspension in TE buffer. 200 ng genomic DNA, Taq DNA polymerase (Qiagen), as well as two primers were put in the PCR reactions . Following PCR, the products were separated on 1% agarose gel. The original PCR product (with intron) was 1192 bp, while LINE-1 retrotransposition generate intron-removed DNA which gives rise to a short PCR product (292 bp)  and reversed-transcribed with iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s instructions. Indicated gene induction was analyzed by PCR for 25–28 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s. Primers are listed in Supplementary Table RT-PCR was performed as previously described3VO4 by sonication. Extracts were centrifuged at 14,000 r.p.m  for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 4–12% CriterionTM XT Bis-Tris Protein Gel (Biorad), transferred by Trans-Blot-Turbo (Biorad), and blocked with EveryBlot Blocking Buffer (Biorad). Primary antibodies were incubated with membranes overnight at 4 °C. Antibody against ORF1p  was used and validated as previously published60, anti-β-actin is commercially available . Membranes were developed with ECL solution  and imaged by Image Lab. The relative expression of the ORF1p protein was measured by densitometry using Image J and normalized for the β-actin intensity of the corresponding lane.Cells were lysed in RIPA buffer with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Nat-test (GraphPad Prism 9.0) for all studies unless otherwise indicated. We considered P < 0.05 to be statistically significant as previously described94.We performed statistical analysis by using an unpaired Student’s 59. In brief, HEK293FT cells were maintained in 10% FBS-containing DMEM. To generate lentiviral particles, 5 × 106 HEK293FT cells were plated per 10 cm dish. The following day, cells were transfected by calcium phosphate transfection method with 7.2 μg of indicated lentiCRISPR v2 plasmid, 3.6 μg of VSVG, 3.6 μg of RSV-REV, and 3.6 μg of PMDLg/pPRE. The media was changed 8 h after transfection. The viral supernatant (10 mL/10 cm dish) was collected 36 h after transfection, passed through a 0.45-μm filter, pooled, and used either fresh or snap-frozen. For lentivirus infection, 2 mL of viral supernatant with polybrene (6 μg/ml) was added to one well of 6-well plates of cells with at ~60–70% confluency. The viral supernatant was exchanged for fresh medium 8 h later. After 48 h, cells were selected with 1–2 μg/mL (for HEK293T/U2OS cells) or 2–3 μg/mL (for HeLa cells) puromycin to enrich CRISPR/Cas9 lentiviruses infected cells.The production and transduction with CRISPR/Cas9 lentiviruses were performed as previously describedAmplicon sequencing is a method of targeted next-generation sequencing. Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific) was used to amplify selected target sites from 5 μg template genomic DNA within 5 PCR reactions as follows: 98 °C for 3 min, then 35 cycles of , followed by a final 72 °C extension for 5 min. Multiple reactions were pooled and size fractionated for DNA fragments between 300 and 1000 base pairs (exclude the strong main band) on 1% agarose gel. DNA were purified using Gel extraction kit (Qiagen) following the manufacturer’s protocol and sequenced bi-directionally 250 bp (PE250) in a Miseq sequencing platform at the Molecular Biology Core Facilities of the Dana-Farber Cancer Institute. Primers used for amplicon sequencing are listed in Supplementary Table 59. Briefly, reads for each experimental condition were demultiplexed by designed forward and reverse barcodes . To enhance specificity of target sequences, reads were further filtered by the presence of primer plus additional 10 downstream bases.Sequences in library were processed as previously described35, L1RP(L1PA1), and relatively younger inactive L1PA2, L1PA3,…, L1PA8, to identify endogenous L1 insertions we particularly mapped sequences against the youngest reference elements annotated as L1Hs in RepeatMasker Repbase95 (http://www.repeatmasker.org). As for LINE1 (ORFeus) alignment, we specially excluded the alignments where contain the common sequences with lentiCRISPR v2 plasmid and attributed those reads to lentiCRISPR v2 plasmid; excluded the alignments where contain the common sequences with endogenous L1Hs and attributed those reads to L1Hs reference. As for plasmids alignment, the indicated plasmid was chosen for corresponding experiment, i.e., lentiCRISPR v2 for canonical CRISPR/Cas9 editing analysis, pCMV-PE2 and pU6-pegRNA for PE editing analysis, pCMV-BE2 for BE2 editing analysis, pCMV-BE3 for BE3 editing analysis, pCMV-BE4-Gam for BE4-Gam editing analysis, pCMV-AncBE4max for AncBE4max editing analysis, pCMV-ABE8e for ABE8e editing analysis, respectively , exogenous LINE1 (ORFeus), and indicated plasmids, respectively. Because of high similarity among younger L1 members of L1PA subfamily including youngest active L1HsTo understand how L1-ORFeus mechanically joins to break point of CRISPR/Cas9-mediated DSBs to complete its insertion, we analyzed the position distribution and microhomology of insertion junction. First, the raw paired-end sequences that contain L1-ORFeus insertions identified by above insertion sources analysis obtained by amplicon sequencing were particularly selected and aligned to amplicon target reference sequence and LINE1 (ORFeus) by using BLAST v2.11, respectively. The forward sequences alignments records against target reference and LINE1 (ORFeus) were used to determine the left end of DSB and insertion junction, the overlapped base pairs between break end and insertion junction were calculated as microhomology, while the gap between them was considered as “other sources” of insertions (include genomic fragments or plasmid insertions). Likewise, the reverse alignments records were calculated for right end of DSB and insertion junction. Both forward and reverse sequences alignment against LINE1 (ORFeus) were analyzed to determine the L1 insertion fragments between two ends of break. Finally, a map of L1-ORFeus insertions at break point and insertion junction position distribution surrounding CRISPR/Cas9-mediated cutting site (three base pairs before PAM) were plotted by using R ggplot to illustrate the mechanisms of insertions at DSBs. BLASTN parameters ‘-reward’ and ‘-penalty’ were set as 1 and −2 for more conserved result. Only alignments record with alignment start in PCR target reference no more than 8 bp from 5′ end for forward and 3′ end for reverse sequence were considered as valid portion of PCR target reference. Furthermore, sequences with PCR target reference alignment length <18 bp or LINE1 (ORFeus) alignment length <30 bp were further eliminated as well. For PE experiments, the raw sequences from pegRNA nick strand were also aligned to pU6-pegRNA-GG-acceptor plasmid to identify pegRNA portion required to have edited base, minus alignment orientation and at least 10 bp alignment length. Then by taking into account the LINE1 (ORFeus) alignment records, pegRNA portion’s junction and microhomology with L1 were finally calculated.59 and a similar L1 retrotransposition capturing technique43. Briefly, 50 μg genomic DNA was digested overnight with HaeIII enzyme (recognizes and cuts at 5′-GG/CC-3′), followed by 3′A addition by Klenow polymerase  and adapter ligation (composed of an upper linker and a lower 3′-modified linker) by T4 DNA Ligase (Promega). To reduce sequencing background, ligation reactions were digested with Xba I to remove the exogenous LINE-1 sequence from plasmid (first blocking). In the first round of PCR, DNA was amplified using Biotin-LEAP, together with adapter-specific reverse primer (AP1) and Phusion polymerase (Thermo Fisher Scientific). 20 PCR cycles were performed in the following conditions: 98 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min. Multiple reactions were performed in generating large-scale libraries. Biotinylated PCR products were captured by Dynabeads MyOne StreptavidinC1 kit (Invitrogen) for 2–3 h at room temperature with rotation, followed by 2 h digestion with Xba I (second blocking). PCR products were then eluted in 95% formamide/10 mM EDTA and purified by PCR extraction kit (Qiagen). The second round emulsion-PCR (EM-PCR) was performed in an oil-surfactant mixture by nested forward primer (SV40-polyA-F) and nested adapter-specific reverse primer (AP2) using the following conditions: 20 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min. The PCR products were then extracted three times with diethyl ether and DNA was re-purified by PCR extraction kit (Qiagen). The third round of PCR was performed with barcoded forward and reverse primer . PCR products were size fractionated for DNA fragments between 300 and 1000 bp on a 1% agarose gel and purified by Gel extraction kit (Qiagen).PolyA-seq was developed based on HTGTSTwelve to eighteen samples were pooled and sequenced through Illumina MiSeq PE250) by the Molecular Biology Core Facilities of the Dana-Farber Cancer Institute. Oligonucleotides and primers used for PolyA-seq are listed in Supplementary Table 0 by the 59. Briefly, reads for each experimental condition were demultiplexed by designed barcodes . Then reads were further filtered by the presence of primer (7 bp Poly(A) was imposed to primer directly) plus additional 8 bp Poly(A), and the barcode and primer portion of the remained sequences were masked for alignment analysis. Next, the processed sequences were aligned to human genome (GRCh38/hg38) using BLAT, and finally aligned sequences were further cleaned by removing PCR repeats , invalid alignments  and ligation artifacts . L1 insertion junction position was determined based on the genomic position of the 5′ end of the aligned read.Sequences in library were similarly processed as previously describedThe genomic DNA sequence flanking L1 insertion positions −3 (upstream) and +8 (downstream) was retrieved to analyze the consensus sequence motif at the L1 insertion sites obtained by PolyA-seq. R package ‘Biostrings’ and ‘ggseqlogo’ were performed to generate sequence logos. The corresponding position-weighted matrix (PWM) was calculated by R package ‘seqLogo’. The motif score is defined as the sum of information content at each position.To identify the 3′ Poly(A) tract additionally added to L1 intact 3′ end, we generated 200 bp Poly(A) reference sequence and BLASTN all reads with L1 insertion to such Poly(A) reference, restricting query start in read mapped to Poly(A) reference less than query start in read to Human genome to ensure that the identified Poly(A) was inserted and followed by genomic sequence at insertion junction. The default parameter ‘word_size’ of BLAST is 11, so Poly(A) size <11 was ignored accordingly. The above approach was applied to PolyA-seq.96. Briefly, sequences with mean quality score <20 and length <50 were excluded. The remained sequences were aligned to target reference sequence using BLASTN. Only sequences with alignment length >80 were considered for calculation of editing efficiency (mutation rate). The real mutations were determined by passing a Neighborhood Quality Standard (NQS) filter criterion that requires a minimum Phred score of 30 for the mutation itself, and 20 for the five adjacent bases on either side. Finally, the percentage of reads that mutated at target base site represents editing efficiency.Nucleotide mutations were calculated from raw sequences as previously describedNo statistical methods were used to predetermine sample size.Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Dust surface chemistry plays an important role in explaining the formation of interstellar molecules. However, many of the dust surface chemical parameters, such as the adsorption energies, are still of uncertainty. Here we present a study of the adsorption of water (H2O), ammonia (NH3), and carbon dioxide (CO2) on graphene-like substrate within the framework of density functional theory (DFT). We used Gaussian 16 software and adopted the corrected generalized gradient approximation (GGA) with the Perdew–Burke–Ernzerhof (PBE) functions. We determined the optimal accretion position of the studied molecules on the graphene-like surface and calculated the adsorption energies. Furthermore, according to the density of states and molecular orbitals of the adsorbed states, we analyzed the charge transfer between the molecules and the graphene-like surface. These results can provide more accurate parameters for calculating the chemical reaction rates on the dust surface, thus contributing to the understanding of dust-surface reactions in ISM.Since the first detection of CH molecule in interstellar medium (ISM), more than 270 molecules have been identified in various astronomical sources in ISM. These molecules include big complex ones, such as fullerene (C To date, more than 270 molecules and ions have been detected in interstellar spaceThe finding of interstellar molecules is one of the four major discoveries of astronomy in the 1960’s. Astronomers used large radio telescopes and detected NHhe Milky . These mky (2CO) . It is w60, hydrogenated amorphous carbon and other dust particles are important research objects of interstellar medium . Carbonaceous dust such as graphite, nanodiamond, PAHs, Cr medium . Indeed,r medium , and somr medium . After tinfrared .2 and CO) are deposited on the dust surface, and the new molecules are formed through the cold surface chemical reaction, due to the thermal fluctuations on the dust surface, the matter can be desorbed to the gas phase again  on the graphene-like surface. In In recent years, astronomers have calculated and studied the binding energy of species on interstellar various substrates . In this2O, NH3, CO2 and graphene-like structures. The graphene-like surface contains 48 carbon atoms, and H is added to the edge to eliminate the edge effect. In addition, the PBE functional was mostly used for 2D material correlation calculations , and two hydrogen atoms pointing vertically to the graphene-like surface (denoted by v2). Coupled with the three graphene adsorption sites, so H2O has six adsorption modes; For NH3, there are two forms, the nitrogen atom pointing vertically to the surface (denoted by v1) and the three hydrogen atoms pointing vertically (denoted by v2), with a total of six adsorption modes; For CO2, there are two forms: parallel (denoted by p) and vertical (denoted by v), so it has six modes. The different forms of these molecules are shown in For each of the molecules, we considered three adsorption sites on graphene-like surfaces See , namely,Eads) is used to determine the stability of the structure before and after adsorpting interstellar molecules, and the equation is:Egr is the energy of graphene-like surface, Emol is the energy of interstellar molecules H2O, NH3, and CO2, and Egr+mol is the energy of the whole adsorption system. According to this definition, when the adsorption energy is negative, the adsorption process is exothermic, and the larger the absolute value of adsorption energy, the stronger the adsorption of molecules on the graphene-like surface and the more stable the configuration; On the other hand, when the adsorption energy is positive, the process is endothermic, and the larger the value, the weaker the adsorption. , or two hydrogen atoms point vertically to the surface (v2). Depending on the binding site , followed by the orientation of the molecule . Furthermore, the farther the distance in between the molecule with the binding site, the smaller the adsorption energy. All the absolute value of adsorption energy in the table are less than 0.8 eV, the length of the H-O bond and the angle of H-O-H are almost unchanged. In combination with the charge density of H2O adsorbed on the graphene-like surface , and N atoms pointing to graphene-like surface (v2). All properties are once again found to be almost invariant to the rotation around the axis perpendicular to the surface and through the nitrogen atoms. The calculated results are shown in For NH3 and the graphene-like surface is the largest in v2 mode, where the hydrogen bond is easily formed between H in NH3 and the atom with greater electronegativity (such as C). Therefore the adsorption energy is relatively large. As also shown in 3 does not overlap with the electron cloud of the graphene-like surface, and the adsorption of NH3 is physical adsorption, with no electron transfer.It can be seen from 3, and show them in 3 molecule (concentrated around the N atoms), not on the N-H line. This indicates that the electrons of HOMO are uninvolved in the N-H bonding of the NH3 molecule, and that HOMO is occupied by a lone pair of electrons. In LUMO, the electron density is mainly concentrated in the space in between the N and H atoms (near N atom), but not along the N-H bond, indicating that LUMO is in the N-H antibonding position and is unoccupied. Therefore, the electronic properties before and after adsorption are almost unchanged.We calculated the DOS of the graphene-like surface before and after the adsorption of NH2 molecules and the selected binding sites, there are 6 different adsorption configurations of CO2 molecules on the graphene-like surface. The calculated adsorption energies of CO2 on the graphene-like surface are listed in According to the structural characteristics of CO2 on the graphene-like surface is an endothermic process. The magnitude of adsorption energy mainly depends on the distance. When the distance is the same, the adsorption energy is also the same. The larger the distance, the greater the adsorption energy. Also, the adsorption distances of all modes are greater than 0.34 Å, which is greater than the sum of the covalent bond radii of the corresponding atoms. In combination with the charge density of CO2 adsorbed on the graphene-like surface  and the graphene-like surface. Based on the DFT, the adsorption energy, adsorption distance, charge density, DOS and other properties of interstellar molecules H2O, NH3, and CO2 on the graphene-like surface were calculated, and their adsorption properties were studied.We established the stability models of three interstellar molecules  for calculating the chemical reaction rates on dust surfaces, which in return can provide a more accurate theoretical reference for the models and observations of interstellar molecules, thus contributing to the understanding of dust-surface reactions in ISM.2O and other molecules  as ice mantles to establish a dust particle model, simulate the adsorption of interstellar molecules on the ice surface, and calculate adsorption energies.On the basis of this work, we will further take carbonaceous materials as the core, and use amorphous phase H


It substantially overcomes the poor reliability of previous sensors with suspended graphene and exhibits excellent mechanoelectrical coupling performance, as graphene is placed on the root of the crossbeam. By performing basic mechanical electrical measurements, a preferable gauge factor of ~1.35 is obtained. The sensitivity of the graphene pressure sensor based on the crossbeam structure chip is 33.13 mV/V/MPa in a wide range of 0~20 MPa. Other static specifications, including hysteresis error, nonlinear error, and repeatability error, are 2.0119%, 3.3622%, and 4.0271%, respectively. We conclude that a crossbeam structure with a graphene sensing element can be an application for the N/MEMS mechanical sensor.A graphene membrane acts as a highly sensitive element in a nano/micro–electro–mechanical system (N/MEMS) due to its unique physical and chemical properties. Here, a novel crossbeam structure with a graphene varistor protected by Si With its Young’s modulus of up to 1 TPa [3 W/m·K , light t·V−1·s−1 , it has ·V−1·s−1 ,6.2/Si substrate, and a sensitivity of 3.95 μV/V/mmHg was obtained as pressure ranged from 200 to 1000 mbar. There is also an unfavorable phenomenon where liquid remains in the square cavity during the graphene wet transferring process. Subsequently, the electrical property of graphene is inevitably affected, which leads to poor stability of the suspended graphene pressure sensor. A new type of graphene pressure sensor was developed by Zhu et al. [3N4 film, and sensitivity reached up to 8.5 mV/bar with pressure ranging from 0 to 700 mbar. In addition, a more sensitive graphene pressure sensor was developed by Wang et al. [3N4 for supporting graphene was etched to form substantial numbers of through hole arrays. Owing to the increased strain on the graphene membrane, the pressure sensor showed a sensitivity of 2.8 × 10−5 mbar−1 under a pressure of 0~400 mbar. Although residual liquid in the cavity can be resolved by the suspended Si3N4 approach, the measurement range is below 1 MPa due to the process limitations of the suspended Si3N4 structure. In the past few years, some researchers have also made great progress in graphene based accelerometers. A suspended graphene high–g accelerometer made by Hurst et al. has a high repeatable response to a wide range of 1000~3000 g [In previous research, graphene sensors have been used to detect multiple different mechanical parameters, such as pressure ,8, accelu et al. . A folde0~3000 g . Another0~3000 g , and an 0~3000 g ,19. In a2, O2, CO2, H2O, etc.) or other impurities, causing n–/p–type doping in graphene [2O3, and Si3N4, are used to protect the graphene sensing element and can improve the electrical properties and long–term stability of sensors [2O3, depositing and etching Si3N4 are relatively convenient methods [3N4 processing is also highly compatible with N/MEMS technology, avoiding risk of oxidation under high temperature [3N4, rather than organic polymer or h–BN, on graphene to reduce organic residue pollution in subsequent processing. In this paper, a novel crossbeam structure with a graphene sensing element is presented for N/MEMS mechanical sensors. The core graphene varistors are encapsulated with Si3N4 film to achieve highly sensitive pressure detection.As the core element of a pressure sensor, the quality of graphene directly affects the piezoresistive performance of the device. Improper operation during the graphene transferring process can easily introduce some defects and residues, such as wrinkling, cracking, and photoresistance . This cagraphene ,25. As agraphene . General sensors ,30,31,32 methods ,34, and perature ,36,37. A3N4 with a thickness of 200 nm was first deposited on 2″ Si wafer by plasma enhanced chemical vapor deposition (PECVD). The electrode, 15 nm/25 nm thick Cr/Au, was followed by magnetron sputtering [3N4 film was also deposited by PECVD to protect the graphene from undesired doping and pollution in the ambient environment. Reactive ion etching (RIE) was used to remove Si3N4 on the Pad, and Cr/Au with a thickness of 25/100 nm was sputtered on the Pad [The fabrication process is shown in uttering a. A squauttering b. Subsequttering c. A thin the Pad d. Finall the Pad e.The crossbeam structure with the graphene sensing element was firstly measured using a scanning electron microscope . 2 plasma, and the Cu substrate of supporting Gra was etched with 40% FeCl3 solution for about 6 h. After, the PMMA/Gra sample was transferred to the target substrate and heated on a hot plate at 85 °C for 30 min. The PMMA layer was dissolved in acetone (CP) solution at 50 °C for 10 min, and the target was cleaned with alcohol (EA) solution and deionized (DI) water. Finally, photolithography technology and O2 plasma etching were used for patterning graphene. The parameters of the O2 plasma etching were as follows: the power was 60 W, gas flow rate was 30 sccm, and etching time was 3 min. The schematic of transferring the graphene layer to the target substrate is shown in Single–layer graphene (Gra) was grown on a Cu substrate. After being cropped into a 1.5 × 1.5 cm sample, we coated the graphene on Cu foil with polymethyl methacrylate (PMMA) using a spin coater at speeds of 600 rpm/5 s and 4000 rpm/30 s. The PMMA/Gra/Cu/Gra sample was placed on a hot plate at 130 °C for 20 min. Then, the backside graphene of the sample was etched by O3N4. As shown in 3N4–protected layer, the intensity of the D peak rapidly increased; meanwhile, the intensity ratio of the 2D peak to the G peak (I2D/IG) decreased immensely from 3.08 to 0.60. Moreover, a slight shift in the G and 2D peaks was also observed [−1, Si3N4−encapsulated graphene resistance ranged from 566.1 to 570.8 Ω·sq−1. This indicated that graphene can retain high quality and consistency after overlaying a Si3N4 protective layer.Raman spectra  were used to analyze the stacking and defects of graphene before and after depositing Siobserved ,39. The 3N4 layer on the stability of the graphene sensing element, samples with and without the top Si3N4 layer were placed in ambient air (20~25 °C and relative humidity 40~60%). From 3N4 increased by ∆R/R0 = 25.8%, where R0 is the initial resistance. In contrast, the relative resistance protected with a Si3N4 layer was almost unchanged, showing higher stability. The main reason is that Si3N4 can effectively isolate graphene and avoid water or air doping [3N4 protecting graphene from serious environmental pollution or doping [To further investigate the effect of a Sir doping . After 3r doping .Tests of the piezoresistive effect for the graphene sensing element on the crossbeam structure were then performed in an uncomplicated experiment. The setup schematic is shown in 0/ε). Finally, G = 1.35 was obtained in this work, which is similar to the G = 1.6 for CVD graphene obtained in previous research [The piezoresistive effect of the graphene sensing element, through the strain−induced resistance change, was further investigated by finite element analysis (FEA). During FEA simulation, differential displacement load, ranged from 0 to 4.5 μm with a step of 0.5 μm, was applied on the center of the crossbeam structure. Although the maximum displacement was located at the center of the crossbeam, the maximum stress  appeared at the root of the crossbeam. As shown in research . This al3N4, but also isolated from the environment by multistack bonding. Double protection for graphene can greatly improve the repeatability and stability of a sensor. The results of three reciprocating cycles were extracted, as shown in A new type of graphene pressure sensor was developed by stacking an elastic diaphragm, crossbeam chip, and substrate through bonding technology. 3N4 protection can prolong the working life of graphene devices. The piezoresistive effect of the graphene sensing element was gradually confirmed through displacement resistance measurements and strain–resistance analysis. In the end, a gauge factor of 1.35, near to that of CVD graphene, was reached. Based on the crossbeam structure chip, the sensitivity of the graphene pressure sensor was as high as 33.13 mV/V/MPa under a wide range of conditions. Other static specifications also demonstrated high repeatability and reliability. This indicates that the crossbeam structure is an extremely useful application for graphene N/MEMS mechanical sensors.In conclusion, a novel crossbeam structure was employed for graphene N/MEMS mechanical sensors in this work, which significantly overcomes some disadvantages in the performance stability and fabrication process of a suspended structure. Defect free structure and high–quality graphene was demonstrated by a SEM inspection, Raman analysis, and I–V measurement. The results of stability tests further confirmed that Si


SAB is common, serious, and potentially lethal. Antibiotic options are limited, especially for MRSA. Ceftobiprole is an advanced-generation cephalosporin with bactericidal activity against Gram-positive (including MRSA) and Gram-negative pathogens, with efficacy and safety demonstrated in previous Phase 3 studies in acute bacterial skin infections and pneumonia. The present study evaluated ceftobiprole in patients with complicated SAB.ERADICATE was a randomized (1:1), double-blind, multicenter, Phase 3, non-inferiority trial comparing ceftobiprole (BPR) vs daptomycin (DAP) ± optional aztreonam, for up to 42 days of treatment, in patients with complicated SAB (NCT03138733). The primary efficacy endpoint was overall clinical success 70 days post-randomization, adjudicated by a blinded independent Data Review Committee. Success required survival, no new SAB complications, symptom improvement, SAB clearance, and no receipt of other potentially effective antibiotics. The non-inferiority margin for the difference in success rates was -15% . Safety was assessed through adverse events (AE) and laboratory data.S. aureus (94 MRSA). Median treatment duration was 21 days for both groups. Key baseline characteristics were balanced . In the BPR group 69.8% experienced success, compared to 68.7% for DAP . There were no significant differences in mortality, microbiological eradication, or in key subgroup analyses . The proportion of patients experiencing ≥1 AE was 63% for BPR and 59% for DAP. Treatment-related severe or serious AEs were infrequent. Gastrointestinal AEs, mostly mild nausea, were more frequent with BPR, consistent with data from previous Phase 3 studies.Of 390 patients randomized, 387  were in the modified intent-to-treat (mITT) population who received study medication and had a positive baseline blood culture for Ceftobiprole is non-inferior to daptomycin for overall success in patients with complicated SAB. All-cause mortality, microbiological eradication rates and new SAB complications were similar between treatment groups. Both treatments were well tolerated.Thomas L. Holland, MD, Aridis: Advisor/Consultant|Basilea Pharmaceutica: Advisor/Consultant|Karius: Advisor/Consultant|Lysovant: Advisor/Consultant Sara E. Cosgrove, MD, Basilea: Advisor/Consultant|Debiopharma: Advisor/Consultant Sarah B. Doernberg, MD, MAS, Basilea: Advisor/Consultant|Genentech: Advisor/Consultant|Gilead: Grant/Research Support|Johnson and Johnson: Advisor/Consultant|NIH: Grant/Research Support|Regeneron: Grant/Research Support Maziar Assadi Gehr, MD, Basilea Pharmaceutica: full time employee of Basilea Pharmaceutica International Ltd Marc Engelhardt, MD, Basilea Pharmaceutica: full time employee of Basilea Pharmaceutica International Ltd Kamal Hamed, MD, Basilea Pharmaceutica: previous full time employee of Basilea Pharmaceutica International Ltd|Lysovant: full time employee of Lysovant Daniel Ionescu, MD, Basilea Pharmaceutica: full time employee of Basilea Pharmaceutica International Ltd Mark Jones, PhD, Basilea Pharmaceutica: full time employee of Basilea Pharmaceutica International Ltd Mikael Sauley, MSc, Basilea Pharmaceutica: full time employee of Basilea Pharmaceutica International Ltd Jennifer Smart, PhD, Basilea Pharmaceutica: full time employee of Basilea Pharmaceutica International Ltd Harald Seifert, MD, Basilea Pharmaceutica: Advisor/Consultant|Debiopharm: Advisor/Consultant|Eumedica: Advisor/Consultant|Gilead: Advisor/Consultant|MSD: Advisor/Consultant|Shionogi: Advisor/Consultant Timothy C. Jenkins, MD, Basilea: Clinical outcomes adjudication committee Vance G. Fowler, Jr, MD, MHS, Armata Valanbio Akagera Aridis Roche: Advisor/Consultant|BASILEA: Grant/Research Support|Basilea Novartis Debiopharm Genentech: Advisor/Consultant|MedImmune Bayer Janssen Contrafect Regeneron Destiny Amphliphi Integrated Bioth: Advisor/Consultant|NIH MedImmune Allergan Theravance Novartis Merck Contrafect Karius Genentech Regeneron Janssen: Grant/Research Support.


A considerable percentage of Health Care Workers (HCW) have experienced psychological distress during the COVID-19 pandemic. Data from previous pandemics suggest that HCW might develop psychiatric disorders. Psychosocial and workplace measures can improve mental wellbeing of the MHW. As part of the program of the Hospital to give support to the HCW, five support weekly open dynamic groups have been carried out with HCW from the COVID Areas of our Hospital including the ICUIdentify recurrent contents in the group that express areas ofconcern Identify HCW in risk of develop a psuchiatric disorder and refer them to their apropiate level oThe sessions were carried out in a freely open group and the contents expressed in the sessions were recorded and analyzed ina narrative way. Special attention was given to the the more stresfull activities identified, to Signs of overload and to the ability to seek relief, as well as signs of disruption of personal life outside of work. Four sessions of 90 minutes, with staff of the same area were established and after these four sessions booster sessions was offered through continuity groups to members with need of more long term care as well as individual care.The recurrent areas identified were Concern about inadequite Personal Protective Equipment Concern about spreading the infection in their own families Need for relief and avoid double turn Uncertainty about the course of the illness Exposure to patients suffering and dyingHCW need nor only psychological support but also pragmatic measuresNo significant relationships.


Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy in patients older than 50 years of age. sIBM is hardly responds to any immunosuppressing theraphies, and its pathophysiology remains elusive. This study aims to explore pathogenic pathways underlying sIBM and identify novel therapeutic targets using metabolomic and transcriptomic analyses.In this retrospective observational study, we analyzed biopsied muscle samples from 14 sIBM patients and six non‐diseased subjects to identify metabolic profiles. Frozen muscle samples were used to measure metabolites with cation and anion modes of capillary electrophoresis time of flight mass spectrometry. We validated the metabolic pathway altered in muscles of sIBM patients through RNA sequencing and histopathological studies.A total of 198 metabolites were identified. Metabolomic and transcriptomic analyses identified specific metabolite changes in sIBM muscle samples. The pathways of histamine biosynthesis and certain glycosaminoglycan biosynthesis were upregulated in sIBM patients, whereas those of carnitine metabolism and creatine metabolism were downregulated. Histopathological examination showed infiltration of mast cells and deposition of chondroitin sulfate in skeletal muscle samples, supporting the results of metabolomic and transcriptomic analyses.We identified alterations of several metabolic pathways in muscle samples of sIBM patients. These results suggest that mast cells, chondroitin sulfate biosynthesis, carnitine, and creatine play roles in sIBM pathophysiology. Sporadic inclusion body myositis (sIBM) is the most common inflammatory myopathy in the elderly,VCP, have been identified,The pathogenesis of sIBM is thought to have two mechanisms: inflammation and degeneration. Inflammatory mechanisms are supported by infiltration of CD8‐positive cells in histopathology and anti‐cytosolic 5′‐nucleotidase 1A (cN1A) autoantibodies in the sera of a certain population of sIBM patients.Metabolomics is a method for identifying changes in metabolic pathways by identifying small molecules in a biological system, but few studies have explored the metabolomics of muscular diseases. A study of the metabolomic analysis of various myopathies revealed alterations in several metabolic pathways, including transsulfuration, creatine, and niacinamide, in the blood of sIBM patients,The present study aimed to elucidate the metabolic profiles of sIBM via metabolomic and transcriptomic analyses of biopsied muscle specimens of sIBM patients and identify novel therapeutic targets of this devastating age‐related myopathy.To identify the metabolic profile of sIBM muscle, we analyzed muscle samples biopsied from 14 sIBM patients (10 men and four women) and age‐ and sex‐matched six healthy subjects. In an attempt to perform RNA sequencing, we also analyzed muscle samples biopsied from 12 sIBM patients (eight men and four women) and age‐ and sex‐matched five non‐diseased subjects. Of those, seven sIBM and five non‐diseased subjects underwent both metabolomic and transcriptomic analyses. All sIBM patients were diagnosed as clinicopathological defined IBM according to the European Neuromuscular Centre IBM research diagnostic criteria 2011.We performed an open muscle biopsy for diagnostic purposes, as previously described.g and 4°C for 5 min. Subsequently, 400 μL of the upper aqueous layer was centrifugally filtered through a Millipore 5 kDa cutoff filter at 9100g and 4°C for 120 min to remove proteins. The filtrate was centrifugally concentrated and resuspended in 50 μL of Milli‐Q water for CE‐TOFMS analysis.Metabolite extraction was conducted at Human Metabolome Technologies (HMT), Japan. For capillary electrophoresis time of flight mass spectrometry (CE‐TOFMS) analysis, approximately 30 mg of frozen muscle tissue was plunged into 750 μL of 50% acetonitrile/Milli‐Q water containing internal standards  at 0°C to deactivate enzymes. Tissues were homogenized 10 times at 3500 rpm for 60 sec using a tissue homogenizer, and then, the homogenate was centrifuged at 2300m/z 50 to 1000. Other conditions were performed as described previously.Metabolome measurements were performed through a facility service at HMT. CE‐TOFMS was performed using an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE‐MS adapter kit, and Agilent G1607A CE‐ESI‐MS sprayer kit . The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE . Metabolites were analyzed using a fused silica capillary , with commercial electrophoresis buffer  as the electrolyte. Samples were injected at a pressure of 50 mbar for 10 sec (approximately 10 nL) for cation analysis and 25 sec (approximately 25 nL) for anion analysis. The spectrometer was scanned from m/z, migration time for CE‐TOFMS measurement (MT), and peak area.m/z values determined via TOFMS. The tolerance range for peak annotation was configured at ±0.5 min for MT and ±10 ppm for m/z. Furthermore, peak areas were normalized against internal standards; then, resultant relative area values were further normalized according to sample amount. Multiple possible metabolites were noted if several annotations were assigned to a single peak.Peaks were extracted using automatic integration software MasterHands  to obtain peak information, including We extracted RNA from frozen biopsied muscle tissues of 12 sIBM patients and five non‐diseased subjects using the miRNeasy Mini Kit (Qiagen). Total RNA was quantified using a 2100 Bioanalyzer with an RNA 6000 Pico Kit (Agilent Technologies). Libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Prep according to the manufacturer's protocol (Illumina) and then sequenced with Illumina HiSeq 2500 (PE‐150 nt).https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The quality of sequence reads was assessed using FastQC v. 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) before and after trimming. Processed reads were mapped on human genome version hg19 using HISAT2 v. 2.1.0Adapter sequence trimming and low‐quality  base removal were processed with Trim Galore v. 0.5.0 .To determine mast cells, sections of muscle samples were fixed and stained with toluidine blue according to a previously reported procedure.Immunohistochemical studies were performed as previously described.https://www.reactome.org/) on the 20 metabolites with high VIP scores. RNA‐seq data were assessed using Prism 8 (GraphPad Software). Values were presented as mean ± standard deviation. Student's t‐test or Welch's t‐test was used for comparing two groups.Targeted metabolomics data were analyzed using MetaboAnalyst 3.0.m/z values and migration time, a total of 198 metabolites were identified in CE‐TOFMS analysis. PLS‐DA showed that non‐diseased subjects and sIBM groups were clearly separated , increased more in sIBM skeletal muscle than in non‐diseased subjects Fig. . In suppcle Fig. . The mRNcle Fig. . Collectcle Fig. . To valicle Fig.  and sIBMcle Fig. , their nHYAL1, HYAL2, and HYAL4, genes associated with chondroitin sulfate degradation were downregulated , SLC25A20 (carnitine‐acylcarnitine carrier), and SLC6A8 (creatine transporter) were lower in sIBM skeletal muscle than non‐diseased subjects Within the nucleotide sugar metabolism pathway, we revealed enhanced biosynthesis of some nucleotide sugars, which are components of glycosaminoglycans. We also demonstrated insufficient degradation of chondroitin sulfate in metabolomic and transcriptomic analyses, as well as its deposition in the interstitium of sIBM muscle specimens from histopathological studies. By contrast, neither heparan sulfate nor the keratan sulfate pathways were affected in histopathology. These pathological and transcriptomic findings suggest that excessive chondroitin sulfate is involved in sIBM pathophysiology. Chondroitin sulfate proteoglycan 4 plays a role in both fibrogenic differentiation induced by transforming growth factor‐β (TGF‐β) and adipogenic differentiation from basic fibroblast growth factor in skeletal muscle.We also found carnitine and creatine deficiency in sIBM skeletal muscle. Both carnitine and creatine play important roles in energy metabolism. Carnitine deficiency in skeletal muscle has been described in various myopathiesThe present study has several limitations. First, this was an exploratory study with a limited sample size. Second, since participants did not fast before the biopsy, the effect of metabolic changes due to dietary content cannot be excluded. Furthermore, only Japanese subjects were included in the study, although the effects of ethnicity on sIBM have not been clarified.In summary, our results indicate that metabolic pathways for histamine, chondroitin sulfate, creatine, and carnitine are altered in the skeletal muscle of sIBM patients and are potential therapeutic targets for this devastating myopathy for which no effective treatment has been available.Nothing to report.A.M., S.N., and M.K. contributed to the conception and design of the study; A.M., S.N., T.K., S.H., S.Ki., H.N., K.M., K.T., M.I., H.K., K.S., and Y.H. contributed to the acquisition and analysis of the data; A.M. and S.N. contributed to drafting the text and preparing the figure; and S.Ku., K.K., T.S., T.O., and M.K. revised the manuscript critically for important intellectual content.


Research on the synthesis of water-soluble polymers has accelerated in recent years, as they are employed in many bio-applications. Herein, the synthesis of poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (PSB) by free radical polymerization in a sonication bath is described. PSB and iron oxide nanoparticles (IONPs) were simultaneously stabilized on the graphene surface. Graphene surfaces with PSB (GPSB) and graphene surfaces with PSB and IONPs (GPSBI) were prepared. Since PSB is a water-soluble polymer, the hydrophobic nature of graphene surfaces converts to hydrophilic nature. Subsequently, the prepared graphene composites, GPSB and GPSBI, were well-dispersed in water. The preparation of GPSB and GPSBI was confirmed by X-ray diffraction, Raman spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis. The impacts of PSB and IONPs on the graphene surfaces were studied systematically. Continuous research is ongoing towards the development of diagnosis and therapeutic agents for detecting and treating cancer cells. Nanostructures and hybrid nanostructures are used for diagnosis and therapeutic applications ,2,3,4. T2 carbon atoms dimethyl-(3-sulfopropyl)ammonium hydroxide (SB) monomer was polymerized on the graphene surface, and graphite (G) was exfoliated into thin-layered graphene sheets by sonication. Two composites were prepared in the absence and presence of IONPs, GPSB and GPSBI, respectively. The prepared GPSB and GPSBI composites were characterized using various studies.SB (95%), G, and 4,4′-azobis  were purchased from Sigma-Aldrich, South Korea, and used as received. Deionized water (DI) was used in all experiments. Using a bath sonicator , in situ polymerization of SB monomer was performed on the graphene surface at 70 °C. The composites were centrifuged using VS-18000M, VISION Scientific Co., Ltd., Daejeon-Si, Korea.−1. X-ray diffraction (XRD) studies were carried out using the PANalytical X’Pert3 MRD diffractometer with monochromatized Cu Kα radiation (λ = 1.54 Å) at 40 kV and 30 mA and were recorded in the range from 20° to 80° (2θ). Field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy (EDS) was used to evaluate the surface morphology of the composites. Using the Hitachi S-4800 equipped with EDX at an accelerating voltage of 10 kV, FESEM and EDS measurements were carried out. Transmission electron microscopy (TEM) images were obtained from JEOL JEM with an operating accelerating voltage of 120 kV. X-ray photoelectron spectroscopy (XPS) spectra were recorded using K-Alpha , and CasaXPS software was used for the deconvolution of the high-resolution XPS spectra. Thermogravimetric analysis (TGA) measurements were carried out on SDT Q600 with nitrogen atmosphere over 0–900 °C with 10 °C/min.PSB, GPSB, and GPSBI were characterized using various physicochemical techniques. Raman spectra for composites were obtained on the XploRA Micro-Raman spectrophotometer (Horiba) in the range between 1000 and 3000 cmThe preparation of graphene-poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] composite (GPSB) was prepared as shown in The preparation of the graphene-poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide]–iron oxide nanoparticle composite (GPSBI) was prepared as shown in In situ polymerizations of SB monomer on the graphene surface were carried out, and the graphite was exfoliated into thin layers with the smaller size of the graphene sheets. To know the molecular weight of PSB on the graphene surface, PSB was prepared by adopting the same procedure as GPSB and GPSBI by excluding graphene powder. The prepared PSB was characterized using size-exclusion chromatography (SEC) and TGA. The molecular number of PSB was measured as 24,536 g/mol using SEC (refer to supporting information). The GPSB and GPSBI composites were characterized using various physicochemical techniques. The composites were compared with G and IONPs.X-ray diffraction (XRD) patterns have been utilized to study the defect polymer-stabilized graphene sheets in composites. The XRD patterns obtained for G, IONPs, GPSB, and GPSBI can be seen in −1 corresponding to D, G, and 2D bands, respectively. The D band represents the defect sites in the graphene sheets at edges and surfaces and the size of the graphitic crystals [sp2 carbon–carbon bond from the first-order scattering of the E2g phonon [D/IG ratios of G, GPSB, and GPSBI were calculated as 0.13, 0.15, and 0.29, respectively. The ID/IG ratio represents the degree of disorder and inversely relates to the size of graphene sheets [D/IG ratio result suggests that the size of graphene is decreasing in the order of G, GPSB, and GPSBI. In addition, the disorder in the composite increases, which confirms the functionalization of the graphene surface with PSB in GPSB and with PSB and IONPs in GPSBI. The 2D band around 2700 cm−1 indicates the layer of graphene sheets in the composites [−1, attributed to the IONPs, confirmed by the Raman spectra of IONPs shown in crystals ,53. The g phonon . ID/IG re sheets . The ID/mposites ,57. CompThe surface morphology of the G, IONPs, GPSB, and GPSBI was examined by FESEM and TEM measurements. The FESEM images of G and corresponding elemental mapping are shown in FESEM images, along with the elemental mapping of GPSBI, are depicted in The morphologies of G, IONPs, GPSB, and GPSBI were further investigated using TEM measurements. The TEM images of G depicted in 3/2 and Fe 2p1/2, respectively, consistent with the reported IONPs [3/2 shows three peaks at 710.98, 714.54, and 719.01 eV attributed to Fe3+, Fe2+, and satellite peaks of Fe 2p3/2, respectively. Three peaks observed on deconvolution of Fe 2p1/2 at 724.08, 727.13, and 732.36 eV correspond to Fe3+, Fe2+, and satellite Fe peak of Fe 2p1/2, respectively. This result reveals that the major component of IONPs is Fe3+ with a minor component of Fe2+.The XPS data provide further information about the chemical composition and formation of GPSB and GPSBI. The survey XPS spectrum of G is depicted in ed IONPs ,59. Deco3, C-O, C=O, and O=C-O/O-C-SO3, respectively. The SO3 arises from the sulfonate group in PSB [3)3+ (402.65 eV) [32− 2p3/2, and C-SO32− 2p1/2, respectively. The presence of −SO3 and −N(CH3)3+ confirms the functionalization of the graphene surface with PSB.The XPS spectra of GPSB are shown in p in PSB ,61. The 2.65 eV) . The O 13, C-OH/C-O-C, C=O, and O=C-O/O=C-SO3, respectively. Deconvolution of the N 1s level results in two peaks at 400.14 and 402.78 eV, attributed to the presence of −N(CH3)3+ and −N- groups in GPSBI, respectively (32− 2p3/2 and C-SO32− 2p1/2, respectively. At the Fe 2p level, GPSBI showed six peaks on deconvolution, which is clear from 3/2 level at 711.41, 714.46, and 719.35 eV, which are attributed to Fe3+, Fe2+, and Fe 2p3/2 satellite peaks, respectively. Similarly, the Fe 2p1/2 level showed peaks at 724.47, 727.35, and 732.81 eV, which are responsible for Fe3+, Fe2+, and Fe 2p1/2 satellite peaks, respectively. This result reveals that the major composition of IONPs in GPSBI is Fe3+. XPS studies reveal that the major component of IONPs is Fe3O4, and it has a minor component, α-Fe2O3. Moreover, the structure of IONPS remains the same in GPSB, and GPSBI structural changes were not observed.ectively c. Four pectively d, responThe thermal behaviors of G, IONPs, PSB, and prepared composites were studied by TGA. G, IONPs, GPSB, GPSBI, and PSB samples were subjected to thermal decomposition up to 900 °C in the inert atmosphere, as shown in The overall studies revealed that PSB interacts with graphene surfaces through ester, quaternary ammonium, and oxygen from sulfonate units. Furthermore, sulfonate units from PSB interact simultaneously with IONPs and graphene surfaces. These interactions help to thoroughly disperse GPSB and GPSBI composites in water. Thus, the hydrophobic nature of the graphene surface can be converted to hydrophilic nature and can be applied to bio-applications.We successfully prepared GPSB and GPSBI composites using a simple method. XRD and Raman studies confirm the partial exfoliation of graphite into thin-layered graphene sheets in GPSB and GPSBI composites. TEM and SEM measurements revealed the size of graphene sheets in GPSB and GPSBI as 3 ± 0.2 µm. These graphene sheets are smaller than the graphene sheets that were in G. From XRD and TEM studies, IONP size was measured as ~20 nm. The elemental mapping further showed the uniform distribution of PSB and IONPs on graphene sheets. XPS and TGA revealed the presence of PSB and IONPs in the prepared composites. TGA showed the residual weight as 60.5% and 58.8% for GPSB and GPSBI, respectively. This shows the amount of graphene that exists in the prepared composites. The combination of PSB with graphene surface showed remarkable structural and physicochemical properties with IONPs. These composites with outstanding functionalities can be potential candidates for biomedicine applications.


Background: Idiopathic pulmonary fibrosis (IPF) needs a precise prediction method for its prognosis. This study took advantage of artificial intelligence (AI) deep learning to develop a new mortality risk prediction model for IPF patients.Methods: We established an artificial intelligence honeycomb segmentation system that segmented the honeycomb tissue area automatically from 102 manually labeled (by radiologists) cases of IPF patients’ CT images. The percentage of honeycomb in the lung was calculated as the CT fibrosis score (CTS). The severity of the patients was evaluated by pulmonary function and physiological feature (PF) parameters . Another 206 IPF cases were randomly divided into a training set (n = 165) and a verification set (n = 41) to calculate the fibrosis percentage in each case by the AI system mentioned previously. Then, using a competing risk (Fine–Gray) proportional hazards model, a risk score model was created according to the training set’s patient data and used the validation data set to validate this model.Result: The final risk prediction model (CTPF) was established, and it included the CT stages and the PF  grades. The CT stages were defined into three stages: stage I (CTS≤5), stage II (5 < CTS<25), and stage III (≥25). The PF grades were classified into mild , moderate , and severe . The AUC index and Briers scores at 1, 2, and 3 years in the training set were as follows: 74.3 , 8.6 ; 78 , 16.0 ; and 72.8 , 18.2 . The results of the validation sets were similar and suggested that high-risk patients had significantly higher mortality rates.Conclusion: This CTPF model with AI technology can predict mortality risk in IPF precisely. The survival of IPF patients varies considerably. Some are stable for a long time, some progress slowly, and some exacerbate acutely, leading to short-term death . A widel1), intrathoracic gas volume (Vtg), diffusing capacity of the lung for carbon monoxide (DLco) and lung volume (VA) ratio (DLco/VA), and the alveolar–arterial oxygen partial pressure difference (AaPO2) in the resting state. The CRP model includes numerous parameters, the calculation is complex, and it is difficult to identify fibrotic lesions from chest X-ray images. Currently, severity assessment models of IPF mainly include the following: 1)The CRP  scoring model proposed by Leslie C. Watters et al.  in 1986,In recent years, artificial intelligence (AI), especially deep learning, has been evolving rapidly and has achieved remarkable results in computer vision (CV). Traditional computer-based CT analysis provided objective quantitation of IPF disease programs such as CALIPER. via phone, including the patient’s survival status, time of death (the year and month), cause of death, whether there were other complications, whether undergoing lung transplantation, and the time of lung transplantation. The deadline for follow-up was 1 August 2020. After follow-up, finally, 206 qualified cases were involved in the study. The patient screening process and follow-up are shown in For testing the AI system that we established (patent application no. 202010985175.8), we did a retrospective analysis of 232 patients diagnosed with IPF from 1 January 2011 to 31 January 2020 in the Department of Respiratory Medicine, Shanghai Pulmonary Hospital. IPF diagnosis of these cases was confirmed by the criteria of the 2018 IPF International Guidelines . Data ofBased on the IPF diagnosis guidelines in 2018 , the honThe patient’s PF parameters are indispensable for prognosis estimation . After aThe Spearman correlation coefficient was used to analyze the correlation between CTS and lung function parameters, namely, FVC%pred, DLco%pred, SpO2%, and CPI. The patient’s survival time was calculated from the evaluation time to the endpoint event, which was death due to lung disease or lung transplantation, measured in months. According to survival time, X-tile software  was used to find the optimal CT score threshold to classify patients in three CT stages.Lung transplantation was considered the most effective treatment for IPF , so lungThe statistical software used in this study was IBM SPSS24.0, Stata/MP14.0 X-Tile, and R3.4.3.Following the process shown in p < 0.01), DLco%pred , and SpO2%  and positively correlated with the existing CPI  which reflects the severity of the patient’s disease. In addition, the CTS was closely related to manual-CT scores by radiologists and Spearman correlation coefficient rs = 0.80, p < 0.01 (The fibrosis segmentation network (FSN) was the essential component of deep learning, which performed the semantic segmentation of fibrosis regions in the CT images, and is the basis of further calculation, such as CT scores (CTS) and FSN’s performance, shown in Supplemental Material 2. p < 0.01 .. It indp < 0.05). To facilitate clinical application, we tried to take integer cut-off points, i.e., 5 and 25. We used the two cut-off point CT scores  and  to establish the prediction model and found that both scores have the same prediction efficiency. For ease of clinical use, we chose the latter. So, the three groups were as follows: stage I (CTS<5), stage II (5 < CTS<25), and stage III (CTS>25).According to the survival time of all patients, we used X-tile software to find the cut-off points of CT scores and are calculated as 5.6 and 25.4, which divide the patients into three groups, and the survival rates of the three groups are statistically different  shown in A nomogram of death risk prediction for a CTPF prediction model and calibration curve are shown in As the flow in A total of two representative cases are displayed as follows: As some IPF patients had also developed emphysema , we traiCurrent IPF staging evaluation methods shown in This method has the advantages of fast incremental learning, objective and accurate quantitative calculation, efficient complete lung scanning, and high repeatability. The DSC of the model reached 77.26%, which is 8.39% higher than that of the benchmark  that is 68.78% . CompareWe selected FVC%pred, DLco%pred, SpO2%, age, and gender as five essential indicators that have been proved to have a good prognostic value and are easy to be accessed clinically to evaluate the severity of IPF. In both univariate and multivariate regression analyses, the results suggested that PF classification was an independent risk factor for predicting IPF patients’ mortality risk. The severity of each patient’s disease stage  was calculated according to these five parameters. The CTPF evaluation system combines the results from CT pulmonary fibrosis staging  and severity grading  to form a complementary pulmonary fibrosis staging/severity grading model CTPF . The assIn the task of mortality risk prediction, the CTPF model has better AUC, Brier score, and stability than any other model . Lung transplantation is an effective way to improve the prognosis of IPF patients . HoweverThe prognostic evaluation of pulmonary fibrosis with emphysema needs to be further analyzed in additional cases.The major limitation of this study is lack of an external validation cohort to further evaluate the CTPF model. We are planning a multicenter clinical study in the future and hope to verify its clinical significance.The deep learning-based model calculated the percentage of fibrosis lesions of the whole lung quantitatively by segmenting the fibrosis region from chest CT images automatically, combined with the IPF severity determined by five important physiological and pulmonary function indicators. The CTPF model predicted the mortality risk for IPF patients more precisely.


The use of cellular models is a common means to investigate the potency of therapeutics in pre-clinical drug discovery. However, there is currently no consensus on which model most accurately replicates key aspects of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathology, such as accumulation of insoluble, cytoplasmic transactive response DNA-binding protein (TDP-43) and the formation of insoluble stress granules. Given this, we characterised two TDP-43 proteinopathy cellular models that were based on different aetiologies of disease. The first was a sodium arsenite-induced chronic oxidative stress model and the second expressed a disease-relevant TDP-43 mutation (TDP-43 M337V). The sodium arsenite model displayed most aspects of TDP-43, stress granule and ubiquitin pathology seen in human ALS/FTD donor tissue, whereas the mutant cell line only modelled some aspects. When these two cellular models were exposed to small molecule chemical probes, different effects were observed across the two models. For example, a previously disclosed sulfonamide compound decreased cytoplasmic TDP-43 and increased soluble levels of stress granule marker TIA-1 in the cellular stress model without impacting these levels in the mutant cell line. This study highlights the challenges of using cellular models in lead development during drug discovery for ALS and FTD and reinforces the need to perform assessments of novel therapeutics across a variety of cell lines and aetiological models. Proteinopathy relating to the transactive response DNA-binding protein 43 (TDP-43) is a pathological hallmark in almost all cases of amyotrophic lateral sclerosis (ALS)  and apprOne pathomechanism proposed to be implicated in TDP-43 proteinopathy is the formation of stress granules. Stress granules are membraneless cytoplasmic mRNA-protein granules formed under conditions of cellular stress, such as oxidative stress or heat shock . TDP-43,In vitro models of acute oxidative stress, such as exposure to the oxidative phosphorylation uncoupler sodium arsenite, induce reactive oxygen species and free radicals that cause a temporary accumulation of TDP-43-positive stress granules. These stress granules quickly dissipate after removal of the stressor, highlighting their dynamic nature . Under cIn the typical ALS and FTD drug discovery process, high-throughput screens targeting TDP-43 aggregation, cleavage or motor neuron survival have been used as an initial read-out of activity ,16. ThesGiven this, we conducted an in-depth characterisation of two different common cellular models of ALS and FTD. The first was an oxidative stress model using HEK-293 cells overexpressing TDP-43 WT treated with a chronic dose of sodium arsenite. The second used SH-SY5Y neuroblastoma cells stably expressing a disease-relevant mutant form of TDP-43 (TDP-43 M337V). We used Western blots to assess whether TDP-43, stress granule and ubiquitin localisation and solubility in these models mirrored that seen in human ALS and FTD. The sodium arsenite model recapitulated most aspects of TDP-43, stress granule and ubiquitin pathology, whereas the mutant model only displayed some aspects. As this may suggest different aetiologies vary in their pathology, both models were used to test the ability of two small molecules to correct aberrant changes.HEK-293 cells produce endogenous TDP-43, but in order to induce overexpression of TDP-43, the Flp-In™ system was used . This reOnce established, cells were exposed to tetracycline  for various time periods  to determine optimal TDP-43 overexpression levels. Treatment with 2 μg/mL of tetracycline for 48 h was sufficient to induce overexpression of TDP-43 in the cytoplasm and nucleus, as well as an increase in both soluble and insoluble TDP-43, as compared to endogenous TDP-43 produced in HEK-293 cells . It was To better model stress granule formation, tetracycline-treated TDP-43-overexpressing cells were stressed with sodium arsenite. Sodium arsenite is an oxidative stressor which generates reactive oxidative species and free radicals . A numbeGiven this precedent, we opted to establish an oxidative stress model by treating tetracycline -induced TDP-43-over-expressing cells with a mild, chronic dose of sodium arsenite  rat PC-12 cells transfected with TDP-43:GFP under control of a tetracycline-off system. This effect was dose dependent, with an IC50 of approximately 300 nM. Western blots on whole-cell soluble and insoluble fractions of PC-12 cells treated with 1 showed a global reduction in monomeric and higher molecular weight TDP-43:GFP in insoluble fractions . In. In1, 35ractions . Compari1 and 2 had no effect on TDP-43 expression in the SH-SY5Y TDP-43 M337V mutant cellular model, but 1 reduced cytoplasmic TDP-43 in the oxidative stress model. Compound 1 locally reduced TDP-43 levels in the cytoplasm of HEK-293 TDP-43 cells treated with sodium arsenite, without altering nuclear levels of TDP-43  of 1 as determined in Boyd et al. in the present study. Furthermore, our HEK-293 oxidative stress model was stressed with the same mild, chronic dose of sodium arsenite  as Boyd in their high-throughput screen. However, when they performed Western blots to determine the effect of 1 on insoluble TDP-43, cells were acutely treated with a high dose of sodium arsenite . It is possible that different doses of sodium arsenite induce different cell morphology and alter TDP-43 recruitment ability into stress granules; therefore, the different oxidative stress intensities between the studies may have contributed to the difference in findings. Other discrepancies that may have contributed include the difference in TDP-43 type  and the cellular background (HEK-293 in the present study vs PC-12 cells in the Boyd paper). This may further highlight the need to perform the same assays across multiple cell lines to build an accurate picture of the effect of drugs on TDP-43 in vitro.The lack of effect of compound us study , which f1 had no effect on the nucleocytoplasmic distribution of TIA-1 in TDP-43 WT HEK-293 cells .2.Human neuroblastoma SH-SY5Y cells stably transfected with TDP-43 M337V were kindly provided by Lars Ittner . Cells were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12) with 10% fetal bovine serum , 1% penicillin-streptomycin and 25 μg/mL blasticidin S hydrochloride. Cells were grown at 37 °C in 5% COE. coli  by adding 50 ng of plasmid to competent E. coli and incubating on ice for 30 min. Cells were heat shocked at 42 °C for 45 s then incubated with Luria–Bertani (LB) media for 1 h. Cultures were then incubated for 16 h at 37 °C on LB agar plates (Life Technologies) containing 100 μg/mL ampicillin. Antibiotic-resistant positive transformants were inoculated into LB broth. Both pcDNA5/FRT/TO and pOG44 plasmids were purified using the Plasmid Midiprep System  according to manufacturer’s protocols. Generation of wild-type TDP-43-expressing HEK-293 cells.HEK-293 cells stably expressing human wild-type TDP-43 (TDP-43 WT HEK-293 cells) were established using the Flp-In™ system . Briefly, a pcDNATM5/FRT/TO plasmid containing human TDP-43 cDNA and a pOG44 plasmid (Life Technologies) were propagated in endonuclease and recombinase-deficient competent ® transfection reagent (Promega) according to the manufacturer’s protocol by co-transfecting pcDNA5 plasmid containing wild-type TDP-43 cDNA sequence and pOG44 plasmid containing Flp-recombinase cDNA at a 1:10 ratio. Hygromycin  was used to select positive clones. Stably-transfected cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 1% penicillin-streptomycin and 15 μg/mL blasticidin S hydrochloride. The overexpression of TDP-43 was induced by the addition of 2 μg/mL of tetracycline for 48 h prior to protein extraction.Flp-In™ T-REx™ HEK293 cells (Life Technologies) were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) with 10% fetal bovine serum, 1% penicillin-streptomycin, 15 μg/mL blasticidin S hydrochloride and 100 μg/mL zeocin selection reagent (Life Technologies). Stable transfections were performed using FuGENE HDTDP-43 WT HEK-293 cells were treated with sodium arsenite (15 μM) for 18 h, with or without test ligands (300 nM) or MG-132 . TDP-43 M337V SH-SY5Y cells were only treated with test ligands (300 nM) for 18 h or with 0.1% DMSO as a control. TDP-43 WT HEK-293 cells were grown until 80–90% confluency in a T75 before use and TDP-43 M337V SH-SY5Y cells were grown until 80–90% confluency in a T175 before use.2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, 0.5 mM MgCl2) with 1X cOmplete EDTA-free protease inhibitors  and 1X phosSTOP phosphatase inhibitors (Roche). After incubating on ice for 10 min, cells were sonicated using a probe sonicator at 60% power for 5 s at 4 °C, repeated 5 times. A 2.5 M sucrose solution was added and centrifuged at 6300× g for 10 min at 4 °C. The supernatant containing the cytoplasmic fraction was stored at −20 °C. The pellet was resuspended in tris/sucrose/EDTA buffer  with 1X cOmplete EDTA-free protease inhibitors and 1X phosSTOP phosphatase inhibitors. The resuspended solution was centrifuged at 1000× g for 5 min at 4 °C. The pellet was washed and centrifuged 4 times. The final pellet, corresponding to the nuclear fraction, was resuspended in 100 μL of 2% SDS in cold RIPA buffer and stored in −20 °C until further analysis.To examine the nucleocytoplasmic distribution of TDP-43, the stress granule marker TIA-1 and ubiquitin, cells were treated with test ligands according to the method described above, washed with warm Dulbecco’s phosphate buffered saline (PBS) and trypsinised with 0.25% trypsin with 0.5 mM EDTA in PBS. Cells were lysed in cold cytoplasmic extraction buffer  using a probe sonicator at 60% power for 5 s at 25 °C, repeated 5 times. The resuspended solution was centrifuged at 100,000× g for 30 min at 25 °C. The supernatant contained the urea-soluble fraction.To examine the effect of drug treatments on the solubility profile of TDP-43 WT HEK-293 cells and TDP-43 M337V SH-SY5Y cells, the RIPA-soluble and urea-soluble fractions were extracted based on established methods . Brieflyp < 0.05 was considered statistically significant.The protein concentration of the nuclear, cytosolic, soluble and insoluble fractions was determined using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. Samples (15 μg) for immunoblotting were made up in 2× Laemelli buffer containing 10% β-mecaptoethanol, heat-denatured at 95 °C for 5 min then immediately placed on ice. Protein was electrophoresed on 4–12% Bis-Tris gels (Invitrogen) then dry-transferred onto nitrocellulose (Invitrogen). Blocking was performed by submerging the blots into 5% skim milk in tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h. Primary antibodies were made up in either TBS-T or 5% bovine serum albumin (BSA) and incubated at 4 °C overnight. Primary antibodies included rabbit anti-TDP-43 , rabbit anti-ubiquitin , mouse anti-TIA , mouse anti-tubulin , rabbit anti-lamin B1 . Blots were washed three times in tri-suffered saline, followed by secondary antibody incubation in TBS-T for 1 h at room temperature. Secondary antibodies include goat anti-rabbit-HRP  and rabbit anti-mouse-HRP . The detection of chemiluminescence was performed using enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific). Densitometry analyses was performed with ImageLab 6.1 . TDP-43, TIA-1 and ubiquitin band intensities were normalised against α-tubulin for RIPA, urea and cytoplasmic fractions and lamin B1 for nuclear fractions. Data represent mean ± SD of n ≥ 3 repetitions. Significant differences between mean protein expression levels in the different treatment conditions were analysed using GraphPad Prism 8.4.3 software  by a one-way ANOVA with Dunnett’s post hoc test. 3 cells per well) were plated for 24 h prior to fixing. HEK-293 cells (3 × 103 cells per well) were plated for 24 h prior to treatment with tetracycline (2 μg/mL) for 48 h and sodium arsenite (15 μM) for 18 h. Following treatment, both cell lines were washed twice with warm PBS, then fixed with 4% paraformaldehyde for 10 min. Cells were washed with PBS three times to remove residue paraformaldehyde, each incubating for 5 min. Blocking was performed with 10% donkey serum in PBS for 1 h. Primary antibodies were diluted in BSA solution  and incubated at 4 °C overnight. The primary antibodies used were rabbit anti-TDP-43  and mouse anti TIA-1 . Cells were washed three times in PBS, each incubating for 10 min. Secondary antibodies were diluted in BSA solution and incubated for 1 h, covered from light. The secondary antibodies were donkey anti-rabbit IgG Alexa Fluor 594  and donkey anti-mouse IgG Alexa Fluor 488 . Cells were washed three times in PBS, each incubating for 10 min and mounted in Fluoroshield™ with DAPI (Sigma). Microscopy was performed on an ECLIPSE Ni-E fluorescence microscope  carrying lasers 395, 470, 640 nm. Images were captured using a 40× lens. Images were combined into figures using ImageJ and Adobe Illustrator .Cells were grown on poly-D-Lysine-coated 8-well chambered coverglass . SH-SY5Y cells (1.5 × 10® cell viability assay (Promega) was performed to measure cell viability after drug treatments. Both TDP-43 WT HEK-293 cells and TDP-43 M337V SH-SY5Y cells were plated at 4 × 104 cells per well of a 96-well plate. After 24 h, the media was replaced with fresh media with or without drug treatment (300 nM) for 18 h. CellTitre-Blue® (20 μL) was then added and incubated for 4 h at 37 °C. The fluorescence was measured at 560/590 nm using a POLARstar Omega plate reader. Significant differences were analysed with GraphPad Prism 8.4.3 software using a one-way ANOVA with Dunnett’s post hoc test. p < 0.05 was considered statistically significant.The CellTitre-Blue1, and a novel bioisosteric analogue, 2, reduce insoluble stress granule levels and ubiquitin expression. These effects do not influence cell viability suggesting 1 and 2 are not toxic to cells. However, they do not reduce stress-induced cell death, suggesting increasing stress granule solubility may not confer cell survival benefits in TDP-43 models.The work in this study characterised two ALS and FTD cellular models commonly used in the literature. Using these models, we demonstrated a previously disclosed compound, 1 in TDP-43 WT HEK-293 oxidative stress model compared to a previous study in PC-12 cells. At present, there is no cellular model that perfectly demonstrates the pathological changes seen in ALS or FTD. Even motor neurons derived from ALS patient iPSCs vary in their ability to show TDP-43 mislocalisation and aggregation, do not show stress granule formation without exposure to chemical stressors and show line-to-line transcriptomic differences [This study highlights the challenges of using cellular models in lead development during drug discovery for ALS and FTD. We identified differing pathomechanisms in the stress and mutant cellular models, and the conflicting effect of ferences ,46. It i