Patients with muscle-invasive bladder cancer face a poor prognosis due to rapid disease progression and chemoresistance. Thus, there is an urgent need for a new therapeutic treatment. The tumor microenvironment (TME) has crucial roles in tumor development, growth, progression, and therapy resistance. TME cells may also survive standard treatment of care and fire up disease recurrence. However, whether specific TME components have tumor-promoting or tumor-inhibitory properties depends on cell type and cancer entity. Thus, a deeper understanding of the interaction mechanisms between the TME and cancer cells is needed to develop new cancer treatment approaches that overcome therapy resistance. Little is known about the function and interaction between mesenchymal stromal cells (MSC) or fibroblasts (FB) as TME components and bladder cancer cells.We investigated the functional impact of conditioned media (CM) from primary cultures of different donors of MSC or FB on urothelial carcinoma cell lines (UCC) representing advanced disease stages, namely, BFTC-905, VMCUB-1, and UMUC-3. Underlying mechanisms were identified by RNA sequencing and protein analyses of cancer cells and of conditioned media by oncoarrays.Both FB- and MSC-CM had tumor-promoting effects on UCC. In some experiments, the impact of MSC-CM was more pronounced. CM augmented the aggressive phenotype of UCC, particularly of those with epithelial phenotype. Proliferation and migratory and invasive capacity were significantly increased; cisplatin sensitivity was reduced. RNA sequencing identified underlying mechanisms and molecules contributing to the observed phenotype changes. NRF2 and NF-κB signaling was affected, contributing to improved cisplatin detoxification. Likewise, interferon type I signaling was downregulated and regulators of epithelial mesenchymal transition (EMT) were increased. Altered protein abundance of CXCR4, hyaluronan receptor CD44, or TGFβ-signaling was induced by CM in cancer cells and may contribute to phenotypical changes. CM contained high levels of CCL2/MCP-1, MMPs, and interleukins which are well known for their impact on other cancer entities.The CM of two different TME components had overlapping tumor-promoting effects and increased chemoresistance. We identified underlying mechanisms and molecules contributing to the aggressiveness of bladder cancer cells. These need to be further investigated for targeting the TME to improve cancer therapy. Fibroblasts (FB), mesenchymal stromal cells (MSC), pericytes, endothelial cells, and immune cells, as functional components of the TME, can affect cancer initiation, angiogenesis, invasion, and metastasis –8. TME cecretion  and in hecretion . For blaecretion , 12. Thunsferred . Concurrnsferred –19. ThusIn bladder cancer, recently defined molecular subtypes that are associated with differences in patients’ prognosis and chemotherapy response differ in the extent of stromal differentiation and abundance of stromal cell types . DiffereSince FB are an essential component of the connective tissue , 23, whiin vitro studies on UC cell lines 5637 and HT-1376 with partially contradictory results. Therefore, the present study aimed to investigate the cellular and molecular effects of multi-donor pooled conditioned media (CM) from MSC or FB on tumorgenicity, epithelial mesenchymal transition (EMT), and migration and invasion capacity of urothelial carcinoma cell lines (UCC). Furthermore, analysis of secreted proteins and RNA sequencing analysis were performed with UCC treated with MSC- and FB-CM to analyze the underlying mechanisms and players that could be targeted in the future.Since CAFs may also originate from tumor-induced differentiation of MSC , we cons22.12.MSC and FB were cultivated in Dulbecco’s Modified Eagle’s Medium  with 2 mM α-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum (FBS) . The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.22) and digested with collagenase solution type I  at 37°C for 45 min with constant shaking. The ratio of tissue to enzyme was 1:2. After filtration (100 µm), the suspension was centrifuged at 300g for 10 min. The fat layer was removed, and the cell suspension was centrifuged again at 300g for 10 min. After resuspension, the cells were seeded in cell culture flasks and cultured in a standard cell culture medium (as mentioned above). Validation of the MSC culture technique by the analysis of characteristic marker expression was reported earlier (MSC were isolated following an established protocol from human abdominoplasty . Briefly earlier .2.32) and digested overnight with 0.2% dispase II solution. The samples were treated with 0.2% collagenase type I (type: CLS 255 U/mg) buffer  for 2 h at 37°C in a shaking water bath to release the cells from the tissue matrix. Following digestion, the suspension was passed through a filter (100 µm), washed with PBS, and centrifuged at 300g for 5 min. The cell pellet was resuspended in a standard culture medium and incubated at 37°C, 5% CO2.FB were isolated as described previously . Briefly2.42 cell culture flasks in 35 mL standard cell culture medium. MSC were plated in a seeding density of 9 × 105 and FB in a cell concentration of 7 × 105. After 72 h, media from six different donors in cell culture passages 3–8 were collected and sterile-filtrated (0.2 µm). The CM was aliquoted and frozen at -80°C until pooling on a parity basis and usage. For further experiments, CM of six different donors were pooled prior to application to UC cell lines.MSC and FB were seeded in T175 cm2.52. For the experiments, UCC were seeded in undiluted CM for 72 h. The exceptions were Western blot, gene expression analysis, and the additional invasion assay for BFTC-905. For these experiments, UCC were preincubated for 6 days in CM since we observed in the course of our study that certain effects took a longer incubation time to establish pronounced effects in UCC.Three urothelial cancer cell lines (UCC), namely, UMUC-3 , BFTC905 , and VMCUB-1 , were selected since they originate from rather progressive tumors and represent the heterogeneity of the disease. The characteristics and genomics of commercially available UC lines were summarized in , 50. Sin2.6Ex/590Em) was measured in a 1420 Multilabel Counter . For the analysis of cisplatin resistance, UCC were seeded in standard cell culture medium or with CM for 24 h. Then, the respective cisplatin concentrations (0.4–6 µM) were added. After 72 h, cell viability assay was performed.UCC were seeded with a standard cell culture medium or with CM. After 72 h, cell viability was determined using CellTiter-Blue . CellTiter-Blue’s working solution was diluted in medium (1:20). CellTiter-Blue uses an indicator dye to measure the metabolic activity of cells, as indirect evidence for cell viability. After 1 h of incubation, fluorescence  were used. The ibidi inserts were placed in 12-well culture plates using sterile tweezers. Cell suspensions were prepared at 2.3 × 102.82), followed by a further coating step with 20 µL Matrigel for 1 h. For VMCUB-1 and UMUC-3, 3 × 104 cells in 100 μL Opti-MEM® Reduced-Serum medium were added to the upper chamber. For BFTC905, 5 × 104 cells were seeded, and 500 μL of conditioned media was added into the lower chamber. Then, 20 h (VMCUB-1 and UMUC-3) to 24 h (BFTC905) later, non-adherent cells were cautiously removed using a cotton swab. BFTC905 were additionally preincubated with CM media (PCM) for 6 days before the experimental setup. The cells in the chambers were washed with PBS (4°C) and fixed for 10 min with ice-cold methanol and then stained with crystal violet for 25 min. After a further washing step and 20 min of drying time, the membrane was removed with a scalpel and embedded in xylol. The membrane was positioned on a microscope slide and coated with DePeX . Three representative images of each membrane were taken with a Zeiss Axiovert 200. The area covered with invaded cells was analyzed using ImageJ, and an average value was calculated. For each cell line, nine differently conditioned media were analyzed, with each of them representing a pool of six donors.In total, 24 Transwell chambers (8 μm in pore size) were pre-coated with 20 µL Matrigel  and incubated for 20 min , and 100 µL of the respective samples was added in duplicate into 96 wells. Analysis was performed according to the manufacturer’s specifications . The optical density was determined using Victor X3 Multilabel-Reader .2.10α-SMA) or 20 μg protein was mixed with 5 µL of Laemmli buffer , centrifuged , denatured for 5 min at 95°C, and separated on 12% sodium dodecyl sulphate-polyacryl-amide gel (SDS-PAGE). The separated proteins were transferred using BioRad Trans-Blot Turbo to a nitrocellulose membrane . Antigens were detected with the following antibodies: E-cadherin , vimentin , SMAD4 , CXCR-4 , and α-SMA . Anti-rabbit or anti-mouse conjugated with horseradish peroxidase (HRP) served  as secondary antibodies, and according to the manufacturer’s guidance, the protein concentration was normalized to that of total protein. Western blots were visualized with ChemiDoc MP Imaging system and analyzed with Image Lab, version 6.0.1, build 34, 2017, standard edition, BioRad Laboratories.To evaluate protein abundance, Western blot analysis was performed. After the UCC were treated for 6 days with a standard cell culture medium or CM, the protein concentration was analyzed with the Pierce BCA Protein Assay Kit . For electrophoresis, 10 μg  containing 3% FBS and centrifuged at 300g for 5 min, the supernatant was removed, and then UCC were blocked in 50 µL FBS on ice for 20 min. The cells were then resuspended and stained for 30 min with fluorophore-conjugated antibodies (10%). After two further washing steps with CellWash + FBS (3%), the samples were analyzed.CD44  protein abundance in UCC was analyzed after UCC had been treated for 6 days with a standard cell culture medium or with CM with a flow cytometer  and BD FACSsuite Software, BD Biosciences, Heidelberg, Germany). An appropriate isotype-matched control antibody was used as control in all analyses. For the flow cytometer analysis, UCC were washed with PBS and then detached with a cell scraper. Afterward, these were washed again with CellWash2.12BTC905, VMCUB-1, and UMUC3 cells were pre-incubated with CM for 6 days and seeded on coverslips for 72 h with or without (w/wo) CM. The cells were washed twice with PBS and fixed with Roti-Histofix (4%). After a further washing step, anti-E-cadherin  and anti-vimentin  antibodies were applied in Triton X-100 with normal goat serum overnight at 4°C. The cells were rewashed and stained with secondary antibodies conjugated with Alexa-594 or Alexa-488 for 30 min at room temperature (RT). Finally, nuclear staining was performed with 0.1% 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at RT. The cells were washed again, covered with Fluoromount, and examined microscopically with Zeiss Axiovert 200.2.13TBP) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were used. qRT-PCR was performed using initial denaturation at 95°C for 2 min and 45 cycles of amplification, including denaturation at 95°C for 10 s, annealing and elongation for 30 s at 60°C, and a melting curve analysis.RNA was extracted from UCC treated w/wo CM for 6 days using the RNeasy Mini Kit according to the manufacturer’s specifications . One microgram of RNA was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen), with an extended incubation time of 30 min at 42°C. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with PowerUp™ SYBR® Green Master Mix  according to the manufacturer’s instructions on the StepOne Real-Time PCR System . RNA expression was measured using the primers given in 2.14p-values for multiple testing. A p-value of ≤0.05 was considered as significant. The cutoff for differential gene expression was set to 1.5-fold. Further analysis and data visualization were performed using Microsoft Excel and Graph Pad Prism 8. Venn diagrams were prepared with the online tool Venny 2.0  were used for RNA quantification. The RNA qualities were confirmed by capillary electrophoresis using the fragment analyzer with total RNA Standard Sensitivity Assays . Library preparation and next-generation sequencing were performed as described . Multigrenny 2.0 . GO grouenny 2.0 .2.15MSC-conditioned media from two different donors and FB-conditioned media from two different donors were applied to the Proteome Profiler Human XL Oncology Array membranes according to the manufacturer’s instructions . Dot blots were visualized with the ChemiDoc MP Imaging system and analyzed with Image Lab, version 6.0.1, build 34, 2017, standard edition, from BioRad Laboratories. The value of the negative control was subtracted from all other values. Values were further normalized to the values of the reference spots.2.16t-test or two-way ANOVA was used, and a p-value less than 0.05 was considered significant. The values shown are mean ± standard deviation (SD).The statistical analysis was carried out with GraphPad Prism 8.0 . Two-tailed Student’s 33.1Initially, we determined whether CM from stromal cells (MSC or FB) had inhibitory or augmenting effects on the cellular growth of three different UC cell lines used as models for MIBC. Since we later aimed also to determine the effects on motility and EMT, we selected two cell lines with an epithelial phenotype, namely, BFTC-905 and VM-CUB1, compared to UMUC-3 cells with a mesenchymal phenotype. MSC-CM strongly augmented the cell growth of all three cell lines  were in the same range over different treatment conditions, with the exception of BFTC-905 which responded more strongly by altered gene expression to FB-CM than VM-CUB1 cells Table 1.In the first step of data analysis, we aimed to identify the cell line-independent effects of MSC-CM. To this end, we compared the MSC DEGs between BFTC-905 and VMCUB-1 cell lines , 59. FurSnail 1 (SNAI1) and SQSTM1 encoding for p62 protein involved in the reduction of cisplatin which reduced cell stress via NRF-2 signaling-mediated detoxification and activation of NF-κB signaling  repair genes like SNAI1, TWIST1, and CLDN4 also demonstrated stronger expression changes in BFTC-905 cells , glutathione peroxidase (GPX) 1, GPX2, and saliva level of solute carrier family 3 member 2 (SLC3A2), which are all involved in detoxification.Likewise, FB DEGs that were unique for the investigated cell lines were also subjected to GO analysis 10. Whil3.5Taken together, treatment with both MSC- or FB-CM often induced similar changes in cellular processes in UCC. Concurringly, 954 and 810 DEGs in VMCUB-1 cells overlapped between FB- and MSC-regulated genes , and response to reactive oxygen species , an enzymatic agent that can rapidly reduce the HA level  regulating drug uptake/efflux and antioxidant response via glutathione metabolism  signaling may be involved in the mediation of chemoresistance. Intact IFN type I signaling impacts on the efficacy of cytotoxic drug therapy, radiotherapy, and targeted immunotherapies and depends on both direct tumor cell inhibition and indirect anti-tumor immune response. Thus, malfunctions of IFN signaling, as observed in hypoxic TME or in immune cells, may be a causative factor behind the therapeutic resistance in cancer patients , 123. Invia the cyclic GMP-AMP synthase (cGAS)-STING pathway. cGAS senses cytosolic double-stranded DNA (dsDNA), resulting in the synthesis of cyclic GMP-AMP, a secondary messenger binding to the adaptor protein STING. It is known that TME can impact the DNA repair pathways in cancer cells . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.Conception and design: VG and MH. Administrative support: JW and BB. Provision of study materials or patients: VG and MH. Collection and assembly of data: LF, BF, KK, and PP. Data analysis and interpretation: LF, BF, KK, PP, VG, and MH. Manuscript writing: VG and MH. All authors contributed to the article and approved the submitted version.


Late-stage clicking of thioacetals and acetalsallows simultaneous attachment of targeting units and interfacersand exploration of the critical chalcogen-bonding donor at the sametime. Initial studies focus on plasma membrane targeting and developthe chemical space of acetals and thioacetals, from acyclic aminoacids to cyclic 1,3-heterocycles covering dioxanes as well as dithiolanes,dithianes, and dithiepanes, derived also from classics in biologylike cysteine, lipoic acid, asparagusic acid, DTT, and epidithiodiketopiperazines.From the functional point of view, the sensitivity of membrane tensionimaging in living cells could be doubled, with lifetime differencesin FLIM images increasing from 0.55 to 1.11 ns. From a theoreticalpoint of view, the complexity of mechanically coupled chalcogen bondingis explored, revealing, among others, intriguing bifurcated chalcogenbonds.Progress with fluorescentflippers, small-molecule probes to imagemembrane tension in living systems, has been limited by the effortneeded to synthesize the twisted push–pull mechanophore. Here,we move to a higher oxidation level to introduce a new design paradigmthat allows the screening of flipper probes rapidly, at best  In this study, thio/acetalization is designedto turn on, rather than turn off, the fluorescence of flipper probeseasily, at best in situ, to simultaneously installvariable targeting groups and screen for new chalcogen-bonding donors.Thio/acetals are popularfunctional groups in fluorescent probes.Thioacetals have been used for mercury sensing,33 For the imaging of biomembrane function,47 membrane tension50 is particularly interesting but also particularlydemanding because physical forces are detectable only through thesuprastructural changes they cause.33 Inspiredby nature,51 we have considered the conceptof planarizable push–pull probes to tackle this challenge.52 The resulting flipper probes are built aroundtwo dithienothiophenes,54 one electron rich andone electron poor. In the relaxed ground state I, theyare twisted out of coplanarity by chalcogen-bonding repulsion  to liquid-ordered (Lo) and solid-ordered (So) membranes  incells and has been made available for the community as Flipper-TR.Progress with flipper probes has been limited bythe syntheticeffort required for their preparation. Particularly challenging isthe donor terminus. Located at the membrane surface, variable targetingunits R have to be attached to this terminus 1.33,55−5s reason 1d. Sulfiiminated 1g. This 5, increased stability, but the addedaromatic system perturbed spectroscopic properties.57 Both thenyl ether and triazole have been proposed to act as noncovalentturn-on donors, forming a chalcogen bond63 as soon as probe planarization deepens the σ hole on the endocyclicsulfur ,69 and so on promise access to rich chemical space. This flipper diversitywill allow for the screening for improvements of the original chalcogen-bondingdonors V together with an additional, less powerful noncovalentCH···X bonding donor for a refined push–pullsystem VI (IV wereexpected to prevent the thenyl ether elimination VII byintramolecular tethering of the leaving group to enable reverse ringclosure (VIII–X). Overall, donor junctions couldthus be expected to secure facile synthetic access to a broad varietyof turn-on donors with attached targeting units of free choice, allin one single final step, at best possible in situ.Thio/acetal donor junctions could be installedby late-stage modification of common aldehyde precursor hols III 1e. Satur III  surface on the sulfurincreased from Epot = 21.5 to Epot = 23.5 kcal mol–1, whilethe C–H donor increased only by Epot = +0.2 kcal mol–1  was also facilitated by thedoubled push–pull dipole of 7b-II .Computational evaluation of potentialchalcogen-bonding donors was performed at the PBE0-D3/def2-TZVP levelof theory. The virtual flipper al mol–1 2a. The chan 7a-I 2b. The u7 into minimalistthenylether 9 and acetal 10 increased the push–pulldipole from μ = 6.5 D over μ = 9.9 to μ = 10.9 D,which in turn stabilized planar high-energy conformer II from Erel = +1.82 kcal mol–1 over Erel = +1.75 kcal mol–1 to Erel = +1.69 kcal mol–1 relative to the twisted conformer I  radii (d = 3.32 Å). Moreover, NBO analysis indicated that electron transferfrom the oxygen lone pair (LP) to the antibonding σ* orbitalof the sulfur starts to contribute significantly to chalcogen bondingonly with the 1,3-dioxane donor 10.Conversion of aldehyde II increased from acyclic to cyclic thioacetals. With cyclicthioacetals, dipoles increased with the ring size from μ = 9.4D for 1,3-dithiolane 12 over μ = 10.5 D for dithiane 13 to record μ = 11.1 D for dithiepane 14  andmaximal NBO electron transfer  and polarization (μ = 10.5D) of planar high-energy conformer 13 compared to ring-contracted 12 implied that unorthodox bifurcated chalcogen bonds canbe at least as powerful as optimized conventional chalcogen singlebonds. The significance of these bifurcated chalcogen bonds couldonly be identified and appreciated in the context of the coupled processesin planarizable push–pull probes. They will be of interestin future design strategies in general.Almost equal stabilization . Flipper 15 was synthesized in 12 steps fromcommercially available starting materials following reported procedures.33 Weakening of the push–pull system in 15 by the aldehyde acceptor in 7 was correctlyreflected by the emission changing from orange to green. As initialtargets for donor junctions, 1,3-dioxolane 16 and thioacetals 17–20 were selected to probe for accessibilityof the central motifs from popular starting materials like reducedasparagusic acid66 or dithiothreitol68 . The in situ synthesis of 1,3-dithiolane 18 showed similar kinetics, while the formation of dithiane 19 was faster (t50 ∼ 2min) and dithiepane 20 formed instantaneously (t50 < 2 min). Thioacetal formation was confirmedby LC-MS  and NMRanalyses, performed after purification of some compounds. Added toHeLa Kyoto (HK) cells with or without short work-up, in situ produced thioacetals were not toxic and afforded FLIM images thatwere indistinguishable from images obtained from isolated and purifiedflippers . The complementary acetal flipper 16 showed identicalstability at pH = 7 but hydrolyzed at pH = 5 with t50 = 4 h . Usually,the replacement of oxygen by sulfur in functional groups decreasesstability and enables dynamic covalent chemistry because the largersize gives weaker bonds, stronger acids, and better leaving groups.The exception with thioacetals, long known, originates from the differentmechanism at work, with C=S+ intermediates thatare harder to access than C=O+ intermediates, forthe same reason.Thioacetal flipper d LUVs (large unilamellarvesicles) of all new flippers were roughly the same as for the originalFlipper-TR 4, characterized by a broad excitation maximumat λex ∼ 440 nm . In Lo LUVs, the excitation maxima red-shiftedaround +80 nm to a 0–0 transition at λex ∼520 nm, with distinct differences between the different flippers,particularly with regard to vibrational fine structure . These results indicatedthat, remarkably, all new donor junctions produced operational, mechanosensitiveflippers.The spectroscopic properties in LS23–S29, av or τ1 for the longer componentwere extracted from FLIM images by biexponential fitting of decaycurves. Reported from GUVs are τ1 in Lo membranes, termed τLo ,and τLom, τ1 in Lo domains of mixed membranes containing Lo and Ld domains of GUVs (giant unilamellar vesicles) and HK cells S23–S29, 2. Lifetinditions e,f, top nditions 2.16–20, selectedto implement computational guidelines with readily accessible reagents,fluorescence lifetimes were generally higher for S–O chalcogenbonds, that is, ether 4 and acetal 16, thanfor thioacetals 17–20 (17 (20 with a DTT dithiepane junction that misses the charge forplasma membrane targeting (18 (19 (S29), demonstrating that there is much room for improvement for membranetargeting, which should improve fluorescent lifetimes at the sametime.Within the series argeting 4d, top. ting .In agreement with this emerging understanding, I1/2 values did indeed increase significantly with increasingresponsiveness from dithianes 19 to 23 and 6 .Determinationof tension sensitivity Δτferences4g,h. Com0.11 ns 4h.The s69 were considered as clickable donor junctions. This natural-product-derivedmotif yields disulfides at maximal tension that are of interest topenetrate cells.69 The parent ETP was reduced prior to in situ thioacetalizationwith aldehyde 7 (Scheme S9). The resultingthioacetal junction 24 features a seven-membered ringlike the dysfunctional dithiepane 20 but performed muchbetter, more like dithiolane 17, including high ΔτTR = −0.6 at similarly impressive τLo, τLom, τiso, and ΔτGUV  and a solution of the respective dithiol/thiolin DMF  to a solution of 7  in CH2Cl2 at 25°C (Scheme S8). Absorption spectra(l = 0.1 cm) of the reaction mixture were recordedevery 1–5 min, and the corresponding mixture without 7 was used as the background (S9). Basedon the time-dependent absorption spectra, the kinetics of conversionfrom 7 to 17, 18, 19, and 20 were determined . The complete consumption of 7 and the generationof the desired products were confirmed by LC-MS of the reaction mixtures. For direct usein FLIM imaging of GUVs and living cells, the obtained product mixtureswere extracted with brine and CH2Cl2, and theorganic phase was dried over Na2SO4 and filteredthrough a cotton plug in a pipet. The filtrate was concentrated, andthe residue was dissolved in DMSO .Flippers ckground 3d and S924, solutions of the parent ETP69  and tris(hydroxypropyl)phosphine in DMF were combined and stirred for 10 min(Scheme S9). The resulting solution ofreduced ETP and HCl  was added to asolution of 7  in CH2Cl2. LC-MS confirmed the formation of 24in situ  and showedthe presence of residual 7 . For direct use in the FLIM imaging of GUVs and livingcells, the reaction mixture was stirred for 48 h. Then, the productmixture was extracted with brine and CH2Cl2,dried over Na2SO4, filtered through a cottonplug in a pipet, concentrated in vacuo, and dissolvedin DMSO (0.5 mL).For flipper 17, 19, 20, 0.3mM 18, 0.5 mM 24) were added to 190 μLof Tris buffer . Theobtained suspensions were placed on a 35 mm glass bottom dish  and left for 15 min at room temperaturebefore imaging . Leica ApplicationSuite Software LASX FLIM 4.5.0 Stellaris or SymPhoTime 64 softwarefrom PicoQuant was used to analyze the FLIM images. The lifetimesτ1 were calculated from a biexponential fit of thesignal coming from GUVs (selected as the ROI by “painting”).For FLIM imaging of GUVs, 10 μL ofstock solutionsof GUVs and 0.2 to 0.4 μL of stock solutions of flippers inDMSO (0.6 mM 4 cells mL–1) were seeded in FluoroBriteDMEM  medium containing10% fetal bovine serum (FBS), 1% penicillin/streptomycin (PS), and1% glutamine and kept at 37 °C at 5% CO2 overnight.Then, the cells were washed (3 × 1 mL) with PBS buffer and incubatedwith DMEM medium containing the corresponding probe  for 10 minat 37 °C at 5% CO2. The images were acquired withoutexchanging the incubation medium or additional washing. The hypertonicshock was achieved by adding 1 mL of 1 M sucrose medium containingthe corresponding probe  in the dish containing 1 mL of isotonic medium for30 min (S29). FLIM data were analyzed by usingSymPhoTime 64 software (PicoQuant) that fit fluorescent decay data to a biexponentialdeconvolution model from the plasma membrane only.For FLIM measurements in HeLa Kyoto cells, the cells (8 ×10


The high reaction capability and stability of the polysulfide cathode result from the high polysulfide retention and smooth lithium-ion diffusion of the polyacrylonitrile film, which endows the lithium–sulfur cells with high areal capacities (7.0–8.6 mA·h cm−2) and energy densities (14.7–18.1 mW·h cm−2).The high theoretical charge-storage capacity and energy density of lithium–sulfur batteries make them a promising next-generation energy-storage system. However, liquid polysulfides are highly soluble in the electrolytes used in lithium–sulfur batteries, which results in irreversible loss of their active materials and rapid capacity degradation. In this study, we adopt the widely applied electrospinning method to fabricate an electrospun polyacrylonitrile film containing non-nanoporous fibers bearing continuous electrolyte tunnels and demonstrate that this serves as an effective separator in lithium–sulfur batteries. This polyacrylonitrile film exhibits high mechanical strength and supports a stable lithium stripping and plating reaction that persists for 1000 h, thereby protecting a lithium-metal electrode. The polyacrylonitrile film also enables a polysulfide cathode to attain high sulfur loadings (4–16 mg cm Li/Li+). Accordingly, the rechargeable lithium–sulfur battery offers 10 times the capacity and 3–5 times the energy density of current rechargeable lithium-ion batteries [Lithium-ion battery technology is mature and approaching the theoretical limits of the charge-storage capacities and energy densities of the active materials. Thus, in addition to the studies focused on optimizing the active material, device configuration, and fabrication method of lithium-ion batteries, there are increasing efforts to develop novel energy-storage systems with next-generation electrode materials featuring either high operating voltages or high charge-storage capacities. Next-generation electrode materials are needed to meet the requirements of the current electrical devices for batteries that have high energy densities, low toxicity, and low cost ,2,3. Amoatteries ,5,6. Thiatteries ,8,9. Theatteries .−30 and 1 × 10−14 S cm−1, respectively) [However, despite the above-mentioned electrochemical and device advantages of lithium–sulfur cells, the low conductivity and poor stability of sulfur cathodes need to be ameliorated. The low conductivity of their active material in its solid state and their high solubility in its liquid state result in a sluggish reaction and a high loss of liquid active material. Specifically, the electrochemical utilization and reversibility of a lithium–sulfur cell are limited by the insulating nature of sulfur in the charged state and lithium sulfide in the discharged state, which lead to cells having high resistance in these states 1 × 10ctively) ,12,13. Tctively) ,16, metactively) ,18,19, cctively) ,21,22, octively) ,24,25 asctively) ,24,25. Tctively) ,27. To sctively ,ctively) ,12,13. Actively) ,30,31,32−2). Most importantly, we applied the polyacrylonitrile film directly to a high-loading polysulfide cathode with a high sulfur content and loading  to demonstrate the designed configuration enabling the high electrochemical utilization and stability of a large amount of polysulfides, which overcomes chemical and engineering problems [−2) and energy densities (8.6–18.1 mW·h cm−2), which meant that the cell performance of a lithium–sulfur battery containing the polyacrylonitrile film is comparable to or higher than those of commercially available lithium-ion batteries, such as the areal capacity for electric vehicle batteries (2–4 mA·h cm−2) and the energy densities of lithium-ion batteries (10–15 mW·h cm−2) [In this work, we adopted electrospinning—which is widely used to prepare polymeric fiber films and has been used to make lithium-ion battery separators consisting of polyacrylonitrile ,34, polyproblems ,40. The ·h cm−2) ,38,39,40−1, Sigma-Aldrich, Darmstadt, Germany) in N,N-dimethylformamide  to form an 11 wt% solution. The electrospinning was conducted at an applied voltage of 18 kV and a flow rate of 2 mL h−1 as the optimal fabrication condition for the preparation of a smooth polyacrylonitrile film with a low number of beaded fibers. The electrospun polyacrylonitrile film was dried at 50 °C for one day and cut into a disk of 19 mm in diameter and 200 µm in thickness, which was used as a lithium–sulfur battery separator.The electrospinning solution was prepared by dissolving polyacrylonitrile  spectrometry  to determine its electrolyte affinity. The 7Li NMR data were used to calculate and fit the diffusion coefficient (D), according to: I/0I = exp(-Dg2δ2σ2g2Δ′), where I and 0I are the intensity and initial intensity of the selected resonance, respectively; Δ′ is the diffusion delay; δ is the gradient pulse length; g is the gradient pulse strength; and σ is the gradient shape factor [W1 − W0)/W0 × 100% and (xW − W0)/(W1 − W0) × 100%, respectively, where W1 is the weight of the wetting separators, W0 is the weight of the origin separators, and Wx is the weight of the wetting separators after drying for an hour [The microstructure and morphology of the electrospun polyacrylonitrile nanofiber film before and after cycling were observed by field-emission scanning electron microscopy . The polyacrylonitrile film was subjected to Fourier-transform infrared spectrometry  to determine its chemical structure, gas sorption analysis  to determine its gas adsorption–desorption behavior, microtensile testing  to determine its mechanical strength, and e factor . The ele an hour ,43.−2 to analyze how the polyacrylonitrile film supported the lithium stripping/plating reaction and protected the lithium electrodes. The lithium–sulfur cells contained a lithium counter/reference electrode and a polysulfide cathode to enable the direct analysis of polysulfide inhibition and retention. The catholyte comprised 1.5 M polysulfide that was fabricated by dissolving sulfur  and lithium sulfide  in a blank electrolyte. The blank electrolyte was composed of 1.85 M lithium bis(trifluoromethanesulfonyl)imide and 0.5 M lithium nitrate  dissolved in 1,2-dimethoxyethane  and 1,3-dioxolane . This catholyte was dropped onto the current collector to form cells with high sulfur loadings and high sulfur contents  in the consideration of the total mass of the cathode. A reference cell with a conventional polypropylene separator (19 mm in diameter and 25 µm in thickness) was fabricated at the same high sulfur loadings , and only the cell with a sulfur loading of 4 mg cm−2 afforded sufficient experimental data.The polyacrylonitrile film was used as a separator in a lithium//lithium symmetrical cell and a lithium–sulfur electrochemical cell, respectively, and the performance of these cells was analyzed. The performance of the symmetrical cells was examined at a current density of 1 mA cm−1. Cyclic voltammetry (CV) analyses were performed using a potentiostat  at the aforementioned voltage range and at various scan rates (0.010 to 0.030 mV s−1). Electrochemical impedance spectra were recorded using the aforementioned potentiostat from 1 MHz to 10 mHz. The ionic conductivity of polyacrylonitrile films was analyzed from 1 MHz to 100 Hz at various temperatures (from room temperature to 70 °C in increments of 10 °C), and was calculated by σ = d/A × bR, where σ, d, A, and bR are the ionic conductivity, thickness, area, and bulk resistance of a polyacrylonitrile film, respectively [The assembled lithium–sulfur cells were analyzed at room temperature (25 °C). Their cycling performances and discharge–charge curves were determined using a programmable battery cycler  at 1.8–2.6 V and at C/20–1C rates with 1C = 1672 mA gectively .3 g−1 and 16–23 m2 g−1, respectively. The closely packed fibric network built up by non-nanoporous electrospun fibers aims to suppress the free diffusion of polysulfides and to maintain the fast penetration of liquid electrolyte. To support the latter feature, −1 represent nitrile (C≡N), carbonyl, and alkyl bonds, respectively, confirming the presence of polyacrylonitrile. 7Li NMR spectra of the electrolyte, conventional polypropylene separator, and polyacrylonitrile film separator, demonstrating the effect of the highly electronegative C≡N on the chemical shift of the 7Li peak (as discussed below). The diffusion-ordered spectroscopy (DOSY) calculation shown in 7Li NMR spectrum of the electrolyte-wetted polyacrylonitrile film was upfield from its position in the 7Li NMR spectra of the electrolyte and electrolyte-wetted conventional separator. This demonstrated that the polyacrylonitrile film interacted with lithium ions and accounted for its high lithium-ion diffusion coefficient (1.72 × 10−6 cm2 s−1), which is similar to that of the liquid electrolyte (1.80 × 10−6 cm2 s−1) but higher than that of electrolyte-wetted conventional polypropylene separator (1.58 × 10−6 cm2 s−1). This indicates that the thick polyacrylonitrile film would not block the penetration of electrolyte and slow down the transfer of lithium ions. As a result, the highly safe polyacrylonitrile film has excellent lithium-ion transfer capability for the high-performance conversion lithium–sulfur battery chemistry [hemistry ,12.Overall, the abovementioned material analyses show that the polyacrylonitrile film features complex non-interwoven fibric networks with a layered structure consisting of non-nanoporous fibers. This indicates that the polyacrylonitrile film is well penetrated by electrolytes and thus can enable the high diffusion of lithium ions, while also exhibiting good mechanical strength, thermostability, and low flammability.−1) [−2) equipped with a polyacrylonitrile film separator and a conventional separator at C/10 rate as a reference, respectively. The resulting lithium–sulfur cell using the polyacrylonitrile film revealed high peak discharge capacities of 929 mA·h g−1 when cycled at C/10. At the same cycling rate, the reference cell with the commercial polypropylene separator showed a decrease in charge-storage capacity by 160 mA·h g−1 and had only 769 mA·h g−1. The polyacrylonitrile film and the commercial polypropylene separator showed capacity retention of approaching 60% and only 25% after 200 cycles, respectively. These demonstrate the significant improvement brought about by the use of the polyacrylonitrile film in the electrochemical utilization and stability of lithium–sulfur cells. In addition to the improved charge-storage and reversible capacities of cells containing a polyacrylonitrile film, those that also contained a high-loading polysulfide cathode with 4 mg cm−2 sulfur exhibited high discharge capacities of 1017, 737, 587, 422, and 411 mA·h g−1 when cycled at C/20, C/5, C/3, C/2, and 1C, respectively. After 200 cycles, the cells equipped with a polyacrylonitrile film retained excellent discharge–charge efficiencies (approaching 99%) and high capacity retentions (50–80%). The long cycle life of these polyacrylonitrile film-equipped cells at various current densities indicates the excellent rate performance, fast reaction kinetics, and stable electrochemical reactions of these cells [−1)  necessarse cells .−1. As the scan rate increased, the same redox reactions were maintained in the cell, which also exhibited almost unchanged discharge–charge redox peaks. The chemical reaction of the polysulfide cathode during the discharge process showed two cathodic peaks, which show the reduction conversion of sulfur to polysulfides (black box: S → Li2S4–8) and of polysulfides to sulfides (red box: Li2S4–8 → Li2S2/Li2S). The chemical reaction of the cathode during the charge process displayed two anodic peaks, which represent the charge conversion of sulfides to polysulfides and sulfur (blue box: Li2S2/Li2S → Li2S4–8/S) [−8 to 9.28 × 10−9 cm2 s−1 [2S4–8/S) ,7,8. At  cm2 s−1 ,45. Over−2. The cell equipped with a polyacrylonitrile film exhibited stable and smooth lithium plating and stripping behavior for 1000 h, which was significantly longer  than that of the cell equipped with a commercial separator (368 h). Moreover, after 1000 h of testing, the symmetrical lithium cell equipped with the polyacrylonitrile film remained stable with no apparent short circuit. The lithium-metal electrodes were subsequently retrieved from these cycled symmetrical lithium cells and examined. −2) and high sulfur contents . The polyacrylonitrile film enabled the high-loading cathodes with sulfur loadings of 4, 8, 12, and 16 mg cm−2 to attain high discharge capacities of 1017, 867, 718, and 533 mA·h g−1 at C/20, respectively. −2) and high energy densities (8.6–18.1 mW·h cm−2). eR, ctR, iR, and wZ represent the cathodes’ bulk resistance, charge-transfer resistance, interface resistance, and lithium-ion diffusion Warburg impedance, respectively [−2 sulfur showed ctR values of 170.3, 187.3, 208.1, and 321.3 Ω, respectively. However, compared with the corresponding uncycled high-loading cathode, the reference cathode with 4 (50 wt%) mg cm−2 sulfur showed a much higher ctR value (238.2 Ω). After cycling, the relocation of active material in the cathode region of the cells protected with a polyacrylonitrile film resulted in a decrease in their ctR values to 5.8, 6.4, 15.5, and 40.8 Ω, respectively. Moreover, the smooth interface between the electrode and electrolyte in these cells led to iR values of only 1.1, 3.7, 5.0, and 28.6 Ω, respectively; in contrast, the reference cell showed high resistances (14.4 Ω (ctR) and 14.6 Ω (iR)). The consistently low impedances of the cells equipped with the polyacrylonitrile film demonstrate that the film enhanced the electrochemical reaction and, crucially, maintained this good redox-reaction capability as the sulfur loading increased and after cell operation. −2) polysulfide cathode containing 80 wt% sulfur, which maintained good electrochemical utilization for achieving the high areal specific capacity (8.6 mA·h cm−2) and energy density (18.1 mW·h cm−2). These cell-design parameters and the electrochemical performance exceed the requirements for the electric vehicle power source  and commercial cathode  [Furthermore, the polyacrylonitrile film enabled excellent active-material utilization and efficiency at the polysulfide cathode and high electrochemical stability at the lithium-metal electrode in cells. ectively ,47,48. T∙h cm−2) ,38,39,40−2 and 50, 68, 75, and 80 wt%). Moreover, the polyacrylonitrile film stabilized a high-loading polysulfide cathode, such that it achieved an impressively high areal capacity and high energy density .In this study, we electrospun a polyacrylonitrile nanofiber film and used it as a separator in a lithium–sulfur cell. The polyacrylonitrile film had high mechanical strength and high electrolyte affinity but low nanoporosity. It endowed a high-loading polysulfide cathode with excellent rate performance from C/20 to 1C and a long cycle life (200 cycles). The presence of the polyacrylonitrile film led to a lithium-metal electrode exhibiting stable lithium stripping and plating reactivity for 1000 h. The stabilized polysulfide cathode and lithium electrode generated by the use of the polyacrylonitrile film enabled a lithium–sulfur cell to have high sulfur loadings and contents (4, 8, 12, and 16 mg cm


NAD+ is an essential molecule involved in numerous cellular pathways, including genome integrity and DNA repair, and thus, NAD+ supplementation might be beneficial for mitigating mustard-induced (geno)toxicity. In this study, the role of NAD+ depletion and elevation in the genotoxic stress response to SM derivatives, i.e., the monofunctional agent 2-chloroethyl-ethyl sulfide (CEES) and the crosslinking agent mechlorethamine (HN2), was investigated with the use of NAD+ booster nicotinamide riboside (NR) and NAD+ synthesis inhibitor FK866. The effects were analyzed in immortalized human keratinocytes (HaCaT) or monocyte-like cell line THP-1. In HaCaT cells, NR supplementation, increased NAD+ levels, and elevated PAR response, however, did not affect ATP levels or DNA damage repair, nor did it attenuate long- and short-term cytotoxicities. On the other hand, the depletion of cellular NAD+ via FK866 sensitized HaCaT cells to genotoxic stress, particularly CEES exposure, whereas NR supplementation, by increasing cellular NAD+ levels, rescued the sensitizing FK866 effect. Intriguingly, in THP-1 cells, the NR-induced elevation of cellular NAD+ levels did attenuate toxicity of the mustard compounds, especially upon CEES exposure. Together, our results reveal that NAD+ is an important molecule in the pathomechanism of SM derivatives, exhibiting compound-specificity. Moreover, the cell line-dependent protective effects of NR are indicative of system-specificity of the application of this NAD+ booster.Sulfur mustard (SM) and its derivatives are potent genotoxic agents, which have been shown to trigger the activation of poly (ADP-ribose) polymerases (PARPs) and the depletion of their substrate, nicotinamide adenine dinucleotide (NAD Sulfur mustard , also known as mustard gas, is a chemical warfare agent, initially used during WWI. Despite being banned by the Chemical Weapons Convention (CWC), it is still occasionally released in asymmetric conflicts or terrorist attacks. Furthermore, old SM depots pose a threat due to potential release and accidental exposure . SM has +) as a substrate . I. I+ is p pathway . To date pathway . Since N studies ,25. Moreor FK866  prior toor FK866 . Our groor FK866 . In the TM, Thermo Fisher Scientific, Waltham, MA, USA). Moreover, human monocytic leukemia cell line THP-1 [TM, Thermo Fisher Scientific). Cells were tested for mycoplasma contamination, and experiments were performed on cells between passages 3 and 15 after thawing. Growth media were supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin . Cells were cultured at standard conditions: 37 °C, 95% humidity, and 5% CO2. For detachment, HaCaT cells were incubated with 0.25% trypsin-EDTA  for 5–10 min at 37 °C.As the skin is one of the primary targets of SM exposure, we used the human keratinocyte cell line HaCaT as an experimental model system of human keratinocytes  , nicotinic acid , nicotinamide riboside , and nicotinamide mononucleotide  were prepared in MiliQ-H2O stock solutions (10 mM) and stored at −80 °C. Compounds were added to the culture medium  and incubated for varying durations (1–24 h) under standard conditions.The NAD+ levels as a result of NAD+ turnover. Stock solutions of 10 mM FK866 were prepared in DMSO and stored at −80 °C. The compound was added to the cells in fresh culture medium both 24 h before and immediately after mustard treatment. Control samples were incubated with 0.5% (v/v) DMSO.The pharmacological compound daporinad  was used to inhibit the salvage pathway enzyme, NAMPT, and thus decrease cellular NADv/v) directly before cell treatment as described previously [2O2 (100 µM) was used as a positive control of genotoxicity and PARP1 activation. After toxic treatment, cells were washed twice with PBS and supplemented with fresh culture medium or harvested immediately for analysis.Premixes (100×) of CEES (Sigma-Aldrich) or HN2 (Sigma-Aldrich) solutions were freshly prepared in a solvent containing 95% EtOH and 0.5% HCl  per well in 6-well plates 24 h prior to the experiment. THP-1 cells (2 × 106 cells/mL) were incubated in 12-well plates during the treatments. After the treatments, cells were harvested (including detached cells in culture media), resuspended in 0.5 mL cold PBS, and placed on ice. Subsequently, 24 µL of perchloric acid (3.5 M) was added, samples were incubated for 15 min on ice, and centrifuged . The supernatant was mixed with 350 µL of 0.33 M K2HPO4 (pH 7.5), incubated for 15 min on ice, snap-frozen in liquid nitrogen, and stored at −80 °C until NAD+ analysis. The pellet was stored at −20 °C until analysis of the total protein content. For the NAD+ assay, after thawing and centrifugation , 40 µL of each sample  was used for the enzymatic reaction in a 96-well plate format. To each sample, 160 µL of buffer  and 100 µL of the reaction mixture  were added. The reaction mixture was also added to wells containing 200 µL of NAD+ standard (Sigma-Aldrich) dissolved in the buffer  in a concentration range of 0.05–0.5 µM. After a 30 min incubation at 30 °C, absorption was measured at 550 nm with 690 nm as a reference wavelength in a microplate reader . The results enabled the calculation of NAD+ concentrations in the samples, which were subsequently normalized to the total protein level. For the protein assay, sample pellets were dissolved in 500 μL 0.1 M NaOH, and the protein level was determined via a PierceTM BCA Protein Assay Kit  according to the manufacturer’s protocol.Cellular NADng assay . HaCaT c6 cells/well in 12-well plates 24 h before treatment. Cell samples were collected immediately after the treatment (0 h) or after 24 h incubation with fresh culture media. Harvested cells were centrifuged , the pellet was resuspended in fresh culture medium, and ATP levels were measured with the Cellular ATP Kit HTS  according to manufacturer’s protocol in a 96-well plate format. The luminescence signal was measured with a microplate reader . The values were normalized to protein levels measured via a PierceTM BCA Protein Assay Kit  according to the manufacturer’s protocol.To measure cellular ATP levels, HaCaT cells were seeded 0.2 × 106 cells/well in 12-well plates 24 h before the treatment. After the exposure to genotoxicants, cells were supplemented with fresh culture medium and incubated for 24 h. Thereafter, cells and culture medium containing detached cells were collected and centrifuged . After washing with cold PBS and centrifugation, the cell pellet was resuspended in an Annexin binding buffer (ABB) . A drop of Annexin V-APC solution  and 5 µL of propidium iodide (PI) solution (Sigma-Aldrich) were added to 100 µL of each sample and incubated in the dark for 15 min. Subsequently, 1 mL of ABB was added, samples were assayed by flow cytometry , and results were analyzed by FlowJoTM v.10.9.0 software (BD Biosciences).To analyze cell death, HaCaT cells were seeded at 0.15 × 103 cells/well) 24 h before the treatment with FK866 (0.5–5 nM) for 24 or 48 h. Additionally, repeated exposure was applied (24 + 24 h) where, after 24 h, culture medium with FK866 was renewed, and cells were incubated for an additional 24 h. Thereafter, the culture medium was removed, and the fresh medium containing 10% AlamarBlue reagent (Invitrogen) was added to each well, according to the manufacturer’s protocol. Samples were incubated in standard conditions for 4 h, and the fluorescence signal was measured at 550 nm excitation and 590 nm emission wavelength with a microplate reader . Results were expressed as relative fluorescence normalized to the control (0.5% DMSO).To assess FK866 cytotoxicity, HaCaT cells were seeded into 96-well plates  and washed twice with 1 mL PBS. Next, cells were resuspended in 1 mL growth medium containing or not 200 µM NR (post-treatment), transferred into 96-well plates in triplicates (4 × 103 cells/well), and incubated for 20 h at standard cell culture conditions. Thereafter, AlamarBlue reagent was added to each well, according to the manufacturer’s protocol. Samples were incubated in standard conditions for 2 h, and the fluorescence signal was measured at 550 nm excitation and 590 nm emission wavelength with a microplate reader . Results were expressed as relative fluorescence normalized to the control.AlamarBlue assay was also used to assess THP-1 viability following mustard exposure. Briefly, 2 × 106 cells on 100 mm culture dishes 24 h before treatment with FK866 and/or NR. Next, cells were harvested, resuspended at 106 cells/mL culture medium, and treated with genotoxicants. After incubation, 100 µL of each sample was transferred into 9.9 mL of culture medium, resulting in a cell titer of 10 cells/µL. A total of 100 µL of such a sample (containing 103 cells) was seeded into new 6-well plates (three wells per sample) with fresh culture medium and incubated for 7 days in the presence or absence of NR and/or FK866. Then, the medium was removed, and colonies were fixed by 30 min incubation with 10% formaldehyde (Sigma-Aldrich), followed by 30 min staining with 0.1% crystal violet (Sigma-Aldrich). The wells were washed with PBS and air-dried before being counted using OpenCFU software [Clonogenic survival assays were performed as described previously . HaCaT csoftware . For eac2O2 was assessed by the automated fluorimetric alkaline DNA unwinding assay (FADU), as described previously [6 cells on 100 mm culture dishes 24 h before the treatment. After, the toxic exposure samples were harvested immediately (0 h) or incubated for a further 24 h in fresh culture medium. Harvested cells were centrifuged , and the pellet was resuspended in suspension buffer  at a final cell titer of 4 × 106 cells/mL. Then, with the use of a liquid handling device , the FADU protocol was executed. Initially, 70 µL of each sample was transferred in technical triplicates into 96-well plates and lysed with 70 µL of lysis solution  for 12 min at 0 °C. Next, 140 µL of alkaline solution  was added, and alkaline DNA unwinding was performed at 30 °C for 60 min. Unwinding was stopped with 140 µL of neutralization solution . Directly after incubation, at 22 °C for 30 min, 156 µL of SYBR Green solution (Thermo Fischer Scientific) (1:8333 v/v in H2O) was added, samples were mixed by automatic pipetting, and the fluorescence signal was measured at 492 nm excitation and 520 nm emission wavelength in a microplate reader . For each sample, an internal control of the DNA levels was included, where unwinding was prevented by the addition of a neutralization solution prior to the alkaline solution. The relative amount of double-stranded DNA was calculated as (fluorescence of sample/fluorescence of sample without unwinding) × 100%. The HN2-induced interstrand crosslinks (ICLs) were measured via a reversed FADU (rFADU) protocol [The induction of DNA strand breaks following the treatment with CEES and Heviously ,35. HaCaprotocol . Then, p6 cells per well in 12-well plates, on sterile 18 mm glass coverslips 24 h before the treatment. Directly after the toxic exposure, cells were fixed with ice-cold methanol for 7 min at −20 °C. Next, samples were washed 3 × 10 min with PBS and permeabilized with 0.4% Triton X-100 in PBS for 3 min. Then, samples were incubated in blocking solution (BS), containing 5% (w/v) milk powder and 0.5% Tween-20 in PBS, for 1 h at RT, and incubated with anti-PAR antibody (10H) (1:300 in BS) [The immunochemical detection of PAR formation was performed as previously described ,31. HaCa0 in BS)  overnigh0 in BS) . All experiments were performed in replicate numbers as indicated in figure legends. Data were analyzed with GraphPad Prism v.9.2.0 software, using statistical tests as indicated.+ boosting in HaCaT cells, we applied a selection of NAD+ boosters, i.e., NA, NAM, NR, and NMN, for up to 24 h to the cell culture medium. Among them, NR resulted in the highest increase in NAD+ levels, up to 3-fold, within 4 h of exposure  at 4 h post-ingestion [+ levels in human PBMCs on average by around 60% when compared to the placebo group [To identify the most efficient strategy of NADexposure B. Other h as NMN D or NA  during 10 min exposure, which was additionally elevated 2.4-fold by NR  cellular NAD+ decline after H2O2 treatment   , which aon (67%) .+ levels .In agreement with results reported in the previous section, NR had also no effect on the cytotoxicity of the genotoxicants tested. Both CEES A and HN2+ levels  or ATP l+ levels  at 24 h.+ levels . Clonoge+ levels  or cell + levels  followin+ levels poorly attenuated the toxicity of mustards, as shown in the present and previous studies [+ in general plays crucial roles in cellular physiology [+ participates in mustard-induced mechanisms of toxicity was further examined by experimentally depleting NAD+ levels. To address this view, prior to genotoxicant exposure, we decreased cellular NAD+ levels via FK866 treatment, which is a specific inhibitor of NAMPT, the key enzyme in the salvage pathway, which converts NAM to NMN    after twctively) A,B. Alrectively) A in compctively) B or H2O2ly) . Yet, the 24 h incubation did not result in a similar increase , which authors interpreted as the result of the induction of NAD+ degrading enzymes [+ decline with time would be frequent NR re-application. However, one needs to be careful, as the biological effects of prolonged NAD+ boosting are unclear and might possibly cause adverse effects on cellular homeostasis [+ elevation may have particularly detrimental effects on systems where intracellular NAD+ levels normally oscillate according to the circadian rhythm [The fact that NR produced the highest increase in cellular NADn humans ,25,51. Hincrease , suggesteostasis . For exaeostasis . Moreoven rhythm . + levels provided a window of opportunity to investigate possible protective effects of NR against early events of genotoxic stress induced by mono- and bi-functional mustard agents. Hence, in our experimental conditions, NR, despite significantly elevating NAD+ (+ precursors (NA and NAM) in mustard toxicity, as reviewed elsewhere [+ increase within the first hours of NR supplementation, at 24 h, NAD+ matched the control levels  reduced the induction of necrosis and ensured the appropriate execution of caspase-mediated apoptosis in HaCaT cells after SM treatment [+ levels and subsequent energy crisis trigger apoptosis in those cells. For example, CEES has been shown to induce apoptosis via Erk1/2 mitogen–activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K/Akt) signaling cascades in mouse keratinocytes JB6 [Similarly, maintaining NADll death ; althougsurvival . Moreoveval [2O2 , NR suppobserved C. Overalobserved C but a dobserved E,G). Thireatment . Here, wytes JB6 ,60. Addiytes JB6 .+ levels might be particularly beneficial in the case of CEES-induced toxicity. Additionally, these findings demonstrate that NR supplementation attenuates genotoxic stress in a system-specific manner. The reason behind the differential response of HaCaT and THP-1 is not clear, but data obtained suggest the following explanation: THP-1 cells seem to have lower basal NAD+ content, as the untreated cells exhibited on average 1.48 nmol/mg of protein (+ levels might provide room for further elevation via supplementation, whereas a highly saturated system, such as HaCaT, might be less susceptible to further NAD+ elevation. Additionally, the NAD+ metabolism seems to be slower in THP-1 cells where NAD+ levels remained elevated for up to 5 h after NR removal from the growth medium (+ levels at 5 h were not different from the untreated control (+ elevation in THP-1, following mustard exposure (+ levels, but not in young healthy animals/individuals exhibiting higher NAD+ levels [Unlike in HaCaT, in THP-1, we observed a protective NR effect on cell viability, particularly upon CEES exposure A. These  protein , whereas protein A. Lower h medium , whereas control . Thus, lexposure B, might exposure A. These + levels ,62,63,64+ levels with NR provides system-specific protection against genotoxicity induced by the sulfur mustard analogue CEES, suggesting that NR is a promising intervention for certain types of cells exposed to mustards. NR showed positive outcomes particularly in the conditions of initially lower NAD+ levels, providing evidence of the compound-specific functional role of NAD+ in the mustards’ toxicity pathomechanism. Apart from new insight into the status of NAD+ in mustard toxicity, our study suggests the particularly beneficial therapeutic application of NR supplementation in the context of reduced basal NAD+ levels, which is the phenomenon associated with certain diseases and ageing [In conclusion, this study shows that elevating cellular NADd ageing . Further


Engineering oncolytic vaccinia virus to express a potent TGFβR inhibitor results in local neutralization of TGFβ, increased T While checkpoint blockade immunotherapies have widespread success, they rely on a responsive immune infiltrate; as such, treatments enhancing immune infiltration and preventing immunosuppression are of critical need. We previously generated αPD-1 resistant variants of the murine HNSCC model MEER. While entirely αPD-1 resistant, these tumors regress after single dose of oncolytic vaccinia virus (VV). We then generated a VV-resistant MEER line to dissect the immunologic features of sensitive and resistant tumors. While treatment of both tumor types induced immune infiltration and IFNγ, we found a defining feature of resistance was elevation of immunosuppressive cytokines like TGFβ, which blunted IFNγ signaling, especially in regulatory T cells. We engineered VV to express a genetically encoded TGFβRII inhibitor. Inhibitor-expressing VV produced regressions in resistant tumor models and showed impressive synergy with checkpoint blockade. Importantly, tumor-specific, viral delivery of TGFβ inhibition had no toxicities associated with systemic TGFβ/TGFβR inhibition. Our data suggest that aside from stimulating immune infiltration, oncolytic viruses are attractive means to deliver agents to limit immunosuppression in cancer.  As cancer progresses, it establishes an immunosuppressive tumor microenvironment (TME) that alters local stromal and immune cells to prevent immune infiltration, recognition, and function. These include physical barriers to infiltration, alterations in the local metabolic milieu, the recruitment of immunosuppressive cell types, and elevation of soluble factors that can dampen immunity . Many ofIndeed, checkpoint blockade immunotherapies have had widespread success in a variety of solid tumors, prolonging the lives of millions of patients. However, these therapies rely on the presence of a pre-existing immune infiltrate, and in patients that carry immunologically inert tumors, checkpoint blockade has shown little success. Thus, there remains a critical need for treatment modalities that can promote immune infiltration as well as limit suppression within the TME.Oncolytic viruses (OVs) preferentially infect tumor cells and can be utilized for cancer therapy. These viruses, by natural tropisms and engineered selectivity, replicate in tumor cells leading to tumor lysis, release of pathogen-associated molecular patterns and damage-associated molecular patterns, and ultimately T cell priming with tumor and viral antigen . The adaA key defining feature of OVs is their ability to be engineered not only to promote tumor selectivity but also to deliver gene therapy to the TME. T-VEC, for instance, delivers the gene for GM-CSF to the tumor  to suppo+ T cells after treatment, the role of immunoregulatory factors is less apparent in OV therapy despite controlling resistance mechanisms in the TME. Key among these resistance mechanisms is the recruitment of regulatory T (Treg) cells to the TME and the heightened concentration of inhibitory cytokines like TGFβ. Treg cells, among other factors present in the TME, including soluble, cellular, and structural factors, can prevent immune infiltration and activity, and consequently immunotherapy efficacy  treatment to generate paired isogenic tumor cell lines resistant or sensitive to therapy. This allowed direct comparison for deep understanding of the immunologic mechanisms underlying OV-responsive tumors. Analysis of the infiltrate from these various tumor models revealed that while tumors treated with OVs showed infiltration of new T cells, a defining feature within the TME of resistant tumors was the phenotypic stability of TRAS and HPV E6/E7, is partially sensitive to anti-PD-1 therapy (5 PFU/mouse) of oncolytic vaccinia . This oncolytic, “double deleted” vaccinia virus has a luciferase reporter, an insertion of GFP in the thymidine kinase (TK) locus, and a deletion of virulence growth factor, which make the virus more selective for replication in tumor cells  head and neck squamous cell carcinoma (HNSCC), MEER, a mouse oropharyngeal line transformed with h therapy . Our lab therapy . Mice beor cells . Remarkaor cells . Thus, t2, these tumors were treated with VV  and monitored for response to therapy. Treatment-resistant tumors were again resected, and this process was repeated three times until a MEER derivative (termed MEERvvR) stably resistant to VV therapy was generated  of 0.1 and supernatant was harvested at 24 and 48 h. This supernatant was tittered on HeLa cells using a crystal violet plaque assay. No significant difference in virus production was observed . We found that MTE-LXSN cells retained p53 expression that was lost in both MEER lines  with HPV16 E6/E7 and hRas . To ensuER lines . This shER lines . The MEESN cells . We also MEERvvR . These cvvS tumors with VV as in previous studies. We found no difference in tumor growth between PBS control and VV-treated tumors in this model  to profile the balance of suppressive and stimulatory cytokines in the MEERvvS and MEERvvR tumors. In untreated tumors, we observed significantly higher levels of the protumorigenic IL-6 –TGFβ1 , which stabilizes Treg cell function in cancer , and TGFβ1, as well as other activation markers, and are superior at suppressing CD8 T cell proliferation and tumor control –TGFβ1  and the P)–TGFβ1 , which iP)–TGFβ1 . Both LAeg cells . CD103, eg cells . ExpressR tumors  and was R tumors . Treg cen cancer . InteresS tumors . In Treg control . We findpression  comparedpression , markers+ Foxp3− conventional T cells (Tconv) cells in MEERvvR tumors compared with MEERvvS  in the two models. We found that in MEERvvR tumors, there was a significant reduction in effector T cells  and increase in effector Tconv cells in the dLN of MEERvvR  within the TK locus. This dnTGFβmm was derived from the original variant of a mutant form of TGFβ containing structurally guided mutations to prevent dimerization, generated by 1-3 for receptor binding  than PBS or VVctrl-treated tumors . These clinical trial failures point to a need for a deeper understanding of the unique mechanisms of resistance to oncolytics as resistance mechanisms may not apply broadly across immunotherapies. In this study, we used paired sensitive or resistant tumors  to dissect the “common” features of OV treatment versus those that ultimately produce durable responses. In doing so, we uncovered tumor-derived resistance mechanisms paving the way for a more potent therapy.OVs, which can inflame and lyse tumors, promote T cell infiltration, and deliver payload to the local environment, carry the potential to immunologically activate otherwise “cold” tumors. However, despite substantial investigation and investment into OVs, clinical trials have not shown broad success apart from the initial approval of T-VEC in 2014. Recently, a trial of T-VEC in combination with pembrolizumab failed in phase Ib/III in HNSCC  and in preg cells, which we found to be directly related to TGFβ in the TME. We found that in mice bearing contralateral MEERvvS and MEERvvR, the Treg cells infiltrating the sensitive tumor are primed for fragility, harboring lower surface Nrp1 expression, lower TGFβ signatures , and a higher sensitivity to IFNγ via STAT1 signaling. Treg cells in MEERvvS were also less activated , and these tumors had higher infiltration of effector Tconv and CD8+ cells. After OV treatment, IFNγ increases in the tumor interstitial fluid in both MEERvvR and MEERvvS; however, in accordance with a more fragile phenotype, only MEERvvS infiltrating Treg cells have increased IFNγ signaling and consequent IFNγ production, consistent with previous data  resulted in a striking 67% of tumors completely regressing. In fact, in an abscopal model of C24, we found that this combination was even able to significantly reduce tumor growth in the uninjected lesions compared with VVdnTGFβmm + isotype control. We found in the C24 model that while these tumors had high TGFβ like MEERvvR, treatment with VV alone did not increase IFNγ in the tumor. Treating with αPD-1 led to increased IFNγ production by all T cells in the tumor and increased responses when combined with VVdnTGFβmm. This confirmed the rationale for combining therapies: oncolytic vaccinia to increase tumor-infiltrating T cells, dnTGFβmm to reduce TME suppression, and αPD-1 to improve the anti-tumor T cell response and increase intratumoral IFNγ.We found that VVotherapy . By combmm. Our mice were treated with one IT dose; however, patients are often given multiple doses over a period of many weeks when treated with OVs. Additional preclinical studies will ultimately evaluate the effect of a long-term dosing schedule with this inhibitor.It has been shown by other groups that TGFβ inhibition may have an effect on viral replication  while otOV immunotherapy has immense promise to act as a potent immunologic adjuvant in cancer. However, the immunologic mechanism of action has not been rigorously studied. While the majority of prior work has focused on CD8 and conventional CD4 T cells, our study highlights the importance of suppressive populations in OV therapy. Much of the focus within OVs has been on increasing the immune stimulatory effects of the virus with such mechanisms as virally produced GM-CSF and IL-12 or a combination with checkpoint blockade. Shifting the focus of the field toward understanding the mechanisms of resistance against OVs and rationally designing combination therapies to alleviate the tolerogenic mechanisms in the TME may be more beneficial to improve responses to OVs. Further, OVs may also be a more attractive way to therapeutically target TGFβ signaling, as their local and temporally restricted delivery allows for high potency without systemic toxicity. In this way, the therapeutic efficacy of many forms of immunomodulatory therapy for cancer hindered by TGFβ may be similarly improved.Foxp3tm1Flv/J), Foxp3-Ametrine reporter (C57BL/6.Foxp3flpo-mAmetrine), and RAG1 knockout (C57BL/6-Rag1tm1Mom/J) mice were bred in-house and off-site at Charles River. Foxp3Cre-ERT2.GFP × Rosa26LSL.Td.Tomato mice were bred in-house. Thy1.1 mice were obtained from Jackson Laboratories. Mice used in experiments were male and female between 6 and 10 wk old at the initiation of the study. All animal work and protocols in this study were approved by the University of Pittsburgh Institutional Animal Care and Use Committee, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.C57/BL6 mice were obtained from Jackson Laboratories, bred in-house, and bred off-site at Charles River. Foxp3-RFP reporter mice  coated plate. All cells were maintained at 37°C with 5% CO2.Tumor experiments were conducted with MEER, C24, LLC, B16-F10, MC38, and PanCO2 tumors. The MEER tumor line is an MTE that stably expresses E6/E7 and H-ras and has been rendered resistant to anti-PD-1 therapy through serial treatment in vivo . C24 is ER mouse . HeLa ceew Hinck . MTE-LXSew Hinck . C24, B1ew Hinck . MEERvvRvvR was generated from the MEERvvS line. A MEERvvS tumor  three times per week with αPD-1 (J43) or isotype control (Armenian Hamster IgG) at 2 mg/kg. Tumor growth was monitored until tumors reached 15 mm in any direction. In some experiments, two MEERvvR or two C24 were implanted on opposing ends of the back of the same mouse. In these experiments, one tumor was treated the same as in single-tumor experiments and αPD-1 treatments occurred the same as well. In some experiments, single MEERvvR tumors were treated multiple times with PBS or VV. In these experiments, mice were treated with IT PBS or VV as previously described, and at 4 and 8 d after treatment were treated again.C57BL/6 were implanted intradermally with 250,000 C24, MEERvvS, and MEERvvR) were implanted and treated the same way. At 4 or 7 d after treatment, tumors were harvested and digested as previously described  analysis, tumors  were implanted and treated with VV at 2.5 × 105 PFU per mouse. Starting at 24 h after treatment, mice were given an IP dose of luciferin (30 mg/ml). After 10 min, mice were imaged in the IVIS Spectrum In Vivo Imaging System. Mice were imaged every day for 5 d after treatment.For in vivo viral imaging, tumors  were harvested at ∼75 mm2 into an empty 15-ml conical tube. Tumors were then placed onto a 5-μm nylon membrane filter in a fresh conical tube and spun at 3,000 RPM for 20 min. The volume of interstitial fluid was recorded and then stored at −80°C until analysis via IFNγ ELISA, Luminex, or TGFβ reporter assay.To harvest tumor interstitial fluid, tumors (MEEREscherichia coli (NEB). The colonies were mini-prepped  and sent for Sanger Sequencing to Genewiz to confirm. The bacteria carrying the desired plasmid was then isolated with NucleoBond Xtra Midi Plus EF .The lentiviral vectors pHIV-RFP720 and pHIV-RFP720-E2A-mTGFβ were produced using the Addgene plasmid pHIV-iRFP720-E2A-Luc  as backbone. For the pHIV-RFP720 vector, two segments of the original plasmid were produced by PCR designed to produce overhangs. Then the segments were used for a HiFi DNA Assembly (NEB). The pHIV-RPF720-E2A-mTGFβ vector was produced using a segment from the original vector produced with PCR and a gBlock from IDT. These plasmid DNA fragments were used for a HiFi DNA Assembly. Briefly, the PCR fragments were digested with DpnI  to remove the original plasmid without overhangs and gel isolated . 25 ng of each DNA fragment was added into a reaction with HiFi DNA Assembly , and the resulting plasmid was transformed into 5-α competent vvS cells at 10% confluency for 2 h at 2,000 × g with 1:2,000 polybrene (stock solution 10 mg/ml). The cells were allowed to rest overnight; viral media was removed in the morning and fresh media was added. After 2 d, transfected cells were sorted by RFP720 expression. TGFβ overexpression was then confirmed by Western blot.After plasmid generation, LentiX 293T cells  were transfected with three plasmids: the viral plasmid (pHIV-RFP720 or pHIV-RFP720-E2A-mTGFβ), psPAX2 , and pMD2.G  at 4:3:1 ratio and the transfection reagent Xfect . After 4 h, the transfection mix was removed and fresh media was added. Viral supernatants were collected after 2 d and filtered. This fresh virus was used to spin-transduce MEERdnTGFbmm was generated as previously described in VVThe dnTGFβmm was developed and produced in the lab of A.P. Hinck. It is described in ctrl or VVdnTGFβmm at an MOI of 0.1 for 2 h in DMEM supplemented with 10% FBS (vol/vol). Virus was then removed and fresh media was added. Once cytopathic effect was visible (∼48 h after infection), cells were harvested into their supernatant by gentle rinsing. Cells were then pelleted, resuspended into 10 ml of 10 mM Tris-HCl (pH 7.0), and lysed by three cycles of freeze–thaw. The resulting supernatant was layered onto a sucrose cushion  and spun for 2 h at 14,500 RPM at 4°C. The viral pellet was resuspended in 200 μl of 10 mM Tris-HCl and stored at −80°C. Purified virus was tittered on HeLa cells using a crystal violet plaque assay.HeLa cells were infected with VVCell surface staining was performed on ice in PBS for 20 min with surface antibodies and Zombie Viability Dye (BioLegend). Cells were then washed in PBS and either run for live panels or fixed with 4% paraformaldehyde for 5 min at room temperature and then washed in PBS. For phospho staining, cells were fixed with ice-cold 90% methanol at −20°C for 20 min, then washed with permeabilization wash buffer (eBioscience), and stained overnight at 4°C. For nuclear staining, cells were fixed with the Foxp3 Transcription factor fixation kit (eBioscience) for 20 min at room temperature and then stained overnight at 4°C. For cytokine restimulation, lymphocytes were restimulated with PMA and ionomycin for 5.5 h in the presence of a Golgi plug (BD Biosciences). Surface staining was performed as above followed by fixation with the CytoFix/CytoPerm kit (BD Biosciences) and stained overnight at 4°C for intracellular cytokine staining.The following antibody clones were utilized for flow cytometry experiments: CD4 , CD8 , Nrp1 , IFNγ Receptor 1 , IFNγ Receptor β chain , PD-1 , Tim3 , CD45 , LAP-TGFβ1 , GARP , CD103 , TCF1/7 , CD44 , CD62L , CD25 , CD122 , Granzyme B , Foxp3 , TNFa , IFNγ , pSTAT1 ser727 , and pSTAT1 tyr701 .vvS and MEERvvR were cultured in vitro as described above, and cDNA was generated using SMART-seq HT kit  using 1,000 cells. cDNA product was checked by Tape Station D5000 from Agilent Technologies 2200 to make sure cDNA was successfully generated. Library construction was done using Nextrera XT kit # 15031942 from Illumina. 1 ng cDNA was used in a total volume 5 µl. Sequencing was done using NextSeq 500 System. High Output 75 Cycles kit with run Parameter Paired Read 150 cycles (2 × 75).MEERMus musculus reference genome (Mus_musculus_ensembl_v80_Sequence) using CLC Genomics Workbench. Using CLC Genomics Workbench, differential gene expression was found with statistical cut-offs of P value <0.05, max group means >1, and |foldchange| >1.5. Heatmaps were generated using R package pheatmap with log2 transformed transcript counts per million .Sequencing reads were trimmed for adapters and then aligned to Gene set enrichment analysis  was perf2SO4 was added to stop the reaction. The plate was read at 450 nm.The ELISA plate was coated with 50 μl of capture antibody  at 4°C overnight. The next morning the plate was washed three times with wash buffer (PBS + 0.05% Tween-20). The plate was blocked with 200 μl blocking buffer (PBS + 1% BSA) for 1 h at room temperature. After washing three times, the samples and standard curve were added (50 μl), diluted in blocking buffer, and incubated at room temp for 1 h. The plate was washed five times and the detection antibody was added  for 1 h at room temperature After five washes, HRP streptavidin  was added and incubated for 30 min at room temperature After seven washes, tumor mutational burden substrate A (50 μl) and tumor mutational burden substrate B (50 μl) were added to develop the samples, and 50 μl of HThe reporter assay protocol was followed from The inhibitory activity (IC50) of the dnTGFβmm was determined using the protocol previously described in reg cells were sorted from spleen and lymph nodes of Foxp3RFP or Foxp3Ametrine mice by Foxp3-reporter expression  and then 250,000 cells per condition were cultured in serum-free media for 3 h followed by culture in 100 ng/ml IFNγ (Peprotech), 100 ng/ml TGFβ (R&D systems), or both for 30 min. Cells were then harvested for Western blotting.TvvR, MEERvvS, and MTE-LXSN cells were cultured at 50,000 per well of a 24-well plate. 100,000 cells were harvested 24–248 h later for lysis.MEERCells were lysed in 1% NP-40 lysis buffer, cell debris was removed by centrifugation, and samples were boiled for 10 min in NuPAGE LDS sample buffer (Thermo Fisher Scientific) and dithiolthreitol (Thermo Fisher Scientific). Lysates were then separated by SDS-PAGE using 4–12% Bio-Rad gels. Samples were then transferred from the gel to a polyvinylidene difluoride membrane and blocked in 5% milk in Tris-buffered saline 0.1% Tween-20 (TBST). The membrane was then incubated overnight at 4°C with primary antibodies diluted in a blocking buffer. The membrane was washed five times for 5 min in TBST. The membrane was incubated with secondary antibody  in blocking buffer for 1 h at room temperature and subsequently washed five times for 5 min with TBST. Protein was visualized by chemiluminescence by using Western Lightning ECL (PerkinElmer). Antibodies were obtained from the following companies: STAT1 , pSTAT1 Y701 , β-actin (Santa Cruz), pSMAD2 , SMAD2 , Ras G12V , and p53 .reg cells were sorted from spleen and lymph nodes of Foxp3RFP or Foxp3Ametrine mice by Foxp3-reporter expression , then spun down and sorted again to ensure purity. Cells were then stimulated overnight with PMA (0.1 µg/ml), ionomycin (1 µg/ml), and IL2 (100 U/ml) at 1 × 106 cells/ml. The next morning, stimulation was washed off and cells were placed into no cytokine, IFNγ (100 ng/ml), TGFβ (100 ng/ml), or both, with IL2 (100 U/ml). After 48 h, Treg cells were sorted from their conditions by Foxp3-reporter. CD4+ responder cells and APCs were also sorted from a Thy1.1+ mouse. CD4+ responder cells were stained with Cell Trace Violet proliferation dye (Thermo Fisher Scientific). CD4+ responder cells were then mixed 1:1 with APCs and αCD3 . 30k Treg cells were plated in the top row of a 96-well round bottom plate and serially diluted. The APC:responder mix was added into these wells so that the first well contained a 1:2 Treg:responder ratio and that was diluted by half at each dilution. After 72 h, the cells were analyzed by live flow cytometry to read out the proliferation of the CD4+ Thy1.1+ responder cells compared with no Treg cell control.TvvR and MEERvvS tumors (conv cells in the tumor (TGFb-OE tumor line (The supplementary information contains data confirming similar viral kinetics in the MEERS tumors  as well he tumor , and S4.mor line  as well mor line .SourceData F4is the source file for Fig. 4.Click here for additional data file.SourceData F5is the source file for Fig. 5.Click here for additional data file.SourceData FS1is the source file for Fig. S1.Click here for additional data file.SourceData FS4is the source file for Fig. S4.Click here for additional data file.


The emphasis here is on theory-aided evaluation of available experimental data with a view to establishing standardised cross-section database for production of the relevant radionuclide in high purity. Evaluated cross section data of the positron emitters have been already extensively reported; so here they are only briefly reviewed. More attention is given to the data of the 68Zn67Cu intermediate energy reaction which is rather commonly used for production of 67Cu.Copper has several clinically relevant radioisotopes and versatile coordination chemistry, allowing attachment of its radionuclides to biological molecules. This characteristic makes it suitable for applications in molecular imaging or radionuclide targeted therapy. Of particular interest in nuclear medicine today is the theranostic approach. This brief review considers five radionuclides of copper. These are Cu-60, Cu-61, Cu-62, Cu-64, and Cu-67. The first four are positron emitters for imaging, and the last one Cu-67 is a β The two nuclides are denoted as “matched pair” (60Cu(T1/2 = 23.7 min), 61Cu(T1/2 = 3.33 h), 62Cu(T1/2 = 9.67 min) and 64Cu(T1/2 = 12.7 h), and the β–emitting radionuclide 67Cu (T1/2 = 61.83 h). Whereas 60Cu and 62Cu, being rather short-lived, have found only limited application in PET measurements  energies and their intensities. They were taken from NUDAT which is based on evaluated mass decay chains. Yet some uncertainties do exist. The intensity of the weak 1,345.7 keV γ-ray of 64Cu, for example, is slightly controversial. The reported standardised value is (0.47 ± 0.01) %  % . A lateris γ-ray . But an is γ-ray , utiliziβ+). In most cases this intensity (% of decay) is derived from a balance of various γ-transitions described in the decay scheme. But often there are uncertainties in the reported values. A direct experimental determination of the positron emission intensity involving a spectrometric analysis of the 511 keV annihilation radiation and Kα X-rays, as developed at FZJ, appears to provide more accurate results ].In contrast to neutron-induced reactions, the standardisation of charged-particle induced reaction cross sections, needed for production of radionuclides at cyclotrons/accelerators, remained initially somewhat neglected . From 19σev(E), σmodel(E) and f (E) are the evaluated cross section, model calculated cross section and the energy-dependent normalisation factor, respectively. The ratio of experimental to calculated data is plotted as a function of energy, followed by a polynomial fitting to estimate the Standardisation work on charged-particle induced reaction cross sections has also been done outside the IAEA-CRPs mentioned above. In those studies extensive use was made of nuclear model calculations to ascertain the most concordant set of data for a given nuclear reaction, followed by polynomial fitting of the selected data. Based on a suggestion made by For nuclear model calculations, in our collaboration four major codes, namely, STAPRE, ALICE-IPPE, EMPIRE and TALYS  were useFor all 5 radionuclides under consideration, several production routes were studied (see below). However, in 60Ni60Cu process. However, no attempt has been made to standardise its cross-section data either in the IAEA-CRP or by us. On the other hand, a detailed experimental and theoretical study of the excitation function of this reaction has been carried out by p = 15→7 MeV) for the production of 60Cu, and also calculated its expected thick target yield. This process has been developed to produce high-purity 60Cu on a clinical scale 61Cu, 62Ni61Cu, 60Ni61Cu and 58Ni61Cu were standardised by 64Zn61Cu, 64Zn61Cu, 59Co61Cu and 59Co61Cu by 59Co61Cu reaction can be used if an α-particle beam of about 40 MeV is available 61Cu and 64Zn61Cu have been more commonly used for production. The suitable energy ranges for those two reactions were deduced. The calculated yields of 61Cu from standardised curves by us -reaction and about 10% for the 64Zn-reaction. For both reactions, using highly-enriched targets, clinical scale production of high-purity 61Cu has been reported 62Zn→62Cu (generator)a) 62Ni62Cub) This short-lived positron emitting radionuclide is obtained via two routes:natCu62Zn have been very well standardised because it is an important monitor reaction  is given in 62Cu 62Cu were standardised by us 64Cu, 64Ni64Cu, 68Zn64Cu, 66Zn64Cu, 64Zn64Cu, 66Zn64Cu, natZn64Cu and a few others. Out of all those reactions, however, the 64Ni64Cu process on highly-enriched target is the most interesting. Its thick target yields reported by us and the IAEA-CRP agree within 1%. Initially proposed by the Jülich group 67Cu process. Its excitation function is known fairly well. We concentrated on four charged-particle induced reactions. Three of them have been investigated in the low-energy range. They are 70Zn67Cu 67Cu 67Cu 67Cu reaction under an IAEA-CRP 67Cu process up to proton energy of about 70 MeV 67Cu, is presently often used for production purposes. The standardisation of production cross sections was attempted under an IAEA-CRP 67Cu , 70Zn67Cu  and 64Niα,p)67Cu . An evalIAEA-CRP . The newclotrons . Very ret 70 MeV . The croIAEA-CRP . AnotherIAEA-CRP . Jung ethttp://www-nds.iaea.org/exfor/exfor.htm) over the proton energy range up to 430 MeV. For evaluation, however, the data only up to 100 MeV are interesting, i.e., leaving out some data points in the higher energy region (68Zn (98.3%) thin target samples and radiochemical separation of 67Cu. The experimental data were generally consistent but a few data points showed a systematic lower trend in the energy range of 35–45 MeV investigated at the cyclotron JULIC. 68Zn (≥99%) as the target material. Recently two detailed measurements have been reported for this reaction using enriched 68Zn targets, one by Medical Portal (iaea.org)) are also shown in 67Cu via this route, however, the energy range Ep = 80→30 MeV remains the most suitable 67Cu are available up to 30 MeV which consists of the  reaction. In the higher energy region up to 70 MeV, however, the cross section increases rapidly, possibly due to the onset of the many nucleon emission processes like 70Zn67Cu. But more measurements and a critical evaluation are needed to obtain standardised data for this process. The presently rather commonly used intermediate reaction 68Zn67Cu was evaluated under two IAEA-CRPs. The newest measurements, however, show some deviations from the evaluated data. A new critical evaluation should thus be very meaningful. In order to estimate the specific activity of the radionuclide produced, it is also imperative to determine via model calculation the inactive material, i.e., 65Cu, co-produced with the respective radionuclide.The data for production of


Bio-based polymers can reduce dependence on nonrenewable petrochemical resources and will be beneficial for future sustainable developments due to their low carbon footprint. In this work, the feasibility of bio-based polyamide 56 (PA56) substituting petroleum-based PA66 is systematically investigated. The crystallization, melting, and decomposition temperature of PA56 were all lower than that of PA66. PA56 formed a γ crystal type with larger grain size and took a longer amount of time to complete the crystallization process since its crystallization rate was lower than that of PA66. Compared with PA66, PA56 exhibited a higher tensile strength of 71.3 ± 1.9 MPa and specific strength of 64.8 ± 2.0 MPa but lower notched impact strength. More importantly, the limited oxygen index and vertical combustion measurement results indicated that the flame retardancy of PA56 was better than PA66, and the LOI values and the UL94 result of PA56 were 27.6% ± 0.9% and V-2. It is worth noting that the PA56 fiber had superior biodegradability compared to the PA66 fiber. PA56 showed significant biodegradation from the eighth week, whereas PA66 remained clean until the sixteenth week (without obvious biodegradation taking place). Eventually, PA56 did not show significant differences compared to PA66 in terms of thermal and mechanical properties. However, PA56 had great advantages in flame retardancy and biodegradability, indicating that the bio-based PA56 could potentially replace petroleum-based PA66 in many fields. Recently, renewable polymers from biomass have attracted significant attention all over the world . These bThe preparation, characterization, and modification of PA56 have all been reported in last decade ,13. For Numerous efforts have been made to develop degradable or recyclable polymers by introducing various natural fillers or monomers ,22. SyntPA66 and bio-based PA56 pellets were kindly supplied by EPPEN Biotech Co., Ltd. . 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was purchased from TCI . All the chemicals were used without further purification. Fourier-transform infrared spectroscopy−1 to 400 cm−1.Infrared spectra of PA56 and PA66 were determined using a Nicolet 6700 FT-IR spectrophotometer  with an attenuated total reflection (ATR) attachment. Wavenumber range from 4000 cmThermogravimetric analysis2 (20 mL/min). Test temperatures from 40 °C to 600 °C.Thermogravimetric analysis (TGA) was carried out using Discovery TGA  at a heating rate of 10 °C/min under NDifferential scanning calorimetryDifferential scanning calorimetry (DSC) was performed on Netzsch DSC 204 Phoenix  under nitrogen atmosphere with a gas flow rate of 60 mL/min. The data were collected in a range of 40–310 °C at a heating rate of 10 °C/min. Crystallization behavior at different cooling rates, including 20, 10, and 5 °C/min, were also evaluated via DSC.Wide-angle X-ray diffractionbkl measures the average dimension of the particles in a direction perpendicular to the Bragg planes giving rise to the reflection [Wide-angle X-ray diffraction (WAXD) analysis was conducted on a Rigaku D/max-2550 PC  at room temperature. The Cu/Kα radiation (λ = 1.54 Å) was operated at 40 kV and 200 mA. The 2θ scan data were collected at 0.02° intervals over the range of 3°–45°.flection .Melt index testMelt index test was carried out to evaluate the melt flow properties. ASTM standard D 1238 was followed for the measurements, with barrel temperature of 275 °C and load of 0.325 kg (as recommended).Rheological testRheological test was performed using a double-tube capillary rheometer  with a capillary  under 275 °C.Gel Permeation Chromatography testThe relative molecular weights and distribution of PA56 were determined via Malvern gel chromatography  with high-performance liquid chromatography grade hexafluoroisopropanol as solvent and PMMA polymers with molecular weights of 1000~300,000 as standard; PA56 and PA66 were prepared as 0.1 mg/mL solution, fully dissolved, and then filtered for testing.Tensile and flexural properties testTensile and flexural properties were measured according to the national standard GB/T 1040-92 and GB/T 9341-2000 at testing speeds of 15 mm/min and 1 mm/min, respectively. The V-notches impact strength with diameter of 2.5 ± 0.05 cm was measured under 15 °C according to the standard GB/T 1843-2008/ISO180:2000.Combustion performances testCombustion performances of the polymers were measured by means of testing limiting oxygen index (LOI) on the basis of national standard (GB/T 2406.2–2009/ISO 4589-2:1996) and vertical flammability (UL94) on the basis of national standard (GB/T 2408-2008/EIC 60695-11-10:1999).Biodegradability testThe biodegradability of PA56 and PA66 fibers were tested in a culture fluid enriched with phanerochaete chrysosporium and by extracting the samples from the culture fluid at regular intervals to measure the molecular weight and distribution.Scanning Electron MicroscopeSurface morphology images of PA56 and PA66 fibers after biodegradation were tested by using a Scanning Electron Microscope (SEM). Fibers were sprayed gold 120 s before testing.−1/1461 cm−1 (CH2 symmetric scissors deformation), 1543 cm−1/1539 cm−1 (N-H bend and C-N stretch), 1637 cm−1/1637 cm−1 (amide C=O stretch), 2859 cm−1/2859 cm−1 (CH2 symmetric stretch), 2930 cm−1/2931 cm−1 (CH2 asymmetric stretch), 3082 cm−1/3080 cm−1 (NH bend overtone), and 3300 cm−1/3307 cm−1 (NH stretch), respectively  analysis provided detailed information and revealed the fine crystal structure of the polymers. PA66 showed a broad characteristic peak that was formed by two overlapped peaks . The fulNon-isothermal crystallization process was performed on PA56 and PA66 using DSC analysis at different cooling rates. Both the crystallization onset and endset temperature of PA56 were lower than that of PA66, as shown in Our DSC analysis revealed that the melting temperature of PA56 (252.9–253.9 °C) was slightly lower than that of PA66 (258.2 °C), as shown in d5%) of PA56 was about 414 °C, which was slightly lower than that of PA66 (430 °C). The temperature of maximum degradation (Tdmax) of both PA56 and PA66 was about 478 °C.TGA analysis  revealedMelt flow index (MFI) plays an important role in the plastics industry and is a useful tool for quality control and assurance . In partα (Pa·s), n, γ (s−1), and K represent shear viscosity, non-Newtonian index, shear rate, and constant, respectively [Polymer melts exhibit elastic and viscous properties, so the rheological measurements of materials are important for optimizing processing conditions such as extrusion, injection molding, and melt spinning, which are highly dependent on temperature, pressure, molecular weight, and shear rate . Howeverectively .When the non-Newtonian index approaches 1, the polymer is closer to Newtonian polymers. Both the shear viscosity and shear stress of the polymer are less sensitive to shear rate. The n values of PA56 and PA66 were 0.77 and 0.70, respectively. This indicates that the shear viscosity and shear stress of PA56 are less sensitive to shear rate compared to PA66, suggesting that PA56 is less likely to suffer from the Barus effect during the extruding process.Mechanical properties are important for practical application. The tensile strength, yield strength, and specific strength of PA56 were all higher than that of PA66, as shown in The higher tensile strength and lower yield strain of PA56 contributed to its mechanical strength and dimensional stability, which could help broaden the range of nylon applications in engineering fields, such as in the automotive industry. However, the tensile properties of PA56 are far from sufficient to meet the requirements for the engineering of plastics. Currently, pultruded profiles are widely applied as structural elements in different areas due to their high strength, low weight, and improved corrosion properties, as well as their fatigue resistance . Alexandw/w flame retardant composed of melamine and pentaerythritol with PA66 to increase the LOI from 22% to 27.9% [The results of combustion performance demonstrated that the LOI values of PA56 and PA66 are 27.6% ± 0.9% and 22.9% ± 0.5%, respectively, and the UL94 results of both polymers are V-2. It is clear that the LOI of PA56 was much higher than that of PA66. Lyu reported that the LOI was increased to about 28% with the addition of 5 wt.% N-benzoic acid (ethyl-N-benzoic acid formamide) phosphamide (NENP) prepolymer into PA66 . Li mixeto 27.9% . In summm and Tc of both fibers did not change much and biodegradation did not affect their melting-crystallization behavior, which was consistent with the TGA results. The Td5% and char yield of PA56 and PA66 fibers remained almost the same for different biodegradation times . Compared to PA66, PA56 had superior biodegradability, benefiting from a shorter biodegradation cycle (8 weeks). Our systematic and comprehensive investigation indicates that PA56, based on the bio-source monomer , has superior integrated performance compared to PA66, meaning that it could be a promising alternative to PA66.