Arabidopsis thaliana.Plants are continuously challenged with biotic stress from environmental pathogens, and precise regulation of defense responses is critical for plant survival. Defense systems require considerable amounts of energy and resources, impairing plant growth, and plant hormones controlling transcriptional regulation play essential roles in establishing the appropriate balance between defense response to pathogens and growth. Chromatin regulators modulating gene transcription are broadly involved in regulating stress-responsive genes. However, which chromatin factors are involved in coordinating hormone signaling and immune responses in plants, and their functional mechanisms, remains unclear. Here, we identified a role of bromodomain-containing protein GTE4 in negatively regulating defense responses in Pseudomonas syringe. Genome-wide profiling of GTE4 occupancy shows that GTE4 tends to bind to active genes, including ribosome biogenesis related genes and maintains their high expression levels during pathogen infection. However, GTE4 is also able to repress gene expression. GTE4 binds to and represses jasmonate biosynthesis gene OPR3. Disruption of GTE4 results in overaccumulation of jasmonic acid (JA) and enhanced JA-responsive gene expression. Unexpectedly, over-accumulated JA content in gte4 mutant is coupled with downregulation of JA-mediated immune defense genes and upregulation of salicylic acid (SA)-mediated immune defense genes, and enhanced resistance to Pseudomonas, likely through a noncanonical pathway.GTE4 mainly functions as activator of gene expression upon infection with Pseudomonas. These findings provide new knowledge of chromatic regulation of plant hormone signaling during defense responses.Overall, we identified a new role of the chromatin factor GTE4 as negative regulator of plant immune response through inhibition of JA biosynthesis, which in turn noncanonically activates the defense system against The online version contains supplementary material available at 10.1186/s12915-022-01454-5. Plant diseases caused by phytopathogens are the major threats for plant survival during life cycle. To counteract the pathogens, plants have evolved two layers of innate immune systems, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) . Both imJasmonic acid (JA), one of the most studied phytohormones, plays essential roles in both plant growth and stress response . Under nAlternaria brassicicola [Pseudomonas syringae. pv. tomato (Pst) DC3000. However, JA and SA also act synergistically in certain circumstances. JA could positively regulate ETI through a noncanonical pathway to defend Arabidopsis plants against biotrophic pathogens [JA normally protects plants from necrotrophic fungal pathogen, such as sicicola . It antaathogens . JA coulathogens , which mathogens .Arabidopsis thaliana, the chromatin remodeler BRAHMA interacts with three bromodomain-containing proteins, BRD1, BRD2, and BRD13 [. They are also named as global transcription factor group E (GTE) proteins [Histone modifications play critical roles in transcriptional regulation , 15. Lysnd BRD13 , 33. Altnd BRD13 , 33. Anond BRD13 . Absencend BRD13 . The brond BRD13 . There aproteins . GTE4 isproteins –38. GTE1proteins , 39. Besgte4 mutant, indicating that GTE4 mainly functions as a transcriptional activator. Within the GTE4-bound genes, we identified numerous ribosome biogenesis related genes that are downregulated in gte4 during pathogen infection, suggesting that GTE4 tends to maintain active cellular activity under pathogen stress. GTE4 also functions as repressor of gene expression. A 12-oxophytodienoate reductase gene OPR3, required for JA biosynthesis, is bound and repressed by GTE4. GTE4 disruption-resulted overexpression of OPR3 leads to over-accumulation of JA and enhanced JA-responsive genes expression. Surprisingly, elevated JA content in gte4 is coupled with downregulation of PDF1.2a/b/c and upregulation of PR1, which represent JA- and SA-mediated immune response, respectively. Consistently, gte4 mutants show more resistance to bacterial pathogen Pseudomonas syringe pv. tomato (Pst) DC3000. In conclusion, we characterize a new chromatic regulator that attenuates plant defense response through decreasing JA content, which acts noncanonically to activate SA-mediated immune response.In this study, we provide evidences showing that bromodomain-containing protein GTE4 negatively regulates plant immune response through repressing JA biosynthesis. Genome-widely, GTE4 binds to highly-expressed genes. The overall expression levels of GTE4-bound genes are decreased in gte4 T-DNA insertion mutant  DC3000  in gte4 mutants  using GTE4-HA and non-transgenic WT plants in parallel as a control. The overall distribution pattern of GTE4 is similar as euchromatic histone modification H3K9ac, which is enriched in euchromatic regions while depleted in centromeric heterochromatin regions Fig. A, B. Totgte4 mutant displayed deficient embryo and root development [gte4 mutant  analysis, and found that GTE4-bound genes were significantly enriched in vesicle-mediated transport, ncRNA (mainly rRNA) and mRNA metabolic process, and ribosomal protein biogenesis  were also found downregulated under pathogen inoculation  with WT and gte4 but with a lower degree than in WT -qPCR  and found that JA level was significantly increased in gte4 and restored in GTE4-HA  and SA-mediated immune response (PR1 and PBS3). We found that PDF1.2a/b/c and COI1 were significantly downregulated, while PR1 and PBS3 were dramatically increased in gte4 , which encodes a 12-oxophytodienoate reductase required for JA biosynthesis  was used as wild type (WT) for all experiments. The T-DNA insertion line of gte4 (SALK_083697) was obtained from Arabidopsis Biological Resource Center (https://abrc.osu.edu/). All plants were grown at 22 °C under long-day condition (16 h light/8 h dark) and 50% humidity conditions unless otherwise specified.Agrobacterium-dipping into gte4 mutants. Detailed information for primers can be found in Additional file Genomic DNA of GTE4 with its 1 kb promoter was cloned into PC1300-TB-bHA vector modified from pCAMBIA1300 to create epitope-tagged HA fusions and then transformed by Pst DC3000 was grown on King’s B medium plate for 2 days at 28 °C. Single clone was inoculated in King’s B broth and cultured overnight at 28 °C. In the next morning, bacteria were suspended in 10 mM MgCl2 to OD600 of 0.2 then diluted 100 times. Four-week-old plants grown under long-day photoperiods and 50% humidity conditions were injected the bacterial suspension with needless syringe. Afterwards, plants were covered to keep humidity and incubated in growth chamber. Bacteria growth was analyzed at 3 days post inoculation (dpi). Proper amount of plants was pooled together as one biological replicate and six such biological replicates were measured at the same time.MON1 , instead of ACTIN, was used as internal control [Four-week-old plants were used for total RNA extraction by Trizol Reagent . RNA was treated with DNase before used for cDNA synthesis by QPCR cDNA synthesis kit . RT-qPCR was performed on CFX96™Real-Time System (BioRad) using 2 × Universal SYBR Green Fast QPCR Mix .  control , 57.gte4 mutant.For RNA sequencing, total RNA was extracted from 4-week-old plants using Trizol Reagent and then purified by Quick-RNATM Plant MiniPrep Kit . cDNA libraries were constructed using Library Preparation VAHTS mRNA Capture Beads  and VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina . All samples were run on Illumina NovaSeq 6000 platform. Three biological replicates were performed for both WT and 2, 0.6% Triton X-100, 0.4 mM PMSF, and 1 × Mini-Complete cocktail) with 1% formaldehyde for 15 min at room temperature with rotation. Chromatin pellet was washed with 1 mL ChIP Buffer 2  then resuspended with 300 μL nuclear lysis buffer  and kept on ice for 10 min. The lysates were diluted tenfold with ChIP dilution buffer  and sheared by sonication. After centrifugation at 12,000 rpm for 10 min, the supernatant was incubated with 3 μL anti-HA antibody  overnight with rotation at 4 °C. Then, add 40 μL Protein A/G beads  into supernatant and incubated 4 h with rotation at 4 °C. High salt wash was replaced by another low salt buffer wash. DNA–protein complex was released and reverse cross-linked by boiling at 95 °C for 10 min in 100 μL 10% Chelex  with 1000 rpm shaking. After proteinase K and RNase treatment, DNA was purified by standard phenol–chloroform method. Ten and 2 g of leaves were used for sequencing and qPCR assay, respectively.ChIP of GTE4-HA was performed as previously described  with mod2JA) was added as an internal standard. The samples were vortexed for 1 min and then extracted with 5 mL methanol at 4 °C for 24 h. After centrifugation at 13,000 rpm at 4 °C for 10 min, the supernatant was transferred to a new tube, and dried with nitrogen, and then re-dissolved with 10 mL dichloromethane/methanol (v/v = 99:1) by vortexing for 1 min. JA was further purified with SPE-NH2 Cartridges . Column was activated with 6 mL dichloromethane/methanol (v/v = 99:1) before use. JA was eluted twice with 2% acetic acid/methanol. After being dried with nitrogen, JA was dissolved with 1 mL 0.05% acetic acid aqueous solution/acetonitrile (v/v = 80:20) by vortexing for 2 min and then filtered with 0.22 μm syringe filter.One-gram powder of well-ground leaves was transferred into 15 mL centrifuge tube, and 20 ng dihydro-JA  using a Dionex UltiMate 3000 HPLC system (Thermo Scientific) at a flow rate of 0.5 mL/min. A-eluent was Milli-Q water containing 1 ‰ (v/v) formic acid, and the B-eluent was acetonitrile containing 1 ‰ (v/v) formic acid. The program of elution started at 95% (v/v) Solvent A and ramped from 5 to 20% (v/v) Solvent B over 7 min, 20 to 50% (v/v) Solvent B over 5 min, 50 to 100% (v/v) Solvent B over 5 min, remained isocratic at 100% (v/v) Solvent B over 4 min, ramping from 100 to 5% (v/v) Solvent B over 0.1 min, and remaining isocratic at 95% (v/v) Solvent A over 3.9 min. The total run time was 25 min and data were collected for the first 17 min. The chromatograms were acquired at 280 nm, and photodiode array spectra were recorded from 180 to 400 nm. Heated ESI source and interface conditions were operated in positive ion mode as follows: vaporizer temperature 400 °C, source voltage 3 kV, sheath gas 60 au, auxiliary gas 20 au, capillary temperature 380 °C, capillary voltage 6 V, and tube lens 45 V. An LTQ-Orbitrap-XL (Thermo Fisher) mass spectrometer was operated using LTQ Tune Plus v. 2.5.5 SP1 and Xcalibur software (version 2.1.0.1140), with additional analyses using the QualBrowser feature of Xcalibur.2JA as internal standard. The measured JA/H2JA ratio and theoretic JA/H2JA ratio of each standard sample was used to draw standard curve. Three biological replicates were performed for both WT, gte4 mutant and GTE4-HA transgenic plants.JA standard was serially diluted to 2, 5, 10, 20, 50, and 100 ng/mL and mixed with 20 ng/mL HP = 1.0e − 5. DNA motif identification was performed with Homer (v4.11). In GTE4 binding profiling, the number of mapped ChIP-seq reads in 50-bp bins normalized to the total number of reads. The metaplots were built by Deeptools (v3.5.0). The R package clusterProfiler (v4.0.5) was used to perform the GO enrichment analysis involved in the biological processes. Other analysis and figures were done using R (v4.0.3). Accession numbers of published ChIP-seq data of histone modifications used in this study are listed in Additional file P < 0.05 were considered as significantly differential expressed genes.Raw data were filtered using Cutadapt (v2.7) and were checked for quality scores and other metrics using FastQC (v0.11.9). ChIP-seq data were aligned by Bowtie2 v2.3.5) with the default parameters. The mapped reads were sorted, and duplicates were removed using SAMtools (v1.9). MACS2 (v2.2.7) was used for Peak calling with .5 with tAdditional file 1: Fig. S1. Characterizations of gte4 mutant and GTE4-HA rescue plants. (A)Diagram of gte4 T-DNA insertionmutant. (B) Genotyping of gte4 T-DNAinsertion mutant. (C) RT-PCR verification of GTE4 transcripts in gte4T-DNA insertion mutant. MON1 servesas internal control. (D) Diagram of GTE4-HA construct for rescuing gte4 mutant. (E) Immunoblots of GTE4-HAprotein in GTE-HA rescue plants.Additional file 2: Table S1. List of peaks and geneswith GTE4 enrichment.Additional file 3: Table S2. Published ChIP-seq dataused in this study.Additional file 4: Fig. S2. Representative snapshots ofIGV views of GTE4 enrichment on ribosome biogenesis related genes.Additional file 5: Table S3. List of differentiallyexpressed genes in gte4 under controlcondition.Additional file 6: Table S4. List of differentiallyexpressed genes in gte4 under Pst DC3000 condition.Additional file 7: Fig. S3. RT-qPCR verifying theexpression of GTE4-bound and downregulated genes in gte4 under normal condition. Data are presented as mean±SEM of 2biological replicates.Additional file 8: Fig. S4. JA-responsive genes areover-activated while growth pathways are impaired in gte4. (A) Biological repeat of RT-qPCR verification ofJA-responsive gene expression in gte4. GO analysis of downregulated genes in gte4 under control (B) and PstDC3000 treatment (C) conditions.Additional file 9: Table S5. List of genes with GTE4enrichment and co-upregulated in gte4under both conditions.Additional file 10: Fig. S5. RT-qPCR verification ofcell cycle related gene expression in gte4.Data are presented as mean±SEM of 2 biological replicates.Additional file 11: Fig. S6. Examples of conservedtranscription factor binding motifs identified in GTE4-enriched peaks.Additional file 12: Table S6. Primers used in thisstudy.Additional file 13: Fig. S7. Uncropped pictures for Additional file 1_Fig S1. A Picture for Fig S1B. B Pictures for Fig S1C. Dash-lineboxes indicate samples presented in Fig S1C. C Pictures for Fig S1E.


X-box binding protein l (XBP-1) is activated under endoplasmic reticulum (ER) stress to form an important transcription factor XBP-1 splicing (XBP-1s). The cerebral ischemia/reperfusion (CI/R) causes cytotoxicity, which correlates with the activation of splicing XBP-1 mRNA and NLRP3  inflammasomes, along with increases in the expression and secretion of proinflammatory cytokines and upregulation of pyroptosis-related genes in HT22 cells and in the middle cerebral artery occlusion (MCAO) rat model. However, whether XBP-1 plays a role in regulating pyroptosis involved in CI/R is still unknown. Our present study showed that behavior deficits, cerebral ischemic lesions, and neuronal death resulted from CI/R. CI/R increased the mRNA level of XBP-1s, NLRP3, IL-1β, and IL-18 and the expressions of XBP-1s, NLRP3, Caspase-1, GSDMD-N, IL-1β, and IL-18. We further repeated this process in HT22 cells and C8-B4 cells and found that OGD/R decreased cell viability and increased LDH release, XBP-1s, NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and especially the ratio of pyroptosis, which were reversed by Z-YVAD-FMK and downregulated XBP-1. Our results suggest that downregulated XBP-1 inhibited pyroptosis through the classical NLRP3/Caspase-1/GSDMD pathway to protect the neurons.Ischemic stroke is a major condition that remains extremely problematic to treat. A cerebral reperfusion injury becomes apparent after an ischemic accident when reoxygenation of the afflicted area produces pathological side effects that are different than those induced by the initial oxygen and nutrient deprivation insult. Pyroptosis is a form of lytic programmed cell death that is distinct from apoptosis, which is initiated by inflammasomes and depends on the activation of Caspase-1. Then, Caspase-1 mobilizes the N-domain of gasdermin D (GSDMD), resulting in the release of cytokines, such as interleukin-1 They can evoke dangerous signaling inflammatory mediators such as Caspase-1/Gasdermin, JAK/STAT-1, and TLR4/MyD88/NF-κB [Stroke is a leading cause of permanent disability and death that affects about 15 million people around the world , 3. At p88/NF-κB .β (IL-1β) to the mature, active form and is also implicated with the pyroptosis [Shigella flexneri, intracellular bacteria [β and interleukin-18 (IL-18) and also cleaves gasdermin D (GSDMD) to release the membrane pore-forming N-terminal GSDMD domain (GSDMD-N). To trigger inflammation, GSDMD-N pores promote the release of activated IL-1β and IL-18 [β, and Caspase-1, has been reported to play critical roles in rodent models of the ischemic brain injury [β [An inflammasome is a type of a tissue-damage sensor that is necessary for the conversion of the proform of interleukin-1roptosis –10. Pyrobacteria  that enabacteria . Caspasend IL-18 . The NLRn injury , and strnjury [β .X-box binding protein l splicing (XBP-1s) serves as a significant transcription factor and is activated under endoplasmic reticulum (ER) stress, which can positively regulate both cell proliferation and angiogenesis. Previous studies have shown that transient cerebral ischemia can activate XBP-1 mRNA splicing to protect cells from ischemia/reperfusion-induced cell damage and apoptosis , 17. XBPα inhibitor STF-083010 or genetic silencing of XBP-1 can selectively inhibit the IRE-1α/XBP-1s branch and then attenuate Cd-induced NLRP3 inflammasome activation and pyroptosis in HK-2 cells. Sirtuin-1 can ameliorate cadmium-induced ER stress and pyroptosis through XBP-1s deacetylation [A recent study indicated that NLRP3 inflammasome deficiency or using its selective inhibitor (MCC950) can improve cerebral injury after ischemic stroke . The IREtylation . Howevertylation .β, and IL-18, which were restored by downregulated XBP-1 expression.In the present study, we explored the effects of XBP-1 on pyroptosis in HT22 cells, C8-B4 cells, and rats treated by OGD/R or middle cerebral artery occlusion (MCAO). We found that OGD/R decreased the cell viability, whereas and pyroptosis increased, which could be mostly inhibited by knockdown of XBP-1. OGD/R or MCAO increased the levels of XBP-1s, NLRP3, Caspase-1, GSDMD, IL-1β was purchased from Affinity Biosciences . Antibody against beta-actin was purchased form Proteintech Group, Inc. (USA). Z-YVAD-FMK was purchased from Selleckchem . Polyphyllin VI was purchased from MCE .HT22 cells and C8-B4 cells were obtained from the Kunming Institute of Zoology . Antibodies against NLRP3, GSDMD-N, IL-18, and XBP-1 were purchased from Abcam . Antibodies against XBP-1s and Cleaved Caspase-1 were purchased from Cell Signaling Technology . Antibody against Cleaved IL-1Male Sprague-Dawley rats (weighing 250-280 g) were provided by the Shanghai Laboratory Animal Center, Chinese Academy of Sciences, and were housed under a under a 12 : 12 h light/dark cycle at 21 ± 1°C with ad libitum access to food and water. Animals were divided into four experimental groups: (i) sham group (sham), rats without carotid occlusion (S); (ii) I/R group, carotid artery occlusion was performed for 60 minutes followed by reperfusion for 24 hours (24 h); (iii) I/R group, carotid artery occlusion was performed for 60 minutes followed by reperfusion for 48 hours (48 h); and (iv) I/R group, carotid artery occlusion was performed for 60 minutes followed by reperfusion for 72 hours (72 h).We followed the methods of Deng et al. . Rats weGait was measured using the footprint test to assess limb coordination and stride length. For gait analyses, rats were habituated to the experimenter and the behavior room from 2 to 3 days before the test. Then, the rats were habituated to walk straight to their home cage. The paws of the rats were recorded by software ; the rats were allowed to walk freely on the track. The mean stride length and mean stride width were analyzed by measuring the distance between paw prints.We followed the methods of Li et al. . BehavioThe criteria for scoring were as follows:Grade 0: no neurological deficitsGrade 1: the contralateral forelimb cannot be stretched completely when the rat is lifted by its tailGrade 2: the rat spontaneously circles to the paralytic side when walkingGrade 3: the rat involuntarily falls down to the contralateral side when walkingGrade 4: the rat cannot walk automatically and loses consciousnessTwo, three, five-triphenyltetrazolium chloride- (TTC-) stained brain sections were used to assess cerebral infarct volume. Twenty-four hours, forty-eight hours, and seventy-two hours after reperfusion, immediately after sacrifice, the brains were removed from the rats and cut coronally into five serial 2 mm slices. Samples were then incubated for 15 min in 2% TTC  at 37°C and fixed in 4% paraformaldehyde overnight. Infarctions remained unstained by TTC. Each brain section of each rat was stained with TTC (unstained areas were recognized as infarctions) and was evaluated quantitatively using Image-Pro Plus software to calculate the percentage infarct volume.μm thick sections. The sections were dewaxed in 2 changes of xylene (10 min each) and rehydrated in 2 changes of absolute ethanol (5 min each) and then rinsed by running tap water orderly. Haematoxylin-eosin (H&E) staining was performed to observe the histomorphology. Histology assessment was performed by blinded investigator.Deeply anaesthetized rats were transcardially perfused with PBS followed by 4% paraformaldehyde. The brains were quickly removed by decapitation and carefully postfixed. Then, the samples were paraffinized and sliced to 5 μm) were immersed in xylene , rehydrated in absolute ethanol  followed by 95%, 75%, and 50% solutions of ethanol in water (5 min each), and then washed in distilled water for 2 times, 5 min each. Slides were stained in FD cresyl violet solution  for 10 min and, then, briefly rinsed in 100% ethanol and differentiated in 100% ethanol containing 0.1% glacial acetic acid for 1 min. The slides were then dehydrated in absolute ethanol  followed by clearance in xylene . Coverslips were mounted with resinous mounting medium. The staining of the hippocampal CA1 region was routinely analyzed by a researcher blinded to the experimental protocol.For the Nissl staining, paraffin-embedded brain tissue sections (5 2. C8-B4 cells (cell line) were cultured in DMEM nutrient medium, containing 25 mM glucose  supplemented with 200 mM glutamine , 10% fetal bovine serum (Gibco) and 0.5% penicillin–streptomycin , in a humidified atmosphere at 37°C under 5% CO2. Both cells were seeded in 6-well culture plate at a density of 4 × 104 per well. The cells were pretreated with Z-YVAD-FMK (20 μM) for 30 min before stimulation with OGD/R. The cells were pretreated with polyphyllin VI (4 μM) for 30 min before stimulation with OGD/R.HT22 cells (cell line) were cultured in F12 nutrient medium  supplemented with 10% fetal bovine serum (Gibco) and 0.5% penicillin–streptomycin , in a humidified atmosphere at 37°C under 5% COWe followed the methods of Bai et al. . XBP-1 sμg siRNA and 5 μl siTran transfection reagent  per well were diluted separately in serum free Opti MEM for a final volume of 250 μl, gently mixed, and incubated for 5 min at room temperature. Then, the diluted siRNA solution and the diluted siTran transfection reagent were mixed gently and incubated for 20 min at room temperature. The diluted siRNA/siTran transfection reagent complex was added to the plates. After transfection with siRNA for 24 h, the cells were exposed to OGD/R and then harvested for assay.HT22 cells and C8-B4 cells were seeded in 6-well plate at a density of 4 × 104 per well. The contents of 0.33 2, 92% N2, and 5% CO2) at 37°C for 2 h. After that, the medium was discarded and the cells were cultured in normal medium under normoxic conditions for another 24 h reperfusion.To mimic the CI/R conditions in vitro, the OGD/R modelling method was used, and the HT22 cells and C8-B4 cells were cultured under normal conditions for 24 h, then moved to glucose-free DMEM (Gibco), and placed under ischemic conditions (3% OμM) and polyphyllin VI (4 μM) for 30 min before OGD/R and then were incubated for 24 h after exposure to OGD/R. The cell viability was measured by using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. Twenty-four hours later, 10 μl CCK-8 was added into every well, followed by 2 h 37°C incubation. Absorbance at 450 nm/630 nm was detected using an enzyme-labeled instrument. The results were obtained from three independent experiments, and each experiment was performed in triplicate. The mean OD of one group/mean OD of the control was used to calculate the viability.We followed the methods of Bai et al. . HT22 ceCell death was evaluated by the quantification of plasma membrane damage which resulted in the release of lactate dehydrogenase (LDH). The level of LDH released in the cell culture supernatant was detected by LDH cytotoxicity assay detection kit  following the manufacturer's instructions.g for 5 min after OGD/R. The supernatant was discarded to collect the cells that were then resuspended in PBS. The cells in the suspension were counted and labeled with Annexin V (AV) and propidium iodide (PI) briefly, after which flow cytometry  was employed to quantitatively determine and meticulously analyze the pyroptosis cells.HT22 cells and C8-B4 cells were washed with PBS  and then digested with pancreatin  and next centrifuged at 168 × 3VO4, 1 mM beta-glycerolphosphate, 1 mg/ml leupeptinglycerolphosphate, and 1 mg/ml leupeptin). Protein concentration was determined by using Bio-Rad protein assay reagent . An equal quantity of proteins was separated by SDS-PAGE of 10% for NLRP3 (1 : 1000) and 12% for GSDMD-N (1 : 1000), Cleaved IL-1β (1 : 500), IL-18 (1 : 1000), XBP-1s (1 : 1000), XBP-1 (1 : 2000), Cleaved Caspase-1 (1 : 1000), and β-actin (1 : 10000) and then transferred to PVDF membrane . The membrane was soaked in 5% skimmed milk  overnight at 4°C and then incubated with primary antibody followed by peroxidase conjugated anti-mouse or anti-rabbit IgG (1 : 10000) . The epitope was visualized by an ECL Western blot detection kit . Other steps followed the instructions of each antibody. The detailed catalog numbers of all antibodies are listed in Protein lysates  were prepared using a solubilizing solution (20 mM Tris HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF, 1 mM EGTA, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 1 mM Naβ, and IL-18, q-PCR was performed on a CFX96 Touch thermocycler (Applied Biosystems) using SYBR® Premix Ex Taq™ II . Primer sequences for Xbp1 spliced, Xbp1 unspliced, NLRP3, IL-1β, IL-18, and GAPDH  were used as follows in We followed the methods of Bai et al. . Rat tisΔΔCT– method.PCR amplification was carried out at 95°C for 30 s, followed by 45 cycles of 95°C for 5 s and 55°C for 30 s. GAPDH was used as an endogenous control to normalize differences. All fluorescence data were processed by a PCR postdata analysis software program. The differences in gene expression levels were analyzed with the 2p value less than 0.05 was considered statistically significant. All blots are representative of experiments that were performed at least three times.Data were expressed as mean ± SEM. Statistical analysis was performed by using SPSS software. The one-way ANOVA followed by a post hoc Bonferroni multiple comparison test was used to compare control and treated groups. F = 60.3, p < 0.001). Meanwhile, the neurological deficit scores of the 24 h group, 48 h group, and 72 h group were significantly higher than that of the sham group  = 136.1, p < 0.001). TTC staining was used to evaluate the ipsilateral volume  = 75.3, p < 0.001). To investigate the presence of CI/R-induced cerebral injury, the haematoxylin and eosin (H&E) staining and Nissl staining were used to observe changes in the model. The forms of neuronal death occurring in the hippocampal CA1 region of the 24 h group, 48 h group, and 72 h group were documented, including apoptotic cells  and pyroptotic cells , while the sham group exhibited a normal appearance  = 132.5, p < 0.001), and the expression of XBP-1s was also significantly increased in the 24 h group, 48 h group, and 72 h group  = 57.07, p < 0.001). XBP-1s can activate NLRP3 inflammatory bodies [NLRP3 was significantly increased in the 24 h group, 48 h group, and 72 h group  = 164, p < 0.001), and the expression level of NLRP3 was also significantly increased in the 24 h group, 48 h group, and 72 h group  = 45.78, p < 0.001). The NLRP3 inflammasomes assemble and trigger the autocleavage of pro-Caspase-1 into Caspase-1 [F = 49.37, p < 0.001). Then, the active Caspase-1 generates an N-terminal GSDMD fragment (GSDMD-N) by cleaving GSDMD, which is recognized as a crucial executor of pyroptosis by inducing the formation of membrane pores [F = 63.88, p < 0.001). IL-1β and IL-18 were also evaluated and determined to be consistent with previous studies in which the mRNA expression levels of IL-1β  = 47.90, p < 0.001) and IL-18  = 47.99, p < 0.001), were significantly increased in the 24 h group, 48 h group, and 72 h group  = 33.82, p < 0.001) and IL-18  = 106.3, p < 0.001) were significantly increased in the 24 h group and 48 h group  = 91.9, p < 0.001) and showed obvious toxic effects  = 83.82, p < 0.001). To investigate which type of cell death was induced by OGD/R, Z-YVAD-FMK  and polyphyllin VI  were used as a positive control. In previous studies involving OGD/R and polyphyllin VI, cell death was induced, whereas the Caspase-1 inhibitor suppressed this response. OGD/R had similar effects to those of polyphyllin VI  = 14.57, p < 0.001), NLRP3  = 29.12, p < 0.001), Caspase-1  = 24.75, p < 0.001), GSDMD-N  = 35.33, p < 0.001), IL-1β  = 20.43, p < 0.001), and IL-18  = 39.19, p < 0.001), which mostly could have been inhibited by Z-YVAD-FMK and was similar to the effects of polyphyllin VI  = 48.22, p < 0.001) and increased the release of LDH  = 59.77, p < 0.001). OGD/R increased the expression of slicing XBP-1  = 10.66, p < 0.001), NLRP3  = 388.1, p < 0.001), Caspase-1  = 95.57, p < 0.001), GSDMD-N  = 218.4, p < 0.001), IL-1β  = 20.62, p < 0.001), and IL-18  = 27.05, p < 0.001), which mostly could have been inhibited by Z-YVAD-FMK and was similar to the effects of polyphyllin VI  = 53.14, p < 0.001) and increased LDH release  = 33.81, p < 0.001), which could have mostly been inhibited by downregulated XBP-1 in a similar manner to the effects of Z-YVAD-FMK. In this experiment, PI and Annexin V double staining were used to exhibit pyroptotic cells. The OGD/R group had a higher Annv/PI double-positive cell population (pyroptosis rate) than the control group  = 372.3, p < 0.001) and was the same as the polyphyllin VI group; however, downregulated XBP-1 can decrease the rate similarly to the effects of Z-YVAD-FMK. Downregulated XBP-1 also decreased the expression of slicing XBP-1  = 10.42, p < 0.001), NLRP3  = 140.4, p < 0.001), Caspase-1  = 48.79, p < 0.001), GSDMD-N  = 35.67, p < 0.001), IL-1β  = 28.59, p < 0.001), and IL-18  = 52.55, p < 0.001), like the effects of Z-YVAD-FMK  = 58.27, p < 0.001) and was the same as the polyphyllin VI group; downregulated XBP-1 may decrease the rate, in a similar manner as the effects of Z-YVAD-FMK. Downregulated XBP-1 also decreased the expression of slicing XBP-1  = 46.63, p < 0.001), NLRP3  = 31.18, p < 0.001), Caspase-1  = 22.88, p < 0.001), GSDMD-N  = 24.5, p < 0.001), IL-1β  = 25.53, p < 0.001), and IL-18  = 26.76, p < 0.001) similar to Z-YVAD-FMK  activation, inositol-requiring transmembrane kinase/endonuclease-1 (IRE1) activates its endoribonuclease (RNase) activity through dimerization and autophosphorylation . The IRE-2 cells . Our resnd IL-18 . DownregOur results showed that XBP-1 may be involved in pyroptosis and could play an important role in regulating pyroptosis in CI/R injury. Downregulated XBP-1 can inhibit NLRP3 inflammasome activation and potentially pyroptosis induced by OGD/R or MCAO. These effects may protect the neurons through NLRP3/Caspase-1/GSDMD pathway . These fThis study demonstrated that downregulated XBP-1 could inhibit pyroptosis by inhibiting the NLRP3/Caspase-1/GSDMD pathway in the hippocampus which rescues cerebral ischemia/reperfusion injury.


Peak aerobic capacity declines with age concomitant with greater fatigability and slower gait speed. We explored these relationships with cross-sectional SOMMA data . Participants completed a treadmill peak oxygen consumption test (VO2peak) and the self-administered Pittsburgh Fatigability Scale . Mean VO2peak was 19.4±4.2 ml/kg/min, PFS Physical score was 16.9±8.5 points, and PFS Mental score was 8.1±8.2 points. Pearson correlations between VO2peak and PFS Physical and Mental scores were r=-0.36 and r=-0.23, respectively. For every one standard deviation higher VO2peak, PFS Physical and Mental scores were lower by 2.07 points  and 1.10 points , respectively, when adjusted for clinic site, age, race, sex, and self-reported physical activity. Future SOMMA analyses will evaluate a likely bidirectional association between VO2peak and fatigability, as well as examine their potential mediating effects on physical function.


These nanoparticles were synthesized employing a citrate-based thermal decomplexing precipitation method, testing a nominal Tb3+ doping concentration between 0.001 M to 0.020 M, and a maturation time from 4 h to 7 days. This approach allowed to prepare apatite nanoparticles as a single hydroxyapatite phase when the used Tb3+ concentrations were (i) ≤ 0.005 M at all maturation times or (ii) = 0.010 M with 4 h of maturation. At higher Tb3+ concentrations, amorphous TbPO4·nH2O formed at short maturation times, while materials consisting of a mixture of carbonated apatite prisms, TbPO4·H2O (rhabdophane) nanocrystals, and an amorphous phase formed at longer times. The Tb3+ content of the samples reached a maximum of 21.71 wt%. The relative luminescence intensity revealed an almost linear dependence with Tb3+ up to a maximum of 850 units. Neither pH, nor ionic strength, nor temperature significantly affected the luminescence properties. All precipitates were cytocompatible against A375, MCF7, and HeLa carcinogenic cells, and also against healthy fibroblast cells. Moreover, the luminescence properties of these nanoparticles allowed to visualize their intracellular cytoplasmic uptake at 12 h of treatment through flow cytometry and fluorescence confocal microscopy (green fluorescence) when incubated with A375 cells. This demonstrates for the first time the potential of these materials as nanophosphors for living cell imaging compatible with flow cytometry and fluorescence confocal microscopy without the need to introduce an additional fluorescence dye. Overall, our results demonstrated that Tb3+-doped citrate-functionalized apatite nanoparticles are excellent candidates for bioimaging applications.This work explores the preparation of luminescent and biomimetic Tb Luminescent nanoparticles are attractive materials with great potential for nanomedicine as they allow to track their fate in vivo when used as drug carriers, or for applications related to tissue engineering to provide the fluorescent contrast needed to visualize tissues, cells, and tumors . Several3+ [2+-doped lanthanide phosphate  nanophosphors [To maximize apatite biocompatibility, it is necessary to synthesize biomimetic nanocrystalline apatites , which m3+ ,31, and hosphors ,33.3+ has been used as a luminescence activator in different host structures using an excitation wavelength from 300 to 380 nm. Its emission spectra, based on 5D3→7Fj (blue) and 5D4→7Fj (green) transitions [3+ emission, which possesses a mostly red luminescence. Thanks to its green luminescence, Tb3+ doping can be a good alternative to fluorescein-5-isothiocyanate (FITC) and other common fluorescent organic dyes used for nanoparticle surface labeling. Indeed, the use of FITC-labeled apatites and other FITC-labeled nanoparticles poses the risk of FITC detachment in vivo or in vitro [3+ doping. Fluorescence labeling is necessary when using flow cytometry and confocal fluorescence microscopy to quantify and image nanoparticle uptake into cells [3+-doped citrate-functionalized carbonated apatite (Tb3+:cit-cAp) nanoparticles can therefore be a good alternative to surface labeled apatites, preserving, in addition, the biomimetic surface properties of non-doped nanoparticles. In addition to the above features, Tb3+ has been found to have bactericidal activity [3+:cit-cAp nanoparticles are also a promising biomimetic, lanthanide-doped apatitic filler for bone reconstruction.Tbnsitions , is compin vitro , leadingin vitro . All theto cells ,36. The activity  and to pactivity , so Tb3+3+:cit-cAp nanocrystals via the thermal decomplexing precipitation method; (ii) to study their physico-chemical, luminescence and cytocompatibility properties; and (iii) to assess nanoparticle cell uptake and the feasibility for cytoplasmatic fluorescence imaging using flow cytometry and confocal fluorescence microscopy.The aims of the present contribution are: (i) to investigate the possibility to obtain luminescent biomimetic and highly biocompatible Tb3+/Ca2+/citrate/phosphate/carbonate solutions and thoroughly characterized using a large set of complementary techniques. Special attention was paid to conditions that allowed to obtain nanoparticles with biomimetic features. Afterward, the luminescent properties of the produced particles were explored. Finally, the cytocompatibility of such particles and the possibility to use them to image cells was investigated via in vitro cell tests.In a first step, precipitates were generated by thermal decomplexing of Tb3(cit)·2H2O, cit = citrate = C6H5O7, ACS reagent, ≥99,0% pure), terbium (III) chloride anhydrous , calcium chloride dihydrate , and sodium phosphate dibasic  were provided by Sigma-Aldrich . Sodium carbonate monohydrate  was supplied by Merck  and hydrochloric acid  by Panreac . Solutions were prepared with ultrapure deionized Milli-Q water . The precipitation experiments were carried out by thermal decomplexing of M(2+/3+)/Ca2+/cit/phosphate/carbonate solutions at 80 °C [3+ as doping lanthanide ion  and decreasing concentrations of Ca2+ in the precipitating solution  to set x + y = 0.100 M. The experiments lasted 4 h, 24 h, 96 h, and 7 days. The precipitates were subjected to washing by centrifugation with ultrapure water  and freeze-dried overnight at −50 °C under vacuum (3 mbar).Sodium citrate tribasic dihydrate  and deposited on copper microgrids coated with FORMVAR carbon film. High resolution TEM (HRTEM) and selected area electron diffraction (SAED) analyses were performed with a TITAN G2 60–300 FEI Instrument operating at 300 kV . The instrument was equipped with an EDX Super X detector to perform elemental microanalysis and element mappings, as well as scanning transmission electron microscope (STEM) type high-angle annular dark-field imaging (HAADF).Precipitates were characterized using different techniques. X-ray powder diffraction (XRD) data were collected using a BrukerD8 Advance Vario diffractometer  with a Bragg Brentano parafocusing geometry and Cu Kα1 radiation (1.5406 Å). Transmission electron microscopy (TEM) observations were performed with a TEM Libra 120 Plus  instrument operated at 80 kV. Samples were dispersed in absolute ethanol . Pellets with ~1 wt% sample in anhydrous KBr were prepared and pressed using a hydraulic pump at 10 tons. Pure KBr pellets were used to record the background. Raman spectra were recorded with a LabRAMHR spectrometer . The excitation line was provided by a diode laser emitting at a wavelength of 532 nm while a Peltier cooled charge-coupled device (CCD) (1064 × 256 pixels) was used as the detector.Fourier transform infrared spectra (FTIR) were recorded in transmittance mode within a wavenumber range from 4000 cmx = 0, 0.001, 0.005, and 0.010 M Tb3+, with those measured for the same particles prepared after immersion in a stirred 0.1 M NaOH solution for 1 h, since this procedure allowed us to remove the citrate ions [Particle size distribution (PSD) and electrophoretic mobility  were analyzed with a Zetasizer Nano ZS analyzer  in aqueous suspensions  in disposable polystyrene cuvettes. For ζ-potential versus pH measurements, an MPT-2 autotitrator  was employed to adjust the pH of the suspensions. Diluted HCl and NaOH solutions  were used as titration agents without any additional electrolytes. To analyze the effect of citrate in terms of ζ-potential versus pH measurements, we compared the values obtained for samples prepared with ate ions ,27.3 aqueous solution.Elemental composition of the samples was measured by inductively-coupled plasma optical emission spectrometry (ICP-OES) using an Agilent 5100 instrument . Before analysis, 10 mg of sample was dissolved in triplicate in 50 mL of a 1 wt% HNO−1 under airflow.The thermal behavior of the samples was studied using a simultaneous thermogravimetric differential thermal analyzer (TG-DTA), using a STA 449F3 Jupiter device . The samples were placed in an alumina pan, and an empty crucible was used as a reference. The heating profile was set from room temperature to 1100 °C with a ramp of 10 °C min3+-doped particles and their aqueous suspensions (~0.5 mg/mL) were recorded using a Cary Eclipse Varian Fluorescence Spectrophotometer . A front surface accessory was used to obtain the luminescence spectra, the luminescence lifetime (τ), and the relative luminescence intensities (R.L.I.) of the powders and a commercial Peltier cell holder connected to a temperature control module  was used to obtain the luminescence spectra and the R.L.I. of the liquid suspensions. The instrumental parameters for measuring the excitation and emission spectra of both the powder and aqueous suspension (~0.5 mg/mL) samples were excλ = 375 nm, emλ = 545 nm, delay time (dt) = 120 µs, and gate time (gt) = 5 ms. For powder samples, the photomultiplier voltage was of 470 V and slit widthexc/em 5/5 nm, and for aqueous suspension particles photomultiplier voltage was of 800 V and slit widthexc/em 10/10 nm. The excitation and emission spectra were recorded within wavelength ranges 250–500 nm and 500–750 nm, respectively. Luminescence lifetime measurements were also recorded using a Cary Eclipse Varian Fluorescence Spectrophotometer  using exc/emλ = 375/475 nm, 100 µs delay time (dt), 0.010 ms gate time (gt), photomultiplier voltage of 600 V, slit widthexc/em 10/10 nm, and 100 cycles.The luminescence properties (excitation and emission spectra and lifetime) of solid Tb2 at 37 °C.HeLa, human cervical cancer cell line, was obtained from ATCC. MCF7 human breast cancer and A375 melanoma cancer cell lines were obtained from Biobanco . Human fibroblasts were isolated from skin tissue from abdominoplasty surgery using an enzymatic digestion with collagenase I . Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), and 1% penicillin/streptomycin (P/S) (Sigma-Aldrich), in an atmosphere containing 5% CO2. Twenty-four hours after seeding, cells were treated with 100, 10, 1, and 0.1 µg/mL Tb-containing samples prepared with x = 0.001, 0.010, 0.015, and 0.020 M Tb3+. Additionally, a non-doped sample was added (undoped). Seventy-two hours after treatment, the medium was removed and 100 µL of MTT -2,5-diphenyltetrazolium bromide) was added. After 3 h of incubation at 37 °C and 5% CO2, the MTT was removed and 100 µL DMSO were added. Immediately, the plate was measured using an absorbance spectrophotometer at 570 nm.HeLa, MCF7, A375, and fibroblast cells lines were seeded onto 96 wells plate at a confluence of 4 × 103 cells/wells in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), and 1% penicillin/streptomycin (P/S) . Plates were incubated at 37 °C in 5% CO3+-containing samples (x = 0.01 M and 0.02 M Tb3+) at 100 µg/mL for 12 h and 24 h. Next, cells were fixed in paraformaldehyde (PFA) at 4% for 20 min at room temperature and washed with PBS. Images were obtained using a spectral confocal Leica TCS SP2 AOBS microscope.Cell line A375 was seeded in an eight-well chamber slide  at a density of 8000 cells/well. They were treated after 24 h with Tb3+-doped samples (x = 0.01 M and 0.02 M Tb3+) at 100, 10, 1, and 0.1 µg/mL during 12 h and 24 h. Afterwards, cells were fixed with PFA 4% for 20 min and washed with PBS. Cells were then processed with a FACScan flow cytometer .Cell lines were treated with Tb3+-free (x = 0) and Tb3+-doped samples prepared with nominal Tb3+ doping concentrations from x = 0.001 to 0.010 M are plotted in x = 0.005 M Tb3+ show a similar evolution with time. At 4 hours of maturation, the major peaks of hydroxyapatite phase are already present (PDF 01-1008), i.e., at 2θ 25.87° (plane (002)), 31.77° (211), 32.19° (112), 32.90° (300), 33.9° (202), and 39.81° (310) [The XRD patterns of Tb1° (310) ,30. The x = 0.010 M Tb3+, the evolution is different. At 24 h, the diffractogram presents narrower peaks characteristic of a more crystalline material with larger dimensions and at 7 days, in addition, are also present the peaks of hexagonal TbPO4·H2O . By increasing x to 0.015 and 0.020 M Tb3+  was lower than that of stoichiometric hydroxyapatite (Ca/P = 1.67). The calcium deficiency of the samples is expected, as is characteristic of biomimetic apatites. For samples prepared with higher x, the (Ca + Tb)/P ratios were lower due to the presence of TbPO4 phases.The chemical composition of most matured precipitates  revealedx = 0.015 M and x = 0.020 M at 96 h, all samples displayed a TGA profile similar to that reported for other biomimetic carbonate apatites prepared using the same method [2 (from ≈ 600 °C to ≈ 1100 °C). In samples x = 0.015 M and x = 0.020 M Tb3+, especially at 96 h, the curves present additional weight losses due to the presence of the amorphous TbPO4·nH2O phase. For these samples, the contents of water, citrate, and carbonate could not be accurately assessed; thus, for the sake of comparison we have given data only for samples prepared at 7 days. x, particularly in samples x > 0.010 M Tb3+, due to the presence of hydrated TbPO4 phases. However, the citrate and carbonate content showed little variability. This could be due to the presence in these precipitates of hydrated TbPO4 in both amorphous and rhabdophane phases. With maturation, for the samples x = 0.001 M and x = 0.005 M Tb3+ there is no variation in water, citrate, or carbonate content between 96 h and 7 days, suggesting that progressive apatite crystallization did not affect the chemical composition. On the other hand, in sample x = 0.010 M Tb3+ at 7 days there was a slight increase of Ca, P, and Tb content paired with a decrease of adsorbed water content in comparison to 96 h sample  shows a homogeneous composition of P, Ca and Tb  and Tb-doped apatite specimens in the x range from 0.001 to 0.010 M Tb3+, up to 96 h, exhibited broad O–H stretching of physisorbed water between 3600 cm−1 and 2800 cm−1. The apatitic OH bands around 3580 cm−1 and 620 cm−1 were not clearly detected, as is usually found in bio-inspired apatites. The 400–1800 cm−1 region displays the typical spectral features of apatitic compounds, i.e., the asymmetric stretching mode of PO43− groups (υ3PO4) at 1000–1100 cm−1 , the bending modes υ4PO4 at ~608, and 564 cm−1 and υ2PO4 at ~470 cm−1. The shoulder at ~535 cm−1 in the υ4PO4 domain is attributed to surface HPO42− (non-apatitic) and points to the biomimetic character of these compounds [100 cm−1 , the symompounds .32−) is attested by the bands at ~1414 cm−1 and 1473 cm−1 (υ3CO3), and the bulging peak around 873 cm−1 (υ2CO3). From the area ratio between the υ3CO3 and the υ1υ3PO4 contributions (rc/p) the estimated overall degree of carbonation [x = 0.010 M at 96 h is 4.02 ± 0.5 wt%, which fully agrees with the value determined by TGA. This degree of carbonation is slightly lower than that determined (up to 5.9 wt%) in Tb3+-free samples [The presence of carbonate (CObonation  for samp samples . x = 0.010 M Tb3+) the deconvoluted υ2CO3 region  [−1 (υ2PO4) and ~530 cm−1 (υ4PO4). The signal at ~1072 cm−1 is due to a combination of the υ1CO3 mode at 1070 cm–1 with the υ3PO4 mode at 1076 cm–1 [−1, clearly visible in − mode of citrate ions [x = 0.015 M at 4 h of maturation  , as well076 cm–1 . The smaate ions . For x =turation  and x = ion time , the υ1Pe sample ,44.The PSD and ζ-potential of colloidal particles at pH of physiological interest are useful parameters to evaluate the suitability of the particles as nanocarriers in nanomedicine. Indeed, the dispersion/aggregation behavior of the colloid at pH around 7.4 (representing the blood) or pH 5–6 (representing the tumor microenvironment) depends on the size and surface charge of the particles ,30. Thes3+:cit-cAp was plotted both as volume distribution . Accordingly, D10 is closer to the individual particle size whereas D50, the median size, is to some extent influenced by particle aggregation and D90 is completely dominated by aggregates. The percentiles corresponding to samples x = 0, 0.001, 0.005, and 0.010 M Tb3+ at 96 h were D10 59, 59, 111, and 103 nm, respectively, and D50 745, 186, 153, and 210 nm, respectively . An inversed correlation between ζ-potential and pH was observed. In addition, Tb3+:cit-cAp samples always had more negative ζ-potential values in comparison to Tb3+:cAp. At higher x values, the inverse correlation between ζ-potential and pH was stronger. For example, at pH 7 the increase of x from 0 to 0.010 M Tb3+ induced a ζ-potential decrease from 0 to −18.9 mV for Tb3+:cAp samples and from −10.6 to −31.9 mV in Tb3+:cit-cAp particles. These strongly negative ζ-potential values favor the dispersion of both types of colloids. However, the increase in x had little impact on the ζ-potential at pHs of 5–6. We can conclude that citrate and Tb3+ doping act synergistically to decrease the ζ-potential, particularly at pH ≥ 7.To assess the effect of surface citrate and Tbersus pH c–f of Tbexcλ and emλ are the same as those depicted in excλ were 230, 283, 302, 319, 340, 352, 369, and 375 nm. The broad bands between 200 and 300 nm, centered at 230 nm, correspond to charge transfer bands (CTB), which occur by electron delocalization from the filled 2p shell of O2– to the partially filled 4f shell of Tb3+. In addition, this band can be partially attributed to the charge transfer transition X5+–O2– [7F6→5I8 and 5F4,5→5H4, 7F6→5H5,6, 7F6→5H7, and 7F6→5L7,8, 7F6→5L9,5D2,5G5, 7F6→5L10, and 7F6→5G6,5D3 transitions [3+ is visibleThe luminescence properties of the particles are shown in the  X5+–O2– ,48. The nsitions . Figure x = 0.020 M Tb3+ at 96 h using exλ = 230 (UV) and 375 nm (near visible). The emission spectra are the same and only the relative intensities being affected by exλ. Thus, for nanomedicine applications, we selected excλ = 375 nm, as it is close to the visible spectra. emλ, the emission bands are centered at 491, 545, 585 and 621 nm, which correspond to the Tb3+ 5D4→7F6, 5D4→7F5, 5D4→7F4 and 5D4→7F3 transitions, respectively [emλ corresponding to the hypersensitive transition without an inversion center  produces the highest R.L.I., we selected this emλ for the study.Concerning the ectively . As the x on the R.L.I. Only samples x = 0.010 M Tb3+ show a significant variation of the R.L.I. with maturation time while the rest of samples with x < 0.010 M Tb3+ show a time-independent R.L.I. This difference can be due to the existence of rhabdophane, which shows lower luminescence emission [x = 0.015 M and x = 0.020 M Tb3+ of 7 days of maturation but in lower amounts; in addition, these materials contain the amorphous TbPO4·nH2O phase which contributes positively to the R.L.I. [emission . This phe R.L.I. .x it is observed that the increase of x increases the luminescence in all cases with a linear trend, as expected [Analyzing the evolution of the R.L.I at a fixed maturation time versus expected .τ), in τ versus both maturation time and x, respectively. It is shown that, only when x = 0.010 M Tb3+ does the maturation time affect τ , τ is practically the same. The luminescence properties of all samples in aqueous suspensions are similar to those depicted in excλ, emλ, R.L.I. and τ) are the same at any of the tested pHs, so it is likely that changing the pH in a biological environment does not affect these properties.Concerning the luminescence lifetime  as the rest of experiments were performed at room temperature (25 °C).3+:cit-cAp nanoparticles at different concentrations were tested in three carcinogenic cells lines  and in a non-carcinogenic cell line (Fibroblast). As expected, the nanoparticles did not show cytotoxic effects; only a decrease in confluence appeared at the highest concentrations, but cell viability was always greater than 70% .As shown in 3+:cit-cAp nanoparticles with different Tb3+ doping, confirming the MTT results. Our results are similar to those previously obtained by using Eu3+:cit-cAp [:cit-cAp .x 0.010 M and 0.020 M Tb3+ at 96 h. We decided to analyze concentrations at 100 µg/mL and 0.1 µg/mL at two different times: 12 h and 24 h  and 7.33 ± 0.13 wt%  were obtained in these conditions. After 96 h of maturation, aggregation and agglomeration prevailed and the most mature crystals were found forming sheaf wheat or spherical agglomerates. The formation of sheaf wheat is a previous step in the formation of spherical shapes following the mechanism of spherulitic crystal growth [x = 0.010 M Tb3+, nanocrystals were produced only at 4 h of maturation. At higher times (96 h) the method yielded Tb3+:cit-cAp prisms up to 300 nm length, containing 12.80 ± 0.18 wt% Tb3+, and a carbonate content of 4.02 ± 0.18 wt%. The Tb3+ content of the particles is in agreement with those previously reported for Tb3+-doped apatites prepared using the sol-gel method, i.e., 8 and 12 wt% [3+:cit-cAp prisms prevailed, leading to spherical agglomerates and, in addition, TbPO4·H2O nanocrystals formed, raising the total Tb3+ content of the precipitate to 14.20 ± 0.16 wt%. Increasing x to 0.015 M, the amorphous phase was found at 4 h, while the Tb-doped apatite started to appear at 24 h, and the TbPO4·H2O (rhabdophane) nanoparticles at 7 days. At x = 0.020 M Tb3+ the amorphous phase persisted at all maturation times, while Tb3+:cit-cAp microcrystals and TbPO4·H2O nanocrystals formed at 96 h. The higher Tb3+ content of these precipitates, i.e., 19.10 ± 0.40 wt% for x = 0.015 M Tb3+ and 21.71 ± 0.77 for x = 0.020 M Tb3+, is explained by the simultaneous presence of amorphous TbPO4·nH2O and rhabdophane phases besides the highly-doped Tb3+:cit-cAp microcrystals. The above findings confirm that the herein reported bioinspired route succeeded in preparing Tbl growth . For x =d 12 wt% , or fromd 12 wt% , and in d 12 wt% . At 7 da3+ content of the apatite is ~12 wt%, and the heterogeneity of the samples  is beneficial to increase the Tb3+ content, thus increasing the luminescence intensity. In fact, R.L.I. was almost linearly dependent on x, and thus, on the global Tb wt% of the precipitates, regardless the sample composition. In this respect, when x ≤ 0.010 M, Tb3+:cit-cAp nanocrystals reached an R.L.I. ~300, and for x > 0.010 M Tb3+ the R.L.I. reached 850. The presence of amorphous TbPO4·nH2O contributed positively to this significant increase, as previously reported [The findings suggest that the maximum Tbreported . In addireported , proving3+ content. This phase was identified as amorphous TbPO4·nH2O, likely doped with Ca2+ ions by considering the following arguments: a) the bulging of the XRD baseline is similar to that reported for amorphous rare earth phosphates [−1 (υ1PO4), similarly to found in [−1 [x = 0.015 M Tb3+, and at 96 h for x = 0.020 M Tb3+, might also indicate the presence in the mixture of an ACP, likely doped with Tb3+, acting as precursor of the apatitic phase. The presence of Tb3+-doped ACP and amorphous TbPO4·nH2O in different proportions in the amorphous mixture can explain the increase in R.L.I. when increasing x from 0.015 M Tb3+ to 0.020 M Tb3+ at 4 h of maturation time. The co-precipitation of terbium phosphate and apatite for x ≥ 0.010 M Tb3+ in such Ca2+-rich solutions is possible, because the ionic activity product largely exceeds the solubility product of both compounds. The saturation indices (S.I.) for TbPO4 were 10.11 for x = 0.010 M, 10.6 for x = 0.015 M Tb3+, and 10.40 for x = 0.020 M Tb3+. For the same solutions, the S.I.s of apatite were 17.5 for x = 0.010 M, 17.3 for x = 0.015 M Tb3+ and 17.2 for x = 0.020 M Tb3+ [A key point to discuss is the nature of the amorphous phase observed in the samples with highest Tbosphates , and quiosphates ); b) thefound in , while fd in [−1 . Neverth0 M Tb3+ . 3+ content on the ζ-potential, the main findings were: the important decrease of ζ-potential in function of pH in all samples as well as the more negative ζ-potential values for particles with adsorbed citrate (Tb3+:cit-cAp) in comparison to citrate-free ones (Tb3+:cAp). Finally, , it is possible to deduce that at these pH values Ca2+ and Tb3+ had a negligible influence in the ζ-potential, as these species are occupying crystal lattice positions and were not adsorbed to the particle surface. Only H+ adsorption influenced the ζ-potential at these pHs. At pHs higher than the isoelectric point, the phosphates, HCO3−/CO32−, OH− and their negatively charged complexes were likely the responsible for the decrease of the ζ-potential. For Tb3+:cit-cAp, it is assumed that citrate coating acted as a barrier for the dissolution of the particles. For these particles, besides OH−, the deprotonated –COO− groups of adsorbed citrate molecules, pointing upward toward the solution, were the ones responsible for the decrease of the ζ-potential. In a previous study [1 = 3.13, pK2 = 4.76, and pK3 = 6.40), allowing the citrate species to interact with the apatite in different ways. Thus, the cit3+ ions interact weakly in a bidentate manner (1 citrate per 2 Ca sites), whereas Hcit2− interacts strongly in a monodentate manner (1 citrate per 1 Ca site). For both citrate species, the free –COO− groups pointing toward the solution are those influencing the ζ-potential. The synergistic effect of Tb3+ doping with citrate coating is, thus beneficial for the stability of suspensions of these particles in physiological conditions, and thus for their in vivo applications.Concerning the impact of citrate and Tbsundaram , for salus study  we put fSumming up, all the above properties are positive relative to the applications of these nanoparticles as luminescent labeling agents, or as nanocarriers for drug delivery, and highlight the potential of the thermal decomplexing synthetic method to prepare this kind of nanophosphor.3+:cit-cAp nanocrystals in a range of Tb3+ doping concentrations and maturation times, with a maximum Tb3+ content of ~12.8 wt% . Tb3+ content increased to 19.10 wt% for mixtures with TbPO4 phases  and to 21.71 wt% for mixture x = 0.020 M Tb3+, 96 h. The R.L.I. increased almost linearly with Tb3+ content, regardless of the sample composition, thus revealing that the presence of TbPO4 phases in the sample is beneficial to increase luminescence. The particles showed good luminescence properties, large Stokes shifts, high luminescence lifetime, similar optical properties in powder and dispersed in water, unaffected by pH , ionic strength, and temperature. Moreover, they were not cytotoxic against A375, MCF7, and HeLa carcinogenic cell lines, as well as against fibroblast healthy cells, and their luminescence was maintained after cell internalization of the particles, allowing intracellular localization by flow cytometry and fluorescence confocal microscopy. The particles are, thus, excellent candidates for bioimaging.The citrate-based thermal decomplexing method succeeded in preparing biomimetic (bonelike) Tb


The aim of the present study was to describe the dynamics of serum IgG  and total protein  concentrations during the first 16 d of life. Secondary objectives were to evaluate the transfer of passive immunity (TPI) classification at d 1 of life as a conditional factor for the aforementioned dynamics, and to describe over time changes on calves' TPI classification. At a commercial raising operation, 36 calves  were sampled immediately after arrival (d 1) and at d 4, 8, 12 and 16 of life, for serum IgG and TP concentration, and hematocrit determination (HCT). Transfer of passive immunity was categorized based on serum IgG  and TP concentrations . Multiple linear regression was used to evaluate serum IgG and TP changes over time, considering the effects of time after birth, breed, HCT, and TPI classification at d 1 of life. At d 1, median serum IgG and TP concentrations were 29.9 g/L and 6.3 g/dL, respectively . Dynamics of serum IgG and TP concentrations were conditional to TPI at d 1 of life. Serum IgG concentration declined over time for IgG-Excellent and IgG-Good calves , but remained constant for IgG-Poor calves. Serum TP concentration declined over time in the 3 TPI groups but it was more marked for TP-Excellent (27%) and TP-Good (19%) than for TP-Poor (14%) calves. At d 1, 83.3% of the calves were classified as IgG-Excellent or IgG-Good, whereas 77.8, 55.6, 41.7, and 58.3% of calves were classified within these categories at d 4, 8, 12, and 16 of life, respectively. Similarly, at d 1, 66.7% of calves were classified as TP-Excellent or TP-Good, whereas 47.2, 36.1, 25.0, and 2.8% were classified within these categories at d 4, 8, 12, and 16 of life, respectively. In summary, our results indicate that serum IgG and TP concentrations decline over 16 d of life, and the decline is associated with TPI classification at d 1 of life. Further studies are needed to determine the biological implications of serum IgG and TP decline after d 1 of life, and to elucidate the factors determining the different dynamics. Our results suggest that current thresholds for TPI classification should be interpreted carefully when the age of calves is unknown or outside the age range used to define those thresholds (>24 h to 7 d). Calves are born agammaglobulinemic  as the cow's cotyledonary synepitheliochorial placenta prevents transfer of passive immunity . FurtherRID); TPI) classification and a wider time window for its determination. Serum IgG concentrations within 1 to 7 d of life of <10.0, 10.0 to 17.9, 18.0 to 24.9, and ≥25.0 g/L are considered to indicate poor, fair, good, and excellent TPI, respectively (TP) can be measured calf-side using refractometry as an indirect means to estimate serum IgG concentrations  and indirect (TP determined with refractometry) measurements.2 of patio space , bedded with almond and rice husks, and bottle-fed twice a day 1.9 L of milk replacer  prepared to reach a 14% Brix reading. Water and starter  were bucket-fed ad libitum starting at 1 and 3 d of life, respectively. From 3 to 15 d of life, calves were bottle-fed 1.9 L of oral electrolyte solution in between milk feedings  as part of the calf raising operation management procedures.All procedures carried out in this study were approved by the University of California Davis Institutional Animal Care and Use Committee (#21615). The study was conducted from December 2019 to February 2020 on a commercial operation raising 1,000 preweaning Holstein and Jersey female calves. All enrolled calves were born at the same source dairy, located <1 km away from the raising operation. Newborn calves were separated from their dams within 3 h after birth and housed in group pens bedded with almond and rice husks for 24 h before being transported to the calf raising operation. Within the first 24 h of life, calves were fed 6 L of pasteurized colostrum from multiparous cows (three 2-L feedings), and their navels were dipped with a 7% iodine solution. At the calf raising operation, calves were placed in individual plastic hutches wire-fenced to allow for 1.73 m2-EDTA for hematocrit (HCT) determination. Samples were stored on ice and transported to the laboratory within 4 h after collection. Blood samples for serum IgG and TP determination were centrifuged at 1,430 × g for 20 min at 24°C. Indirect measurements of serum TP were performed using the TP scale on a handheld digital Brix refractometer (Misco DD-2). The remaining serum was transferred into 1.7-mL aliquots and frozen at −20°C for later IgG determination. Serum IgG concentration was determined using a single RID technique in accordance with manufacturer directions (Triple J Farms) within 4 mo after collection. Samples that exceeded the highest bovine reference serum IgG concentration were diluted with deionized water in a 1:1 proportion. Diameters of precipitin rings were measured using a digital caliper  after 24 h of incubation at room temperature. The intra-assay CV was 4.8% for IgG RID determination. Samples for HCT determination were transferred into microhematocrit capillary tubes (Jorgensen Laboratories Inc.) and centrifuged at 2,000 × g at 24°C for 5 min at room temperature. The HCT value was measured using a reader microhematocrit card (LW Scientific).A convenience sample of 36 female calves (19 Holstein and 17 Jersey) were sequentially enrolled in the experiment as they arrived at the raising operation . Blood samples were collected at the same time (1100 h) on d 1 , 4, 8, 12, and 16 of life by jugular venipuncture into 2 vacuum tubes : 1 without anticoagulant for serum IgG and TP determinations and 1 containing KIgG-Poor: IgG <18 g/L), good (IgG-Good: IgG 18 to <25 g/L), or excellent (IgG-Excellent: IgG ≥25 g/L). Using serum TP concentration, TPI was categorized as follows: fair to poor (TP-Poor: <5.8 g/dL), good (TP-Good: 5.8 to <6.2 g/dL), or excellent (TP-Excellent: ≥6.2 g/dL). Changes in serum IgG and TP concentration over time were evaluated through multiple linear regression using the MIXED procedure. Variables considered as fixed effects in the multiple linear regression models were time after birth , breed (Holstein and Jersey), HCT, and TPI classification at d 1 of life . Repeated measures were modeled with time as the repeated measure, calf as the subject, and the variance-covariance structure selected based on the lowest Akaike's information criterion. Independent explanatory variables not statistically associated with the outcome (P > 0.10) were removed by stepwise backward selection. Pairwise interactions among breed, TPI classification at d 1 of life, and time were evaluated and removed at P > 0.10 when appropriate. Least squares means comparisons were conducted with the PLM procedure adjusted by Bonferroni or Dunnett (with d 1 of life as reference). Final model fit was assessed with residual plots. Statistical significance was declared at P ≤ 0.05. The accompanying figures were created with SigmaPlot . The interquartile range (IQR: 25th percentile–75th percentile) was used to describe the variables.All statistical analyses were performed in SAS (SAS Institute Inc.). Based on P < 0.001), TPI classification at d 1 of life (P < 0.001), and time by TPI classification at d 1 of life (P = 0.005). Compared with that at d 1 of life (26.63 ± 2.63 g/L), serum IgG concentration was lower at d 4 , d 8 , d 12 , and d 16 of life . Relative to that at d 1 of life, serum IgG concentration declined by 5.55 ± 2.04 g/L (20.8%) at d 4, 8.10 ± 1.83 g/L (30.4%) at d 8, 10.73 ± 1.92 g/L (40.3%) at d 12, and 8.88 ± 2.34 g/L (33.4%) at d 16 of life. Overall, serum IgG concentration within the first 16 d of life was higher for IgG-Excellent  than for IgG-Good  or IgG-Poor calves . No significant differences in serum IgG concentration were observed between IgG-Good and IgG-Poor calves (P = 0.34). Within the first 16 d of life, time effect on serum IgG concentration was conditional on TPI classification at d 1 of life (P < 0.001) and 6.27 ± 4.10 g/L for IgG-Good calves , but the decline was not significant for those classified as IgG-Poor at d 1 of life . No associations between serum IgG concentration and breed or HCT were observed.Median serum IgG concentration at d 1 of life was 29.87 g/L (IQR: 21.32–42.27 g/L). Serum IgG concentration was associated with time after birth , TPI classification at d 1 of life (P < 0.001), and time by TPI classification at d 1 of life (P < 0.001) while adjusting by the HCT linear (P = 0.11) and quadratic (P = 0.02) effects. Compared with that at d 1 of life (6.08 ± 0.17 g/dL), serum TP concentration was lower at d 4 , d 8 , d 12 , and d 16 of life . Relative to that at d 1 of life, serum TP concentration declined by 0.33 ± 0.11 g/dL (5.4%) at d 4, 0.50 ± 0.10 g/dL (8.2%) at d 8, 0.84 ± 0.14 g/dL (13.8%) at d 12, and 1.26 ± 0.14 g/dL (20.7%) at d 16 of life. Overall, serum TP concentration within the first 16 d of life was 5.91 ± 0.11 g/dL for TP-Excellent (n = 20), 5.63 ± 0.24 g/dL for TP-Good (n = 4), and 4.95 ± 0.14 g/dL for TP-Poor (n = 12). Serum TP was significantly higher for TP-Excellent than for TP-Poor calves (P < 0.001); however, no significant differences on serum TP concentration were observed between TP-Good and TP-Poor (P = 0.06) or TP-Excellent calves (P = 0.92). Within the first 16 d of life, the time effect on serum TP concentration was conditional on TPI classification at d 1 of life (P < 0.001), 1.18 ± 0.32 g/dL for TP-Good , and 0.74 ± 0.19 g/dL for TP-Poor calves , respectively. No statistical association between serum TP concentration and breed was observed.Median serum TP concentration at d 1 of life was 6.3 g/dL (IQR: 5.6–6.7 g/dL). Similar to findings on serum IgG, serum TP concentration was associated with time , TP-Good and TP-Poor calves (+0.7 g/dL), and TP-Good and TP-Excellent calves (−0.3 g/dL). Consistent with our results, In our study, the proportion of calves classified as TPI-Poor (<18 g/L) for IgG was 17% at d 1 and 44% at d 8; consistent with our results, Classifying calves based on TPI at d 1 of life led to categories represented by few calves, given our initial sample size, and we caution the reader about this limitation. Also, it should be noted that calf management may have influenced the results presented herein. Regardless, the observed serum IgG and TP dynamics conditional on TPI classification at d 1 of life have potential to be biologically relevant and warrant further study. Currently accepted TPI thresholds were validated with calves from 24 h to d 7 of life. Thus, validation of current thresholds for IgG and TP at different ages will be informative, especially considering that large calf raising operations are assessing TPI on a specific day .We conclude that serum IgG and TP concentrations decline within the first weeks of life, and this decline is associated with TPI classification at 1 d of life. Current TPI classifications should be interpreted carefully when calf age is unknown or is outside the age range used in their initial validation.


Purpose: There is a paucity of data on physiological heart adaptation in elite-level judo female athletes. This study aimed to assess left ventricular morphology and function in highly trained elite female judokas.Methods: The study prospectively included 18 females aged 23.5 ± 2.25 years, nine elite level judokas, and nine healthy non-athlete volunteers. All participants underwent a medical examination, electrocardiogram, and transthoracic 2D echocardiogram. Left ventricular diastolic and systolic diameters and volumes were determined, and parameters of left heart geometry and function (systolic and diastolic) were measured, calculated, and compared between groups.Results: When groups were compared, judokas had significantly increased left ventricular cavity dimensions p < 0.01, left ventricular wall thickness p < 0.01, and volumes p < 0.01. Elite female judokas exhibited left ventricular dilatation demonstrated as high prevalence increased end-diastolic volume/index, and increased end-systolic volume/index in 88.9% of judokas vs. 0% in controls, p < 0.01. Left ventricle mass/index was significantly increased in judokas, p < 0.01), with a 43.3% difference between groups. The majority (77.7%) of judokas had normal left ventricular geometry, although eccentric hypertrophy was revealed in 2 (22.2%) of judokas.Conclusion: Elite, highly trained female judokas exhibit significant changes in left heart morphology as a result of vigorous training compared to non-athletes. These findings suggest that female judokas athletes’ heart follows a pattern toward chamber dilatation rather than left ventricular wall hypertrophy. Judo is a popular combat sport worldwide. As reports from International Judo Federation (IJF) claim, there are 1,181 female judokas competing on an elite level, spread across all weight categories. Despite many proven positive physiological benefits of practicing judo, it inevitably leads to the exhibition of various adaptations of the cardiovascular system . Combat Over time these complex adaptive changes usually lead to the development of the “athlete’s heart” . Early rUnderstanding the specific features of the athlete’s heart in specific populations is of great importance in order to know what to expect of a specific athlete’s sports discipline and gender. Initial and follow-up examinations help us to differentiate physiological exercise that induces cardiac remodeling from pathological conditions. Differentiating exercise-induced cardiac remodeling of abnormal cardiovascular pathology is important in screening and diagnosing potentially life-threatening conditions. Essentially, there is limited data regarding female athletes’ heart response to long-term elite-level judo participation and training stimulus . To our This study aimed to assess left ventricular morphology and function in highly trained elite female judokas.The study prospectively included 18 females aged 23.5 ± 2.25 years. The Judo group consisted of nine elite-level judo athletes  and nine healthy non-athlete female student volunteers previously not involved in regular exercise training for the last 5 years in the control group. All judokas were a part of the national team, participating in both national and international contests. Subjects started with judo practicing between six and 8 years old, the average duration of the judo training experience was 16.55 ± 3.16 years, with twenty to twenty-five (20-25) hours of training volume per week. Judokas spent 1,5-2 h of training in the weight room two to three times a week. The participants competed for the Serbia senior national team in international competitions and scored for the IJF rank, and they achieved the minimum of one ranking. Two competitors were category up to 48 kg, two −52, one −57, two −63, one −78, and one +78 kg. They are all still competing. Four of them achieved the Olympics norm , and two were close to the Olympics norm and the 25th place. Three of them participated in the Olympics, one didn’t because two were in the same category −48 kg, but only one could participate.All participants underwent a medical examination, electrocardiogram (ECG), and transthoracic 2D echocardiogram at the Institute for Cardiovascular Diseases of Vojvodina, Serbia. All participants underwent a standard resting 12-lead surface ECG record at a paper speed of 25 mm/s and gain of 10 mm/mV. One cardiologist interpreted each ECG to exclude potential abnormalities. Exclusion criteria were: A previous history of arterial hypertension, diabetes mellitus, heart, kidney, hepatic, infectious, psychiatric, malignant, disorders, taking drugs that can influence ECG intervals, and electrolyte imbalance. The study was performed corresponding to the Helsinki Declaration with ethical approval obtained from the local ethics committee of the University of Novi Sad, Serbia (Ref. No. 46-06-02/2020-1). Measurements were done in the early morning hours before any water or food intake. For the judo group at least 24 h after the last training. Body height (BH) measurement was performed in a standing position with heels together, toes apart, and hands close to the body with an anthropometer, according to Martin , with an accuracy of 0.1 cm. Body mass (BM) was determined using an Omron weight scale BF511  with an accuracy of 0.1 kg. Body surface area (BSA) and body mass index (BMI) were calculated using the following formulas:.All subjects underwent two-dimensional transthoracic echocardiography (ECHO) using Vivid E9  machine that is equipped with an M5S-D, 1.5–4.6 MHz transducer with continuous ECG monitoring during the examination. Subjects were instructed to take a left lateral decubital position for examination. One investigator, blinded to the clinical characteristics of the participants, without blinding was evaluating echocardiographic parameters . All measurements and calculations were performed by previously described methods according to standardized procedures by the European Association of Cardiovascular Imaging  and the t)–(5/6A2L), where A1 is epicardial area at end-diastole (cm2), A2 endocardial area at end-diastole (cm2), L-ventricular length at end-diastole (cm), t = average wall thickness (cm), 1, 05-specific gravity of the muscle (g/ml). To obtain the indexed value (LVMi) the LVM was divided by BSA. Calculation of left ventricular hypertrophy level (LVHL) was calculated by adding intraventricular septum diameter (IVSDd) and posterior wall thickness (PLWd) and dividing the sum by two (LVHL= (IVSDd + PLWd)/2).Parasternal long-axis view (2D) was used to measure LV end-diastolic (LVEDd) and systolic (LVEDs) diameter, the diastolic thickness of the inter-ventricular septum (IVS), posterolateral wall (PLW), aortic root, ascending aorta dimension and cusp separation. Volumes of the left atrium (LA) were measured by the Simpson method using apical 4-chamber and 2-chamber views at end-systole in maximum LA size (LAVs) and then normalized for body surface area (BSA) as LAVs index (LAVsI). Using the biplane method of disks, left ventricular end-diastolic (EDV) and end-systolic volumes (ESV) were measured, and EF was calculated. Left ventricular mass (LVM) was calculated by the software automatically via area–length method, according to measures obtained from parasternal short-axis view in which mid-ventricular systolic and diastolic epicardial and endocardial surfaces were traced (with the exclusion of papillary muscles), including measuring the systolic and diastolic mitral-to-apical distance in apical 4-chamber view. LVM = 1, 05 (5/6A2 (L + According to left ventricular mass index (LVMI) and relative wall thickness (RWT), left ventricular morphology is classified into one of four structural groups: 1) normal geometry, characterized by normal LVMI ≤95 g/m2 and RWT ≤42 mm; 2) concentric remodeling, LVMI ≤95 g/m2 and RWT >42 mm; 3) Concentric hypertrophy, LVMI>95 g/m2 and RWT>42 mm; and 4) Eccentric hypertrophy LVMI>95 g/m2 and RWT ≤42 mm (8).In the apical 4-chamber view, using transmitral pulsed-wave (PW) Doppler at the tips of mitral leaflets peak early wave (E), atrial late (A) diastolic filling velocities were measured, and E/A ratio was calculated. Tissue Doppler on the mitral annulus was obtained at the septal and lateral positions, where peak early (e′) velocities were measured and averaged. E/e' ratio was calculated from E velocity and averaged e' velocities obtained from septal and lateral positions of the mitral annulus.t-test or Chi-square test where appropriate. A t-test was used to compare continuous variables with normal distribution, and a chi-square test was used to compare categorical variables. A p-value < 0.05 was considered statistically significant. All calculations and interpretations were made using Statistical Package for Social Sciences–SPSS Version 20.0 .The analysis included descriptive statistics. To test the normal distribution, the Kolmogorov-Smirnov test was used. Continuous variables data are presented as mean ± standard deviation. Categorical variables are expressed as absolute numbers and percentages. The data were compared using the Students p = 0.84, BW 63.92 ± 13.87 vs. 62.67 ± 9.35 kg, p = 0.83, BMI 22.82 ± 6.64 vs. 22.31 ± 3.04 (kg/m2) p = 0.83, and BSA 1.70 ± 0.19 m2 vs. 1.71 ± 0.18 m2, p = 0.93. The mean age of judo athletes was 24.67 ± 3.84 years, while the mean age of the control group was 23.44 ± 0.88 years, p = 0.38.There was no statistically significant difference between judo and control group for: BH 166.56 ± 7.29 vs. 167.44 ± 10.78 cm, p < 0.01, LVEDd/BSA 29.05 ± 2.34 vs. 25.45 ± 2.56 mm/m2, p < 0.01, with 22.46% difference, LVEDs 33.11 ± 2.32 vs. 27.00 ± 2.55 mm, p < 0.01, and LVEDs/BSA 19.63 ± 2.04 vs. 16.03 ± 2.58 mm/m2, p < 0.01, with 14.14% difference), LV wall thickness  and volumes EDVI 70.07 ± 7.75 vs. 49.12 ± 6.32ml/m2, p < 0.01, ESVI 28.39 ± 3.14 vs. 17.15 ± 2.40 ml/m2, p < 0.01, (p < 0.01 in controls and increased ESV (>42 ml) and ESVI (>24 ml/m2) in 88.9% vs. 0% of controls, p < 0.01.When groups were compared judokas had significantly increased left ventricular cavity dimensions  exhibited normal left ventricular geometry. Eccentric hypertrophy was found in two judokas (who had an Olympic norm) 22.2% vs. 0%, p = 0.14.Majority of judokas , left ventricular wall thickness didn’t exceed 11mm, but 10.3% exhibited left ventricular internal diameter above 50 mm, and 4.2% above 55 mm . In our Our results confirm that athlete training is associated with enhanced systolic and diastolic function, probably mediated by a combination of enhanced early relaxation and increased LV compliance. The diastolic function shows evidence of enhancement in lower late atrial A wave, higher e' waves, and lower filing velocities E/e.’ Greater stroke volumes and SVI reflect improvement in systolic function. In a large meta-analysis, when groups of endurance-, resistance- and mixed-training athletes and healthy controls were compared, the E/A ratio was either normal or slightly, but not significantly, enhanced in athletes compared to controls . In 1991Whyte et al., in a meta-analysis of 13 studies , including 38 (16.7%) judokas (classified into the strength and sprint group) suggested that female athletes follow a similar pattern of cardiac adaptation as men . As therp < 0.001)  , these f< 0.001) , 3–4 min< 0.001) . We hypo< 0.001)  prior to< 0.001) . Echocar< 0.001) . These rThese findings confirm that an athlete’s physiological cardiac adaptation pattern is much more complicated than first thought and that it can’t be generalized to a sports discipline. It probably depends on numerous individual parameters like individual sport type, duration of practicing, weekly training volume, the athlete’s weight category, and other variables that influence the physiology of heart adaptation. Further longitudinal studies could help us piece the puzzle together and see the whole picture.n = 18 subjects. However, our sample consisted of nine elite-level judokas competing at an international level, where four of our subjects met entry norms for the Olympic games in 2020. Therefore, recruiting a larger number of athletes at this level is constricted. The measurements were only performed once by only one echocardiographist, and parameters are only indexed to BSA, which may not represent the most precise method.Admittedly, the first weakness of this study is the small sample size of only These findings suggest that elite, highly trained female judokas exhibit significant changes in left heart morphology. Furthermore, athletes’ heart follows a pattern toward chamber dilatation rather than left ventricular wall hypertrophy. The study deals with the population of judo elite-female athletes that have not been exclusively previously investigated, furthermore gives us a different insight into the cardiac remodeling in this specific population. These results indicate that cardiac remodeling is complex and depends on undouble different factors. The findings might assist us in perceiving physiological training-induced adaptations from pathological conditions which may predispose adverse cardiovascular events among athletes. Results may encourage us for further research to strengthen our knowledge related to specific heart structure and function adaptations that play a crucial role in heart remodeling classification and the distinction between pathophysiology and individual sport-specific adaptations.


TMEM151A, encoding transmembrane protein 151 A, a protein of undetermined function, have been very recently associated with paroxysmal kinesigenic dyskinesia (PKD) in the Chinese population.TMEM151A is highly expressed in the brain, including the cerebral cortex and the thalamus and is highly conserved among species. To definitively confirm the association between TMEM151A and PKD, other mutations in the same gene should be identified in independent cohorts from different populations.We read with great interest the article by Tian and colleagues.PRRT2 (proline‐rich transmembrane protein 2) mutations as well as their asymptomatic parents. PKD diagnosis was made by movement disorders specialists according to the consensus clinical criteria.TMEM151A in a single patient . This variant was subsequently classified as likely pathogenic (class IV) according to the ACMG criteria (PS2 + PM1 + PM2 + PP2 + PP4).TMEM151A mutation was identified in one of 23 patients of our PRRT2‐negative PKD cohort, which is in accordance with the frequency of 4.8% found by Tian et al.We applied whole exome sequencing (WES) on 23 French patients with sporadic PKD who tested negative for TMEM151A in a patient with PKD. Our findings confirm TMEM151A variants as a genetic cause of PKD and suggest that de novo mutations in this gene are infrequently responsible for sporadic PKD cases.TMEM151A‐related disorders. Whether TMEM151A is a transmembrane protein involved in synaptic function and whether TMEM151A‐related PKD is underpinned by its loss of function also remain to be elucidated.We report on a de novo mutation in (1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing First Draft, B. Review and Critique.T.W.: 1A, 1C, 2C, 3AA.M.: 1A, 1C, 2C, 3BN.D.: 1C, 2CG.R.: 1C, 2CO.L.B.: 1CJ.C.: 1A, 1C, 3BC.T.: 1A, 1C, 3BA.P.: 1A, 1C, 3BE.R.: 1A, 1B, 1C, 3BM.A.: 1A, 1B, 1C, 2C, 3BADCY5.org, Agence Nationale de la Recherche, Societé Française de Médecine Esthétique, and the Dystonia Medical Research Foundation. The other authors declare no competing interest.Thomas Wirth received grants from the Revue Neurologique, the Fondation Planiol, and the Association des Personnes Concernées par le Tremblement Essentiel (APTES) organizations and travel funding from LVL Medical. Aurélie Méneret received speaker honoraria from Abbvie. Emmanuel Roze received honorarium from Orkyn, Aguettant, and Elivie for speeches and for participating in the advisory board of Allergan and received research support from Merz‐Pharma, Orkyn, Aguettant, Elivie, Ipsen, Allergan, Everpharma, Fondation Desmarest, Association des Malades Atteints de Dystonie (AMADYS),