About 25% of patients with acute hepatitis C virus (HCV) infection show spontaneous clearance within the first six months of infection but may remain at risk of inflammaging, aging, and liver and non-liver disease complications. This study evaluated the differences in the plasma levels of immune checkpoints (ICs) and senescence-associated secretory phenotype (SASP) biomarkers between patients who had spontaneously eliminated HCV infection (SC group) and individuals without evidence of HCV infection (C group).We performed a multicenter retrospective study of 56 individuals: 32 in the SC and 24 in the C groups. ICs and SASP proteins were analyzed using a Luminex 200TM analyzer. The statistical analysis used Generalized Linear Models with gamma distribution (log-link) adjusted by significant variables and sex.13 ICs , CD27, CD28, CD80, GITR, HVEM, IDO, LAG-3, PD-1, PD-L1, PD-L2, and TIM-3) and 13 SASP proteins  were significantly higher in SC group after approximately more than two years of HCV clearance. After stratifying by sex, differences remained significant for males, which showed higher levels for 13 ICs and 4 SASP proteins in SC. While only PD-L2 was significantly higher in SC women, and no differences in SASP were found.Higher plasma levels of different IC and SASP proteins were found in individuals after more than two years of HCV clearance, mainly in men. Alterations in these molecules might be associated with an increased risk of developing liver and non-hepatic diseases.The online version contains supplementary material available at 10.1186/s12979-023-00387-z. Panel 2 included ICs that regulate NK cell activation by the balance of inhibitory  and activating signals .Additionally, 26 senescence-associated secretory phenotype (SASP) biomarkers were also evaluated using the Luminex 200™ system : epidermal growth factor (EGF), eotaxin, chemokine growth-regulated protein alpha , granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), IL-1 beta, IL-1alpha, IL-1RA, IL-2, IL-6, IL-7, IL-8(CXCL10), IL-13, IL-15, IL-18, C-X-C motif chemokine ligand 10 (IP-10(CXCL10)), C–C motif chemokine ligand 2 (MCP-1(CCL2)), C–C motif chemokine ligand 5 (RANTES), stromal cell-derived factor 1-alpha ), fibroblast growth factor 2 (FGF-2), hepatocyte growth factor (HGF), nerve growth factor beta (Beta-NGF), placental growth factor (PIGF-1), skp, cullin, F-box containing complex (SCF), tumor necrosis factor (TNF) alpha, and TNF-beta.The measured raw fluorescence intensity (FI) values  were used as previously described .IFN-λ4 genotype (rs12979860), and aspartate transaminase (AST)). These covariates were previously selected by a stepwise method (forward), where at each step, the covariates were considered to enter according to the lowest AKAike information criteria (AIC) for that specific model. Fibrosis-4 index (FIB4) and AST to platelet ratio index (APRI) were not considered covariates in the GLM models because AST was used to calculate these scores. Likewise, alanine transaminase (ALT) was not considered because of its collinearity with AST.For the descriptive study, quantitative variables  were expressed as median , and categorical variables were shown as absolute count (percentage). Independent groups were compared using the Mann–Whitney U and Chi-square tests for quantitative and categorical variables, respectively. Generalized Linear Models (GLM) with gamma distribution (log-link) were used to estimate differences in the ICs and SASP biomarkers levels between SC and C groups. This test provides the arithmetic mean ratio (AMR), the 95% of confidence interval (95% CI), and its level of significance. Moreover, we adjusted the GLM models for the main available epidemiological and clinical characteristics . The time from clearance was also included as covariate when analyses were performed by sex within the SC group.p < 0.05; q-value < 0.10) were considered relevant.Correlation between significant ICs and SASP proteins was performed using the Spearman correlation test. Those suitable correlations (r > 0.5 or r < -0.5) with a significance value  and q-value < 0.10. The statistical analysis was done with R statistical package .All Additional file 1. Biochemical characteristics of 56 individuals stratified by HCV infection status and sex.Additional file 2. Comparison of plasma immune checkpoints between subjects who spontaneously cleared HCV versus the control group.Additional file 3. Comparison of plasma immune checkpoints between males who spontaneously cleared HCV versus the control group.Additional file 4. Comparison of plasma immune checkpoint proteins between females who spontaneously cleared HCV (SC group) versus controls (C group).Additional file 5. Comparison of senescence-associated secretory phenotype (SASP) proteins between subjects who spontaneously cleared HCV (SC group) versus controls (C group).Additional file 6. Comparison of senescence-associated secretory phenotype (SASP) proteins between males who spontaneously cleared HCV (SC group) versus controls (C group).Additional file 7. Comparison of senescence-associated secretory phenotype (SASP) proteins between females who spontaneously cleared HCV (SC group) versus controls (C group).Additional file 8. Comparison of plasma immune checkpoints between sex in both spontaneous clearance (SC) and control (C) groups: A) SC, B) C.Additional file 9. Comparison of senescence-associated secretory phenotype (SASP) proteins between sex in both spontaneous clearance (SC) and control (C) groups: A) SC, B) C.Additional file 10. Spearman correlation plot between significant immune checkpoint and senescence-associated secretory phenotype (SASP) proteins in males: A) ALL males, B) spontaneous clearance (SC), C) control (C) group.Additional file 11. Spearman correlation plot between significant immune checkpoint and senescence-associated secretory phenotype (SASP) proteins in males: A) ALL males, B) spontaneous clearance (SC), C) control (C) group.


Italian Journal of Pediatrics (2023) 49:10510.1186/s13052-023-01516-6The original article [1] contained an error in Table The correct version of Table


We report a newly emerged SARS-CoV-2 Omicron subvariant FY.4 that has mutations Y451H in spike and P42L in open reading frame 3a proteins. FY.4 emergence coincided with increased SARS-CoV-2 cases in coastal Kenya during April–May 2023. Continued SARS-CoV-2 genomic surveillance is needed to identify new lineages to inform COVID-19 outbreak prevention. The last 3 waves were dominated by Omicron BA.1-like, BA.5-like, and BQ-like lineages (in that order), associated with increases in SARS-CoV-2 cases because of virus mutations that conferred transmission advantage or escape from preexisting immunity   (n = 35) or MiSeq  (n = 61) instruments; we recovered 76 (79%) genomes with >70% coverage. We also received 39 SARS-CoV-2–positive samples from health facilities outside the KHDSS and sequenced 32 (82%) of them, yielding 25 (78%) genomes  of 1,612 samples collected from KHDSS health facilities were positive for SARS-CoV-2. We sequenced 96 (80%) of 120 samples that had PCR cycle threshold values <35 by using GridION , FY.4 had been detected in 4 other counties: Mombasa (n = 2), Narok (n = 2), Nairobi (n = 7), and Kiambu (n = 31). The FY.4 subvariant has since been detected in Austria, Belgium, Germany, Italy, India, Sweden, Canada, France, China, Australia, Spain, the United Kingdom, and the United States  and FY.4 (n = 75). The SARS-CoV-2 infection rate increase observed in late March coincided with detection of the new Omicron FY.4 lineage , panel B>1 dose of a COVID-19 vaccine. A serosurveillance study (February–June 2022) found that 67% of unvaccinated KHDSS health facility residents had SARS-CoV-2 IgG, indicating a high percentage of this population might have been naturally infected , fever (78%), and nasal discharge (74%); 7% had difficulty breathing . Only 13Compared with other Omicron lineages, FY.4 had 2 amino acid mutations, Y451H in the spike protein and P42L in the open reading frame (ORF) 3a protein. The effect of the Y451H change is unknown; however, an L452R mutation in the receptor-binding domain of the spike protein (near the Y451H site) increased virus infectivity and fusogenicity by enhancing spike stability and cleavage (>2 consecutive weeks. We compared growth rate estimates from Kenya with those from Germany and the United States, the only other countries reporting FY.4 cases for >2 consecutive weeks during the last weeks of May 2023 (We applied a Bayesian hierarchical model (May 2023 , panel CIn conclusion, we detected emergence of a new Omicron lineage with unique spike and ORF3a gene mutations in coastal Kenya by using SARS-CoV-2 genomic surveillance. FY.4 subvariant detection coincided with an increase in SARS-CoV-2 cases in Kilifi. FY.4 was also detected in other parts of Kenya, and growth estimates suggest potential for continued spread of the FY.4 subvariant. Continued SARS-CoV-2 genomic surveillance is critical for identifying new lineages to inform COVID-19 prevention measures.Appendix. Additional information for new SARS-CoV-2 Omicron variant with spike protein mutation Y451H, Kilifi, Kenya, March–May 2023.


To address this, we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics, and transcriptional profiles of LN-resident virus-specific CD8+ T cells in persons who control HIV without medications. Antigen-induced proliferative and cytolytic potential consistently distinguished spontaneous controllers from noncontrollers. T cell receptor analysis revealed complete clonotypic overlap between peripheral and LN-resident HIV-specific CD8+ T cells. Transcriptional analysis of LN CD8+ T cells revealed gene signatures of inflammatory chemotaxis and antigen-induced effector function. In HIV controllers, the cytotoxic effectors perforin and granzyme B were elevated among virus-specific CXCR5+ fCD8s proximate to foci of HIV RNA within germinal centers. These results provide evidence consistent with cytolytic control of lymphotropic infection supported by inflammatory recruitment, antigen-specific proliferation, and cytotoxicity of fCD8s.Follicular CD8 + T cell cytotoxicity contributes to immune control of HIV replication within germinal centers.Antigen-driven follicular CD8 CXCRar cells , 6.+ T cells (+ T cells (fCD8) has been associated with immune control of pathogenic viral infections and cancers, including LCMV  (+ T (TFH) cells in these individuals  , but HIVividuals , 38, as ividuals –44 and dividuals –49. Limilication , 50–56. variable , 53–69. + T cells from persons with durable spontaneous control of HIV replication to elucidate mechanisms by which these cells sustain immune control of active HIV replication within LNs. We investigated their in situ, ex vivo and antigen-induced functionality in paired LN and PB obtained from long-term spontaneous controllers with a range of ongoing LN HIV replication. In addition, we investigated spatial localization, clonotypic compartmentalization, and transcriptional differences associated with localization of HIV-specific CD8+ T cells within PB and LN, providing insights into regulation of migration and cytotoxicity in LNs during long-term immune control of a chronic lymphotropic viral infection.Here we examined lymphoid CD8We obtained contemporaneous PB and excisional inguinal LN specimens from 43 study participants, including 19 spontaneous HIV controllers, 17 ART-suppressed HIV noncontrollers, and 7 HIV-negative individuals . Among c+ T responses by screening peripheral blood mononuclear cells (PBMCs) for interferon-gamma (IFN-γ) secretion by enzyme-linked immunospot (ELISPOT) assay following stimulation with arrays of HLA-optimal consensus clade B HIV peptides -labeled cells with immunodominant HLA-optimal peptide, specificity was reconfirmed using matched pHLA tetramer staining  and co-expression of perforin and granzyme B among HIV-specific CD8xpansion . Compare both PB  and LN  sequences (TRB) complementarity-determining region 3 (CDR3) sequences per replicate sample  for differential expression, of which 727 genes were upregulated in LN and 620 in PB , and decreased S1PR1, were also differentially expressed in LN relative to PB  and CD45RA+ terminally differentiated effector-memory (TEMRA) subsets expressed higher levels of perforin and granzyme B than central memory (TCM) or naïve/stem-like memory (TNAIVE/TSCM) CD8+ T cells in LN  LN tissue sections stained for HIV gagpol RNA using RNAscope  closer than random versus only 18% of noncytolytic effectors  and noncytolytic (perforin-negative) fCD8s using immunofluorescence segmentation . The proA+ cells , with thffectors , further+ T cells infiltrate follicular sites of ongoing viral replication in LN, where they proliferate and express cytolytic effector molecules in response to antigen recognition. Our results suggest that inflammatory chemotaxis and antigen-driven proliferation can overcome a relative immune privilege that otherwise limits infiltration and cytotoxicity of CD8+ T cells within GCs during homeostasis, highlighting both CXCR5-mediated access and sustained cytolytic effector potential as key features of successful immune control. In contrast, virus-specific CD8+ T cells from noncontroller LNs exhibited low CXCR5 expression, poor antigen-specific proliferation and weak cytolytic potential, highlighting important barriers to widespread HIV control or eradication.This study provides evidence in support of a mechanism of spontaneous immune control of infection by which virus-specific CD8+ T cell specificity against immutable viral epitopes . CXCR5- control –18. Howe control , 63–69,  control . At the  control –95 and f control , spurrin control . We provRM) cells to viral control in LN are also incompletely defined. The extent to which antigen-specific T cells are compartmentalized between LN and PB is variable across reports from different settings. In animal models, transient exposure to antigen via immunization led to clonotypic drift over time between LN and PB in the absence of antigen re-exposure  maintained plasma viral loads below 2,000 HIV RNA copies/mL (median 27 copies/mL) without ART for a median of 23 years (IQR 12 years) before sample collection. Noncontrollers (n = 17) and HIV-negative persons (n = 7) were also included in the study. Noncontrollers treated with a variety of standard ART regimens for a median of 12 years (IQR 10 years) maintained undetectable viral loads across longitudinal measurements. Demographic and clinical characteristics of study participants are reported in HLA genotyping was performed by Dr. Mary Carrington . Sample sizes were determined by specimen availability.Contemporaneous PB and excisional inguinal LN biopsy specimens were collected after written informed consent and study approval by the Institutional Review Board of Massachusetts General Hospital . Viral loads and complete blood count records were obtained from healthcare providers with subject consent. Spontaneous controllers  sections for microscopy, and the remainder was mechanically dissociated for cellular assays. Plasma and density gradient isolated PBMCs and cells dissociated from LN were cryopreserved in liquid nitrogen. Frozen cells were thawed at 37°C and recovered in RPMI supplemented with 10% fetal bovine serum  overnight prior to functional and phenotypic assays.6/mL in RPMI supplemented with 10% FBS (R10) and plated 200 μL per well in Immobilon-P 96-well microtiter plates (Millipore) pre-coated with 2 μg/mL anti-IFN-γ . Individual HLA-optimal HIV peptides matched to each subject’s HLA genotype , cells were washed twice with R10, resuspended at 1×106/mL and plated 200 μL per well in 96-well round-bottom polystyrene plates (Corning). Individual HIV peptides corresponding to IFN-γ ELISPOT responses for each participant were added at 1 μM and incubated at 37°C for 6 days before flow cytometric assessment. Negative control wells did not receive peptide and positive control wells received 1 μg/mL anti-CD3  and anti-CD28  antibodies. On day 6, cells were stained for viability using Live/Dead Violet (Thermo Fisher), AlexaFluor700-anti-CD3 , BUV395-anti-CD8 , and APC-pHLA tetramer matching the peptide used for stimulation, then analyzed by flow cytometry. Representative gating and controls are shown in Mononuclear cells were stained at 37°C for 20 minutes with 0.5 μM CellTrace CFSE (Thermo Fisher) as per manufacturer’s protocol at 1×10Ex vivo (d0) and peptide-expanded (d6) CD8+ T cells were isolated from PBMCs and LNMCs using EasySep Human CD8+ T Cell Isolation Kit (StemCell Technologies) and stimulated with 4 nM APC-pHLA tetramer at 37°C for 4 hours in R10 with BV711-anti-CD107A  to measure cytolytic degranulation. Cells were stained with BUV395-anti-CD8 , Live/Dead Blue (Thermo Fisher), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained for intracellular PE-anti-perforin  and PE-CF594-anti-granzyme B . FMO controls were used to establish gating. Experimental negative controls were pre-treated with 50 nM dasatinib 30 minutes before tetramer stimulation to prevent activation, and experimental positive controls were treated with PMA and ionomycin . Samples were analyzed by flow cytometry. Representative gating and controls are shown in + CD8+ T cells at d0 in both PB and LN were excluded from downstream analyses.in vitro cell activation, degranulation and activation-induced cell death for phenotypic analysis. Cells were then stained with Live/Dead Blue viability dye (Thermo Fisher), BUV395-conjugated anti-CD8 , BV421-anti-CD45RA , BV650-anti-CD62L , BV711-anti-CCR7 , PerCP/Cy5.5-anti-CD56  and PE/Cy7-anti-CXCR5  for 30 minutes, after which cells were fixed and permeabilized  and stained for intracellular markers using PE-anti-perforin  and PE/CF594-anti-granzyme B  for 30 minutes. In some experiments, cells were instead stained with Live/Dead Blue, BUV395-anti-CD8, BV421-anti-CD45RA, BV650-anti-CD62L, AlexaFluor 488-anti-CXCR5 , PE/Cy7-anti-PD-1 , AlexaFluor 700-anti-CD69 , PE-anti-CD103  and PE/eFluor 610-anti-CX3CR1  or with PE-anti-CCR5  and PE/Dazzle 594-anti-CXCR3  before fixation. Cells were analyzed by flow cytometry. Gating thresholds were established using appropriate negative controls, including fluorescence-minus-one and biological negative controls, as shown in + CD8+ T cells were excluded from downstream analyses.Peptide-HLA monomers were obtained from ImmunAware . Tetramers were produced by multimerization with APC-conjugated streptavidin (Biolegend) as per manufacturer’s protocol and stored at 4°C for a maximum of 4 weeks prior to use. Staining was performed using 4 nM individual APC-conjugated pHLA tetramers at 4°C for 30 minutes after 30-minute pre-treatment with 50 nM dasatinib to prevent Flow cytometry and cell sorting were performed at the Ragon Institute flow cytometry and imaging core using BD Fortessa and LSR-II cytometers, FACSAria sorters and FACSDiva software (Becton Dickinson). Flow cytometric analyses were performed using FlowJo v10.8 (TreeStar).+ tetramer availability. Replicate populations of 500 – 2,500 HIV-specific CD8+ T cells were sorted by FACS on the basis of staining with individual pHLA tetramers into RLT Plus lysis buffer then frozen at −80 C. The following were performed as previously described (TRB) complementarity determining region 3 (CDR3) sequences using MiXCR, exclusion of genes with fewer than 5 reads in fewer than 10% of samples, multi-factor linear modeling to isolate biological effects of interest  from potentially confounding sources of variation  via forward model selection using AIC and filtering for co-linear factors for differential expression analysis using DESeq2, gene set enrichment analyses using CERNO ranked by FDR-adjusted q values (signed by direction of fold change) with GO:BP, GO:CC, GO:MF, Reactome, Kegg and Hallmark gene sets from MSigDB, and organization of differentially expressed gene sets into directional nearest-neighbor networks using gene set network analysis (GSNA). TCR clonotypes below 2% of total TRB reads were grouped as “other” due to lack of concordance across replicates below this threshold and the possibility of nonspecific tetramer binding at low frequencies representing potential single cells. Enriched transcription factor (TF) regulation signatures in PB and LN were identified by CERNO pathways analysis on TF-associated activated and repressed target gene sets derived from the dorothea human non-cancer A, B and C confidence database , LN follicular homing [CXCR5, SLAMF6, SELL, TCF7, ID3, CD200, ICOS, IL7R, BCL6  or ALP-conjugated amplifiers with Fast-Red substrate (chromogenic images) as per manufacturer’s protocol (Advanced Cell Diagnostics). Fluorescence specificity and background staining were established using manufacturer’s negative and positive control staining probes (Advanced Cell Diagnostics) and by staining of an HIV-uninfected LN. Stained sections were mounted on SuperFrost microscopy slides (Thermo Fisher) and imaged using the TissueFAXS automated slide scanning system (TissueFAXS USA) consisting of a Zeiss Axio Imager Z2 upright microscope, a Lumencor Spectra 3 light engine , combined with a Zeiss 20X 0.5NA Plan-Neofluar objective and a Maerzhauser automated microscope stage.FFPE inguinal LN tissue sections were stained with DAPI (nuclear marker), mouse IgG1 anti-perforin , mouse IgG2a anti-granzyme B , polyclonal rabbit anti-CD8 (Abcam) and/or polyclonal goat anti-IgD (Southern Biotech) primary antibodies followed by AlexaFluor550-anti-mouse IgG1, AlexaFluor488-anti-mouse IgG2a (Thermo Fisher), AlexaFluor750-anti-rabbit IgG and/or AlexaFluor488-anti-goat IgG (R&D Systems) secondary antibodies. HIV RNA was detected using RNAscope Multiplex Fluorescent Reagent Kit v2 using a HIV + cells expressing HIV gagpol RNA and DAPI+ cells co-expressing CD8, perforin and granzyme B across entire immunostained tissue sections. To evaluate intrafollicular events, a custom StrataQuest application was developed by TissueGnostics. Follicular GC regions of interest (ROIs) were drawn manually based upon morphology, relative CD8 exclusion and IgD staining of mantle-zone B cells in adjacent tissue sections, as illustrated in gagpol RNA detection above background was determined using RNAscope staining of HIV-uninfected LNs as shown in gagpol RNA positive cell, x-y coordinates of HIV gagpol RNA positive cells, perforin+ granzyme B+ CD8+ cells and perforin− CD8+ cells were extracted from image analysis reports. To compare observed effector-to-nearest-target distances against a theoretical uniform distribution, we simulated random placement of CD8+ effector cells 100,000 times within each ROI while retaining HIV RNA+ target cell positions and overall GC geometry. Observed distances were compared to simulated distances for each CD8+ effector cell to determine the probability that randomly positioned effector CD8+ cells would be located at the same distance or closer to the nearest HIV RNA+ target cell than observed.Images were processed using TissueFAXS Viewer (TissueGnostics USA) and analyzed using StrataQuest analysis software (TissueGnostics USA) to segment and quantify DAPIp values were false discovery rate adjusted (q) using the Benjamini-Hochberg method. p or q values less than 0.05 were considered statistically significant. Figures were prepared using GraphPad Prism v9.0.2 and Adobe Illustrator CC v22.0.1. Tables were prepared using Microsoft Word and Excel.Statistical tests were performed using GraphPad Prism v9.0.2 and R. Parametric tests were used for data with normal distributions and nonparametric tests were used for data with non-normal distributions. For data with seven or more replicates, normality was determined using Shapiro-Wilk tests. For data with fewer than seven replicates, normality was estimated by skewness. Where indicated, Supplementary MaterialsSUPPLEMENTARY MATERIALS+ T cells.Fig. S1. Identification of HIV-specific CD8+ T cells.Fig. S2. Gene set network enrichment in LN and PB HIV-specific CD8+ T cells.Fig. S3. Flow cytometric phenotyping of circulating and LN-derived CD8in situ hybridization imaging and quantitation.Fig. S4. HIV RNA Fig. S5. Flow cytometric gating schema and staining controls.Table S2. Immunodominant HIV antigen-specific responses.Table S1Table S1. HLA-optimal HIV peptides.Table S3Table S3. Differentially expressed genes.Table S4Table S4. Differentially expressed pathways and transcription factor signatures.Table S5Table S5. Tabular data for statistical tests.


Correction: Alz Res Ther 15, 9 (2023)10.1186/s13195-022-01158-6Following publication of the original article , there weTable 1. Codes used in the UK Biobank to identify dementia casesThe original article  has been


Besides its mitochondria-based anti-apoptotic role, Bcl-xL also travels to the nucleus to promote cancer metastasis by upregulating global histone H3 trimethyl Lys4 (H3K4me3) and TGFβ transcription. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates H3K4me3 modification are not understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced cell migration and metastasis in mouse models. Furthermore, knockout of CtBP2 suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFβ mRNA upregulation as well as cell invasion. Moreover, cleavage under targets and release using nuclease (CUT&RUN) coupled with next generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor region of genes encoding TGFβ and its signaling pathway in the cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1. Bcl-xL has long been known for its anti-apoptotic function during embryonic development and in pathological conditions . MoreoveWe have previously investigated the interdependence of Bcl-xL’s metastatic function and its canonical anti-apoptotic function. Using multiple mutants, cell lines, and mouse models, we have discovered that Bcl-xL promotes metastasis independent of its canonical anti-apoptotic function and that the metastatic function requires its nuclear translocation .As the C-terminal transmembrane domain of Bcl-xL prevents its nuclear import ; rabbit anti-Bcl-xL 1:200, Cell Signaling Technology, 2764s; MTCO1, 1:500; Abcam, ab14705) overnight at 4°C. The coverslips were then washed three times with PBS for 15 min, followed by incubation with Alexa Fluor® 647 goat anti-mouse , Alexa Fluor® 488 goat anti-mouse , or Alexa Fluor® 647 goat anti-rabbit  at room temperature in the dark for 2 hours, and were washed three times with PBS for 15 min. Coverslips were mounted with Vectashield mounting medium (Vector Labs) and were examined using confocal microscopy (Olympus FLUOVIEW FV10i). The total, nuclear, and cytoplasmic immunofluorescence signals of more than 30 cells per cell line were quantified using ImageJ .Sections of formalin-fixed paraffin-embedded primary breast cancer tissues and lymph node metastases were deparaffinized and rehydrated by passage through a graded xylene/ethanol series before staining. Cells were cultured on poly-D-lysine (PDL)-coated glass coverslips for 24 hours before fixation in 4% paraformaldehyde in PBS for 10 min. After permeabilizing and blocking in 0.5% BSA with PBS plus 0.025 % Triton X-100, 0.02% NaNGenomic DNA was extracted from breast cancer patient lymph node metastases using the DNeasy Blood & Tissue Kit . Exon 1, 2, and 3 of BCL2L1 were amplified by PCR and cloned into pCR-Blunt-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific Cat# 450245) The primers used for PCR are exon 1&2 F1: AACTCTTCCGGGATGGGGTAA; exon 1&2 R1: CCAGCCGCCGTTCTCCT; exon 1&2 F2: ATGGCAGCAGTAAAGCAAGCG; exon 1&2 R2: CCCATCCCGGAAGAGTTCATTCA; exon 1&2 F3: AGAAGGGACTGAATCGGAGATG; exon 1&2 R3: CGAAGGAGAAAAAGGCCACAAT; exon 3 F1: GATACTTTTGTGGAACTCTA; and exon 3 R1: GGTAGAGTGGATGGTCAGT. M13 forward primer GTAAAACGACGGCCAG was used for sequencing.2 with occasional swirl. Crosslinker solution was then removed, and 5 mL of 50 mM Tris (pH 8.0) was added to each plate for an additional 10 min to quench the crosslinker. Following quenching, plates were placed on ice bath and washed one more time with cold PBS and lysed with freshly-prepared CLIP buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% Sodium Doxycholate, 20 mM HEPES, 1% Triton X-100) plus protease inhibitors and phosphatase inhibitors. Lysates were homogenized by end-over-end rotation for 30 min at 4°C and cleared by centrifugation. Clarified lysates were incubated with anti-HA Magnetic Beads  or anti-HA agarose  for 2 hours at 4°C with end-over-end rotation. The beads were then washed 5 times with 1 mL CLIP buffer supplemented with protease and phosphatase inhibitors. Proteins were eluted by incubating the beads with 2X loading buffer (without DTT) at 50°C with gently shaking for 10 min. Eluted proteins were isolated by centrifugation, DTT was added to a final concentration of 100 mM and samples were boiled for 3 min.10-cm dishes of 90% confluent cells were used and cells were washed three times with cold PBS to remove all traces of media. Following removal of PBS, 5 mL of a 1 mM DSP solution in PBS was added to each dish. Cells were incubated for 30 min at 37°C, 5% COWhole cell lysates were prepared by lysing cells with RIPA buffer , 150 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor mixture and PhosSTOP (Roche). Histones were prepared by acid extraction as previously described . Viral supernatants were collected, filtered, and used to infect BON1/TGL cells in the presence of 8 μg/ml polybrene. Cells were selected with 0.8 mg/ml G418  72 hours after infection for 12 days, and then stable cell lines were maintained in 0.4 mg/ml G418. For fluorescence-activated cell sorting (FACS), BON1/HA-Bcl-xL/TR-shRLuc #713, BON1/HA-Bcl-xL/TR-shCtBP2 #2260, and BON1/HA-Bcl-xL/TR-shCtBP2 #2403 cells were pre-treated with 0.5 μg/ml doxycycline for 96 hours and sorted using a BD FACSAria II Cell Sorter System at Weill Cornell Medicine Flow Cytometry Core Facility. All the parental cell lines were authenticated by University of Arizona Genetics Core before use.The shRNAs for human CtBP2 and MLL1 were designed using the SplashRNA algorithm , and MDA-MB-231/CtBP2 knock out (KO) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 0.2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. BON1/TGL/pQCXIP  and BON1/TGL/HA-Bcl-xL (wt or mutants) cells have been described (™ II High-Speed Cell Sorter (λ exc =488nm) in 96-well plates. The cells were grown up to confluency and then replica plated in other 96-well plates. These clones were screened by Western blotting to identify homozygous knockout of CtBP2.V5-tagged CtBP2 constructs (Full length (445 aa), residues 1 to 321, and residues 322 to 445) were described in , Myc-Bcl-xL/B(BH4-loop), Myc-Bcl-xL/B(loop), Myc-Bcl-B/xL(loop), Myc-Bcl-xL/B(BH1-BH2-TM), Myc-Bcl-B/xL(BH4-loop). The plasmids were incubated in 200 μl Opti-MEN medium mixed with X-tremeGENE HP DNA Transfection Reagent (Sigma) (ratio of reagent: DNA = 3 μl : 1 μg) at room temperature for 30 min. Cells at a confluence of 50~60% were used for each transfection. Cells were washed with PBS, then DMEM containing 1% FBS and 0.2 mM L-glutamine (without antibiotics) were added. Transfection complexes were added to cells in a dropwise manner and the dishes were gently swirled. Cells were incubated for 48 hours at 37°C, 5% COBcl-xL/B(BH4-loop) and BclB/xL(BH4-loop) were PCR amplified from Bcl-xL/Bcl-B chimeric constructs , and power SYBR green-based quantitative real-time PCR was performed using primers specific for human BCL2L1 , human CtBP2 , human ACVR1 , human SMAD3 , human NKX2-5, human SMAD5 , human ZNF423 , human MRPL19 , human HMBS , with the comparative CT method . Both MRPL19 and HMBS were used together as endogenous controls for normalization.Cell lines  grown on 6-cm dishes were used to isolate mRNA using RNeasy Plus Mini kit  containing gDNA Eliminator spin columns. cDNA was generated using the SuperScript6 N134 cells in 150 μl PBS were injected into the tail veins of NSG mice at the age of ~10 weeks. Mice were subjected to bioluminescent imaging using the In Vivo Imaging System Spectrum (PerkinElmer) as described  one day before the injection of cells and injected with one million cells that were treated with doxycycline (0.5 μg/ml for 96 hours) in 100 μl PBS via the left ventricle. For tail vein metastasis assay, 1.5 × 10BON1/HA-Bcl-xL/TR-shCtBP2 and BON1/HA-Bcl-xL/TR-shRLuc #713 cells were treated with 1 μg/ml (before sorting) or 0.5 μg/ml (after sorting) doxycycline  for 96 hours, and 2 × 10⁵ of cells were seeded in Matrigel-coated transwell invasion chambers  with DMEM containing 1% FBS, 0.4 mg/ml G418, 0.5 μg/ml puromycin, 0.2 mM L-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin. The lower chambers were filled with DMEM containing 10% FBS, 0.4 mg/ml G418, 0.2 mM L-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin. Fifty thousand cells per cell line were seeded in 1% FBS, 0.4 mg/ml G418, 0.5 μg/ml puromycin, 0.2 mM L-glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin per well of 24-well dish for the proliferation controls. After 24 hours of incubation, cells on the opposite side of the chambers were fixed in 4% paraformaldehyde in PBS for 10 min, washed with PBS−/− for 5 min, stained with 0.1% crystal violet for 30 min and counted in 8 fields under 20X magnification.Subcellular fractionation was performed based on the method previously described  (construct #5), or RCASBP-HA-Bcl-xL/B(BH4-loop) (construct #6)) were seeded in 100 μL culture medium in a 96-well white wall plate. After overnight incubation, cells were treated with DMSO or 10 mM etoposide for 24 hours. Caspase-3 and -7 activities were measured using the Caspase-Glo 3/7 assay kit  according to manufacturer’s instructions. Luminescence was measured using a Glomax Multi Plus Detection System , and caspase 3/7 activities was normalized by the number of cells. Data represented 3 replicates per condition and were expressed as mean of fold change over N134 cells without etoposide ± SEM.A total of 1 × 102, 95% humidity for formation of a single spheroid of cells. After 96 hours, spheroids were then treated with 0.5 μg/ml doxycycline in fresh medium containing Matrigel (Day 0). Fresh medium containing 0.5 μg/ml doxycycline was added every 48 hours. Images were taken at the indicated time points. 6 wells of each cell/condition were used and analyzed. Original images were under × 20 magnification.100 BON1/HA-Bcl-xL/TR-shMLL1 #13408, BON1/HA-Bcl-xL/TR-shMLL1 #13406, and control BON1/HA-Bcl-xL/TR-shRLuc #713 cells in 100 μl growth medium were seeded into 96-well round bottom ultra-low attachment plates  with 2.5% (v/v) Matrigel . Plates were incubated for 96 hours at 37 °C, 5% CO2-induced digestion occurred on ice for 30 min and was stopped by chelation at 37°C for 20 min. Spike-in drosophila DNA  was added in the chelation buffer at 50 pg per condition. DNA was finally isolated using the MinElute PCR Purification Kit  following the manufacturer’s instructions. After quantification of the recovered DNA fragments, libraries were prepared using the ThruPLEX®DNA-Seq kit  following the manufacturer’s instructions, purified with AMPure XP magnetic beads , and quantified using a Qubit fluorometer (Thermo Fisher Scientific).BON1/TGL/pQ and BON1/TGL/HA-Bcl-xL cells were washed with cold PBS and scraped from cell culture dishes (triplicates). Cells were pelleted at 4°C, 3,000g for 5 minutes. CUT&RUN was performed from 500,000 cells per condition in triplicates as previously described in (https://bitbucket.org/qzhudfci/cutruntools/src/default/)  and Agilent Tape Station with High Sensitivity D1000 Screen Tape. The average libraries size was 300 bp. According to the manufacturer’s instructions, denatured and diluted libraries were loaded to the bottom of the cartridge reservoir provided with the Illumina NextSeq 2000 reagents kit, and sequenced at paired-end 50 bps on NextSeq 2000 sequencer (Illumina). The raw sequencing reads in BCL format were processed through bcl2fastq 2.20 (Illumina) for FASTQ conversion and demultiplexing. Data were processed using Cut&RunTools (efault/)  with theefault/) . DiffereIn vitro experiments were performed at least in triplicates.Radiance values of luciferase signals or 3D tumor spheroid sizes (area) were transformed to the natural log scale before analysis. Generalized Estimating Equations (GEE) method was used to test the overall difference in radiance signals or whole area change over time and the analysis were performed in statistical software Statistical Analysis System . In healthy cells, most of the Bcl-xL proteins reside in the mitochondria, while some are localized in the cytosolic and ER , suggesting that the change of subcellular localization of Bcl-xL in the cancer cells was not due to mutations in Bcl-xL. This raised a possibility that Bcl-xL-interacting proteins involve in its subcellular localization changes.in situ prior to isolation  procedure using cell-permeable, thiol-cleavable crosslinkers to stabilize normally labile interactions To verify the interaction between CtBP2 and HA-Bcl-xL identified by IP-mass spectrometry, we performed anti-HA ReCLIP using lysates from BON1/TGL cells overexpressing HA-tagged wt Bcl-xL or the vector, followed by Western blotting using antibodies against CtBP2 and HA. In consistent with the IP-mass spectrometry results, CtBP2 was detected in the anti-HA ReCLIP specifically from cells expressing HA-tagged wt Bcl-xL, but not from the control cells expressing the vector .CTBP2 using doxycycline (dox)-inducible shRNA against CTBP2 in BON1/TGL/HA-Bcl-xL cells. We constructed four dox-inducible shRNAs  against different regions of human CtBP2 and a control shRNA against Renilla Luciferase (shRLuc #713) in a lentivirus vector, LT3RENIR (tet-O-dsRed-shRNA-PGK-rtTA3 (TR-shRNA)” viruses to infect BON1/TGL/HA-Bcl-xL cells (BON1/HA-Bcl-xL). Infected cells were selected with neomycin to obtain stable clones. We investigated knockdown efficiency of CtBP2 by these different shRNAs in the stable cell lines following 96 hours of doxycycline treatment and Western blotting. Two (#2260 and #2403) shRNAs against CtBP2 successfully reduced the levels of CtBP2 proteins . This gave us a unique opportunity to determine whether knockdown of CtBP2 affected Bcl-xL nuclear localization by comparing subcellular localization of HA-Bcl-xL in the dsRed-positive cells and the dsRed-negative cells in the same images. We have previously shown that the nuclear Bcl-xL, but not its cytoplasmic counterpart, accounts for its migration/invasion activity  and 2d. To examine whether the invasion function of Bcl-xL is mediated by CtBP2, we performed transwell invasion assays using dox-treated BON1/HA-Bcl-xL/TR-shCtBP2 #2260 and #2403 cells and BON1/HA-Bcl-xL/TR-shRLuc #713 control cells. We found that invasion activity was significantly reduced in dox-treated shCtBP2 (#2260 and #2403) cells compared with dox-treated shRLuc (#713) control cells . On the As approximately 50% of dox-treated cells showed positive dsRed signals, a reporter for shRNA induction by doxycycline , we enriched shRNA-expressing cells by fluorescence-activated cell sorting (FACS) for dsRed-positive cells. dsRed was observed in >90% sorted cells by fluorescent microscopy . It is oWe next performed biochemical fractionation analysis using these FACS sorted, dsRed-positive cells and found that both soluble nuclear HA-Bcl-xL and chromatin-bound HA-Bcl-xL were greatly reduced in the dox-induced CtBP2 knockdown cells (#2260 and #2403) compared to the dox-induced shRLuc control cells (#713) . Mek1/2 6 dox-treated BON1/HA-Bcl-xL/TR-shRLuc #713 (control cells) and BON1/HA-Bcl-xL/TR-shCtBP2 #2260 cells into NOD/scid-lL2Rgc knockout (NSG) immunodeficient mice. Mice were started on a doxycycline diet one day before tumor injection and kept on doxycycline diet until the end point. The luciferase expression in these cells provided a mean for monitoring the localization and growth of tumor cells through in vivo bioluminescent imaging. Indeed, weekly bioluminescent imaging showed that bioluminescence signals started to increase after 14 days, and metastatic cells were detected at multiple sites on day 28 (p = 0.0366), suggesting that knockdown of CtBP2 reduced metastatic progression of Bcl-xL-overexpressing BON1 cells in mice.As our data from clinical specimens and invasion assays suggested that CtBP2-driven nuclear-localization of Bcl-xL may promote metastasis, we performed experimental metastasis assays in mice using FACS sorted cells. We intracardiacally injected 1 × 10n day 28  and 3e. In addition to shRNA knockdown of CtBP2 in BON1 PNET cells, we used CRISPR-Cas9 to knock out (KO) CtBP2 in the MDA-MB-231 breast cancer cell line to further test the regulation of nuclear translocation of Bcl-xL by CtBP2. We found that CtBP1, a homolog of CtBP2, did not increase to compensate the loss of CtBP2, but CtBP2 KO reduced endogenous Bcl-xL protein levels . BecauseTo determine whether CtBP2 KO also reduced nuclear subcellular localization of endogenous Bcl-xL, we performed biochemical fractionation and immunocytochemical staining in CtBP2 KO cells. The biochemical fractionation analysis showed that the nuclear pool of endogenous Bcl-xL was greatly reduced in the CtBP2 KO cells . ImmunofTo characterize the Bcl-xL/CtBP2 interaction domains, we used deletion constructs of CtBP2 to define the Bcl-xL interaction domain of CtBP2 . DNA conTo further narrow down the Bcl-xL-binding domain in the N-terminal region of CtBP2, we generated a series of CtBP2 N-terminal deletion constructs with a V5 tag , suggestTo determine the CtBP2-binding domain in Bcl-xL, we performed the reciprocal experiments. We initially generated a series of N-terminal deletion Bcl-xL mutants with HA tag, but they did not express well (data not shown). Because we found that Bcl-B, another Bcl-2 anti-apoptotic family member , did notRIP-Tag; RIP-tva mouse, with RCASBP–HA-Bcl-B/xL(BH4-loop) (construct #5) or RCASBP–HA-Bcl-xL/B(BH4-loop) (construct #6). The expression levels of both HA-tagged chimeric proteins in N134 cells were similar by Western blot analysis  and #6 (which does not bind to CtBP2) into an avian retroviral vector, RCASBP, with an HA tag. We infected the N134 cell line   and generated stable BON1/TGL/HA-BcL-xL/tet-O-dsRed-shMLL1-PGK-rtTA3 (BON1/HA-Bcl-xL/TR-shMLL1) cell lines. After 96-hour doxycycline treatment, almost all cells became dsRed-positive . We exam<0.0001) . On the To detect the sites of H3K4me3 histone modification by Bcl-xL, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assay  on the BMetastasis, the dissemination of cancer cells from primary sites to distant organs, accounts for the majority of cancer-associated mortality. Efforts on unraveling the molecular basis of tumorigenesis and metastasis have significantly advanced our understanding of metastasis. During cancer progression, the ability to override apoptosis is a prominent mechanism that cancer cells usually acquire . The Bclin situ, we discovered that CtBP2, a transcriptional regulator, translocates Bcl-xL into the nucleus. CtBP2 contains an KRQR NLS sequence at the N-terminal domain, which is crucial for its accumulation and gene repression functions in the nucleus (in vivo.This study, together with our previous findings . ProteinThe BH1, BH2, and BH3 domains of Bcl-xL create a hydrophobic groove to bind BH3-only pro-apoptotic proteins, inhibit their activity and lead to a pro-survival phenotype . The firEnhanced TGFβ signaling promotes cancer metastasis (


We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity. Hematological malignancies are associated with an increased risk of complications during SARS-CoV-2 infections , 2. AlthFigure S1). nAbs against BA.1 and BA.5 after monovalent or bivalent booster had lower titers compared to WA1, with a reduction in neutralization potency ranging between 7.1–8.2-fold for BA.1 and between 8.9–12.4-fold for BA.5 . Neutralizing antibodies against the newly circulating Omicron variants BQ.1.1 and XBB.1.5 were low or undetectable.The study included two cohorts. The first cohort received anti-SARS-CoV-2 primary immunization and two monovalent booster immunizations, with the last monovalent booster administered 1–6 months after the second dose (n=101). The second cohort received the anti-SARS-CoV-2 primary immunization, two booster immunizations, and a bivalent booster administered 1–6 months after the third dose (n=42). The nAb titers against the ancestral strain (WA1) in patients that received the bivalent booster were 2.3-fold higher than neutralization titers in patients that received the monovalent booster . Next, we compared neutralization and antibody binding titers between individuals with low/undetectable anti-NC antibodies and those with recent SARS-CoV-2 exposure. In the monovalent-boosted group, the neutralization titers against WA1 were 11-fold higher in patients with recent SARS-CoV-2 exposure compared to unexposed patients (Geometric Mean Titer [GMT] 1379 vs 125). Neutralization titers against Omicron variants BA.1 (GMT 134 vs. 28), BA.5 (GMT 95 vs. 24), BQ.1.1 (GMT 38 vs. 20.5), and XBB.1.5 (GMT 30 vs. <20) were higher in SARS-CoV-2 exposed compared to unexposed patients (Detection of anti-nucleocapsid (NC) binding antibodies (900 AU/ml as cutoff) was utilized as evidence of recent SARS-CoV-2 exposure . In the patients . We thenpatients .Figure S2). In the bivalent-boosted group, the neutralization titers against WA1 showed modestly elevated titers (1.3 fold) in patients with recent SARS-CoV-2 exposure compared to unexposed patients (GMT 894 vs. 676). Neutralization titers were elevated against Omicron variants BA.1 (GMT 139 vs. 42), BA.5 (GMT 87 vs. 32), BQ.1.1 (GMT 43 vs. 22), and XBB.1.5 (GMT 33.5 vs. <20) in SARS-CoV-2-exposed compared to unexposed patients  . However, in SARS-CoV-2 unexposed and exposed patients, differences were observed between the IgG antibody response against the Spike protein from the ancestral SARS-CoV-2 strain and the IgG response against most of the Omicron variants .We next determined the IgG binding titers using Spike-specific electrochemiluminescence assays . Nearly Tables S1 and S2). Anti-CD38 therapy correlated with reduced induction of nAb against WA1 in both cohorts of patients that received monovalent and bivalent boosters (Table S2). nAb against XBB-1 were low in all subsets and not impacted by these variables (Table S1).We next analyzed the clinical correlates of neutralizing antibodies against WA1 and the contemporary XBB.1.5 variant as described , 13  were very low or undetectable. Hybrid immunity enabled higher induction of broadly reactive nAbs. However, there was no correlation between Spike-binding antibodies and neutralization against circulating variants, suggesting that vaccination elicited antibodies that are not neutralizing. The inability to elicit antibodies against neutralizing epitopes suggests that a vaccination regimen with repeat boosters or using different vaccine platforms is needed to improve the effect of bivalent booster immunization in MM patients.We have reported that a single monovalent booster vaccine is associated with increased nAb against the ancestral strain and the B.1.617.2 variant but not the Omicron variants in MM patients . It has


Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain detect pathogen effectors to produce TIR-catalyzed signaling molecules for activation of plant immunity. Plant immune signaling by TIR-containing NLR (TNL) proteins converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TNL signaling also require helper NLRs N requirement gene 1 (NRG1) and activated disease resistance 1 (ADR1). In two recent remarkable papers published in Science, the authors show that the TIR-containing proteins catalyze and produce two types of signaling molecules, ADPr-ATP/diADPR and pRib-AMP/ADP. Importantly, they demonstrate that EDS1-SAG101 and EDS1-PAD4 modules are the receptor complexes for ADPr-ATP/diADPRp and Rib-AMP/ADP, respectively, which allosterically promote EDS1-SAG101 interaction with NRG1 and EDS1-PAD4 interaction with ADR1. Thus, two different small molecules catalyzed by TIR-containing proteins selectively activate the downstream two distinct branches of EDS1-mediated immune signalings. These breakthrough studies significantly advance our understanding of TNL downstream signaling pathway. Nicotiana benthamiana ROQ1 form tetramers and function as nicotinamide adenine dinucleotide hydrolase (NADase) enzyme  proteins are intracellular receptors with crucial roles in recognizing pathogen-delivered effectors and activating effector-triggered immunity (ETI). ETI is often associated with strong host defense responses and a localized cell death to restrict pathogen infection  NADase, suggesting that the signaling role of TIR containing proteins require their NADase activity  and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101)  to produce v-cADPR (2′ cADPR) (Wan et al. + hydrolyzate 2′ cADPR can be catalyzed by TIR to produce di-ADPR or ADPr-ATP. TIR can further hydrolyse di-ADPR or ADPr-ATP to generate pRib-AMP or pRib-ADP (Huang et al. It was reported that TIR domain in plants hydrolyze nicotinamide adenine dinucleotide in its oxidized form (NADThe next question is how these small molecules activate two different downstream EDS1 signaling modules. Using biochemical and structural approaches, the authors demonstrated that EDS1-PAD4 is the receptor complex for the pRib-ADP/AMP. The binding of pRib-ADP/AMP allosterically promotes EDS1-PAD4 association with ADR1-L1. In contrast, di-ADPR and ADPr-ATP specifically bind to EDS1-SAG101 and allosterically promote EDS1-SAG101 interaction with NRG1, establishing that EDS1-SAG101 functions as a receptor complex for di-ADPR and ADPr-ATP. Structural analysis reveal specific recognition mechanisms for pRib-ADP/AMP by EDS1-PAD4 or di-ADPR and ADPr-ATP by EDS1-SAG101 (Huang et al. In summary, two remarkable papers published in Science identified the pRib-ADP/AMP and di-ADPR/ADPr-ATP as two types of signaling molecules catalyzed by TIR-containing proteins and revealed that two different small molecules selectively activate the downstream two EDS1-mediated immune branches. Importantly, chemically synthesized pRib-ADP/AMP or di-ADPR/ADPr-ATP can function as second messengers to induce immune signaling (Huang et al.


Satureja is rich in phenolic monoterpenoids, mainly carvacrol, that is of interest due to diverse biological activities including antifungal and antibacterial. However, limited information is available regarding the molecular mechanisms underlying carvacrol biosynthesis and its regulation for this wonderful medicinal herb. To identify the putative genes involved in carvacrol and other monoterpene biosynthesis pathway, we generated a reference transcriptome in two endemic Satureja species of Iran, containing different yields (Satureja khuzistanica and Satureja rechingeri). Cross-species differential expression analysis was conducted between two species of Satureja. 210 and 186 transcripts related to terpenoid backbone biosynthesis were identified for S. khuzistanica and S. rechingeri, respectively. 29 differentially expressed genes (DEGs) involved in terpenoid biosynthesis were identified, and these DEGs were significantly enriched in monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, carotenoid biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways. Expression patterns of S. khuzistanica and S. rechingeri transcripts involved in the terpenoid biosynthetic pathway were evaluated. In addition, we identified 19 differentially expressed transcription factors  that may control terpenoid biosynthesis. We confirmed the altered expression levels of DEGs that encode carvacrol biosynthetic enzymes using quantitative real-time PCR (qRT-PCR). This study is the first report on de novo assembly and transcriptome data analysis in Satureja which could be useful for an understanding of the main constituents of Satureja essential oil and future research in this genus. Terpenoids are the most abundant compounds of plant secondary metabolites and have been interesting due to their pharmacological utility . The uniCarvacrol is a monoterpene compound derived from isoprene hydrocarbure  and formed by the attachment of two or more isoprene molecules . CarvacrSatureja khuzistanica is an aromatic endemic species. This species was first illustrated in 1944 as a new species in the flora of Iran ..S. rechiSatureja. The 179 transcripts of TPS gene candidates were identified based on Swiss-Prot database, while 11 transcripts were differentially expressed in the leaf tissue of Satureja. The 2309 transcripts that encode the DH genes family were annotated, of which 140 transcripts showed differential expression in leaf tissues of Satureja.Some DEGs were putatively involved in the monoterpenoids biosynthesis pathway . The 127Satureja were identified   .In order to understand the regulatory mechanism of terpenoid accumulation patterns, we analyzed the co-expression profiles between differentially expressed TFs and differentially expressed terpene biosynthesis-related enzyme genes  and theiSatureja.As a result, ARF, MYC, bHLH and ERF family genes were predicted to be closely related to terpene biosynthesis genes . The resS. khuzistanica and S. rechingeri were analyzed (S. khuzistanica (sum of approximately 98.96%) and S. rechingeri (sum of approximately 98.18%). Phytochemical analyses of the leaf tissue showed differences in the amount of the main components of the essential oils. It can be due to the terpene synthase, other secondary metabolite, and their regulator genes . It is important to consider that the slight difference in obtained expression values from the qRT-PCR method with the RNA-Seq results is little due to different parameters such as the primers efficiency, quality of the PCR kit, and the integrity of the thermocycler.In this study, since carvacrol is a major compound of Satureja genus. The transcriptomes N50 length was higher than that of other members of this family such as S. miltiorrhiza (1340 bp) ; SrCYP71D181, Cytochrome P450 71D181 [Satureja rechingeri]; TvL3H, limonene-3-hydroxylase [Thymus vulgaris]; TvL6H, limonene-6-hydroxylase [Thymus vulgaris]; MgCYP71D18, (-)-(4S)-Limonene-6-hydroxylase [Mentha x gracilis]; MgCYP71D94, Cytochrome P450 71D94 [Mentha x gracilis]; McL6H, limonene-6-hydroxylase [Mentha canadensis]; Mp(-)-L6H, (-)-limonene 6-hydroxylase-like cytochrome p450-dependent oxygenase [Mentha x piperita], MgCYP71D95, 71D95: (-)-(4S)-Limonene-3-hydroxylase [Mentha x gracilis]; MsCYP71D95, 71D95: Limonene-3-hydroxylase [Mentha spicata]; MpCYP71D15, (-)-(4S)-Limonene-3-hydroxylase; AltName: Full = Cytochrome P450 isoform PM2 [Mentha x piperita]; McL3H, (-)P450 limonene-3-hydroxylase [Mentha canadensis]; MaLH, limonene hydroxylase [Mentha arvensis]; Ms(-)-L3H, (-)-limonene 3-hydroxylase-like cytochrome p450-dependent oxygenase [Mentha spicata], MpCYP71D13, Cytochrome P450 71D13; AltName: Full = (-)-(4S)-Limonene-3-hydroxylase; SiCYP71D95-like, P450 71D95-like [Sesamum indicum]; SmCYP71D374; cytochrome P450 [Salvia miltiorrhiza] B) terpene synthesis.The full name of the sequences are TmTPS, gamma-terpinene synthase [Thymus migricus]; TtTPS gamma-terpinene synthase [Thymus trautvetteri]; TpTPS, gamma-terpinene synthase [Thymus pubescens]; TsTPS, putative gamma-terpinene synthase [Thymus serpyllum]; TdTPS, gamma-terpinene synthase [Thymus daenensis]; TvTPS2, terpene synthase 2 [Thymus vulgaris]; TcTPS, gamma-terpinene synthase [Thymus caespititius]; TfTPS, gamma-terpinene synthase [Thymus fedtschenkoi]; TkTPS, gamma-terpinene synthase [Thymus kotschyanus]; OvTPS2, terpene synthase 2 [O. vulgare]; SkTPS2, terpinene synthase [Satureja khuzistanica]; SrTPS2, terpinene synthase, [Satureja rechingeri]; OvTPS5, terpene synthase 5 [O. vulgare]; PvTPS10, terpene synthase 10 [Prunella vulgaris]; SrPS, pinene synthase [Salvia rosmarinus] The evolutionary history was inferred using the Neighbor-Joining method. Evolutionary analyses were conducted in MEGA7. The bootstrap replications were set to 100.A) Cytochrome P450 71D181. The full name of the sequences are (TIF)Click here for additional data file.S4 FigSatureja TFs genes with high homology of plant species.A) Percent of identified TFs families B) Percent of (TIF)Click here for additional data file.S5 FigS. khuzistanica_1, Sk_2: S. khuzistanica_2, Sk_3: S. khuzistanica_3, Sr_1: S. rechingeri_1, Sr_2: S. rechingeri _2, Sr_3: S. rechingeri _3.Average of FPKM of all transcripts related to each TF gene was used. Ko numbers show the KEGG maps code related to monoterpene biosynthetic pathway. Specie ID of each replicate is Sk_1: (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.S14 Table(XLSX)Click here for additional data file.


The synthesis process yields highly uniform close-packed superlattices with a dominant c-axis orientation. The self-assembled c-oriented Ni(OH)2 films exhibit advantageous electrocatalytic performance in urea oxidation, presenting significantly lower overpotentials and higher current densities compared to randomly distributed Ni(OH)2 particles. In-depth in situ impedance analysis and Raman spectroscopy demonstrate that the c-oriented Ni(OH)2 films possess a higher propensity for a Ni valence transition from +2 to +3 during the urea oxidation process. This finding provides crucial insights into the catalytic behavior and electronic transformations of c-oriented Ni(OH)2 films, shedding light on their superior electrocatalytic activity for urea oxidation. Overall, this study advances our understanding of urea electrooxidation mechanisms and contributes to the design of efficient urea electrocatalysts.This study investigates the electrocatalytic properties of the transparent  c-oriented Ni(OH)2 films exhibit exceptional electrocatalytic performance for urea oxidation compared to randomly distributed Ni(OH)2 particles.The self-assembled  The central process in urea electrolysis, which encompasses hydrogen evolution, direct urea fuel cells, bio-waste remediation, and medical urea detection, is the electrochemical urea oxidation reaction (UOR). However, this process faces significant challenges due to its sluggish reaction kinetics, characterized by a complex six-electron and four-proton mechanism.2) is a standout as a potential catalyst for UOR owing to its advantageous catalytic characteristics, cost-effectiveness, and ubiquity.7–12 Despite extensive research on nickel-based catalysts, uncertainties surrounding catalytic activity, stability, and mechanistic understanding endure. In this regard, a variety of Ni(OH)2-based catalysts, such as atomically thick Ni(OH)2 nanosheets,7 Ni(OH)2 nanoflakes,13 variants of Ni(OH)2 material with diverse heteroatom dopants, exemplified by instances of Co14,15 and Fe16 dopants, and Ni(OH)2–carbon composites17,18 have been investigated. However, the practical application of Ni(OH)2 as an effective electrocatalyst for urea oxidation is thwarted by its restricted electrical conductivity and instability under severe electrochemical conditions.18–22 To circumvent these limitations, novel approaches are being explored to enhance the electrocatalytic efficiency of Ni(OH)2.Nickel hydroxide (Ni(OH)2 nanosheets into highly ordered films.23–26 The self-assembly patterns of colloidal nanocrystals are increasingly recognized as a valuable methodology for investigating the fundamental characteristics of highly uniform nanoparticles. In preceding work,27 we successfully engineered c-oriented NiFe-LDH films and Ni(OH)2 films using a colloid self-assembly method. The resultant films demonstrated superior electrocatalytic properties in the oxygen evolution reaction (OER) and ethanol electrooxidation reaction due to their orderly c-oriented superlattices, which led to elevated proton diffusion coefficients and significantly enhanced electrocatalytic activities.27 The findings from this research emphasize the conceivable potential of these self-assembled c-oriented films from colloidal nano particles for diverse applications in the field of electrocatalysis.28,29One promising approach involves self-assembly of colloidal two-dimensional (2D) Ni(OH)c-oriented Ni(OH)2 films self-assembled from colloidal 2D Ni(OH)2 nanosheets. The primary objective of this paper is to assess the potential of these films to enhance the electrocatalytic activity for the UOR and to investigate the underlying mechanisms that contribute to their superior electrocatalytic performance. In this study, our comprehensive analysis revealed that the c-oriented Ni(OH)2 films (Ni(OH)2-cF) showcased exceptional electrocatalytic efficiency. Ni(OH)2-cF, when applied to the glassy carbon electrode, demonstrated outstanding UOR electrocatalytic activity, resulting in a significant lower value of 180 mV in potential at a current density of 10 mA cm−2. This surpasses randomly distributed Ni(OH)2 films (Ni(OH)2-gPF) by a notable margin of 118 mV. Furthermore, our research has provided valuable insights into the governing factors of UOR electrochemical processes in Ni(OH)2 films, offering valuable guidance for the design and enhancement of next-generation electrocatalytic materials.This current investigation aims to thoroughly examine the UOR performance of transparent 3)2·6H2O, NiCl2·6H2O, NaOH, KOH, ethanol and isopropanol were procured from Sinopharm Chemical Reagent. All experiments employed deionized and decarbonated water.Chemical reagents including Ni2 was rapidly introduced into a 20 mL aqueous solution of 0.15 M NaOH (20% ethanol) under vigorous stirring.The preparation of 2D Ni(OH)2 nanosheets (2 mg mL−1) exhibiting a positive zeta potential of approximately 27 mV.The resultant precipitate underwent several rounds of centrifugation and washing with deionized (DI) water. Subsequently, the precipitate was subjected to overnight sonication in a mixed solution of water and isopropanol (1 : 1 volume ratio) at room temperature. This procedure yielded a homogeneous and stable suspension of colloidal 2D Ni(OH)2 nanosheets were deposited onto diverse substrates to yield c-oriented Ni(OH)2 films (Ni(OH)2-cF) through solution evaporation-driven self-assembly. The substrates encompassed nickel foils, fluorine-doped tin oxide (FTO), glassy carbon, and monocrystalline silicon sample holders for Powder X-ray Diffraction (XRD).Calculated quantities of stable colloidal solutions containing 2D Ni(OH)2-cF films obtained from colloidal solutions of 2D Ni(OH)2-cF nanosheets were removed from the substrates and pulverized into powders, denoted as Ni(OH)2-gP. Subsequently, Ni(OH)2-gP was introduced into a mixed solution of water and isopropanol (1 : 1), followed by sonication to yield a homogeneous dispersion. The resulting dispersion was then deposited onto substrates to generate Ni(OH)2-gPF for subsequent applications.The thick Ni(OH)−1. Monocrystalline silicon sample holders were utilized for sample application, ensuring minimal background diffraction interference. Sample morphologies were scrutinized using a Hitachi S-4800 field emission scanning electron microscope (SEM) and a transmission electron microscope . UV-vis transmittance spectra were captured using a Shanghai Aucy Scientific Instrument UV1902PC UV-vis-NIR spectrophotometer. X-ray photoelectron spectroscopy (XPS) measurements were conducted on a Thermo Fisher-VG Scientific (ESCALAB 250Xi) photoelectron spectrometer. Binding energies were calibrated using adventitious hydrocarbon referencing, with C 1s fixed at 284.60 eV. During the course of UOR, in situ Raman spectra  were employed to monitor the evolution of electrode surface species.X-ray diffraction patterns were acquired using a Bruker D8-Advance X-ray diffractometer, employing Cu Kα radiation with a wavelength of 1.5405 Å and a scanning rate of 0.02° sE(RHE) = E(Hg/HgO) + 0.098 V + 0.0591 × pH. Linear sweep voltammetry (LSV) traces were systematically recorded using a scan rate of 5 mV s−1 in a carefully deoxygenated solution comprising 1 M potassium hydroxide (KOH) blended with 0.33 M urea. All polarization curves were corrected for the iR (95%) drop by considering the ohmic resistance of the solution. Operando electrochemical impedance spectroscopy was meticulously conducted employing the Autolab PGSTAT302N instrument from Eco Chemie, Utrecht, Netherlands. This investigation took place within a three-electrode system, with diverse potentials explored across the frequency spectrum ranging from 1 MHz to 0.1 Hz.Electrochemical assessments were executed using an electrochemical workstation (CHI 760E) coupled with a three-electrode cell setup. The working electrode comprised a glassy carbon electrode coated with the electrocatalyst, while a carbon rod served as the counter electrode, the reference electrode adopted was a saturated Hg/HgO electrode. Employing the Nernst equation, potentials were transformed to values relative to the reversible hydrogen electrode (RHE): −2. The formation of the Ni(OH)2-cF layer on the glassy carbon electrode was achieved via a technique driven by solvent evaporation-induced self-assembly. Subsequently, post-evaporation of the solvent, a volume of 2 μL of a 0.5 wt% Nafion solution was deposited onto the electrodes of Ni(OH)2-gPF. The determination of the double layer capacitance (Cdl) was executed by measuring cyclic voltammograms (CV) across various scan rates, confined within a non-faradaic potential range spanning 1.13–1.23 V. Notably, the calculated Cdl value corresponds to half of the linear slope in the pertinent region. The overpotential, a parameter of crucial importance, was quantitatively assessed using the ensuing For the experimental measurements, a meticulously prepared homogeneous dispersion containing isopropanol, totaling 10 μL in volume, was skillfully deposited onto the surface of a glassy carbon electrode (diameter of 5 mm) bearing an electrocatalyst loading of approximately 0.2 mg cm2-cF used in UOR. The auto-organization procedure of monodispersed and colloidal 2D Ni(OH)2 nanosheets, characterized by their atomically-thin features, resulted in the formation of a highly uniform close-packed superlattice exhibiting a c-oriented feature. The role of colloidal 2D hydroxide nanosheets as rudimentary building blocks for forming c-oriented hydroxide films, as corroborated by numerous scholarly investigations,30–33 is achieved through a layer-by-layer self-assembly technique extensively described in the literature. Surface tension, van der Waals interactions, electrostatic and gravitational forces acting upon the colloidal 2D LDH nanosheets influence the self-assembly operation.33 A solvent evaporation-induced self-assembly approach enabled us to successfully prepare transparent c-oriented films Ni(OH)2-cF27 on varied substrates, such as nickel foils, fluorine-doped tin oxide (FTO) glass, monocrystalline silicon sample holder and glassy carbon, as evidenced in our investigation. In the presence of Ni(OH)2 species as catalysts, urea molecules decompose into H2O, N2, and CO2. The UOR can be represented by CO(NH2)2 + 6OH− → N2 + 5H2O + CO2 + 6e−.342 nanosheets were fabricated using a rapid co-precipitation method with Ni2+ and OH− ions, followed by sonication at ambient temperature. The microstructure analysis of the 2D Ni(OH)2 nanosheets was conducted by using TEM as illustrated in 35 This optical phenomenon indicates uniform dispersion of nanosheets in the aqueous medium, which persists even after a storage period of 5 months. The long-term stability of the colloidal solution is particularly significant as it ensures nanosheet integrity and functionality, rendering them suitable for diverse applications.The colloidal 2D Ni(OH)2 crystal structure. SEM image illustrates in 2-cF on nickel foil substrates, estimated to be 638 nm, featuring a compactly packed structure. Notably, a remarkably smooth surface morphology without discernible grain boundaries or pores within the film structure is clearly visible 2-gP. After ultrasonication, Ni(OH)2-gP samples underwent an irreversible transformation from their original colloidal monodisperse 2D Ni(OH)2 nanosheet configuration, as evident in 2-gP, while 2-gP underwent a dense structural transformation during the self-assembly process. As a result, the resultant films derived from Ni(OH)2-gP, referred to as Ni(OH)2-gPF, showed significant differences from the characteristics of Ni(OH)2-cF. Notably, Ni(OH)2-gPF films revealed an irregular morphology and uneven surface, as visually portrayed in For comparison, the Ni(OH)2-cF on the substrates. The XRD pattern (2-cF exhibits exclusively (001) diffraction peaks, indicative of the c-oriented attribute. Notably, the absence of any non-basal reflections  at high angles denotes an exceptionally well-established c-oriented assembly of 2D Ni(OH)2 nanosheets.29,36–38 In contrast, the XRD pattern of the comparative specimen Ni(OH)2-gPF discloses the presence of (012) and (110) peaks, along with other non-c-axis oriented peaks  at high angles, suggesting the manifestation of diffuse scattering due to irregular stacking.39 Additionally, the diffraction peak intensity of Ni(OH)2-gPF is significantly elevated relative to that of Ni(OH)2-cF, implying good crystallinity in the self-assembled films. These results conclusively ascertain that the crystallites in Ni(OH)2-cF are oriented on the substrate with their c-axis perpendicular to the substrate surface. The XRD patterns of the transparent c-oriented Ni(OH)2-cF with different thickness on the FTO substrate 2-cF samples consistently displayed a lattice parameter of 4.596 nm along [001] direction, closely resembling the value of 4.617 nm reported in the JCPDS database for β-Ni(OH)2 (JCPDS file no. 76-1520).The XRD pattern in the  pattern  of Ni(OH2-cF and Ni(OH)2-gPF. 2-cF film on the FTO substrate exhibits notably enhanced transparency, with an optical transmittance exceeding 90% between 400 nm and 750 nm. This increased transparency can be attributed to the absence or significant decrease in pores amid the orderly self-assembled 2D Ni(OH)2 nanosheets, effectively mitigating Mie and Rayleigh scattering effects.40 Conversely, the Ni(OH)2-gPF film manifests an opaque and rough surface inset in , maintai2-cF film. The XPS spectrum of Ni 2p3/2 2-F film demonstrates the same chemical valence states as previously reported for Ni(OH)2.10,13,27,41X-ray photoelectron spectroscopy (XPS) measurements were performed to acquire insights into the chemical valence states of the Ni(OH)Ni 2p3/2  can be dNi 2p3/2  of Ni2-cF and Ni(OH)2-gPF catalysts on glassy carbon in 1 M KOH electrolytes supplemented with the addition of 0.33 M urea, utilizing a three-electrode configuration at ambient temperature. Importantly, the self-assembled Ni(OH)2-cF film demonstrated excellent adhesion to the substrates, whereas Ni(OH)2-gPF indicated a propensity to detach from the substrates, mandating the necessary use of Nafion solution on these electrodes. The catalyst loading for all films was uniformly maintained at 0.2 mg cm−2.To assess the enhanced UOR electrocatalytic performance of self-assembled 2-cF towards the UOR markedly transcends that of Ni(OH)2-gPF. In the context of overpotentials, Ni(OH)2-cF manifested an exceptional electrocatalytic performance, displaying a significantly lower value of 180 mV at a current density of 10 mA cm−2, exceeding Ni(OH)2-gPF catalysts by 118 mV. Alternatively, within the potential range of 1.4 to 1.55 V for linear sweep voltammetry (LSV) measurements, the oxidation current density of Ni(OH)2-cF exhibited a linear increase with applied voltage, while Ni(OH)2-gPF demonstrated a conspicuous passivation trend after achieving a current density of 12 mA cm−2 at 1.50 V during the UOR. The kinetics of the electrochemical reaction process were scrutinized through Tafel plots  relative to Ni(OH)2-gPF (49.6 mV dec−1), implying that the colloid self-assembly c-oriented hydroxide film effectively expedited the sluggish kinetics of UOR. The turnover frequency (TOF) represents the figure of merit for intrinsic activity, attributed to all the Ni sites of Ni(OH)2-cF. As shown in Fig. S6,−1 at 260 mV overpotential) of Ni(OH)2-cF is almost 8.4 fold higher than that of Ni(OH)2-gPF (0.016 s−1), which indicated the strong enhanced UOR activity.The Linear Sweep Voltammetry (LSV) measurements depicted in el plots . Ni(OH)22-gPF (2.21 mF cm−2), it provides additional evidence supporting the smaller Cdl value of 0.87 mF cm−2 for Ni(OH)2-cF, consistent with the flat surface morphology of Ni(OH)2-cF 2-cF and Ni(OH)2-gPF on the nickel foil substrate were further characterized by the chronoamperometry test. As shown in Fig. S8,2-cF could maintain stable current density for 10 h period of UOR testing, and retain 82% current density at a current density of 10 mA cm−2 after 10 h period. However, the current densities for Ni(OH)2-gPF dropped by 37% respectively over a 10 h period.In addition, the electrochemically active surface area (ECSA) of the electrocatalysts was evaluated using electrical-double-layer capacitances (Cdl) calculated from cyclic voltammograms (CVs). As illustrated in (OH)2-cF . These co 430 nm . This obin situ electrocatalytic operations, Electrochemical Impedance Spectroscopy (EIS) offers a valuable analytical tool for exploring the pivotal parameters directing the UOR electrochemical procedure. Particularly, Nyquist plots within the high-frequency domain deliver crucial intelligence pertaining to solution resistance (Rs) and charge transfer resistance (Rct), as signified by the radius of the semicircle. As indicated in 2-cF, the semicircle's diameter diminishes with the application of a bias voltage, achieving a minimum value of 101 Ohm at 1.38 V and 61 Ohm at 1.43 V, both significantly lower than Ni(OH)2-gPF (c-oriented Ni(OH)2 film. Additionally, a noticeable second semicircle appears at 1.53 V for Ni(OH)2-gPF (44–47 This observation may explain the discernable passivation effect noticed in Ni(OH)2-gPF,45 evidencing that self-assembled c-oriented Ni(OH)2 films proficiently mitigate the genesis of such intermediates and the passivation trend.During OH)2-gPF . These EOH)2-gPF , typical2-cF in urea oxidation, in situ Raman spectroscopy  scale" fill="currentColor" stroke="none"><path d="M160 840 l0 -40 -40 0 -40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 80 0 80 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -80 0 -80 0 0 40 0 40 -80 0 -80 0 0 -40z M80 520 l0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -200 0 -200 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 80 0 80 40 0 40 0 0 80 0 80 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 -80 0 -80 40 0 40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 200 0 200 -40 0 -40 0 0 40 0 40 -80 0 -80 0 0 -40z"/></g></svg> bend Ni3+) within the range of 475–494 cm−1 and the A1g–Ni–O stretching vibration ( str Ni3+) within the range of 558–562 cm−1 are clearly observed, thus confirming the presence of NiOOH,48 in concurrence with the electrochemical findings. As portrayed in 2-cF sample exhibits the NiOOH Raman signal at 1.42 V, whereas the Ni(OH)2-gPF sample displays the NiOOH Raman signal at 1.48 V. This observation implies that Ni(OH)2-cF is relatively more susceptible to transform into NiOOH, thereby generating an increased number of catalytic active sites. It's noteworthy that the oxidation potential of Ni(OH)2 in this environment is larger than in the three-electrode solution, given the electrolyte in the Raman in situ environment is relatively scarce, culminating in a delay in the oxidation potential.In order to fathom the advantageous electrocatalytic activity of Ni(OH)2 films after UOR and OER was undertaken. The XRD data collated from samples at diverse time intervals provided valuable insights. As delineated in 2 manifested minimal modifications.To garner further insights into the electrocatalytic mechanism of urea, a thorough examination of XRD spectra of Ni(OH)49 This comparison further confirms that the UOR activity of Ni(OH)2 films primarily converges at the surface, and due to the sustained reductive influence of urea on trivalent nickel, the internal structure of the film remains unchanged during the catalytic operation.Conversely, a significant change in crystallographic integrity was discerned after a mere 2 minutes of electrocatalytic oxygen evolution, as validated by a diminution in the intensity of the (001) peak and an amplification in peak broadening, signaling structural disruption in the aftermath of the OER catalytic reaction process.c-oriented Ni(OH)2-cF films in the context of UOR, with Ni(OH)2-gPF serving as a comparative reference. The comprehensive analysis showed that the Ni(OH)2-cF samples demonstrated a pronounced electrocatalytic efficiency, especially when compared to Ni(OH)2-gPF, characterized by a significant potential reduction of 180 mV at a current density of 10 mA cm−2, exceeding Ni(OH)2-gPF by a notable margin of 118 mV, and characterized by smaller Tafel slopes. Additionally, through the implementation of in situ EIS analysis and Raman spectroscopy, this investigation has revealed the remarkable propensity of Ni(OH)2-cF to transform into NiOOH under in situ conditions, providing strong evidence for its outstanding catalytic performance. Overall, this research has provided valuable insights into the underlying mechanisms of UOR electrochemical processes with Ni(OH)2, firmly establishing the electrocatalytic advantages of self-assembled c-oriented Ni(OH)2 films.In summary, this study represents a comprehensive exploration of the electrocatalytic activity of self-assembled There are no conflicts to declare.RA-013-D3RA05538H-s001


Within cilia, the dynein-2 complex needs to be transported as an anterograde cargo to achieve its role as a motor to drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. We previously showed that interactions of WDR60 and the DYNC2H1–DYNC2LI1 dimer of dynein-2 with multiple IFT-B subunits, including IFT54, are required for the trafficking of dynein-2 as an IFT cargo. However, specific deletion of the IFT54-binding site from WDR60 demonstrated only a minor effect on dynein-2 trafficking and function. We here show that the C-terminal coiled-coil region of IFT54, which participates in its interaction with the DYNC2H1–DYNC2LI1 dimer of dynein-2 and with IFT20 of the IFT-B complex, is essential for IFT-B function, and suggest that the IFT54 middle linker region between the N-terminal WDR60-binding region and the C-terminal coiled-coil is required for ciliary retrograde trafficking, probably by mediating the effective binding of IFT-B to the dynein-2 complex, and thereby ensuring dynein-2 loading onto the anterograde IFT trains. The results presented here agree with the notion predicted from the previous structural models that the dynein-2 loading onto the anterograde IFT train relies on intricate, multivalent interactions between the dynein-2 and IFT-B complexes. Summary: Direct interaction of the dynein-2 complex with the IFT-B complex as an anterograde intraflagellar transport (IFT) cargo is indirectly required for dynein-2–driven retrograde IFT. Primary cilia are microtubule-based, antenna-like appendages extending from the surface of most eukaryotic cells, and play crucial roles in receiving and integrating mechanical and chemical extracellular signals, such as the Hedgehog (Hh) family of morphogens . For theChlamydomonas flagella , which was first found in flagella , 1993 buflagella . Not onlflagella . The IFTflagella A, right flagella .Anterograde trafficking of the IFT machinery from the base to the tip of cilia is driven by heterotrimeric kinesin-II coupled to the IFT-B complex , whereasChlamydomonas flagella revealed by in situ cryoelectron tomography (cryo-ET)  , and an lar base  suggestelar base . In theslar base . Our bio repeats . Among t repeats . HoweverChlamydomonas IFT54 interacts with IFT dynein via its middle linker region, and that targeted disruption of this interaction impairs anterograde trafficking of IFT dynein  cells expressing various deletion constructs of IFT54. We found that specific deletion of the IFT54 CH domain responsible for WDR60 binding had a marginal effect on dynein-2–driven retrograde trafficking. In clear contrast, the IFT54 C-terminal coiled-coil, which encompasses the region involved in binding to DYNC2H1–DYNC2LI1 and IFT20, was found to be essential for IFT-B function and for effective retrograde trafficking driven by the dynein-2 complex.In the present study, we investigated the role of IFT54 in the dynein-2–IFT-B interaction, and indirectly in dynein-2–driven retrograde trafficking. IFT54 has an N-terminal calponin-homology (CH) domain and a C-terminal coiled-coil region, which are linked via a middle, unstructured linker region. We previously showed that the CH domain and the coiled-coil region of IFT54 participate in its interactions with WDR60 and with the DYNC2H1–DYNC2LI1 dimer, respectively. The coiled-coil region of IFT54 is also essential for its interaction with IFT20 of the IFT-B complex . On the T dynein . Both inanalysis , and in dictions , the lonChlamydomonas study also supported the important role of IFT54 in binding to dynein-2 during anterograde IFT  cells (Chlamydomonas ift54-null mutant (IFT54-KO cells demonstrated a cilia-lacking phenotype depicted by staining for ARL13B (IFT54-KO cells. The stable expression of mCherry-IFT54(WT) restored ciliogenesis  cells . In goodl mutant , IFT54-Kr ARL13B C,D. We togenesis L, excludH domain . Thus, tin vitro .IFT54-KO cells  and IFT54(135–625), the stable expression of mCherry-fused IFT54(1–532), which lacks a part of the coiled-coil region responsible for binding to IFT20 and the DYNC2H1–DYNC2LI1 dimer see , could nKO cells L. This wKO cells F. Secondnd IFT25 A, right nd IFT25 . Note thIFT54-KO cells expressing IFT54 constructs that were further truncated from the N-terminus . With longer truncations of the linker region from the N-terminus, the ciliogenesis efficiency of IFT54-KO cells expressing the IFT54 construct decreased gradually (IFT54-KO cells expressing mCherry-fused IFT54(533–625), which retains the binding abilities to IFT20 and DYNC2H1–DYNC2LI1, virtually no cilia were formed (IFT54-KO cells will be discussed later (see Discussion).We also observed cilia formation in radually L, and thradually M. In IFTe formed L. As mChe formed , lane 9,IFT54-KO cells expressing the various mCherry-IFT54 constructs; note that in these experiments, we omitted cells that did not form cilia [IFT54-KO cells expressing mCherry-IFT54(1–532) and those expressing mCherry-IFT54(533–625)]. In control RPE1 cells and IFT54-KO cells expressing mCherry-IFT54(WT), the IFT-B subunit IFT88 was mainly detected at the ciliary base, and a minor fraction was detected at the tip (IFT54-KO cells expressing mCherry-fused IFT54(135–625) or IFT54(335–625) (IFT54-KO cells expressing mCherry-IFT54(WT) (IFT54-KO cells expressing mCherry-fused IFT54(488–625) or IFT54(507–625), IFT88 was significantly enriched around the tip  of shortened cilia (IFT54-KO cells expressing mCherry-fused IFT54(488–625) or IFT54(507–625)  C,D, the FT54(WT) G,H. By ced cilia G,H. Line507–625) L,M. The 507–625) B–D, wher507–625) N,O.IFT54-KO cells expressing mCherry-fused IFT54(WT), IFT54(135–625), and IFT54(335–625), IFT140 was mainly localized at the ciliary base (IFT54-KO cells expressing mCherry-fused IFT54(488–625) or IFT54(507–625) demonstrated considerable enrichment of IFT140 in the distal region of severely shortened cilia, like as the mCherry-fused IFT54 constructs themselves .Localization of the IFT-A subunit IFT140 also substantially overlapped with that of the mCherry-fused IFT54 constructs. In ary base B–D as inary base A; note tary base . By contemselves G,H. The KO cells  and in DKO cells , but notKO cells . It is tIFT54-KO cells expressing mCherry-IFT54(WT) (IFT54-KO cells expressing mCherry-IFT54(335–625), ciliary GPR161 levels were slightly, but significantly, high in both the basal and SAG-stimulated states compared with the levels in control RPE1 cells and in IFT54(WT)-expressing IFT54-KO cells (IFT54-KO cells expressing either mCherry-fused IFT54(488–625)  B,H, and FT54(WT) C,I. By cKO cells M. The ab488–625) E,K or IF488–625) M.IFT54-KO cells expressing mCherry-IFT54(WT), SMO was not found within cilia under basal conditions (IFT54-KO cells expressing mCherry-IFT54(135–625) (IFT54-KO cells expressing mCherry-IFT54(335–625), the basal ciliary level of SMO tended to be slightly high, although not statistically significant (IFT54-KO cells expressing either mCherry-fused IFT54(488–625) , which is a class F GPCR that positively regulates Hh signaling . In contnditions N,O due tnditions . Howevernditions Z. Simila135–625) P,V,Z. Innificant Q,Z. In I488–625) R,X or IF488–625) Z, the baChlamydomonas anterograde IFT train suggests that the heavy chains of the dynein-2 complex contact the IFT-B2 face of the polymerized IFT-B complexes , which specifically lacks the CH domain that is responsible for its binding to WDR60  (IFT54-KO cells expressing IFT54(335–625) also appeared almost normal, although ciliogenesis efficiency and ciliary length were significantly, although slightly, decreased compared with cells expressing IFT54(WT) or IFT54(135–625). In clear contrast, IFT54-KO cells expressing a construct lacking almost the entire linker region as well as the CH domain, i.e. IFT54(488–625) or IFT54(507–625), demonstrated considerable accumulation of IFT-B and IFT-A components in the distal region of severely shortened cilia. In IFT54(488–625)-expressing and IFT54(507–625)-expressing IFT54-KO cells, dynein-2 is likely to be severely impaired with respect to driving retrograde trafficking due to its impaired trafficking as an anterograde cargo, although the dynein-2 complex itself should be intact, in view of the fact that cells lacking the motor subunit DYNC2H1 and those lacking DYNC2LI1, which stabilizes DYNC2H1  , it is l DYNC2H1 , demonst DYNC2H1 . On the KO cells K,L, evenKO cells , lane 9.KO cells .Chlamydomonas anterograde IFT train  of nephronophthisis (NPHP)-related ciliopathies with IFT54 variations ; NPHP isogenesis F. Howeveis study  and 2. FS-transferase (GST)-tagged anti-mCherry Nb (LaM-2 version) prebound to glutathione–Sepharose 4B beads were prepared as described previously  were cultured in DMEM with high glucose  supplemented with 5% FBS.IFT54 constructs used in this study are listed in eviously . SAG wasIFT54-KO cells were established from hTERT-RPE1 cells by essentially the same strategy using the CRISPR/Cas9 system as we previously described for the establishment of WDR60-KO cells  (Addgene 80767), using X-tremeGENE9 Reagent (Roche Applied Science). After selection in the presence of G418 (600 µg/ml), the cells with blue nuclear fluorescence were isolated. Genomic DNA extracted from the isolated cells was subjected to PCR using PrimeSTAR GXL Premix Fast DNA polymerase (Takara Bio) using three sets of primers  was used in this study.KO cells . The sinl region  was desil region . A doubl primers  to disti primers . Direct r allele . The resg at 4°C for 4 h. Viral particles in the precipitates were resuspended in Opti-MEM (Thermo Fisher Scientific) and stored at −80°C until use. IFT54-KO cells expressing the mCherry-fused IFT54 construct were prepared by addition of the lentiviral suspension to the culture medium and processed for immunofluorescence analysis.Lentiviral vectors for mCherry-fused IFT54 constructs were prepared as described previously . HEK293TTo induce ciliogenesis, hTERT-RPE1 cells were grown to 100% confluence on coverslips, and serum-starved for 24 h in DMEM/F-12 containing 0.2% bovine serum albumin.Immunofluorescence analysis was performed as described previously . Cells wIFT54-KO cells stably expressing an mCherry-fused IFT54 construct were grown to almost confluency on a 15-cm plate. The cells were lysed in 1 mL of lysis buffer  containing EDTA-free protease inhibitor cocktail  by placing on ice for 40 min. After centrifugation of the lysates at 16,000×g at 4°C for 20 min, the supernatants were incubated with 40 µl of GST-tagged anti-mCherry Nb prebound to glutathione-Sepharose 4B beads. The beads were washed five times with the lysis buffer and boiled in SDS-PAGE sample buffer. The proteins bound to the beads were then separated by SDS-PAGE, and electroblotted onto an Immobilon-P membrane (Merck Millipore). The membrane was blocked in 5% skimmed milk and incubated sequentially with the primary antibody and the peroxidase-conjugated secondary antibody. Protein bands were detected with a Chemi-Lumi one L kit, Chemi-Lumi super kit or Chemi-Lumi ultra kit .10.1242/biolopen.059976_sup1Supplementary informationClick here for additional data file.


Pentacta pygmaea (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species Holothuria floridana (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional 1H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors  in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration–response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from  The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), recently provoked a pandemic of vast catastrophic consequences in the world . The delMechanistically speaking, molecular interactions made by the spike (S)-protein, an essential structural protein found on the envelope surface of SARS-CoV-2, are formed during the initial stage of the viral infection. In this stage, S-protein recognizes and interacts specifically with the host cell receptor angiotensin-converting enzyme 2 (ACE2) . In addiBy administering an exogeneous inhibitor or competitor, an intentional disruption of the molecular interactions between the viral S-proteins with one of these molecules can be achieved leading therefore to an anti-SARS-CoV-2 outcome. Indeed, key studies have recently demonstrated the anti-SARS-CoV-2 capacity of numerous molecules, including ACE2 inhibitors, heparin , and othBotryocladia occidentalis [Pentacta pygmaea (PpFucCS)   19]. Th. ThBotryHolothuria floridana (HfFucCS) (1H nuclear magnetic resonance (NMR) spectroscopy. Their anticoagulant effects were measured using activated partial thromboplastin time (aPTT), and inhibitory assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin [AT] and heparin cofactor II [HCII]) in the presence of the exogenous polysaccharides. Anti-SARS-CoV-2 properties were measured using HEK-293T-human ACE2 (HEK-293T-hACE2) cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type (Wuhan-Hu-1) and delta (B.1.617.2) spike (S)-protein variants. Cytotoxicity, which was assessed against the HEK-293T-hACE2 cells, showed no harmful effects of HfFucCS or any of its oligosaccharide preparations. Similar with previously published results about PpFucCS, we were also able to generate HfFucCS oligosaccharides with retained anti-SARS-CoV-2 activities against both wild type and delta, and devoid of high anticoagulant effects. Curiously, however, the oligosaccharides generated in this work had significantly shorter chain lengths than those of the antivirally active PpFucCS oligosaccharides. A structural comparison between PpFucCS and HfFucCS   22]. Ol. OlHolot HfFucCS  enabled H. floridana, was subjected to an anion-exchange chromatography on a DEAE Sephacel column  structural integrity of the holothurian glycan, (2) purity level of our preparation, and (3) potential chemical changes in HfFucCS upon free-radical depolymerization during oligosaccharide production  within the expansion of 5.65–5.20 ppm is diagnostic of the anomeric 1H1 signals from the differently sulfated α-Fuc units: α-Fuc-2,4S (labeled as A unit) with δH1 at 5.64 ppm, α-Fuc-3,4S (labeled as B unit) with δH1 at 5.35 ppm, and α-Fuc-4S (labeled as C unit) with δH1 at 5.22 ppm  are characteristic of the methyl protons (CH3) of both the GalNAc and of Fuc units.HfFucCS is structurally composed of repeating trisaccharide units with a chondroitin sulfate (CS) type-E (CS-E) backbone, characteristic of 4,6-di-sulfation in 3-linked β-N-acetylgalactosamine  units, decorated with α- Fuc branches linked at the C3 position of the backbone 4-linked β-glucuronic acid (GlcA) unit . The Fucoduction . Diagnos2O2) and three concentrations  of copper (II) acetate (Cu[OAC]2). Temperature (60°C) and pH (6.0) were kept constant as successfully employed in our previous work with PpFucCS   and hydrogen peroxide (H2O2) ; at six different time points . Each reaction condition was quenched using chelex resin (50–100 mesh size). The native HfFucCS and hydrolyzed products from each reaction condition obtained within different time points were analyzed by polyacrylamide gel electrophoresis. Samples (10 μg each) were loaded on a 22% polyacrylamide gel and stained by 0.1% (w/v) toluidine blue (in 1% acetic acid) after adequate electrophoretic migration. The electrophoretic mobility was compared against two molecular markers: LMWH (~8 kDa), and the native HfFucCS (~50 kDa).Each 2 mg/ml of HfFucCS was depolymerized using one of the nine different reaction conditions with copper (II) acetate [(Cu(OAC)(TIF)Click here for additional data file.