The role of deregulated methylation in T-LGLL is not well known. We analyzed the epigenetic profile of T-LGLL cells of 11 patients compared to their normal counterparts by array-based DNA methylation profiling. For identification of molecular events driving the pathogenesis of T-LGLL, we compared the differentially methylated loci between the T-LGLL cases and normal T cells with chromatin segmentation data of benign T cells from the BLUEPRINT project. Moreover, we analyzed gene expression data of T-LGLL and benign T cells and validated the results by pyrosequencing in an extended cohort of 17 patients, including five patients with sequential samples.The molecular pathogenesis of T-cell large granular lymphocytic leukemia (T-LGLL), a mature T-cell leukemia arising commonly from T-cell receptor BLC11B and C14orf64 as suggested by in silico data analysis could provide a novel pathogenetic mechanism that needs further experimental investigation.We identified dysregulation of DNA methylation associated with altered gene expression in T-LGLL. Since T-LGLL is a rare disease, the samples size is low. But as confirmed for each sample, hypermethylation of T-LGLL cells at various CpG sites located at enhancer regions is a hallmark of this disease. The interaction of BCL11B is highly significant differentially methylated in T-LGLL cells. Although our results have to be validated in a larger patient cohort, BCL11B could be considered as a potential biomarker for this leukemia. In addition, altered gene expression and hypermethylation of enhancer regions could serve as potential mechanisms for treatment of this disease. Gene interactions of dysregulated genes, like BLC11B and C14orf64, may play an important role in pathogenic mechanisms and should be further analyzed.DNA methylation is altered in T-LGLL cells compared to benign T cells. In particular, The online version contains supplementary material available at 10.1186/s13148-022-01362-z. A Gene Ontology analysis of hypermethylated genes in T-LGL.Additional file 5: Fig. S3B. Gene Ontology analysis of genes hyper- (A) and hypomethylated (B) of T-LGL patients. Significant Biological processes (GO database) enriched in genes associated with significantly differentially methylated CpG loci in T-LGL. Enrichment represented as odds ratio. Point size represents the gene count of each pathway. Enrichment p value obtained by overrepresentation analysis [30], represented by point color. B Gene Ontology analysis of hypomethylated genes in T-LGL.Additional file 6: Fig. S4. Differential gene expression of IL6 between T-LGL and healthy donor-derived CD8+ memory T cells. Differential gene expression for IL6 was measured by qPCR. Bulk CD8+ cells from two healthy donors were used for comparison. In line with previous publications, the T-LGLL cohort analyzed exhibits a higher IL6 expression compared to healthy donor-derived C8+ cells. HD Healthy donor.Additional file 7: Fig. S5A. (A and B): Location of differentially methylated CpG loci in the genes BCL11B and C14orf64 (LINC01550). A Significant differentially methylated CpGs in BCL11B (T-LGLL compared to CD8+. memory T cells) were located in the gene body and assigned as enhancers by ENCODE, which match as enhancers in CD8-positive memory cells.Additional file 8: Fig. S5B. (A and B): Location of differentially methylated CpG loci in the genes BCL11B and C14orf64 (LINC01550). B Significant differentially methylated CpGs in C14orf64 (LINC01550) (T-LGL compared to CD8 pos. memory T cells).Additional file 9: Fig. S1. Correlation of bisulfite Pyrosequencing (BPS) and methylation Array DNA methylation levels. The correlation matrix shows the Pearson correlation coefficient (r: 1 (red) to − 1 (blue) among all CpG loci analyzed by BPS. The candidate genes LINC01550 and BCL11B contained multiple CpG sites. Columns and rows represent one CpG loci of the listed candidate gene.Additional file 10: Fig. S6. Expression correlation between BCL11B & C14ORF64 (LINC01550). Expression correlation between BCL11B & C14ORF64 (LINC01550) in 426 human datasets with 42563 samples from R2: Genomics analysis and visualization platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi).Additional file 11: Material and Methods.


Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). We studied the in vivo process by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue . We report that many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays of the Evening activity under entraining conditions predicted the phase of activity cycles under constant darkness. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior.Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in  Drosophila 150 neurons are dedicated circadian clocks and they all tell the same time. In spite of such strong synchronization, they provide diverse phasic outputs in the form of their discrete, asynchronous neuronal activity patterns. Neuropeptide signaling breaks the clock-generated symmetry and drives many pacemakers away from their preferred activity period in the morning. Each day, neuropeptide PDF is released by Morning pacemakers and delays the phase of activity of specific other pacemakers to later parts of the day or night. When and how the PDF that is released in the morning stops acting is unknown. Furthermore, timing of signal termination is not fixed because day length changes each day, hence the modulatory delay exerted by PDF must itself be regulated. Here we test a canonical model of G protein-coupled receptor physiology to ask how PDF receptor signaling is normally de-activated. We use behavioral measures to define sequence elements of the receptor whose post-translational modifications  may define the duration of receptor signaling.In multi-cellular organisms, circadian pacemakers create output as a series of phase markers across the 24 hour day to allow other cells to pattern diverse aspects of daily rhythmic physiology and behavior. Within circadian pacemaker circuits, neuropeptide signaling is essential to help promote coherent circadian outputs. In the fruit fly  Drosophila, neuropeptide PDF signaling helps pattern the output of the fly circadian pacemaker network that controls rhythmic daily locomotor activity .  displays activity patterns of han mutants that are heterozygous for tim-Gal4; they generally matched prior descriptions of pdfr mutant behavior. Restoration of pdfr function by han; tim> WT-pdfr, restored a Morning peak (anticipatory activity prior to lights-on–) and delayed the evening peak by 1–2 hrs   P{y embryos. For PDFR-tandem fusion protein expression, we made a stable yw; tim(UAS)-Gal4; UAS-pdfr-tandem stock. pdfr mutant flies are described in . Experimental genotypes were tested in the han mutant background, pdfr5304, or in the pdf01 (null) mutant background [w1118 background, as noted.All locomotor activity experiments were conducted with 2–5 days-old male flies at 25°C using Trikinetics Activity Monitors as previously described . We crosanalysis . Arrhythanalysis  to produ [et al. : [, followed by activity during nine or more days of constant darkness (DD). For each genotype, we display locomotor activity during light entrainment in four ways: (i) group eductions , (ii) direct comparisons between two genotypes of the amplitudes of behavioral peaks using bin-by-bin analyses during the last two days of entrainment (days 5–6) ; (iii) derivations of Morning and Evening behavioral phases (in Panels D and E); and (iv) double-plotted group actograms . The bin-by-bin analyses directly compare experimental genotypes to a control one [cf. Figs  one cf. . They prAdditional details on Methods and procedures are found in the text file S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Figth transmembrane domain (TM7) is marked in GREY. The C terminal tail starts with V505 (numbering for the melanogaster protein). The 28 potentially phosphorylated residues in the D.m. C terminal tail are highlighted in color: the 14 residues chosen for analysis are marked by their Cluster (CL) designation (1 to 7) and marked in AQUA; the 14 non-selected residues are marked in YELLOW. See S2 Table for additional sequence information.The predicted 7(PDF)Click here for additional data file.S2 Fighan (pdfr mutant) flies that expressed either no UAS transgene (A), or a UAS-WT pdfr transgene (B) or a variety of Simple pdfr Variants, including 2-3A (C), 4A (D), 5A (E), 6A (F) or 7A (G), or Multiple pdfr variants, including 5-7A (H), 1-4A (I), 1-5A (J), and 1-6A (K). Top Right Box: A schematic of the PDFR C terminal segment for the WT and all variants studied: see Figure Legend 1 for details. Letters to the right of each variant C terminal segment correspond to the Panels in this Figure that display the behavior observed following its expression. Each Panel (A)-(K) contains sub-panels (1) and (2): Sub-Panel (1) displays a daily plot of locomotor activity (a group eduction) averaged over the last two days of entrainment (LD 5–6). Open bars indicate the 8 hr periods of Lights-on and filled bars indicate 16 hr periods of Lights-off. Sub-panel (2) displays a double-plotted group actogram throughout the 6 days of LD entrainment, followed by ~9 days of . Green lines indicate the phase of the dominant activity period in DD. Panel L displays the average Morning activity Phase Onset timepoint, and Panel M displays the average Evening activity Phase Offset (marked by a Blue Arrow) for each genotype over the last two days of entrainment (LD 5–6). The positions of the Red and Blue arrows in panels A-K are representative phase points; panels L and M present their true values respectively. Analyses represent ANOVA followed by Dunnett’s post hoc multiple comparisons of all compared to WT: ns = not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001.All behavioral records were recorded from (PDF)Click here for additional data file.S3 Fighan (pdfr mutant) flies that expressed either no UAS transgene (A–marked in YELLOW), or a WT pdfr cdNA (B–marked in BLACK) or a variety of Simple pdfr Variants  including 2-3A (C), 4A (D), 5A (E), 6A (F), 7A (G), 5-7A (H), 1-4A (I), 1-5A (J), and 1-6A (K). These measures were averaged across all experiments run for each individual genotype -(L) contains sub-panels (1) through (3), each of which displays Bin-by-Bin analyses of activity levels sorted by 30 min bins, for three different time periods: (1) ZT17-23.5; (2) ZT 0.5–8; (3) ZT 8.5–16. The missing bin at timepoint 0 contains the startle response that accompanies the sudden lights-on signal. Blue asterisks indicate significantly-different activity levels according to a Student’s T-test following an ANOVA (p< 0.05). Red arrows highlight elevated Morning activity levels displayed by different PDFR variants. Blue arrows highlight elevated Evening activity levels produced by different PDFR variants.All behavioral records were recorded from (PDF)Click here for additional data file.S4 Fighan (pdfr mutant) flies that expressed either no UAS transgene (A), or a UAS-WT pdfr transgene (B) or a variety of Simple pdfr Variants, including 2-3A (C), 4A (D), 5A (E), 6A (F) or 7A (G), or Multiple pdfr variants, including 5-7A (H), 1-4A (I), 1-5A (J), and 1-6A (K). Top Right Box: A schematic of the PDFR C terminal segment for the WT and all variants studied: see Figure Legend 1 for details. Letters to the right of each variant C terminal segment correspond to the Panels in this Figure that display the behavior observed following its expression. Each Panel (A)-(L) contains sub-panels (1) and (2): Sub-Panel (1) displays a daily plot of locomotor activity (a group eduction) averaged over the last two days of entrainment (LD 5–6). Open bars indicate the 12 hr periods of Lights-on and filled bars indicate 12 hr periods of Lights-off. Sub-panel (2) displays a double-plotted group actogram throughout the 6 days of LD entrainment, followed by ~9 days of . Green lines indicate the phase of the dominant activity period in DD. Panel L displays the average Morning activity Phase Onset timepoint, and Panel M displays the average Evening activity Phase Onset (marked by a Blue Arrow) for each genotype over the last two days of entrainment (LD 5–6). The positions of the Red and Blue arrows in panels A-K are representative phase points; panels L and M present their true values respectively. Analyses represent ANOVA followed by Dunnett’s post hoc multiple comparisons of all compared to WT: ns = not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001.All behavioral records were recorded from (PDF)Click here for additional data file.S5 Fighan (pdfr mutant) flies that expressed either no UAS transgene (A–marked in YELLOW), or a WT pdfr cdNA (B–marked in BLACK) or a variety of Simple pdfr Variants  including 2-3A (C), 4A (D), 5A (E), 6A (F), 7A (G), 5-7A (H), 1-4A (I), 1-5A (J), and 1-6A (K). These measures were averaged across all experiments run for each individual genotype -(L) contains sub-panels (1) through (3), each of which displays Bin-by-Bin analyses of activity levels sorted by 30 min bins, for three different time periods: (1) ZT17-23.5; (2) ZT 0.5–8; (3) ZT 8.5–16. The missing bin at timepoint 0 contains the startle response that accompanies the sudden lights-on signal. Blue asterisks indicate significantly-different activity levels according to a Student’s T-test following an ANOVA (p< 0.05). Red arrows highlight elevated Morning activity levels displayed by different PDFR variants. Blue arrows highlight elevated Evening activity levels produced by different PDFR variants.All behavioral records were recorded from (PDF)Click here for additional data file.S6 Fighan (pdfr mutant) flies that expressed either no UAS transgene (A), or a UAS-WT pdfr transgene (B) or a variety of Simple pdfr Variants, including 2-3A (C), 4A (D), 5A (E), 6A (F) or 7A (G), or Multiple pdfr variants, including 5-7A (H), 1-4A (I), 1-5A (J), and 1-6A (K). Top Right Box: A schematic of the PDFR C terminal segment for the WT and all variants studied: see Figure Legend 1 for details. Letters to the right of each variant C terminal segment correspond to the Panels in this Figure that display the behavior observed following its expression. Each Panel (A)-(L) contains sub-panels (1) and (2): Sub-Panel (1) displays a daily plot of locomotor activity (a group eduction) averaged over the last two days of entrainment (LD 5–6). Open bars indicate the 16 hr periods of Lights-on and filled bars indicate 8 hr periods of Lights-off. Sub-panel (2) displays a double-plotted group actogram throughout the 6 days of LD entrainment, followed by ~9 days of . Green lines indicate the phase of the dominant activity period in DD. Panel L displays the average Morning activity Phase Offset timepoint, and Panel M displays the average Evening activity Phase Onset (marked by a Blue Arrow) for each genotype over the last two days of entrainment (LD 5–6). The positions of the Red and Blue arrow in panels A-K are representative phase points; panels L and M present their true values respectively. Analyses represent ANOVA followed by Dunnett’s post hoc multiple comparisons of all compared to WT: ns = not significant; * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001.All behavioral records were recorded from (PDF)Click here for additional data file.S7 Fighan (pdfr mutant) flies that expressed either no UAS transgene (A–marked in YELLOW), or a WT pdfr cdNA (B–marked in BLACK) or a variety of Simple pdfr Variants  including 2-3A (C), 4A (D), 5A (E), 6A (F), 7A (G), 5-7A (H), 1-4A (I), 1-5A (J), and 1-6A (K). These measures were averaged across all experiments run for each individual genotype -(L) contains sub-panels (1) through (3), each of which displays Bin-by-Bin analyses of activity levels sorted by 30 min bins, for three different time periods: (1) ZT17-23.5; (2) ZT 0.5–8; (3) ZT 8.5–16. The missing bin at timepoint 0 contains the startle response that accompanies the sudden lights-on signal. Blue asterisks indicate significantly-different activity levels according to a Student’s T-test following an ANOVA (p< 0.05). Red arrows highlight elevated Morning activity levels displayed by different PDFR variants. Blue arrows highlight elevated Evening activity levels produced by different PDFR variants.Behavioral records recorded from (PDF)Click here for additional data file.S8 Fighan (pdfr mutant) flies that expressed either no UAS transgene —yellow), or a WT pdfr cdNA —black) or the PDFR 1-7A Multiple Variant –red). Panels (A-F) present data recorded under Short-Day (winter-like) conditions; panels (I-N) present data recorded under Long Day (summer-like) conditions. Each Panel (A—C) and (I -K) contains four sub-panels: Sub-Panel (1) displays an average daily plot of locomotor activity averaged over the final two days of light entrainment (a group eduction): Open bars indicate the periods of Lights-on and filled bars indicate periods of Lights-off. Sub-Panels 2–4 display Bin-by-Bin analyses of activity levels sorted by 30 min bins, for three different time periods: (1) ZT17-23.5; (2) ZT 0.5–8; (3) ZT 8.5–16. The missing bin at timepoint 0 contains the startle response that accompanies the sudden lights-on signal. Blue asterisks indicate significantly-different activity levels according to a Student’s T-test following an ANOVA (p< 0.05). Panels (D—F) and (L—N) display double-plotted group actograms throughout the 6 days of Light: dark entrainment, followed by 9 days of constant darkness . Panels (G) and (H) display the average Phase Onsets and Offsets (respectively) for the Morning and Evening activity periods for each genotype over the last two days of entrainment under short days (LD 5–6). Blue arrows highlight the elevated Evening activity amplitudes. Panels (O) and (P) display the average Phase Offsets and Onsets (respectively) for the Morning and Evening activity periods for each genotype over the last two days of entrainment under long days (LD 5–6). Red Arrows highlight the elevated amplitude of the Morning peak. Ns–not significant; *—p < 0.05; ***—p < 0.005; ****—p < 0.001.All behavioral records were recorded from (PDF)Click here for additional data file.S9 FighEK-293T cells stably expressing WT PDFR or its variants and transiently expressing CRE-Luciferase. The histogram represents basal levels of 2nd messenger signaling, i.e., in the absence of stimulation by neuropeptide PDF. Values represent the mean +/-SEM of three independent measurements, and were analyzed by Student ‘s T-test: * = p < 0.05; ns = not significantly different.Luciferase measurements in (PDF)Click here for additional data file.S10 FighEK-293T cells stably expressing WT PDFR or its variants and transiently expressing CRE-Luciferase. The histograms represent EC50 values for PDF-stimulated 2nd messenger signaling. Values represent the mean +/-SEM of three independent measurements, and were analyzed by Student ‘s T-test: * = p < 0.05; ns = not significantly different.Luciferase measurements in (PDF)Click here for additional data file.S11 FighEK-293T cells stably expressing WT PDFR or its variants and transiently expressing CRE-Luciferase. The histograms represent the top values achieved for 2nd messenger signaling following PDF-stimulation. Values represent the mean +/-SEM of three independent measurements, and were analyzed by Student ‘s T-test: * = p < 0.05; ns = not significantly different.Luciferase measurements in (PDF)Click here for additional data file.S12 FighEK-293T cells stably expressing WT PDFR or its variants fused to β -lactamase at the N terminus. The histograms represent the basal values for surface receptor expression in the absence of stimulation by neuropeptide PDF. Values represent the mean +/-SEM of three independent measurements, and were analyzed by Student ‘s T-test: * = p < 0.05; ns = not significantly different.β -Lactamase activity measurements in (PDF)Click here for additional data file.S13 FighEK-293T cells stably expressing WT PDFR or its variants fused to β -lactamase at the N terminus. The histograms represent the values for surface receptor expression 20 m after exposure to neuropeptide PDF. Values represent the mean +/-SEM of three independent measurements, and were analyzed by Student ‘s T-test: * = p < 0.05; ns = not significantly different.β-Lactamase activity measurements in (PDF)Click here for additional data file.S14 FigPDFR-Tandem construct from eight independent immunoprecipitation experiments.Nine spectra of phosphopeptides derived from a UAS-(PDF)Click here for additional data file.S15 FigGprk1; Gprk2 and β-arr2(kurtz)) using tim(UAS)-Gal4. The Upper panels (D and F) present over-expression experiments. The Middle (E and G) and Lower panels (H and I) present RNAi experiments. UAS-RNAi constructs illustrated in panels E and G were created and shared by the Paul Hardin laboratory. Values for N and n are found in Group eductions of locomotor activity profiles for different genotypes averaged over six days of light entrainment. Open bars indicate periods of Lights-On and filled bars indicate periods of Lights-Off. The column present manipulations for each of four different gene targets Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Bacillus subtilis.In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions.On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.The online version contains supplementary material available at 10.1186/s12915-022-01278-3. Propagation of genomes between generations requires timely and accurate duplication of DNA by well-known  regulatory mechanisms that control the activity of replication initiators and origins , 2. HoweoriC regulators, as replication still responds to metabolism in cells lacking such regulators - at m/z 333.1303 (5 ppm accuracy). B. Zoom on the mass spectrum of legionaminic acid in the negative mode. C. Collision Induced dissociation (CID) spectrum of legionaminic acid in the negative mode at 22% Normalized Collision Energy (NCE). D. CID spectrum of legionaminic acid in the positive mode at 22% NCE. E and F. Zoom on the mass spectrum of the deuterated forms of legionaminic acid in the negative and positive ionization mode, respectively. G and H. Comparison of legionaminic acid (G) and CMP-legionaminic (H) acid contents in wild-type (WT), ΔspsE and ΔspsF cells, respectively. Data correspond to 3 independent extractions (liquid cultures). ***, p < 0.001; **, p < 0.01 (Welch's T-test). Fig. S5. Representative cell cycle results in Cat and PEPut mutants. Top raw: Microscopy of exponentially growing cells stained with FM4-64  and DAPI . Middle raw: Representative runout DNA histograms (experiments were reiterated 3-12 times). Bottom raw: Representative marker frequency analysis along the right arm of the chromosome (experiments were reiterated at least three times). Fig. S6. Cell cycle parameters of wild-type and pykAT>D cells grown in proline and malate, respectively. Left panel: Growth in malate of wild-type (WT), ΔpykA (Δ), pykAT>D (T>D) and other pykA mutants . Right panel: Runout DNA histograms and cell cycle parameters. Fig. S7. PykA-mCherryBSU localization. Strains deleted for the natural pykA gene and encoding the PykA-mCherryBSU fusion from an inducible promoter (Physpank) were grown in MC and microscopy analysis was carried out at OD600nm = 0.1 to 0.2. Top raw: analysis of the mCherryBSU signal produced at different IPTG concentrations. Bottom raw: analysis of cells grown in the absence of IPTG, fixed in a 1x PBS solution supplemented with 1% paraformaldehyde and stained with DAPI. Scale bar: 4 μm. Similar results were obtained with fusions mutated in the Cat or PEPut domain of PykA. Fig. S8. PykA purification and characterization of its function and oligomeric state. A. SDS-PAGE (15% polyacrylamide gel) showing over-expression of the 6His-MBP-PykA in Rosetta (DE3) E. coli. The soluble expressed tagged PykA protein is shown in a red rectangular in lane 4, whereas lanes M, 1, 2 and 3 show molecular weight standards, the control uninduced insoluble fraction, the control uninduced soluble fraction and the IPTG-induced insoluble fraction, respectively. B.1. SDS-PAGE (15% polyacrylamide gel) showing fractions from the first IMAC purification step of PykA. From left to right, lanes represent molecular weight standards (M), the flow-through (1), the eluted tagged PykA (2), the overnight TEV treated tagged PykA (3), the flow-through fractions containing untagged PykA from the second IMAC step after TEV proteolysis (4-10). B.2. SDS-PAGE (15% polyacrylamide gel) showing the final gel filtration column (HiLoad 26/60 Superdex 200 Prep Grade Gel Filtration Column). From left to right, lanes represent molecular weight standards (M) and fractions of the size exclusion chromatography (1-9). C. The graph shows a Hill plot for the activity of PykA at 25°C. The Rate/(Vmax-Rate) (Y-axis) was plotted against the PEP substrate concentration (X-axis) using GraphPad Prism 4 software and the Vmax (19.3 μmol/min), Km (2.7 mM) and the Hill coefficient n (0.8111) values are shown below the graph. The n value is <1 indicating negative cooperative binding of PykA to its PEP substrate. D. The graph shows a Michaelis-Menten plot for the activity of PykA at 25°C. The initial rate of the reaction (Y-axis) was plotted against the PEP substrate concentration (X-axis) using GraphPad Prism 4 software and the Vmax (16.3 μmol/min) and Km (1.7 mM) values are shown below the graph. E. A native mass spectrum showing the PykA tetramer and miniscule amounts of the dimer and monomer. The theoretical mass of the PykA monomer , dimer  and tetramer . Native mass spectrometry showed that PykA was found to be predominantly tetrameric , with very low abundance dimer  and monomer  peaks. F. Collision induced dissociation of the 33+ charge state of the tetramer shows it to be very stable in the gas-phase, with no apparent dissociation to lower-order oligomers. G. Comparative analytical gel filtration of the PykA tetramer against molecular weight standards  through a Superdex 200 10/300 GL prepacked Tricorn gel filtration column . H. Selectivity trendline constructed from the molecular weight standards (shown in graph G) for the estimation of the PykA MW. The x axis is in logarithmic scale. Graphpad was used for plotting the data points. The theoretical value of our PykA  is close to the estimated  which along with the MS data verifies the tetramer in solution. Kd is the equilibrium distribution coefficient. The numbers (1-5) on the data points correspond to the proteins shown in graph G. Fig. S9. Stimulation of DnaE activity by PykA but not by BSA. A. Primer extension assays monitoring the extension of a 5′-32P-radioactively labelled 60mer DNA primer annealed onto M13 ssDNA over time by the B. subtilis DnaE. The activity of DnaE polymerase (10 nM) was monitored in the presence and absence of PykA  through a time course (30-150 sec). Lanes in the gels from left to right indicate: (M): DNA-ladder and then the time course  depicted by the rectangular triangle. B. Primer extension assays as above with or without 10 nM (monomer) BSA instead of PykA. C. DnaE (1 nM) polymerase activity at increasing BSA concentrations , as indicated by the rectangular triangle, monitored by alkaline agarose electrophoresis. The DNA substrate is a labelled 20mer (5′-CAGTGCCAAGCTTGCATGCC-3′) primer annealed onto ssM13 ssDNA (2nM). The primer extension reaction was carried out for a longer time than above (5 min instead of 30-150 sec) and the film was over-exposed to compensate for the lower DnaE concentration. The assay was carried out at 37 °C in 50 mM Tris-HCl 7.5, 50 mM NaCl, 10 mM MgCl2 mM DTT, 1 mM dNTPs. No stimulation of the DnaE polymerase activity was observed in the presence of 5 and 50 nM BSA. The marginal stimulation observed at 500 nM BSA excess is likely because at this high concentration, BSA acts as a blocking agent preventing adhesion of DnaE to the plastic reaction tubes. Fig. S10. Stimulation of DnaE activity by PykA does not result from stimulation of DnaE binding to primed templates. EMSA investigation of the effect of PykA on the DNA binding of DnaE polymerase. The DNA substrate was constructed by annealing a 5′-32P-radioactively labelled 15mer (5′-AAGGGGGTGTGTGTG-3′) primer annealed onto a 30mer (5′-ACACACACACACACACACACACACCCCCTT-3′) oligonucleotide. Binding reactions were carried out with 1 nM DNA substrate, DnaE (500nM) and increasing concentrations  of PykA, as indicated by the rectangular triangle for 10 min at 37°C in 50 mM NaCl, 10 mM MgCl2, 50 mM Tris-HCl pH 7.5. Lanes C and PykA represent the radioactive substrate in the absence of any proteins and in the presence of PykA , respectively, showing that PykA does not bind to the DNA substrate. No stimulation of DnaE binding to DNA was observed in the presence of increasing concentrations of PykA indicating that PykA does not enhance the DNA binding activity of DnaE. Fig. S11. PEPut purification. A. SDS-PAGE showing overexpression of the His-MBP tagged PEPut in Rosetta (DE3) E. coli. From left to right, lanes show protein MW markers (M), the insoluble uninduced (1), soluble uninduced (2), insoluble induced (3) and soluble induced (4) fractions. The expressed soluble His-MBP tagged PEPut is shown by a red rectangular. B. SDS-PAGE showing the final purified untagged PEPut after removal of the His-MBP tag with TEV proteolysis. Lanes from left to right show protein MW markers (M) and fractions from the flow through the HisTrap column containing the pure untagged PEPut . Fig. S12. PEPut does not stimulate DnaE activity. Primer extension time course  assays using a primed DNA substrate (133 pM) constructed by annealing a radioactively labelled 5′-32P 15mer primer (5′-AAGGGGGTGTGTGTG-3′) onto a 110mer oligonucleotide (5′-CACACACACACACACACACACACACACACACACACACACACACACACACACACACACACCCCTTTAAAAAAAAAAAAAAAAGCCAAAAGCAGTGCCAAGCTTGCATGCC-3′), at suboptimal 25 pM DnaE concentration (left), in the presence of 25 pM PykA tetramer (middle) and 25 pM PEPut domain monomer (right). At this suboptimal DnaE concentration, there is no detectable DnaE primer extension activity in the absence of PykA but clear activity is visible in the presence of PykA. By comparison, no DnaE activity is detectable in the presence of the purified PEPut domain. These data show that full length PykA stimulates the DnaE activity while the PEPut domain alone does not.Additional file 2: Tables S1-S4.


In this study, we searched for the origin of Ex4 and investigated the function of the CDS of this exon. We found that the Ex4-CDS is derived from a noncoding portion of the hAT-10 family of DNA transposon. Evolutionary analysis of transposons and determination of the Ex4 sequences from three other species indicated that Ex4 was generated before the diversification of most or all extant allotetraploid species in subgenus Xenopus, during which time we hypothesize that transposase activity of this hAT superfamily was active. Using DNA–protein binding and transfection assays, we further demonstrate that the Ex4-encoded amino acid sequence increases the DNA-binding ability and transrepression activity of DM-W. These findings suggest that the conversion of the noncoding transposon sequence to the CDS of dm-W contributed to neofunctionalization of a new chimeric SDG in the ancestor of the allotetraploid Xenopus species, offering new insights into de novo origin and functional evolution of chimerical genes.Most vertebrate sex-determining genes (SDGs) emerge as neofunctionalized genes through duplication and/or mutation of ancestral genes that are involved with sexual differentiation. We previously demonstrated  Sry (Sex-determining region of Y) evolved through allelic divergence from Sox3 (Sry-box 3) in the ancestor of therian mammals  in ancestors of the teleost fish medaka and the frogs of the subgenus Xenopus, respectively (Sox3 and dmrt1 encode transcription factors with two different DNA-binding domains—HMG (high mobility group) and DM domains, respectively. The SDGs dmy, dm-W, and Sry each have higher substitution rates than their respective paralogous or gametologous genes, dmrt1, dmrt1, and Sox3  evolved independently many times . Most SD mammals , and dmyectively . Sox3 anand Sox3 . Dmrt1, Xenopus, an allopolyploidization event between two closely related diploid species generated the tetraploid ancestor of all species with 36 chromosomes; subsequent allopolyploidization events generated octoploid (72 chromosome) and dodecaploid (108 chromosomes) species (reviewed in Xenopus is estimated to have occurred around 17–18 Ma (million years ago) and resulted in two “subgenomes”—L and S—that are respectively derived from each diploid ancestor (dm-W evolved from the S subgenome homeolog of dmrt1 (dmrt1.S) and is carried by several species in subgenus Xenopus including highly diverged species pairs such as Xenopus clivii and X. laevis (dmrt1.S) and its genomic context (in subgenome L) coupled with its phylogenetic distribution in many species in subgenus Xenopus argues that this gene became established in its current location after allotetraploidization but before the diversification of extant allotetraploids in subgenus Xenopus  became juxtaposed before allopolyploidization in the diploid ancestor of subgenome S and then later tandemly translocated to subgenome L after allotetraploidization, or whether juxtaposition of these components occurred after translocation of Ex2 + Ex3 to subgenome L in the allotetraploid ancestor of subgenus Xenopus.Early during evolution of the subgenus ancestor   or Rag (which arose from retrotransposons and DNA transposons)  are able to change genomic locations or generate new copies in of themselves, and are generally considered to not benefit the host genome . However, TEs may also influence genome architecture, gene expression, and gene content in beneficial ways . For exailencing . Severalsposons) . TEs aresposons) . Howeverfrom TEs , and thifrom TEs . Not surTEs  superfamily. We then performed in vitro and cotransfection assays that demonstrate that the Ex4-derived amino acid sequences both increase the DNA-binding ability of DM-W and the transrepression activity of DM-W on DMRT1. These findings indicate that the evolutionary origin and neofunctionalization of dm-W was achieved by a combination of partial gene duplication of dmrt1 coupled with the recruitment of a non-CDS transposon sequence into a functionally important component of the CDS of dm-W.As discussed above, dm-W, we first compared the genomic sequences of X. laevis dm-W, dmrt1.S, dmrt1.L, and X. tropicalis dmrt1, which were obtained from the X. laevis genome v9.2 and X. tropicalis genome v10.0 (http://www.xenbase.org/entry/). Although X. laevis has an allotetraploid genome consisting of L and S subgenomes, X. tropicalis has a diploid genome whose ancestor diverged from the ancestor of subgenus Xenopus before dm-W originated  was duplicated into one of the two ancestral chromosome 2L. In addition, we estimated a phylogenetic tree of these genes using the homologous sequences of the dm-W’s introns 1–3 in addition to Ex2 and Ex3. We confirmed that dm-W is more closely to dmrt1.S, not dmrt1.L, with higher confidence values  (dm-W-containing region (69.1%) than in the dmrt1.L or dmrt1.S-containing region  as indicated by the red–blue gradient in dm-W , region B , and region C (108 bp of the 3′ portion of Ex4 and 207 bp of the 3′-flanking region downstream of Ex4)  with X. tropicalis hAT-10 (fig. 1B). We then searched for similar nucleotide sequences to these regions containing the CDS of dm-W Ex4 in the X. laevis genome using BLAST, and found four sequences on scaffolds 19, 30, chromosome 2L, and chromosome 7L. All included hAT transposon fragments that had higher sequence similarities with the regions A and B of dm-W  than with X. tropicalis hAT-10 . Therefore, we conclude that the CDS of dm-W Ex4 evolved from a non-CDS of a hAT-10 transposon to now encode the C-terminal region of the DM-W protein.We previously reported the accumulation of TEs in W-specific regions of . laevis . We examdex.php) . There i in dm-W  online. no acids . We founhAT-10 using MUSCLE .To further clarify how the transposon-derived non-CDS evolved into CDS of Ex4, we performed sequence alignment among the sequences around the Ex4-coding region and its corresponding sequences from the four Ex4-like sequences derived from hAT-10 transposons from dm-W Ex4-related sequences, we blasted the hAT-10 sequence in 14 vertebrate genomes, including eight amphibian species (A). All the vertebrate genomes examined had at least one copy of a X. tropicalis (Xt) hAT-10 transposase-like sequence. The caecilian and six nonamphibian species contained far fewer copies, including host–transposase fusion genes . Notably, 9 and 23 copies of the Ex4-CDS-like sequence corresponding to region B were detected in the two allotetraploid species X. laevis and X. borealis, respectively, whereas no copies were found in the diploid species X. tropicalis. As well, ∼2–3 times as many sequences similar to regions A and C were identified in X. laevis and X. borealis compared with X. tropicalis (A). Moreover, in the other allotetraploid X. borealis, we found six sequences of the hAT-10-derived transposon fragments sharing some sequence similarities with the three regions, A, B, and C of dm-W    online, hAT transposon superfamily in Xenopus, we constructed repeat landscape of the hAT superfamily consisting of hAT-Tp100, hAT-Charlie, hAT-Blackjack, hAT-Ac, and hAT, using X. laevis genome database and RepeatMasker (B). The activation peak of the hAT superfamily was observed around 0.05 substitutions per site. The nucleotide substitution rate in Xenopus was estimated to be 3.0 × 10−9 to 3.2 × 10−9 substitutions per year . The Ex4-CDSs in the three species were highly conserved with that of X. laevis and had two of the same mutations consisting of one nucleotide deletion and substitution as those of X. laevis (C and 3A), suggesting that dm-W Ex4 in these species are homologous. Interestingly, the Ex4-CDSs from X. itombwensis and X. largeni or X. petersii encoded 43 or 67 amino acids, whereas X. laevis contained 71 amino acids (B). These differences are caused by a single nucleotide mutation that generated a UAG stop codon in the upstream region of an ancestor of X. itombwensis and X. largeni and a frameshift mutation caused by the deletion of 16 nucleotides in the ancestor of X. petersii (A). Exons 2 and 3 of dm-W were present in the ancestor of all species in subgenus Xenopus   , 2019.hAT-10-derived Ex4 encodes the C-terminal region of DM-W, which consists of 71 amino acid residues in X. laevis. To clarify the function of this portion of the DM-W peptide, we examined the effect of the region on the DNA-binding ability of the DM domain encoded by Ex2 and Ex3. An in vitro DNA–protein binding assay by Electrophoretic Mobility Shift Assay (EMSA) using 30 bp of DM-W/DMRT1-binding DNA sequence . The amount of each protein, which was produced by in vitro transcription/translation, was quantified using western blot analysis . The assay showed that the full-length DM-W had a stronger binding ability to its binding sequence than the truncated DM-W peptide (Δ124–194) (A). These results indicate that the Ex4-encoding 71 amino acid sequence increases the DNA-binding affinity of DM-W to a DM-W/DMRT1-binding DNA sequence, which is consistent with previous assays  A. These s assays .B). In other words, the hAT-10 transposon-derived C-terminal region increases the transrepression activity of DMRT1.S by DM-W, compared with a truncated version of DM-W that lacks the hAT-10 transposon-derived C-terminal region. Collectively, these findings indicate that the hAT-10 transposon-derived Ex4 may enhance the DNA-binding ability of DM-W to DM-W/DMRT1-binding cis-elements.We next examined the effects of full-length and C-terminal truncated DM-W proteins on the transcriptional activity of DMRT1 using a luciferase reporter plasmid carrying four copies of the DM-W/DMRT1-binding cis-element, which were used for the in vitro DNA–protein binding assay. HEK 293 T-cells were transiently cotransfected with the reporter plasmid and expression plasmids for DMRT1.S, DM-W (full length), and/or truncated DM-W (Δ124–194). The reporter assay showed that DMRT1-driven transactivation activity was more repressed by DM-W (full length) than the truncated DM-W (Δ124–194) B. In othXenopus dm-W and medaka fish dmy independently evolved from a broadly conserved, male-related transcription factor dmrt1 into a male repressor and male inducer, respectively , which includes a degron that regulates protein degradation rates  and one gametolog-specific genes, respectively, although apart from dm-W we do not know their function  sex chromosomes more frequently undergo sex chromosome turnover via the origin of new SDGs than do differentiated (heteromorphic) sex chromosomes . Xenopusomosomes . Recentlsequence . We alsofunction . In thison . A less parsimonious possibility is that some or all of these three components were assembled in the diploid ancestor of subgenome S  and then translocated as a unit to subgenome L, with additional components being added there if the transferred gene was partial. The noncoding Ex1 were generated for promoter/enhancer for dm-W expression in gonadal somatic cells during sex determination. Ex1 may have arisen from an insertion DNA containing a repeated sequence, and then the promoter appeared to have evolved de novo. Ex2 and Ex3 evolved from the duplicate of the region covering Ex2, intron 3, and Ex3 of the S subgenome-derived ancestral dmrt1.S, which had been inserted into the ancestral chromosome 2L of the L subgenome probably through TEs  has independently evolved sex chromosomes . Comparative analysis of the dmrt1 subfamily was performed using mVISTA (http://genome.lbl.gov/vista) using the LAGAN alignment program . Nucleotide sequences were aligned using MUSCLE  (www.repeatmasker.org/) with default parameters and the Xenopus TE dataset obtained from Repbase (www.girinst.org/repbase/). DNA transposon hAT-10-derived sequences, including Xt hAT-10 transposase-like, Ex4-like, and Ex4-CDS-like sequences , BLASTN , and BLASTN , respectively. The values were obtained after removing duplicate fragments of 10 kb (Xt hAT-10 transposase-like), 1 kb (Ex4-like), and 150 bp (Ex4-CDS-like).TEs were detected using CENSOR software (dex.php)  and Repeequences  online, Xl hAT-10, which was determined by collecting hAT-10-like sequences among the existing transposons in the X. laevis genome sequence and constructing them using a simple majority rule based on a multiple alignment by BlastViewer. The age of TE families can be roughly estimated from the distances between each sequence and the consensus  under the conditions of  and female or male whole genome sequence, SRR6357672 or SRR6357673, respectively. The two resulting assemblies were merged, and reference-guided scaffolding was performed with RaGOO , X. itombwensis (MCZ-A A136197), and X. petersii, using the phenol–chloroform extraction method. PCR was performed using KOD FX DNA Polymerase . A pair of primers, 5′-AGTTACATTACACCTCATCCTG-3′ and 5′-AGACGAGGAGTGTTATCCCTC-3′, were used for amplification of Ex4. The obtained DNA fragments were inserted into EcoRV site of pBluescript KS (+). DNA sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit . Gene bank accession numbers for the Ex4 sequences from X. largeni, X. itombwensis, and X. petersii are LC699248/LC699249, LC699247, and LC699250, respectively.Genomic DNA was purified from adult livers of three dm-W from 1 to 123 amino acids was amplified from cDNA by PCR using pcDNA3-FLAG-DM-W , 1 mM spermidine) and incubated on ice for 30 min. The samples were subjected to electrophoresis through a nondenaturing 5% polyacrylamide gel containing Tris/glycine/EDTA  and 2.5% glycerol in Tris/glycine/EDTA at room temperature. The dried gel was autoradiographed with a Fuji super RX film (Fujifilm) at −70°C.The two oligonucleotides, 5′-CCATCGAGCAACAATGTATCAAATCTC-3′ and 5′-GGGGAGATTTGATACATTGTTGCATCGATGG-3′, were annealed and labeled with 32P, using the Klenow fragment. Proteins were produced using the TNT Quick Coupled Transcription/Translation System (Promega), using pcDNA3-FLAG-DMRT1, pcDNA3-FLAG-DM-W , and pcDImmunoblotting of in vitro synthesized proteins was performed using an anti-FLAG antibody, M5 (Sigma), followed by an HRP-conjugated antimouse antibody (Sigma). The reaction was developed by enhanced chemiluminescent staining using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce), and the signal intensity was measured using ImageJ .5 cells per well in a 24-well plate. The cells were transfected with the firefly luciferase reporter plasmid p4xDMRT1-luc (100 ng) (Renilla luciferase vector pRL-SV40 (20 ng) (Promega) using TransITTM-LT1 (Mirus). Total DNA was maintained at 500 ng per transfection with the pcDNA3-FLAG empty vector. After 24 h, activities from the two luciferases, which have dissimilar enzyme structures and substrate requirements, were measured in a Luminocounter 700 (NITI-ON) using the dual luciferase assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.HEK293T cells were cultured in DMEM containing 10% fetal calf serum. Twenty-four hours before transfection, cells were plated at 1 × 10(100 ng) , effectomsac138_Supplementary_DataClick here for additional data file.


While this infection is predominantly caused by Gram positive bacteria, some patients are at high risk for intestinal-derived Gram negative bacteria. However, current diagnostic tools require invasive sampling with substantial risks to the patient. Moreover, conventional imaging, including computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-D-sorbitol (18F-FDS) is the first imaging modality specific for a bacterial class. 18F-FDS accumulates selectively in Enterobacterales, but not in Gram positive bacteria or mammalian cells and was safe in phase I-II clinical studies. Here, we developed a spinal infection model utilizing a previously described posterior-approach to implant a titanium Kirschner wire into the L4 spinous process in mice. We compared 18F-FDS with 18F-FDG PET/CT in mice with spinal implants infected with Staphylococcus aureus , Escherichia coli  or without infection .PET/CT with 2-deoxy-2-[Surgical technique was adapted from Dworky et al., . Briefly, a 2 cm midline incision was made in the skin and the L4 spinous process was exposed. An orthopedic-grade titanium Kirschner wire (diameter 0.1 mm) was surgically placed into the L4 spinous process and placed lengthwise along the spine. A bioluminescent S. aureus  in 10 μL) or phosphate-buffered saline (PBS) were pipetted onto the implant. The model was performed with S. aureus (n=5 mice) or PBS (n=1 mouse) and mice were followed for 14 days for in vivo bioluminescent imaging (BLI) before they were sacrificed for ex vivo CFU enumeration. (A) Computed tomography (CT) of the mouse spine indicating the implant in red. (B) Representative in vivo S. aureus BLI signals on a color scale overlaid on top of a grayscale image of the backs of the mice. (C) Mean in vivo BLI . (D-E) On post-operative day 14 the implants were removed and sonicated, and the infected vertebra with surrounding soft tissue were harvested and homogenized. Mean ex vivo CFUs ± s.e.m from tissue and implants are shown (n=5).Similar surgical technique was used as before  and the model was performed with E. coli  or PBS (n=3 mouse). Mice were followed for 14 days before they were sacrificed for ex vivo CFU enumeration. An additional group of mice infected with E. coli (n=4) received antibiotic treatment  before sacrifice. (A) Representative dorsal skin images showing the surgical site. Note the development of skin wounds around the surgical site in the E. coli infected, but not the treated mice. Scale bars: 1 cm. (B-C) On post-operative day 14 the implants were removed and sonicated, and the infected vertebra with surrounding soft tissue were harvested and homogenized. Mean ex vivo CFUs ± s.e.m from tissue and implants are shown. (D) Bone remodeling was evaluated using μCT to measure bone density in Hounsfield units (HU). The mean HU of bone (defined as HU>700) is presented for mice with PBS (n=3 mice and 5 scans), mice imaged on day of surgery , S. aureus , E. coli  and E. coli treated with ciprofloxacin . Note the lower HU for infected animals (S. aureus or E. coli) compared to non-infected (PBS or before infection). Five days of treatment with ciprofloxacin were not sufficient to mitigate bone remodeling in this model. P values are indicated as shown by the 2-tailed Mann-Whitney test (B-C) or the Kruskal-Wallis test adjusted for multiple comparisons to preserve the desired false discovery rate (D).18F-FDS PET/CT could specifically detect implant-associated spinal infections due to E. coli with mean target-to-non-target standard uptake value (SUV) ratio of 9.2 ± 1.5, which was substantially lower in S. aureus (3.0 ± 0.4) and uninfected mice . In contrast, 18F-FDG could not differentiate between the two bacterial infections or the controls . Finally, 18F-FDS monitored the efficacy of antibiotic treatment, demonstrating a signal proportional to bacterial burden .Both bacteria induced substantial bone pathology with reduced bone density . The mouse model was performed with either E. coli (n=15), S. aureus (n=5) or PBS (n=3) and mice were imaged with 18F-FDS PET/CT imaging. (A) Representative images of 18F-FDS PET/CT. (B) Regions of interest (ROIs) were drawn around the implant (Infected target) and in the heart (uninfected non-target). Standard uptake values (SUVs) are presented as ratios between infected and non-infected ROIs. P values are indicated as shown by the Kruskal-Wallis test adjusted for multiple comparisons to preserve the desired false discovery rate.Same mice used for 18F-FDS imaging  were imaged with 18F-FDG PET/CT as follows: E. coli (n=13), S. aureus (n=5) or PBS (n=2). (A) Representative images of 18F-FDG PET/CT. (B) Regions of interest (ROIs) were drawn around the implant (Infected target) and in the liver (uninfected non-target). Standard uptake values (SUVs) are presented as ratios between infected and non-infected ROIs. P values are indicated as shown by the Kruskal-Wallis test adjusted for multiple comparisons to preserve the desired false discovery rate.The mouse implant-associated spinal infection model was performed with E. coli (n=4) and mice were imaged with 18F-FDS PET/CT before and after completion of antibiotic treatment (IP ciprofloxacin 20mg/kg/dose every 12 hours for 5 days). One day later, mice were sacrificed and the infected vertebra with surrounding soft tissue were harvested and homogenized for ex vivo enumeration of CFUs. (A) Representative images of 18F-FDS PET/CT. (B) ROIs were drawn around the implant (Infected target) and in the heart (uninfected non-target). SUVs are presented as ratios between infected and non-infected ROIs and mean ex vivo CFUs ± s.e.m from tissue are shown, before and after antibiotic treatment. P = 0.014 for the difference in CFU counts and P = 0.032 for the difference in SUV ratios, before and after antibiotic treatment as shown by the one-tailed Mann-Whitney test. (C) The mouse implant-associated spine infection model was performed with E. coli or S. aureus and mice were imaged with 18F-FDS PET/CT at days 1, 7 or 14 following infection (n=4-10 mice/time point). P value is indicated as shown by the two-way analysis of variance (ANOVA) test.18F-FDS PET/CT rapidly and specifically detected E.coli implant-associated spinal infection. We are currently conducting a clinical study to evaluate 18F-FDS PET/CT for specific detection of Enterobacterales implant-associated infection which may reduce the need for surgical interventions.In this preclinical study, Nathan Archer, PhD, Janssen: Advisor/Consultant|Pfizer: Grant/Research Support Sanjay K. Jain, MD, Fujirebio Diagnostics, Inc., USA: Grant/Research Support|Novobiotic LLC, USA: Grant/Research Support|T3 Pharma, Switzerland: Grant/Research Support.


MET exon 14 skipping mutations (METex14). However, the emergence of acquired resistance to capmatinib/tepotinib is almost inevitable partially due to D1228X or Y1230X secondary mutations of the MET. In this study, we explored agents that are active against both D1228X and Y1230X MET to propose an ideal sequential treatment after capmatinib/tepotinib treatment failure in NSCLC patients with METex14.Capmatinib and tepotinib are guideline-recommended front-line treatments for non-small-cell lung cancer (NSCLC) patients with METex14 plus MET D1228A/Y secondary mutations. The screen revealed four-candidate type II MET-TKIs . Therefore, we performed further growth inhibitory assays using these four MET-TKIs plus cabozantinib and merestinib in Ba/F3 cells carrying MET D1228A/E/G/H/N/V/Y or Y1230C/D/H/N/S secondary mutations. We also performed analyses using Hs746t cell models carrying METex14  with/without D1228X or Y1230X in vitro and in vivo to confirm the findings. Furthermore, molecular dynamics (MD) simulations were carried out to examine differences in binding between type II MET-TKIs.The inhibitory effects of 300 drugs, including 33 MET-TKIs, were screened in Ba/F3 cells carrying All 6 type II MET-TKIs were active against Y1230X secondary mutations. However, among these 6 agents, only foretinib showed potent activity against D1228X secondary mutations of the MET in the Ba/F3 cell and Hs746t in vitro model and Hs746t in vivo model, and CEP-40783 and altiratinib demonstrated some activity. MD analysis suggested that the long tail of foretinib plays an important role in binding D1228X MET through interaction with a residue at the solvent front (G1163). Tertiary G1163X mutations, together with L1195F/I and F1200I/L, occurred as acquired resistance mechanisms to the second-line treatment foretinib in Ba/F3 cell models.METex14 after campatinib/tepotinib treatment failure by secondary mutations at residue D1228 or Y1230.The type II MET-TKI foretinib may be an appropriate second-line treatment for NSCLCs carrying The online version contains supplementary material available at 10.1186/s13045-022-01299-z. MET exon 14 skipping mutations (METex14). These drugs yielded progression-free survival of 5.4–12.4 months in clinical trials [Two type Ib MET-tyrosine kinase inhibitors (TKIs), capmatinib and tepotinib, were recently approved for the treatment of non-small-cell lung cancer (NSCLC) patients carrying MET-TKIs are usually classified into two types based on their binding mode to MET . Type I Currently approved type Ib MET-TKIs, capmatinib and tepotinib, interact with the residue Y1230 residue; thus, it is reasonable that secondary mutations at Y1230 cause resistance to these drugs. In addition, through analyses of clinical specimens obtained from patients who acquired resistance to type I MET-TKIs, the emergence of secondary mutations at the D1228 position was also found to be frequent after capmatinib/tepotinib treatment failure –13.50 values for type II MET-TKIs than cancer cells with Y1230X through in vitro experiments [As a potential second-line treatment, type II MET-TKIs that bind to the inactive form of MET have been suggested in previous studies to overcome many of the secondary mutations that emerge after front-line capmatinib/tepotinib monotherapy –17. Altheriments , 16, 24.A total of 33 MET-TKIs and other receptor TKIs, multi-kinase inhibitors and cytotoxic inhibitors were evaluated in the present study. A SCADS inhibitor kit was provided by the Screening Committee of Anticancer Drugs supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. In addition, we purchased almost all commercially available MET-TKIs which are under clinical development or were developed clinically in the past. The features of TKIs evaluated in the present study are summarized in the Additional file METex14 and MET amplification, was purchased from the American Type Culture Collection . Gp2-293 packaging cells were purchased from Takara .The interleukin-3 (IL-3) dependent murine pro-B cell line (Ba/F3) and myelomonocytic, macrophage-like, BALB/c mouse leukemia cells (WEHI-3) were provided by the RIKEN Bio Resource Center . WEHI-3 secretes IL-3 into the medium and was thus used as a source of IL-3 for Ba/F3. The human gastric cancer-derived Hs746t cell line carrying 2. Gp2-293 cells and Hs746t cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% PS at 37 °C in an incubator in a humidified atmosphere containing 5% and 10% CO2, respectively.Ba/F3 cells were maintained in RPMI-1640  medium supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin–streptomycin (PS)  at 37 °C in an incubator in a humidified atmosphere containing 5% COMETex14 and MET-TKI-resistant Ba/F3 clones (METex14 plus D1228A/E/G/H/N/Y or Y1230C/D/S/N/H) were established in our previous study [A Ba/F3 clone with us study . These MMETex14 plus one of the secondary mutations (D1228H/N/V/Y or Y1230H) as described previously [METex14 was used as a template, and the retroviral vector constructs encoding METex14 plus point mutations (D1228H/N/V/Y and Y1230H) were generated by a Prime STAR mutagenesis basal kit (Takara). All mutations were confirmed by direct sequencing. The viral particles were generated by co-transfection of these retroviral vector and the pVSV-G vector (Clontech) into Gp2-293 cells and were added to Ba/F3 and Hs746t cells. Ba/F3 cells and Hs746t cells infected with the retrovirus were collected using green fluorescence protein (GFP)-based fluorescence-activated cell sorting by a BD FACS Aria cell sorter .Transfection was applied to Ba/F3 parental cells and Hs746t cells to generate in vitro models of eviously . Briefly3 cells were seeded per well of 96-well plates. After 24 h, MET-TKIs were added at indicated concentrations. After a 72-h incubation, 10 μl of Cell Counting Kit-8 solution  was added to the wells, and the plates were incubated for an additional 3 h. The absorbance at 450 nm was read using a multiplate reader . The percentage of viable cells was evaluated and compared with that in DMSO-treated control samples. The half-maximal inhibitory concentration values (IC50) were determined by fitting a nonlinear regression curve to a variable slope model with normalized response in GraphPad Prism version 8 .A total of 3 × 104 cells were plated in 96-well plates in the presence of foretinib at indicated concentrations. The medium containing individual MET-TKIs was replaced once weekly for three weeks. When cell growth was detected macroscopically, total RNA was isolated from the resistant clones using an RNeasy Plus Mini kit . Then, RNA was converted to cDNA using ReverTra Ace . The MET gene was sequenced from exon 15 to exon 21 using Genetic Analyzers 3130 or 3500XL .MET-TKI-resistant clones were generated by ENU mutagenesis as described previously . Ba/F3 cBa/F3 and Hs746t cells were treated with MET-TKIs at the indicated concentrations for 3 h. The cells were then washed twice with PBS and resuspended in lysis buffer. Lysates were quantified using a BCA protein assay , and proteins were electrophoresed and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed with antibodies purchased from Cell Signaling Technology  according to the manufacturer’s instructions  reached 0.75 cm3, the mice were randomized to three groups and treated once daily with foretinib, cabozantinib, and vehicle via oral gavage. Foretinib and cabozantinib were first dissolved in DMSO and then diluted with ddH2O to a dose of 16.36 mg/kg and 12.3 mg/kg, respectively. The final concentration of DMSO was 2%. The mixture of ddH2O containing 2% DMSO was used as a vehicle. Tumor size and body weight were monitored every two days and twice weekly, respectively, until the average tumor volume reached 2.0 cm3. Statistical analysis was performed using Kruskal–Wallis test with Dunnett’s multiple comparison test.An in vivo study was performed at Kindai University in accordance with the guidelines of the Institutional Animal Care and Use Committee of Kindai University. Five-week-old nude male mice were purchased from CLEA Japan , and 1.0 × 10Formalin-fixed, paraffin-embedded tissue Sects. (4-μm thick) obtained from in vivo experiments were deparaffinized and heated in Antigen Retrieval Citrate Solution, pH 6 . The tissue sections were treated with Protein Block Serum-Free (Dako) and then incubated with a phospho-MET antibody (#3077)  and then Histofine Simple Stain MAX PO(R) . Peroxidase activity was visualized via the DAB reaction using a reagent from Dako. The sections were counterstained with hematoxylin.Using MolDesk Basic ver. 1.1.77 , molecular dynamics (MD) simulations of MET-TKIs and the MET protein were performed. Co-crystal structures of wild type of MET kinase domain in complex with merestinib, foretinib, altiratinib (type II kinase inhibitors) or MET D1228V with foretinib were downloaded from The Protein Data Bank, respectively . The retrieved protein–ligand structures were processed by MolDesk Basic ver. 1.1.77  (1): addition of all hydrogens and deletion of all water molecules for protein. (2) Addition of all hydrogens and partial charge for ligand was performed using MOPAC7 AM1 BCC of MolDesk Basic. A docking pocket of each ligand for c-Met kinase domain was generated specifying the site where the MET inhibitor was originally bound. For foretinib and merestinib, the structurally determined ligands within 6sd9, 4eev were used. For cabozantinib, 3D structure file  was downloaded from PUBchem. Molecular docking simulation of foretinib, cabozantinib and merestinib with MET kinase domain (wild type or D1228V mutant) were carried out and docking score and RMSD were calculated using the sievgene of MolDesk Basic. Ligands (foretinib and merestinib) were used from co-crystal structure model within the above files. PyMOL (ver 2.5.2) was used to draw the 3D structure and to measure the distance between two points.https://pubmed.ncbi.nlm.nih.gov/) was used to review previous publications and summarize the on-target mechanisms of resistance to type I MET-TKIs in NSCLC patients carrying METex14 [PubMed  are summarized in Fig. METex14 plus Y1230X mutations  or Y1230H secondary mutations into Hs746t cells to generate in vitro models with a secondary mutation in a part of the MET protein (with exon 14 skipping). We selected these four variants based on their frequencies in clinical samples  at the Y1230 position using th K1110 , 30, secth K1110 , 31. ThrAs one of the reasons for the different efficacies against D1228X secondary mutations between foretinib and other type II MET-TKIs, in this study, we found that the distances between V1228 and foretinib were larger than the distance between V1228 and cabozantinib/merestinib Fig. . In geneMET [The safety and tolerability of foretinib have been confirmed in two phase I trials (Additional file MET . ConsideIn this study, we explored adequate second-line MET-TKIs that show high activity against secondary mutations that emerge after treatment failure of two approved type Ib MET-TKIs, capmatinib and tepotinib. We showed that many type II MET-TKIs can overcome Y1230X mutations; however, our results suggest that cabozantinib and merestinib, which are currently under clinical development, may be insufficient to overcome resistance conferred by D1228X secondary mutations. On the other hand, foretinib showed potent activity against both D1228X and Y1230X secondary mutations.Additional file 1:Table S1. A list of tyrosine kinase inhibitors used in this study. Table S2. A list of antibodies used in this study. Table S3. Results of drug screening other than tyrosine kinase inhibitors. Table S4. Type II MET-TKIs evaluated in this study. Table S5. Clinical information of two phase I studies of foretinib. Table S6. Clinical information of five phase II studies of foretinib.Additional file 2: Fig. S1. Drug screening against MET D1228A/Y using a drug library. Fig. S2. Summary of growth inhibitory curves of eight MET-TKIs for Ba/F3 cells with each MET mutation. Fig. S3. Evaluation of the effects of secondary mutations on sensitivity to MET-TKIs using the Hs746t cell line. Fig. S4. Analysis of the secondary resistance mutations against 1st line foretinib. Fig. S5. Growth inhibition curves for ENU-derived foretinib resistant Ba/F3 cells carrying MET secondary mutation. Fig. S6. Comparison of the structures of each type II MET-TKI. Fig. S7. Summary of growth inhibitory curves of three MET-TKIs for Ba/F3 cells with MET tertiary mutations. Fig. S8. Structure of MET in the DFG-in and DFG-out conformations.


While tremendous efforts have been dedicated to developing cellulose-based ultraviolet (UV)-blocking films, challenges still remain in simultaneously achieving high transparency, low haze and excellent UV shielding properties via simple and green strategy. Here, we present a facile and eco-friendly route to fabricate flexible, biodegradable and clear UV-shielding nano-MIL-88A(Fe)@carboxymethylated cellulose films (M(Fe)CCFs) via in situ synthesis of nano-MIL-88A(Fe) in carboxymethylated cellulose hydrogel followed by natural drying. The carboxymethylated cellulose film has high transmittance (93.2%) and low haze (1.8%). The introduction of nano-MIL-88A(Fe) endowed M(Fe)CCFs superior UV-shielding ability, while retaining high transmittance (81.5–85.3%) and low haze (2.5–4.9%). Moreover, M(Fe)CCFs showed stable UV blocking performance under UV irradiation, high temperature, acidic or alkaline conditions. Quite encouragingly, the UV-shielding ability of M(Fe)CCFs did not deteriorate, even after 30 days of immersion in aqueous solution, providing films with a long-term use capacity. Thus, M(Fe)CCFs show high potential in the UV protection field. Overall, these UV-blocking films with outstanding performances are a promising candidate to replace conventional film materials made from synthetic polymers in fields such as packaging and flexible electronics. In recent decades, climate change and environmental pollution caused by human activities have resulted in serious damage to the earth’s ozone layer, and the amount of ultraviolet (UV) light reaching the Earth’s surface has increased significantly, causing a series of issues, i.e., sunburn, skin cancer, yellowing of paper and discoloration of dyes, and other problems associated with UV light ,2,3. For2, ZnO, Al2O3 and CeO2) has become a very important approach to realize UV shielding functions [2, the tunability of the structure allows the design of UV shielding functions to be conferred on the assembled MOFs materials. MOF materials inevitably also have some drawbacks, such as the need for high temperature and pressure synthesis and the use of toxic solvents. In view of these problems, there is an urgent need to find MOF materials with efficient UV shielding capacity that can be synthesized by a simple, room temperature, green method and then used to prepare cellulose-based UV protective films.The modification of cellulosic materials using organic derivatives and inorganic nano/micron oxides , MIL-101(Fe), MIL-53(Fe), MIL-88A(Fe) and MIL-88B(Fe) have been used in various applications due to their non-toxicity, low cost and biocompatibility, easy availability of organic linkers and excellent physicochemical properties [Iron is a transition metal with UV photoprotective properties. Researchers have verified the protective effect of iron against UV radiation . Consideoperties . Recentloperties . Unfortuoperties ,27,28,29In this work, a transparent carboxymethylated cellulose hydrogel platform was prepared from filter paper cellulose fibers. Then, based on this platform, flexible, biodegradable, and UV-blocking nano-MIL-88A(Fe)@carboxymethylated cellulose films (M(Fe)CCFs) were fabricated by in situ synthesis of nano-MIL-88A(Fe) and natural drying. The hypothesis is that nano-MIL-88A(Fe) particles are uniformly distributed in the cellulose matrix and can impart excellent and durable UV-blocking functionality to cellulose films while maintaining high transmittance (>80%) and low haze (<5%). The UV blocking properties under certain harsh environmental conditions such as high temperature, UV irradiation, acidic or alkaline conditions are explored, which may open up wider prospects for the application of composite films. Our proposed methodology is green, easy to operate, efficient, simple both in the post-treatment stage and purification and in line with the principles of green chemistry, as well as sustainable development.3·6H2O) were provided by Fuyu Fine Chemical Co., Ltd. . Sodium chloroacetate was provided by Macklin Biochemical Co., Ltd. .Quantitative filter paper was obtained from Hangzhou Special Paper Co., Ltd. . Lithium hydroxide (LiOH) and urea were purchased from Shanghai Chemical Reagent Co., Ltd. . Sodium hydroxide (NaOH) was purchased from Xiya Chemical Industry Co., Ltd. . Fumaric acid, glycerol and Ferric chloride hydrate (FeClw/v) sodium hydroxide at 50 °C for 2 h. The product (carboxymethylated cellulose fibers) was obtained by filtration and washed with tap water to remove residues until neutral pH. Carboxymethylated cellulose fibers then were dried at 65 °C for subsequent experiment and characterization. The methylene blue method was used to determine the content of the carboxyl group of original and carboxymethylated cellulose fibers [Filter paper was ground to powder using a grinder. Then, 10 g of powdery filter paper was added to a 250 mL sodium chloroacetate solution (1 M) prepared in 15% @carboxymethylated cellulose hydrogels were washed with anhydrous ethanol and distilled water, and then soaked in 5 wt% glycerol solution for 10 min. Finally, the MIL-88A(Fe)@carboxymethylated cellulose hydrogels were fixed to the glass plate to prevent contraction and dried at 25 °C. The MIL-88A(Fe)@carboxymethylated cellulose films (0.12 mm) were obtained, called as M(Fe)CCF1, M(Fe)CCF2 and M(Fe)CCF3. The content of MIL-88A(Fe) in M(Fe)CCFs was determined by weight method. A known weight of M(Fe)CCF was immersed in 50 mL of an aqueous solution containing hydrochloric acid (pH = 1) and sonicated to completely dissolve MIL-88A(Fe). The samples were then removed, washed several times with distilled water and then dried under a vacuum. The weight of the sample before and after was recorded and the weight difference was attributed to the content of MIL-88A(Fe). Pure carboxymethylated cellulosic film  was also prepared.The powdery carboxymethylated cellulose fibers were added to the 4.6 wt% of LiOH/15.0 wt% of aqueous urea medium and then frozen in a −20 °C freezer for 12 h. The mixture was thawed at room temperature and stirred vigorously to form 4 wt% cellulose solution. Subsequently, the cellulose solution was sonicated in an ice water bath for 20 min to remove air bubbles. The de-bubbled cellulose solution was cast on a glass mold, anhydrous ethanol was added, and after regeneration, carboxymethylated cellulose hydrogel (thickness approx. 1.2 mm) was obtained and thoroughly washed with water. A given amount of FeCl3·6H2O  was soluric acid  was solu3+ (CCF-Fe3+) were prepared. Powdery MIL-88A(Fe) was synthesized in the same way as M(Fe)CCFs, except for the addition of carboxymethylated cellulose hydrogel. The synthesis of CCF-Fe3+ was as follows: Carboxymethylated cellulose hydrogels were added into FeCl3 solution  powder and carboxymethylated cellulose film-Fesolution  for 3 h,The effect of certain extreme conditions such as UV exposure, pH or temperature on the stability of UV blocking ability was investigated. M(Fe)CCF1 was placed in the oven at different temperatures (120 °C and 150 °C), or under UV light , or under a solution with different pH values  for 6 h. The UV-Vis transmittance curves were measured.m0 and mt are the masses of M(Fe)CCFs before and after enzymatic degradation tests, respectively.The enzymatic degradation method was used to measure the biodegradability of M(Fe)CCFs . The exp−1. X-ray diffraction (XRD) patterns were collected on a D/max 2200PC X-ray diffractometer . ESCALAB 250Xi  with Al-Kα radiation source was used to conduct an X-ray photoelectron spectroscopy (XPS) test. Prior to FTIR, XRD and XPS tests, all films are ground to a powder using an A11 basic grinder . The surface and cross-sectional morphologies of the films were observed using a scanning electron microscope , and the elemental composition was analyzed using an energy dispersive X-ray spectrometer (EDS). The STA449 F3 equipment was used to perform thermogravimetric analysis (TGA) at a heating rate of 10 °C-min−1 under a nitrogen atmosphere. The UTM2203 universal testing machine  was used to test tensile stress–strain curves according to ISO527-3-1995 (E) at a speed of 2 mm min−1. A UV-Vis spectrophotometer equipped with an integrating sphere  was used to test optical transmittance and haze. The haze was tested and calculated according to GBT 2410-2008.Thermo Scientific Nicolet iS5 FTIR spectrometer  was used to collect Fourier transform infrared (FTIR) spectra in the range of 4000–500 cmThe concept of converting cellulose and nano-MIL-88A(Fe) into a UV-blocking composite film is schematically proposed in 3+ onto the cellulose surface. Subsequently, the carboxymethylated cellulose hydrogel adsorbed with Fe3+ is added to ethanol solution containing fumaric acid, and the Fe3+ adsorbed on the cellulose surface forms nano-MIL-88A(Fe) nuclei with fumaric acid. As the reaction time increases, MIL-88A(Fe) nanoparticles are gradually formed and then anchored on the surface of cellulose and within the nanopores. After natural drying, the MIL-88A(Fe) nanoparticles are encapsulated in the composite film.The interaction between nano-MIL-88A(Fe) and cellulose was analyzed, and the mechanism of the interaction is proposed and shown in The cross-sectional SEM images of M(Fe)CCFs verify the presence of MIL-88A(Fe) nanoparticles in M(Fe)CCFs. As shown in 3 with an ethanolic solution of fumaric acid [The current methods for the synthesis of MIL-88A(Fe) are mostly hydrothermal or solvothermal, both of which require high temperature or organic solvents . Thereforic acid . As show−1 is ascribed to the O-H stretching vibration of the water molecule [−1 and 1603 cm−1 ascribed to the symmetric and asymmetric vibrational modes of the carboxylate group, respectively, showing the presence of the dicarboxylic acid ligand in the MIL-88A(Fe) [−1 is ascribed to the Fe−O vibration [−1 is ascribed to the introduction of carboxyl groups on the backbone of cellulose [−1, 1396 cm−1 and 1603 cm−1 were derived from MIL-88A(Fe), confirming the successful synthesis of MIL-88A(Fe) within M(Fe)CCF3. No significant characteristic peaks for MIL-88A(Fe) were observed in the FTIR spectra of M(Fe)CCF1 and M(Fe)CCF2  and M(Fe)CCF3 are displayed in molecule . The cha-88A(Fe) ,35. The ibration . For CCFellulose . The cha(Fe)CCF2 , which mThe XRD patterns of MIL-88A(Fe), CCF and M(Fe)CCF3 are displayed in 3/2 (711.2 eV) and Fe 2p1/2 (725.01 eV). The fitted peak at 715.66 eV is usually ascribed to the satellite peaks of Fe 2p3/2 and Fe 2p1/2 [3+, demonstrating the presence of Fe3+ in the MIL-88A(Fe) structure. A peak spacing of 13.8 eV exists between the Fe 2p3/2 and Fe 2p1/2 peaks, which is in perfect agreement with previous references on Fe2O3, an important feature of Fe(III) present in M(Fe)CCF3 [The chemical composition of the samples was analyzed by XPS. XPS spectra of MIL-88A(Fe), CCF, M(Fe)CCF1, M(Fe)CCF2 and M(Fe)CCF3 are shown in Fe 2p1/2 . In summ(Fe)CCF3 ,41.The thermal stability of CCF and M(Fe)CCFs was investigated by thermogravimetric analysis (TGA), as shown in The excellent UV-shielding capacity of M(Fe)CCFs was also demonstrated by fluorescence pattern of Paper money. As shown in Due to the variability of the natural environment, the durability of UV shielding films under extreme conditions is of paramount importance. The effect of UV radiation on the UV shielding stability of the films was investigated as shown in 3+ films with UV shielding capability by adsorbing Fe3+ [3+, TOCN-COOFe3+ exhibits excellent UV shielding ability. Unfortunately, the UV-protective film may have low water resistance, as Fe3+ washes off easily in water, resulting in reduced UV shielding properties. In this work, we compared the UV shielding stability of M(Fe)CCF1 with a carboxymethylated film adsorbed with Fe3+ (CCF-Fe3+) in an aqueous environment. As shown in 3+1 and CCF-Fe3+2) adsorbed with low Fe3+ concentration was significantly weakened after 10 days of immersion in water (pH = 6.8), indicating that Fe3+ was gradually released into the solution during the immersion process. In contrast, CCF-Fe3+3 still had a high UV shielding capacity, which was due to the adsorption of carboxyl groups partially adsorbed Fe3+ on the cellulose surface without being released. 3+.A number of UV absorbers have been incorporated into cellulose-based films to obtain UV shielding capability. Yang et al. reported the preparation of highly transparent TOCN-COOFeing Fe3+ . The resAlmost all UV absorbers have a significant impact on the light transmittance and haze of the matrix materials, mainly due to the non-uniform distribution of the UV absorbers in the substrate or their large size and the agglomeration of some small-sized nanoparticles. Interestingly, M(Fe)CCFs not only have superior UV blocking performance but also high transmittance and low haze, which is attributed to the fact that the UV absorber nano-MIL-88A(Fe) is mainly produced in the cellulose hydrogel nanopores and is therefore uniformly distributed. Based on the template effect of the cellulose hydrogel, MIL-88A(Fe) was prepared in a small size. The small size of MIL-88A(Fe) facilitates absorption of UV light and reduces the effect on transmittance and haze. Objects and landscapes viewed through M(Fe)CCFs were also clear, showing that M(Fe)CCFs have low optical haze, greatly extending their range of applications a–d. As s3+ into nanocellulose [3+ significantly reduced the mechanical strength of the films (by 28.50%), which may be due to the disruption of hydrogen bonding by Fe3+, resulting in a decrease in the mechanical strength of the films. However, the mechanical strength of M(Fe)CCFs was slightly reduced. The decrease in mechanical strength of M(Fe)CCFs is attributed to the combination of Fe3+ with the organic ligand fumaric acid to generate MIL-88A(Fe) nanoparticles, thus reducing the disruption of hydrogen bonds. The M(Fe)CCFs are also easy to handle and resistant to bending and folding, indicating that M(Fe)CCFs have excellent flexibility.The stress–strain curves of CCF and M(Fe)CCFs are shown in ellulose . It was Polysaccharide rings linked by glycosidic bonds are readily biodegradable by microorganisms and hydrolytic enzymes. The enzymatic degradation expressed as a percentage weight loss is shown in In this work, a flexible and biodegradable cellulose film with comprehensive properties, including high optical transmittance, low haze, UV blocking, and good mechanical strength, was facilely prepared. First, a transparent carboxymethylated cellulose hydrogel platform was prepared from filter paper cellulose fibers. Subsequently, carboxymethylated cellulose gel acts as a nanoreactor to promote growth and anchorage of nano-MIL-88A(Fe). Finally, clear cellulose films were obtained by natural drying. The uniform distribution of nano-MIL-88A(Fe) in M(Fe)CCFs was confirmed by SEM micrographs. The uniform distribution of nano-MIL-88A(Fe) imparts M(Fe)CCF’s excellent UV shielding performance. The pure CCF has high optical transmittance (93.2%) and low haze (1.8%). The introduction of nano-MIL-88A(Fe) endowed M(Fe)CCF’s superior UV-shielding ability, while retaining high transmittance (81.5–85.3%) and low haze (2.5–4.9%). M(Fe)CCFs showed stable UV blocking performance under UV irradiation, high temperature, acidic or alkaline conditions. Meanwhile, the UV-shielding ability of M(Fe)CCFs did not deteriorate even after 30 days of immersion in aqueous solution, providing films with a long-term use capacity. Compared to films made from synthetic polymers, these films are non-toxic, environmentally friendly and biodegradable, reducing the accumulation of microplastics. These films have potential applications in fields such as packaging and flexible electronics.


This review focuses on the feasibility of combining Bruton’s tyrosine kinase (BTK) inhibitors (BTKis) with chimeric antigen receptor (CAR) T-cell therapy in patients with relapsed or refractory (R/R) mantle cell lymphoma (MCL). Potential scenarios for combination treatment with these agents are presented.BTKis and CAR T-cell therapy have revolutionized the treatment paradigm for R/R MCL. Ibrutinib, acalabrutinib, and zanubrutinib are covalent irreversible BTKis approved for R/R MCL. Brexucabtagene autoleucel was the first CAR T-cell therapy approved for R/R MCL based on findings from the ZUMA-2 trial. There is evidence to suggest that combination treatment with BTKis and CAR T-cell therapy may improve CAR T-cell efficacy.As BTKis and CAR T-cell therapy become mainstays in R/R MCL therapy, combination treatment strategies should be evaluated for their potential benefit in R/R MCL. The pathogenesis of mantle cell lymphoma (MCL) is driven by various mechanisms, including anomalous cell cycle regulation, dysregulation of B-cell receptor (BCR) signaling, molecular and genomic changes, DNA damage, and microenvironmental effects . As BrutFollowing the advent of BTKis, the emergence of CD19-targeted chimeric antigen receptor (CAR) T-cell therapy represents another major advance in R/R MCL therapy. Brexucabtagene autoleucel  was the first approved CD19-targeted CAR T-cell therapy for R/R MCL based on findings from the phase 2 ZUMA-2 trial, which demonstrated an ORR of 93% and CR rate of 67% ••. HowevA subset of patients may not derive an adequate benefit from BTKi or CAR T-cell therapy when administered alone. Patients may experience treatment intolerance, frequently seen with ibrutinib, leading to discontinuation . AdditioP<0.0001) . ••. 12••]Evidence supporting the use of concomitant BTKi and CAR T-cell therapy is mostly limited to studies in CLL, as patients with CLL typically have low rates of CR with CAR T-cell therapy , likely Fan et al. observed that CAR T-cells derived from patients with CLL were less proliferative than those derived from healthy donors, and that stimulation with ibrutinib significantly increased the expansion and viability of CD3+ T-cells from healthy donors and CLL patients . Ibrutinn=3) or refractory follicular lymphoma (n=4), ibrutinib in combination with re-administration of CAR T-cell therapy resulted in a CR rate of 86% (n=6/7) [In another study, patients with R/R CLL previously exposed to ibrutinib received ibrutinib at least 2 weeks before leukapheresis and continued treatment until up to at least 3 months after CAR T-cell infusion . Concomi (n=6/7)  Table 1n=3 or rePreclinical evaluation of the combined effect of BTKi and CAR T-cell administration in MCL demonstrates similar results to those observed in CLL. Evidence from a study in an MCL cell line (JeKo-1) demonstrates that the investigational noncovalent BTKi vecabrutinib, significantly enhanced CAR T-cell cytotoxicity against MCL CD19+ tumor cells . In the TP53 mutations, high Ki67 index, CNS involvement), CAR T-cell therapy may be administered before BTKi therapy  evaluating concomitant BTKi and CAR T-cell therapy approaches in R/R MCL will help determine the potential benefit of the BTKi/CAR T-cell combination in R/R MCL. Furthermore, BTKi/CAR T-cell combinations for MCL should be explored in the frontline setting for high-risk disease. As treatment of R/R MCL continues to rapidly evolve with emerging therapies against ROR1 and other key targets, combination treatment strategies with established and novel agents will improve outcomes in R/R MCL.


G135D) increased TCR signaling and altered antigen recognition in human Jurkat T cells and ex vivo mouse T cells. Here, using a strain of LATG135D knockin mice, we showed that the LATG135D mutation modifies thymic development, causing an increase in the percentage of CD4+CD8+ double-positive cells, and a reduction in the percentage of CD4+ and CD8+ single-positive cells. Interestingly, the LATG135D mutation alters thymic development even in a heterozygous state. In the periphery, the LATG135D mutation reduces the percentage of CD8+ T cells and results in a small increment of γδ T cells. Remarkably, the LATG135D mutation dramatically increases the percentage of central memory CD8+ T cells. Finally, analysis of the proliferation and activation of T lymphocytes shows increased responses of T cells from mutant mice. Altogether, our results reinforce the view that the residue preceding Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation.The LAT transmembrane adaptor is essential to transduce intracellular signals triggered by the TCR. Phosphorylation of its four C-terminal tyrosine residues  recruits several proteins resulting in the assembly of the LAT signalosome. Among those tyrosine residues, the one found at position 136 of mouse LAT plays a critical role for T cell development and activation. The kinetics of phosphorylation of this residue is delayed as compared to the three other C-terminal tyrosines due to a conserved glycine residue found at position 135. Mutation of this glycine into an aspartate residue (denoted LAT The stochastic generation of T-cell antigen receptors (TCRs) allows the production of a wide repertoire of antigen receptors. However, due to the stochastic nature of this process, TCR recognizing self-antigens can be generated, which must be regulated by central or peripheral tolerance mechanisms. During T cell development, T cell precursors rearranging TCR genes must pass several checkpoints in which the quality of the assembled receptors is tested. Accordingly, T cells the TCR of which does not match self-peptide MHC (pMHC) products are eliminated together with those that could recognize self-pMHC products with a too high affinity .The recognition of pMHC ligands by T cells generates a cascade of intracellular signals, and one of the earliest is the phosphorylation of ITAMs present in the intracellular domains of CD3 chains . PhosphoY136F) displays a severe but partial block in T cell maturation, but later develop a polyclonal lymphoproliferative disorder involving CD4+ T cells producing massive amounts of T helper type 2 (TH2) cytokines  plays a dual role in this complex signaling cascade. This is the only residue of LAT able to bind to PLC-γ1, and its mutation to Phe prevents PLC-γ1 activation and calcium concentration increase in response to TCR engagement. A LAT-knockin (LAT-KI) strain of mice in which Tyr136 was mutated to phenylalanine  mice in which glycine 135 was mutated to an aspartate (LATG135D). LATG135D mice showed altered thymic development, as well as abnormal peripheral T-cell populations among which CD8+ T cells showed the strongest phenotype. Interestingly, the LATG135D mutation was found to exhibit a dominant behavior, since heterozygous animals showed a phenotype intermediate between wild-type and homozygous mutant mice. Therefore, our in vivo and ex vivo results reinforce the view that the glycine residue preceding the Tyr136 of LAT constitutes a crucial checkpoint in T cell development and activation.It has been shown that ZAP-70 preferentially phosphorylates the tyrosine residues that are found in the LAT and SLP-76 adaptors and are surrounded by negatively-charged residues . This me® Mouse T-Activator CD3/CD28 reagents were from Invitrogen . Biotin anti-mouse CD3ϵ and streptavidin were from BioLegend.The anti-CD3, -CD4, -CD5, -CD8, -CD24, -CD25, -CD44, -CD62L, -CD69, -CD73, -CCR7, FoxP3, and -FR4 fluorochrome-coupled antibodies, and Annexin V-APC reagent used for cytometry were from BioLegend . Antibodies specific for P-PLC-γ1 (Y783), LAT and β-Actin were from Cell Signaling Technologies . Anti-P-Y132-LAT antibody was from Abcam . P-Erk and PLC-γ1 antibodies were from Santa Cruz Biotechnology . Erk antibody was from BioLegend. CellTrace™ Violet and DynabeadsMice were maintained and used at the local Animal Supply Services . Animal care and handling followed the guidelines of the European Union Council  on the use of laboratory animals. Experimental procedures were approved by the local Animal Care and Ethics Committee  and the Ministry of Agriculture, Fisheries and Rural Development .CGG-3’ (the protospacer adjacent motif (PAM) is shown in italics) found in exon 7 of the Lat gene was synthesized. A single-stranded deoxynucleotide (ssDNA) homology-directed repair (HDR) template was designed to convert the GGC codon found in exon 7 of the Lat gene and corresponding to the glycine residue present at position 135 of LAT into a GAC codon coding for an aspartic acid. The 110 nucleotides-long LatG135D HDR template  was synthesized as a ssDNA and purified using polyacrylamide gel electrophoresis . Fertilized eggs from C57BL/6 female mice were microinjected with Cas9 mRNA and the designed sgRNA and HDR template as described (A chimeric single guide RNA (sgRNA) targeting the sequence 5’-GATGAAGACGACTATCCCAA 7 cells/ml in 2X Laemmli buffer, followed by incubation at 95°C for 5 min and sonication. For Western blotting, whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with the indicated primary antibodies, followed by the appropriate secondary antibody conjugated to IRDye 800CW  or horseradish peroxidase (HRP). Reactive proteins were visualized using the Odyssey CLx Infrared Imaging System (Li-Cor) or by enhanced chemiluminescence (ECL) acquired in a ChemiDoc Touch Imaging System (Bio-Rad Laboratories). For reprobing, PVDF membranes were incubated for 10 minutes at room temperature with WB Stripping Solution , followed by a TTBS wash. Western blots were densitometrically quantified, and statistics were performed with Microsoft Excel using a two-tailed t-test.Thymocytes from wild-type or mutant mice were obtained and incubated with 5 µg/ml of biotin-coupled anti-CD3 mAb for 30 min on ice, and then stimulated at 37°C with 10 µg/ml of streptavidin. Cells were then lysed at 5.0 x 106 cells/ml in RPMI1640 medium containing 10% Fetal Bovine Serum, sodium pyruvate 1 mM, β-mercaptoethanol 50 μM, and Penicillin-Streptomycin. Cells were dispatched in a 48 wells plate (1 x 106 cells/well), and stimulated with different ratios of anti-CD3/CD28 coupled micro-beads: 8 x 104 beads (ratio bead:cells = 1:12.5), 2 x 105 beads (ratio = 1: 5), and 4 x 105 beads (ratio = 1:2.5). Wells of unstimulated cells were also added as a control. 24 hours later, 100 μl of cells were recovered to analyze their activation by Flow Cytometry, after staining with specific antibodies for CD4, CD8 and CD69; only viable cells were analyzed (Annexin-V negative). At 72 hours, CD4 and CD8 T cell proliferation was analyzed by flow cytometry in the live cell population only.Splenocytes were obtained, and T lymphocytes were purified by negative selection MojoSort™ Mouse CD3 T Cell Isolation Kit . Purified T cells were then stained with CellTrace Violet™ for 20 min at 37°C. After staining, cells were collected and resuspended at 3.0 x 102+ in total thymocytes was carried out using Indo-1 AM (acetoxymethyl)  as previously described . It has been previously demonstrated that the LATG135D mutation is able to increase and accelerate phosphorylation of the neighboring LAT-Y136 tyrosine residue . Mice homozygous for this mutation LATG135D/G135D were born at expected Mendelian frequencies, and LATG135D/G135D T cells showed levels of LAT comparable to wild-type T cells  thymic populations were decreased in LATG135D mutant mice relative to wild-type  post-selection cells was reduced in both homozygous and heterozygous LATG135D mutant mice (highCD3low/med (DP2) was increased in LATG135D mutant mice as compared to wild-type mice. Interestingly, the percentages of CD5lowCD3low/med (DP1), DP2 and DP3 populations found in heterozygous LAT+/G135D mice were intermediate between those of the wild-type and homozygous mutant mice, showing statistically significant differences with both populations  cells that can be defined using the expression of CD44 and CD25 showed no difference between wild-type and LAT mutant mice  and up-regulation of CD62L have been used to characterize SP cell maturation and defines two distinct populations, corresponding to immature CD24highCD62Lmed, and more mature CD24medCD62Lhigh SP cells  and homozygous (2.3-fold) LAT G135D/G135D mice, relative to wild-type animals (3YF mice) triggers an expansion of γδ T cells , or favoring its phosphorylation (LATG135D mice), both result in an increase in the percentage of the γδ-T cells. As far as we know with the mice analyzed so far, no increase of the γδ-T cell population with age is observed. Three individuals older than 25 weeks have been analyzed and the percentage never reached 5% (not shown). Therefore, this phenotype is completely different from that observed in LAT-3YF mice , this difference reaches statistical significance. However, this NK1.1 negative DX5 positive population cannot be categorized as true NK cells  was decreased in mutant mice. Similar results were also observed in lymph nodes  constitute reliable surface markers of anergic CD4+ T cells . Purified cells were labeled with Cell Trace Violet (CTV) dye in order to test their proliferative capacity following stimulation with anti-CD3/CD28 antibodies coupled micro-beads, as previously described  and the Ministry of Agriculture, Fisheries and Rural Development .G135D mice. FZ and BM contributed to the establishment and phenotyping of the LATG135D mice. EA wrote the manuscript and directed the study. All authors contributed to the article and approved the submitted version.MMA-E, IV-B, and FZ performed most of the experiments and interpreted results. LMF-A, MC, and IN-S helped with cell culture, animal handling and genotyping, and signaling experiments. MMA-E, IV-B, and FZ provided input on study design. LZ and YL generated the LAT