Middle cerebral artery occlusion/reperfusion (MCAO/R) was created in mice. Oxygen-glucose deprivation/reoxygenation (OGD/R) was performed in brain microvascular vessel-derived endothelial cells (bEnd.3) to mimic ischemia/reperfusion injury in vitro. P-gp-specific siRNA and pharmacological inhibitor cyclosporine A were used to inhibit P-gp, whereas pcDNA3.1 was utilized to overexpress P-gp. Twenty-four hours after reperfusion, acute ischemic stroke outcome, BBB integrity and permeability, autophagic proteins and relative signaling pathways were evaluated. P-gp levels were markedly elevated in mouse brain and endothelial cells following MCAO/R and OGD/R, respectively. P-gp siRNA silencing or pharmacologically inhibiting (cyclosporine A) reduced infarct volume and brain edema, attenuated brain pathology, and improved neurological behavior in association with attenuated accumulation of neutrophils and macrophages, reduced expression levels of inflammatory cytokines (TNF-α and IL-1β), matrix metalloproteinases (MMP-2 and MMP-9) and adhesion molecules (ICAM-1 and VCAM-1). P-gp silence also counteracted BBB leakage, restored the expressions of tight junction proteins , activated autophagic proteins , and diminished Akt/mTOR signal activity in mice following MCAO/R. In the endothelial cell OGD/R assay, P-gp silence downregulated the expressions of inflammatory cytokines and adhesion molecules, inhibited leukocytes adhesion and migration, increased tight junction protein levels, and activated autophagy, all were reversible by forceful P-gp expression. Additionally, treatment with an autophagy inhibitor (3-methyladenine) abolished protections against ischemic stroke and tight junction proteins reduction followed by P-gp silence. In conclusion, increased P-gp expression after ischemic injury resulted in BBB dysfunction and hyperpermeability by suppressing Akt/mTOR-induced endothelial autophagy.P-glycoprotein (P-gp) is expressed on brain microvessel endothelial cells of blood-brain barrier (BBB) and elevated after cerebral ischemia. In this study, we explored the influence and potential mechanisms of P-gp on BBB function in experimental ischemic stroke  Stroke is one of leading causes of disability and mortality worldwide, with ischemic stroke accounting for 87% of all stroke-related incidents . InflammP-glycoprotein (P-gp), also known as multidrug resistance transporter-1, is a membrane transport protein belonging to adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily . As a maAutophagy is a self-eating cellular process to maintain cellular homeostasis and normal cellular functions , and is In this study, we investigated the effect and its underlying mechanisms of P-gp on BBB integrity in the mouse model of ischemic stroke. We found that elevated microvascular endothelial P-gp degraded endothelial tight junction, increased BBB hyperpermeability, accelerated brain inflammation and thus worsened ischemic stroke outcomes in association with increased Akt/mTOR activity and reduced endothelial autophagy.Male C57BL/6 mice weighting 22-25 g were purchased from Qinglongshan Animal Breeding Centre  and used in all experiments. All animals were housed and fed freely in a temperature-controlled room (22 ± 2°C) with a 12 h light-dark cycle. All experimental procedures were performed under licenses that granted by China Pharmaceutical University Animal Experimental committee , in compliance with the National Institutes of Health  guidelines for the care and use of animals. Mice were randomly assigned into 6 treatments, including sham treatment, middle cerebral artery occlusion/reperfusion (MCAO/R) treatment, MCAO/R with negative control siRNA (NC siRNA) treatment, MCAO/R with NC siRNA and cyclosporine A treatments (NC siRNA + CsA), MCAO/R with P-gp siRNA treatment (MCAO/R + P-gp siRNA), MCAO/R with P-gp siRNA and 3-MA treatments (MCAO/R + P-gp siRNA + 3-MA). The treatment was administered in a blinded fashion. The researcher conducting the surgeries and behavior tests was unaware of the animal groups, and the researcher analyzing the data did not know the group condition.Mice were anesthetized by 3% isoflurane and maintained using 1.5% isoflurane  and placed in a stereotaxic frame  as previously described . The skiAutophagy inhibitor (3-MA) and P-gp inhibitor (CsA) were purchased from MedChemExpress LLC . 3-MA was prepared in normal saline immediately before use, and intracerebroventricularly injected at a dose of 15 μg per mouse 30 min prior to MCAO surgery . CsA wasFollowing 12 h fasting, MCAO/R was performed as described previously . Mice weTwenty-four hours after MCAO/R surgery, neurological score was graded as 0-4 based on Bederson’s method , with hiThe whole brain was removed after mice were sacrificed at 24 h after reperfusion, and each brain was sliced into coronal sections (2 mm thickness). Brain slices were stained in TTC  at 37°C for 20 min, photographed with a digital camera, and analyzed using the ImageJ software  . HemisphTo estimate brain edema, brains were weighed prior to and 12 h after drying in a 110°C oven. Brain edema was calculated as percent of water content as following : Brain wTo evaluate the integrity of BBB , mice we2) at 37°C. The cells at 70-80% confluence was transfected with mouse P-gp (GGATCCAGTCTAATAAGAA) siRNA (50 pmol per well (6-well), RiboBio, Guangzhou, Guangdong, China) or P-gp pcDNA3.1 (+)  using the lipofectamine 2000 reagent  to silence or overexpress P-gp, respectively. The transfection efficiency was assayed 36 h after transfection by Western blot according to the manufacturer’s recommendation. The control cells were transfected with the same volume of NC siRNA (RiboBio) or NC pcDNA3.1 (+) (Sangon Biotech).bEnd.3 cells were obtained from China Pharmaceutical University and cultured in high-glucose Dulbecco’s Modified Eagle’s Medium  supplied with 10% fetal bovine serum , D-Glucose (4.5g/L), glutamine (2 mmol/L), penicillin (80 U/mL), streptomycin (0.08 mg/mL) and pyruvate (1 mmol/L) in humidified air (5% CO2 and 5% CO2) at 37°C for 2 h [2) and maintained in serum-free high-glucose DMEM for 24 h. In control group, cells were cultured in the chamber with serum-free high-glucose DMEM and normal culture conditions for the same period. The cells were treated with an autophagy inhibitor 3-MA (1 mmol/L) 1 h prior to OGD stimulation [Confluent bEnd.3 cells were washed twice and subjected to OGD/R by replacing with serum-free low-glucose  DMEM in an anoxic incubator  was replenished every 2 d. Approximately 6-7 d after seeded, bEnd.3 monolayers were obtained. Leukocyte (1×106 cells in 0.2 mL of DMEM) were added to upper chamber and incubated for 4 h in bEnd.3 cells. Migrated leukocytes were counted from the lower chamber. Leukocyte transendothelial migration was quantitated as the number of leukocytes per square millimeter.Leukocyte transendothelial cell migration assay was performed as previously described  with sliBrains were harvested, fixed in 4% paraformaldehyde (PFA) for 24 h, dehydrated in graded ethanol, embedded in paraffin, and sectioned (6 μm). After deparaffinization and rehydration, sections were stained with Giles’ hematoxylin and eosin (H&E) and imaged on a fluorescent inverted microscope .Paraffin sections (2 μm in thickness) were used for all staining. Briefly, following blocking endogenous peroxidase with PBS containing 3% hydrogen peroxide for 5 min, sections were stained with primary antibodies for neutrophils , macrophages , intercellular adhesion molecules-1 , vascular adhesion molecule-1 , and P-gp  at 4°C overnight. Then, sections were incubated with Immunohistochemical staining kit  for 1 h at room temperature. All stained sections were counterstained with Giles’ hematoxylin and imaged on a fluorescent inverted microscope . To quantify P-gp expression levels, neutrophil and macrophages, randomly selected fields from the ischemic cortex were analyzed by using ImageJ software.Mice were anesthetized 24 h after reperfusion and perfused intracardially with cold 4% PFA followed by perfusion with PBS. Brains were fixed with 4% PFA and embedded in optimal cutting temperature compound (OCT), and sectioned (12 μm) followed by blocking nonspecific staining with 8% goat serum for 2 h at room temperature. For culture cells, the cells were fixed in 4% PFA for 20 min and incubated with solution containing 0.3% Triton X-100 in PBS (PBST) containing 8% goat serum for 2 h at room temperature for non-specific staining blocking and permeabilization .Tissue sections and cells were incubated with the primary antibodies against CD31  ICAM-1 (1:100), VCAM-1 (1:50), Claudin-5 , Occludin , Zonula occludens-1 , light chain 3  and glucocorticoid receptor  at 4°C overnight. Following 3 washes with PBST, sections or cells were incubated with Cy3-conjugated goat anti-mouse IgG  and FITC-conjugated goat anti-rabbit IgG  for 2 h at room temperature, washed with PBST 3 times and counterstained with 4, 6-diamidino-2-phenylindole  for 30 min at room temperature. Stained images were captured by a laser confocal microscope  or a fluorescent microscope , and analyzed using ImageJ software.Total RNA was extracted using the RNA isolater  and was transcribed into cDNA using a HiScript II Q RT SuperMix (Vazyme Biotech). Real time PCR was performed using quantitative PCR  with a fluorescent dye (SYBR Green I). mRNA levels were normalized to β-actin of the same samples and were reported as fold changes relative to sham or control treatment . The priTotal protein was extracted using RIPA lysis containing protease and phosphatase inhibitor cocktail . Protein concentrations were determined by BCA assay. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1.5 h at room temperature, the membrane was incubated with antibodies against P-gp (1:500), Claudin-5 (1:500), Ocluddin (1:1000), ZO-1 (1:1000), LC3 (1:1000), mTOR , p-mTOR , Akt , p-Akt  and β-actin  at 4°C overnight. After 3 times washes with TBST, the blots were incubated with HRP goat anti-rabbit IgG (H + L)  or HRP-labeled goat anti-mouse IgG (H + L)  for 2 h at room temperature. The bands were visualized using the enhanced chemiluminescence (ECL) reagents (Affinity Biosciences) and were quantified with ImageJ software. The protein expression levels were expressed as the percentage of β-actin.To evaluate the function of P-gp on glucocorticoid transport, mice were injected with exogenous glucocorticoid  through femoral vein at 24 h after MCAO/R. Brains were harvested and ischemic hemisphere was separated at 5 min after the injection. Brain was homogenized with pure water with a mass/volume ratio of 1:3. Dexamethasone concentration in homogenates was determined by a UPLC-MS/MS system  and given as ng/g ischemic brain tissue.P<0.05. All data are presented as the means ± SD.All statistical analyses were performed using IBM SPSS Statistics 19.0 software. The distribution of all data was tested for normality via a One-Sample Kolmogorov-Smirnov test. Statistical differences among the groups were analyzed by One-Way Analysis of Variance (ANOVA) with post hoc Tukey tests if data were normally distributed and consistent with homogeneity of variance among the experimental groups. The Mann-Whitney test was used to the compare ranks if the data were not normally distributed or not consistent with homogeneity of variance. The difference was considered significant at P<0.01). Treatment with NC siRNA did not impact P-gp expression, infarct volume, and brain histopathology. Therefore, NC siRNA + MCAO/R mice were used to observe P-gp function in following experiments as model control. P-gp siRNA pretreatment reduced infarct volume, attenuated neurological impairment, and improved histopathological damage as compared to NC siRNA treatment after MCAO/R (P<0.01).Mice underwent the MCAO surgery were included for successful occlusion. P-gp expression was substantially increased at ischemic hemisphere 24 h following MCAO/R surgery. Treatment with P-gp siRNA significantly reduced P-gp expression in ischemic brain as compared to NC siRNA treatment , P<0.01.r MCAO/R , P<0.01.P<0.01).Ischemic stroke breaks down BBB, leading to the entry of serum proteins into brain fluid and edema formation . Twenty-P<0.05).MMP-2 and MMP-9 degrade collagen IV that is a major component of basal lamina, and ultimately disrupt BBB . In RT-PP<0.01). In tissue immunostaining, neutrophils (MPO) and macrophages (F4/80) were observed in the infarct site 24 h after MCAO/R. However, P-gp siRNA treatment substantially reduced the densities of both neutrophils and macrophages as compared to NC siRNA treatment (P<0.01).ICAM-1 and VCAM-1 are important for recruiting leukocytes into inflamed brain. ICAM-1 and VCAM-1 levels were significantly lower in the ischemic area of mice treated with P-gp siRNA than those in mice treated with NC siRNA , P<0.01.reatment , P<0.01.P<0.05, P<0.01). These results indicate that silencing P-gp reduced BBB permeability by increasing TJPs expression.In the CNS, tight junctions form a barrier to limit paracellular permeability. In Western-blotting analysis, the expression levels of TJPs including Occludin, Claudin-5, and ZO-1 were largely reduced in ischemic brain of NC siRNA-treated MCAO/R mice. In contrast, P-gp siRNA treatment restored the expression levels of all proteins in ischemic area . In immunofluorescence staining, P-gp knockdown also dramatically enhanced LC3 expression in endothelial cells (CD31+) (P<0.01).To determine the influence of P-gp on endothelial autophagy, we assessed the expression of microtubule-associated protein LC3 and autophagy adapter protein P62, two well established markers for autophagy activation , 39. In  (CD31+) , P<0.01.P<0.01). Glucocorticoid binds to glucocorticoid receptor (GR), induces GR nuclear translocation and thus inhibits Akt/mTOR activity [P<0.01).Akt/mTOR activity suppressed autophagy . As indiactivity . In ischP<0.05, P<0.01).To determine whether autophagy contributes to the suppression of ischemic stroke by P-gp inhibition, an autophagy inhibitor 3-MA was given to mice 30 min prior to stoke induction. 3-MA treatment did not alter P-gp expression in ischemic brain of P-gp siRNA-treated mice. However, inhibiting autophagy by 3-MA counteracted the effects of P-gp siRNA treatment on ischemic stroke outcome as indicated by enlarged infraction size, increased brain edema, worsened neurological defects and brain histopathology (P<0.05). Histologically, CsA treatment reduced infarct volume, brain edema, neurological behavior defects and brain histopathology induced by MCAO/R .Additionally, similar to P-gp siRNA silence, pharmacological inhibition of P-gp with CsA significantly attenuated P-gp expression levels in ischemic brain as compared to vehicle treatment , P<0.05.P<0.01). Preincubation with P-gp siRNA inhibited mRNA expressions of TNF-α, IL-1β, MMP-2, and MMP-9 in endothelial cells induced by OGD/R (P<0.05). Consistent with in vivo findings, P-gp knockdown substantially downregulated the expression of ICAM-1 and VCAM-1 on endothelial cells following OGD/R treatment (P<0.01). P-gp silence also mitigated leukocyte adhesion and transendothelial migration (P<0.01), while increased the expression levels of TJPs  in endothelial cells undergoing OGD/R .Under OGD/R condition, P-gp expression was markedly elevated in endothelial cells (bEnd.3 cells). P-gp knockdown dramatically reduced P-gp expression as compared to NC siRNA incubation , P<0.01.by OGD/R , P<0.05.reatment , P<0.01.igration , P<0.01,P<0.01) as compared to NC plasmid pcDNA3.1. P-gp overexpression increased mRNA expression levels of TNF-α, IL-1β, MMP-2, and MMP-9 following OGD/R treatment as compared to NC plasmid pcDNA3.1 (P<0.05). Additionally, P-gp overexpression also increased adhesion molecules expression, augmented leukocytes adhesion and transmigration , whereas reduced the expression levels of Claudin-5, Occludin, and ZO-1 in endothelial cells following OGD/R .To examine the effect of P-gp overexpression, endothelial cells were transfected with P-gp pcDNA3.1 plasmid to forcefully express P-gp or with pcDNA3.1 plasmid serving the NC. P-gp pcDNA3.1 transfection remarkably upregulated P-gp expression , P<0.01 pcDNA3.1 ; P<0.05.P<0.01). These data indicate that P-gp silence led to autophagy activation to protect endothelial cells against OGD/R-induced cellular injury. Further, P-gp silence largely diminished the phosphorylation of Akt and mTOR protein signaling (P<0.05), suggesting that P-gp silence may activate autophagy by inhibiting Akt/mTOR signaling activity.To assess the influence of P-gp on autophagy and potential mechanisms, endothelial cells were transfected with P-gp or NC siRNA followed by OGD/R treatment. In Western-blotting and immunofluorescence staining analyses, P-gp silence dramatically increased the the ratio of LC3-II/LC3-I as well as Beclin 1 levels, whereas reduced P62 levels ; P<0.01.ignaling , P<0.05,P<0.05, P<0.01). When endothelial cells were incubated with a small molecule inhibitor 3-MA, the upregulation of TJPs by P-gp silence was almost ablated . Expectedly, 3-MA treatment reduced the ratio of LC3-II/LC3-I and Beclin 1 expression (P<0.01), whereas increased P62 expression (P<0.01). These results indicate that autophagy activation by P-gp silence may protect OGD/R-induced endothelial dysfunction.Although OGD/R treatment dramatically reduced TJPs levels in endothelial cells, P-gp silence increased the suppression of TJPs expression following OGD/R as compared to NC siRNA treatment (pression , P<0.01,pression , P<0.01.in vivo and in vitro. P-gp silence or pharmacological inhibition alleviated ischemic stroke by improving the integrity and function of BBB. Therefore, P-gp overexpression caused BBB dysfunction and exacerbated stroke outcome by destroying TJPs and worsening inflammatory response.Despite advances in our understanding of P-gp in multiple drug resistance, the role and underlying mechanisms in BBB dysfunction induced by ischemic stroke remain largely unknown. In this study, we found increased expression of P-gp in experimental ischemic stroke both in vitro, or overexpressed P-gp by transfecting endothelial cells with P-gp pcDNA3.1. We showed that P-gp siRNA relived acute stroke injury, including reduction in infarct volume, improvement in neurological behaviors, and decrease in brain edema. Similar effects were also observed using a P-gp inhibitor CsA. Therefore, elevated P-gp expression induced by ischemic stroke is vital for aggravating acute stoke injury.P-gp is involved in the pathogenesis of certain CNS diseases by regulating inflammatory cytokine expression and immune cell infiltration , 12. ThoP-gp is mainly expressed on BMVECs of BBB that functions as a semipermeable barrier between the CNS and the peripheral circulation , 46. In TJPs including ZO-1, Claudin-5, and Occludin are important in regulating the integrity and permeability of BBB and are disrupted and redistributed after ischemic stroke , 54. We Autophagy is a crucial degradation pathway for maintaining cellular and energy homoeostasis and protecting cell death . AutophaIn conclusion, we demonstrated that elevated P-gp levels following ischemic stroke exacerbated BBB breakdown and brain inflammatory response by increasing Akt/mTOR activity and suppressing autophagy activation. Our findings help understand the role and underlying mechanisms of P-gp in brain inflammatory response and BBB integrity following ischemic stroke.www.aginganddisease.org/EN/10.14336/AD.2022.0225.The Supplementary data can be found online at:


Siphoviridae phage that infects Gordonia terrae 3612. The 68,128-bp genome of StarStruck has a GC content of 65.4% and contains 92 protein-coding genes, including the gene for a HicA-like toxin. StarStruck was assigned to subcluster CR2 based on >35% shared gene content with other cluster CR genomes in the Actinobacteriophage Database.Bacteriophage StarStruck is a lytic  Actinobacteria, are extremely abundant and diverse  icosahedral head and a 297.5-nm  flexible, noncontractile tail (n = 3 particles).Actinobacteriophages, which are viruses that infect bacteria of the phylum  diverse –4. By chdiverse –, 6. Starrae 3612 . Soil exe assays . After ilysogens . The parCGCCGCGTAC). StarStruck shares >35% gene content with members of cluster CR in the Phamerator database Actino_Draft and was assigned to subcluster CR2  . Sequencster CR2 , 10, 11.http://cobamide2.bio.pitt.edu) and PECAAN (https://blog.kbrinsgd.org/) (http://phages.wustl.edu/starterator) analyses (The genome of StarStruck was autoannotated using GLIMMER v3.02 and GeneMark v2.5 within DNA Master v5.23.6 (gd.org/) , 13. Traanalyses . Putativanalyses , 15, 16.analyses , 18. Staanalyses . The laranalyses .Like many cluster CR phages, StarStruck encodes a HicA-like toxin (gp7) within the 12,000-bp region separating the small- and large-subunit terminases. Another interesting feature of StarStruck is the location of the lysin B (gp19) within this region, rather than adjacent to the lysin A genes (protease C39 domain [gp49] and glycosyl hydrolase domain [gp50]), which are located downstream of the minor tail proteins.ON456333 and the Sequence Read jhu7 (SRA) accession number SRX14816101.StarStruck is available at GenBank with the accession number


Pancreatic ductal adenocarcinoma (PDAC) is currently the most deadly cancer. Although characterized by 5–20% of neoplastic cells in the highly fibrotic stroma, immunotherapy is not a valid option in PDAC treatment. As CXCR4-CXCL12 regulates tumor invasion and T-cell access and PD-1/PD-L1 controls immune tolerance, 76 PDACs were evaluated for CXCR4-CXCL12-CXCR7 and PD-1/PD-L1 in the epithelial and stromal component. Neoplastic CXCR4 and CXCL12 discriminated PDACs for recurrence-free survival (RFS), while CXCL12 and CXCR7 discriminated patients for cancer-specific survival (CSS). Interestingly, among patients with radical resection (R0), high tumor CXCR4 clustered patients with worse RFS, high CXCL12 identified poor prognostic patients for both RFS and CSS, while stromal lymphocytic-monocytic PD-L1 associated with improved RFS and CSS. PD-1 was only sporadically expressed (<1%) in focal lymphocyte infiltrate and does not impact prognosis. In multivariate analysis, tumoral CXCL12, perineural invasion, and AJCC lymph node status were independent prognostic factors for RFS; tumoral CXCL12, AJCC Stage, and vascular invasion were independent prognostic factors for CSS. CXCL12’s poor prognostic meaning was confirmed in an additional perspective-independent 13 fine-needle aspiration cytology advanced stage-PDACs. Thus, CXCR4-CXCL12 evaluation in PDAC identifies prognostic categories and could orient therapeutic approaches. Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related deaths in economically advanced countries . In the Patients with resectable PDAC  consecutThree-micrometer sections were cut from formalin-fixed paraffin-embedded (FFPE) tissue blocks. Sections were stained with hematoxylin and eosin for adequate tumor representation.2/field) for each ROI, on Olympus BX51 microscope . The evaluation was carried out by 3 qualified observers . For CXCR7 and CXCL12, staining extension was calculated for each case by the average percentage of positively stained cancer cells for the three ROIs. For CXCR4, due to heterogeneous staining patterns, the staining value was calculated by H-score. Staining intensity was based on membrane staining and rated as absent (0), weak/low (1+), and intermediate/moderate (2+), typically with cytoplasmic localization, strong/high (3+), typically cytoplasmic with membrane localization of CXCR4 staining. The % of positive cells at each staining intensity is obtained, and an H-score is determined by the sum of each intensity rating multiplied by its corresponding percentage. CXCR4 was scored as positive at H-score > 50, corresponding to PDAC with multiple subcellular signal accumulation (membrane and cytoplasm). CXCR7 and CXCL12 cells were rated positive when stained regardless of the cellular localization. CXCR7 was scored as positive with >20% positive cells [FFPE sections were dewaxed and rehydrated, and heat-induced epitope retrieval (HIER)  with appropriate Antigen Unmasking Solution was performed. After incubation with the appropriate serum for blocking non-specific background, FFPE tumor tissue slides were incubated overnight at 4 °C using primary antibodies: Mouse Monoclonal anti-human Anti-CXCR7/RDC-1 Antibody ((11G8), HIER citrate buffer pH6, 1:50 dilution, R&D Systems); Mouse Monoclonal anti-human Anti-CXCL12/SDF-1 Antibody ((79018), HIER citrate buffer pH6, 1:50 dilution, #MAB350 R&D Systems); two commercial CXCR4 antibody clones the mouse Monoclonal anti-human Anti-CXCR4/CD184 ((44716) HIER, citrate buffer pH6, 1:100 dilution, #MAB172 R&D Systems) and the rabbit monoclonal anti-human Anti-CXCR4 ((UMB-2) HIER, Tris-EDTA buffer, pH 9.0, 1:200 dilution, Abcam); two commercial PD-L1 antibodies; the rabbit monoclonal anti-human anti-PD-L1/CD274 ((E1L3N) 1:200; diluted; HIER citrate buffer pH6, #13684 Cell Signaling Technology and (22C3) ready to use with HIER Conditioning Solution (CC1) pH9 Ventana Medical Systems); Mouse monoclonal anti-human PD-1/CD279 ((NAT-105) ready to use, HIER Cell Conditioning Solution (CC1) pH9, Ventana Medical Systems). Stained pancreatic cancer cells were assessed in at least three regions of interest (ROI)/slide, recognized at low power (100× magnification), and the cells enumerated in 5 consecutive, not-overlapping high-power fields , and p > 0.1 (remove variable). p-values less than 0.05 were considered significant. All statistical tests and graphs were conducted using SPSS version 20  and MedCalc 12.3.0 .Association between CXCR4, CXCL12, CXCR7, and PD-1/PD-L1 expression cancer cells and patients’ clinic-pathological features were analyzed applying chi-square and Mann–Whitney U tests for categorical and continuous variables, respectively. Recurrence-free survival (RFS) was set as the time from diagnosis to the recurrence or last follow-up. Cancer-specific survival (CSS) was set as the time from diagnosis to death for cancer. Kaplan–Meier method was performed to estimate the survival curve and logrank test for statistical comparison. Cox proportional hazards regression was utilized to test the effect of multiple dichotomous covariates (risk factors) on RFS and CSS; The backward method for variable selection was applied in the final model with a conventional A population of twenty patients diagnosed with advanced PDAC, evaluated through endoscopic-US-guided pancreatic fine-needle aspiration cytology (FNAC), was considered a validation cohort. The samples were collected and analyzed at Pathology Unit, Department of Mental and Physical Health and Preventive Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy, from January 2020 to October 2021. Three-micrometer sections were stained for CXCL12 immunocytochemical evaluation following hematoxylin/eosin reviewing and histological confirmation. All cases were retrospectively reviewed, and the cytology on direct smears showed moderately to richly cellular samples composed of three-dimensional aggregates of epithelial cells with severe cytological atypia. The cyto-block (CB) sections were also re-examined, with a cytological morphology substantially similar to that observed on direct smears; when necessary, immunocytochemical (ICC) analysis was performed, which showed positivity in the neoplastic cells for CK19 and CA 19–9. In all 20 cases selected, the overall morphological analysis was consistent with the diagnosis of pancreatic adenocarcinoma (Category VI sec. Papanicolaou System). ImmunoCytoChemistry (ICC) was carried out on CB sections and stained for CXCL12 as previously described.Clinical pathological characteristics of the 76 patients are presented in CXCR4 was highly expressed in 25 out of 76 tumors (32.9%) . CXCR4 mRepresentative low–moderate and high CXCR4 expressions were reported C–H. CXCRCXCR7 was highly expressed in 26 out of 76 (34.2%) tumors and lowly expressed or undetectable in 50 tumors (65.8%). The CXCR7 mean expression was 13.3 ± 16.7% . Unlike CXCL12 was predominantly identified in the membrane in 20 out of 76 (26.3%) tumors. The CXCL12 mean expression was 3.7 ± 7.20% . RepresePD-1 and its natural ligand PD-L1 were evaluated in 76 PDAC. While PD-1 was not detectable in cancer cells, it was only sporadically expressed (<1%) in focal lymphocyte infiltrate (data not shown). PD-L1 was predominantly identified at the membrane of cancer cells in 29/76 (38.2%) PDAC. PD-L1 mean expression was 3.4 ± 7.1 . FigureC–H. Tumor = 0.42, p = 0.004). The relation among the evaluated markers and clinic pathologic characteristics revealed that CXCR4-positive tumor and CXCR4-positive tumor-infiltrating inflammatory cells were associated with vascular invasion (p = 0.0421 and 0.045) as expected [p = 0.002)  : 20.19–29.85); 33.60 months for cancer-specific survival CSS : 28.907–38.998). A total of 71 PDAC were analyzed for RFS; 58/71 (81.7%) patients experienced recurrence, with mean RFS durations of 17.30 months, and 13 (18.3%) did not experience recurrence up to 60.52 months. Out of 76, 4 patients were excluded for cancer-unrelated death; 72 PDAC were analyzed for CSS, 56/72 (77.8%) with mean CSS durations of 25.30 months, and 16/72 (22.2%) patients were alive at a mean of 62.65 months. Univariate analyses of RFS and CSS are summarized in p = 0.0024; CSS: 21.9 months vs. 36.6 months p = 0.08, not significant) (Patients with high CXCR4 expression had a significantly worse outcome (RFS: 11.76 months vs. 26.0 months ificant) A.n = 48) (RFS: 11.05 months vs. 33.70 months p = 0.0071) (p = 0.0001) and CSS  (p = 0.0001)  and CSS (p = 0.017)  in R0 patient’s subgroup (p = 0.0441) but not CSS (p = 0.0605)  ( 0.0071) A. CXCL12 0.0024) B. Moreovsubgroup B. Patien 0.0605) C. PD-L1  0.0605) D.p = 0.016) and CSS (0.047) in R0 patients, , perineural invasion (p = 0.0107), and CXCL12 (p = 0.00002) but not CXCR4, predicted shorter poor RFS (p = 0.00007), vascular invasion (p = 0.0067), and CXCL12 (p= 0.0062) but not CXCR7 nor CXCR4 expression predicted shorter poor CSS . The median age was 72 ± 10 years (range 50–85), with 8 (40%) male and 12 (60%) female, and 17 (85%) of patients ≥60 years old. At diagnosis, the majority of patients (85%) had an advanced stage. CXCL12 was detected in 70% (14/20) PDACs predominantly at the membrane and cytoplasm of cancer cells  , showing a median overall survival of 6 months. The patients with a high sharp, consistent distribution of CXCL12 expression showed short CSS with a median survival of 3 months, while low/negative CXCL12 expressing displayed short CSS with a median survival of 12 months (= 0.029) .p ≥ 0.05) as for NCCN 2022 guidelines. In our analysis, chronic pancreatitis was a “potential risk factor”  not retained, for irrelevance or redundancy, in the multivariate analysis. As for PD-L1, early studies showed prognostic significance for overall survival with >5 or 10% cut-off [The role of the CXCR4-CXCL12-CXCR7 axis and PD-1/PDL-1 was addressed in tumor/stromal cells in 76 consecutive single-center patients undergone surgery between January 2014/March 2015 and followed for 5 years until January 2021. We demonstrated that the entire axis CXCR4-CXCL12-CXCR7 was overexpressed in PDAC neoplastic cells as compared to TME cells. Moreover, CXCL12 significantly correlated with poor prognosis in an unrelated cohort of 20 FNAC from PDAC patients. CXCR4 was expressed by acinar cells and islets of Langerhans, surrounded by extensive dense fibrotic stroma or desmoplasia . Trefoil cut-off ,50. Alth cut-off .MMR) proteins) and sample size. In agreement with the herein reported data, Diana et al. examined the prognostic value of PD-1 and PD-L1, together with CD8+ tumor-infiltrating lymphocytes (TILs) and FOXP3+ Tregs in 145 PDAC samples, describing PD-L1 expression not prognostic [Karamitopoulou reported PD-L1 expression in about one-third of 349 samples. The authors concomitantly analyzed PD-L1, PD-1, CD3, CD4, CD8, FoxP3, and CD68, reporting that PD-L1, present with T-cell infiltration, improved overall survival . Herein,ognostic . Wang etognostic . As relaPD-L1 was recently reported in stromal cells in PDAC , and als


Accurate polyp size measurement is important for guideline conforming choice of polypectomy techniques and subsequent surveillance interval assignments. Some endoscopic tools (forceps or endoscopic rulers [ER]) exist to help with visual size estimation. A virtual scale endoscope (VSE) has been developed that allows superimposing a virtual measurement scale during live endoscopies.Our aim was to evaluate the performance of VSE when compared to ER and forceps-based measurement.We conducted a randomized trial to evaluate the relative accuracy of size measurement of simulated colorectal polyps when using: VSE, ER, and forceps. Six endoscopists performed 60 measurements randomized at a 1:1:1 ratio using each method. Primary outcome was relative accuracy in polyp size measurement. Secondary outcomes included misclassification of sizes at the 5, 10, and 20mm thresholds.A total of 360 measurements were performed. The relative accuracy of biopsy forceps, ER, and VSE was 78.9% (95%CI=76.2-81.5), 78.4% (95%CI=76.0-80.8), and 82.7% (95%CI=80.8-84.8). VSE had significantly higher accuracy compared to biopsy forceps (p=0.02) and ER (p=0.006). VSE misclassified a lower percentage of polyps >5mm as ≤5mm (9.4%) compared to forceps (15.7%) and ER (20.9%). VSE misclassified a lower percentage of ≥20mm polyps as <20mm (8.3%) compared with forceps (66.7%) and ER (75.0%). 25.6%, 25.5%, and 22.5% of polyps ≥10mm were misclassified as <10mm with ER, forceps, and VSE, respectively.VSE had significantly higher relative accuracy in measuring polyps compared to ER or forceps assisted measurement. VSE improves correct classification of polyps at clinically important size thresholds.NoneR. Djinbachian: None Declared, M. Taghiakbari: None Declared, C. Haumesser: None Declared, M. Zarandi-Nowroozi: None Declared, M. Abou-Khalil: None Declared, S. Sidani: None Declared, J. Liu: None Declared, B. Panzini: None Declared, D. von Renteln Grant / Research support from: Daniel von Renteln is supported by a “Fonds de Recherche du Québec Santé” (FRQS) career development award and has received research funding from ERBE, Ventage, Pendopharm, Fujifilm, and Pentax., Consultant of: Boston Scientific and Pendopharm,


All authors agree with this retraction.1. Kang, H., Ma, D., Zhang, J. et al. Long non-coding RNA GATA6-AS1 upregulates GATA6 to regulate the biological behaviors of lung adenocarcinoma cells. BMC Pulm Med 21, 166 (2021).


Cytochrome P450 family 8 subfamily B member 1 (CYP8B1) generates 12α-hydroxylated bile acids (BAs) that are associated with insulin resistance in humans.CYP8B1 in individuals without diabetes and identified carriers of complete loss-of-function (CLOF) mutations utilizing functional assays.To determine whether reduced CYP8B1 activity improves insulin sensitivity, we sequenced CYP8B1 mutation associated with lower fasting insulin in the AMP-T2D-GENES study. Exposure of primary human muscle cells to mutation-carrier CA/CDCA ratios demonstrated increased FOXO1 activity, and upregulation of both insulin signaling and glucose uptake, which were mediated by increased CDCA. Inhibition of FOXO1 attenuated the CDCA-mediated increase in muscle insulin signaling and glucose uptake. We found that reduced CYP8B1 activity associates with increased insulin sensitivity in humans.Mutation carriers had lower plasma 12α-hydroxylated/non–12α-hydroxylated BA and cholic acid (CA)/chenodeoxycholic acid (CDCA) ratios compared with age-, sex-, and BMI-matched controls. During insulin clamps, hepatic glucose production was suppressed to a similar magnitude by insulin, but glucose infusion rates to maintain euglycemia were higher in mutation carriers, indicating increased peripheral insulin sensitivity. Consistently, a polymorphic CLOF Our findings suggest that increased circulatory CDCA due to reduced CYP8B1 activity increases skeletal muscle insulin sensitivity, contributing to increased whole-body insulin sensitization.Biomedical Research Council/National Medical Research Council of Singapore.  Cyp8b1 show almost no 12α-hydroxylated BAs and elevated non–12α-hydroxylated BAs (Bile acids (BAs) are products of cholesterol catabolism, accounting for approximately 50% of daily cholesterol turnover in humans, and act as surfactants to promote intestinal lipid absorption . The priNR1H4) and membrane G protein–coupled BA receptor  . Of these, 58 were predicted to be possibly or probably damaging by the functional prediction tool Polyphen 2.0 , and 41 were predicted to be damaging by SIFT . We generated all 100 variants in human CYP8B1 cDNA, quantified the product generated by each variant, and found a spectrum of defective CYP8B1 activities, classified as complete loss of function (CLOF) (<15% activity of wild-type), partial loss of function (PLOF) (15% to 85% activity of wild-type), or benign (>85% activity of wild-type)  . We idenCYP8B1 mutation carriers, and 41 age-, sex-, BMI-, and race-matched controls to our clinical study. P = 0.04) and APOB/APOA-I (P = 0.04) ratios, suggesting reduced risk for atherosclerosis. In addition, high-sensitivity C-reactive protein (hs-CRP) levels were decreased by approximately 50% in the mutation carriers (P = 0.06), suggesting reduced systemic inflammatory status and lower atherosclerotic risk in carriers  (P = 0.02), resulting in a 63% decrease in the CYP8B1 product/substrate ratio  ( 0.0002) . CYP8B1’ 0.0001) .P = 0.003) (P = 0.01), and both glycine- (P = 0.01) and taurine-conjugated (P = 0.048) CA were decreased (P = 0.03) (P = 0.01)  . Unconjuecreased . CDCA di = 0.03) . The CA/ = 0.01) . The BA CYP8B1 mutation carriers. Thus, we assessed whether CYP8B1 mutation carriers showed improved insulin sensitivity. Although fasting glucose was unchanged, fasting insulin was decreased by 28% in carriers  (P = 0.03) (P = 0.03), as well as QUICKI, another measure of insulin sensitivity (P = 0.04) , insulin levels were decreased in carriers  (P = 0.02) and the glucose infusion rate (GIR) was increased by 30% in carriers  (P = 0.02) (P = 0.04) and GIR by 25%  in carriers.12α-Hydroxylated BAs correlate with insulin resistance in humans , and 12α = 0.03) . Accordi = 0.03) . The Mat = 0.04) , were in = 0.03) . During = 0.046) . The ins = 0.02) . The aboP = 0.38) (P = 0.48) (P = 0.58) (P = 0.19)  , glucone = 0.48) , and gly = 0.58)   , indicatCYP8B1 mutations have improved insulin sensitivity, we performed association analyses using the Type 2 Diabetes Knowledge Portal (http://www.type2diabetesgenetics.org), which enables association analyses between coding variation and glycemic traits in 45,231 exomes (CYP8B1 variants (R26X) was both a CLOF mutation (0.6% activity compared with wild-type CYP8B1), common in our study cohort , and present in several copies in the AMP-T2D-GENES data set. Association analyses showed that carriers of R26X had significantly lower fasting insulin levels after adjusting for BMI . These data are consistent with our findings of increased insulin sensitivity in the face of decreased CYP8B1 activity in the CYP8B1 mutation carriers.To confirm that humans with CLOF 1 exomes . Of the variants , only 1 P = 0.65) (P = 0.04) and GLP-1 during MMTT  were decreased  . Glucago = 0.65) . Both faecreased , in lineIRB being the predominant isoform in insulin target tissues, including muscle   (256) is cytoplasmic, whereas dephosphorylated FOXO1 is retained in the nucleus where it functions as a transcription factor (256)FOXO1 was decreased in muscle cells exposed to the carrier BA mix in response to insulin, suggesting increased nuclear localization and transcriptional activity of FOXO1 in carriers  (473)AKT was increased by 116% in muscle cells treated with the carrier CA/CDCA ratio in response to insulin  (P = 0.004)  , suggest= 0.002) . FOXO1 e = 0.04) . Phosphon factor . Phospho= 0.036) . Phospho= 0.036) . Phospho= 0.029) . Additio= 0.004) , confirm473)AKT was increased approximately 2-fold in muscle cells treated with the median carrier CA/CDCA ratio in response to insulin  (P = 0.009)  and lowest insulin sensitivities (CA/CDCA = 21:29). Thus, we assessed whether the median CA/CDCA ratio of the mutation carriers (CA/CDCA = 4.5:45.5) would increase muscle cell insulin signaling and glucose uptake when compared with the median CA/CDCA ratio of noncarrier controls (CA/CDCA = 8.3:41.7). Phospho(S 0.0008) . In line= 0.009) . These dCYP8B1 mutation carriers, suggesting that increased CDCA may be sufficient to increase muscle insulin sensitivity. Thus, we next assessed whether CDCA alone was sufficient to increase muscle insulin signaling. Exposure of muscle cells to CDCA, but not CA, increased phospho(S473)AKT in response to insulin   (Insr and Foxo1 expression (256)FOXO1 levels  (473)AKT levels  , indicatP = 0.8) . Moreovepression , and decP = 0.2) . To furtCYP8B1 mutation carriers (473)AKT or glucose uptake AKT/AKT ratio    , and on P = 0.6) . These dCYP8B1 improves insulin sensitivity in humans. Although BA synthesis involves several enzymes, and complex feedback and feedforward mechanisms are involved in the regulation of BA metabolism . It is possible that CDCA modulates intracellular signaling through as yet unidentified muscle-specific cell surface receptors or BA transporters. Our data suggest that CDCA modulates muscle insulin signaling through increasing muscle FOXO1 activity, and inhibition of FOXO1 reversed the increased CDCA-mediated muscle insulin signaling. How CDCA decreases muscle FOXO1 phosphorylation, thus increasing its nuclear retention and transcriptional activity, remains unclear. One possible mechanism by which CDCA may reduce phospho-FOXO1 is by increasing the activity of phosphatases acting on FOXO1. BAs do modulate other phosphatases such as Src-homology 2 domain–containing tyrosine phosphatase 2 (SHP2)  .The reason for the potential selectivity of CDCA for skeletal muscle in regulating insulin sensitivity is unclear. However, FOXOs show tissue-specific protein interactions to modulate their functions in metabolic regulation . DistincCYP8B1 mutation carriers showed decreased total cholesterol/HDL-C and APOB/APOA-I ratios, suggesting that CYP8B1 inhibition may reduce type 2 diabetes without proatherogenic lipid changes. In addition, hs-CRP levels were decreased by approximately 50%, and hepatic fat was decreased by 30% in the CYP8B1 mutation carriers. These observations are consistent with those in –/–Cyp8b1 mice, which showed increased HDL-C, decreased LDL-C, and reduced atherosclerotic lesions when fed atherogenic diets , an analog of CDCA, was assessed in type 2 diabetics . Increasic diets , 27. Addgression , suggestCYP8B1 mutation carriers.The CYP8B1 product, 7α,12α-dihydroxy-4-cholesten-3-one, was not decreased in plasma of mutation carriers. However, downstream 12α-BAs were decreased by 52%, suggesting that the 12α-hydroxylase function of CYP8B1 in the liver was indeed reduced. Significantly increased unconjugated CDCA was also not observed in mutation carriers, although the ratio of CDCA and its conjugates to total BAs was increased. The reasons for this are unclear. In the mutation carriers, plasma BAs were quantified after an overnight fast. However, it has been shown that circulatory BA levels increase postprandially, with CDCA showing the largest increase (up to 5-fold) . Further–/–Cyp8b1 mice are viable, with no apparent adverse phenotypes, suggesting that the absence of CYP8B1 may not be harmful. We did not identify compound heterozygous or homozygous carriers of CLOF mutations, and almost all CLOF mutations were identified in only 1 or 2 heterozygotes. Thus, homozygous or compound heterozygous CLOF mutation carriers are likely to be extremely rare. One CLOF mutation, R26X, was found at 1.6% frequency in Malays. Assuming Hardy-Weinberg equilibrium, sequencing of approximately 13,000 Malays is required to identify a single R26X homozygote. Thus, it is unsurprising that we identified no homozygous or compound heterozygous CLOF mutation carriers. Additionally, in a large publicly aggregated database (gnomAD), no individuals harboring homozygous predicted CLOF mutations were reported, confirming that these individuals are extremely rare.We establish a fundamental role for CYP8B1-mediated changes in BA composition in the regulation of peripheral insulin sensitivity. We show that reduced activity of CYP8B1 is efficacious in increasing insulin sensitivity in humans, and mechanistically link CDCA signaling to muscle insulin sensitivity. We demonstrate here a target for future therapeutic intervention for diabetes.Detailed methods are provided in the supplementary material.CYP8B1 coding region was Sanger sequenced in population cohorts of nondiabetics from the Singapore Eye Research Institute and the Saw Swee Hock School of Public Health, National University of Singapore. Sequences were assembled in Sequencher (Gene Codes) and aligned to the human CYP8B1 reference sequence (GenBank AF090320.1). All nonsynonymous variants, frameshifts, and insertions/deletions were generated in human CYP8B1 cDNA and functionally characterized.The CYP8B1 cDNA inserted into pcDNA3.1(+) (Invitrogen) was used as the template for site-directed mutagenesis. The neomycin resistance cassette in pcDNA3.1(+) was replaced with green fluorescent protein (Gfp) in order to assess transfection efficiency of the variants. Mutagenesis primers were designed for each of the CYP8B1 variants, and PCR performed with conditions adjusted for each primer pair, amplifying the entire plasmid. PCR products were isolated from the template by Dpn1 digestion, transformed into DH5α competent cells, and amplified plasmids were extracted by E.Z.N.A. Plasmid Mini Kit II (Omega Bio-tek Inc). The mutant CYP8B1 cDNAs were sequence confirmed, subcloned into the pcDNA3.1(+)-Gfp vector, and used for the in vitro assay. For the in vitro functional assay, HEK293T (ATCC) cells in 6-well plates were transiently transfected with the plasmids harboring wild-type CYP8B1, variant CYP8B1, or empty vector using X-tremeGENE HP (Roche). After 24 hours, fresh DMEM with 10% FBS containing 10 μmol/L of the CYP8B1 substrate 7α-hydroxy-4-cholesten-3-one  was added to the cells at 3 mL/well. After 4 hours, media were collected, centrifuged, and frozen until quantification of 7α,12α-dihydroxy-4-cholesten-3-one by LC/MS . Cells were washed, centrifuged, and frozen for Western immunoblotting.CYP8B1 mutations and age-, sex-, race-, and BMI-matched nonmutation carrier controls from the same cohorts were recruited at a ratio of 1 carrier to 2 controls for metabolic studies. Five participants only had 1 matched control. The studies of BAs and insulin phenotypes excluded individuals with BMI greater than 30, the World Health Organization definition of obesity, since obesity modulates insulin sensitivity. Individuals were also excluded if they had type 2 diabetes mellitus, renal impairment, elevated serum aspartate aminotransferase or alanine aminotransferase, chronic liver disease, or medications or previous gastrointestinal surgery that may alter glucose or BA metabolism  were measured in plasma following overnight fasts using ultraperformance liquid chromatography–multiple reaction monitoring/mass spectroscopy (UPLC-MRM/MS) (University of Victoria-Genome BC Proteomics Centre) as described previously .After 8-hour fasts, venous blood was collected for plasma glucose, insulin, and GLP-1 measurements at 0, 15, 30, 60, 90, and 120 minutes after ingestion of liquid mixed meal .2H2O  for quantification of gluconeogenesis and glycogenolysis. To measure hepatic glucose production, primed-constant infusion of -glucose was performed. Insulin was infused at 40 mU/m2 body surface area/min for 180 minutes. Blood glucose was measured every 5 minutes. Blood for plasma insulin measurement was obtained every 30 minutes. Dextrose 20% (wt/vol) enriched with -glucose was infused at a variable rate to maintain blood glucose at 100 mg/dL with a coefficient of variation of less than 5%.Participants ingested 2 doses of CYP8B1 mutant R26X, selected because it was the most frequent in our study cohort, were performed using the publicly available Type 2 Diabetes Knowledge Portal (http://www.type2diabetesgenetics.org). Associations between R26X and fasting insulin, adjusted for BMI, age, and sex were calculated using the portal’s Genetic Association Interactive Tool (GAIT), in which single-variant and gene-level association analysis can be conducted in 45,231 exomes from the AMP-T2D-GENES study.Association analyses for the CLOF Human adult skeletal muscle cells (Cell Applications) were differentiated following manufacturer’s instructions and treated with a 50 μmol/L CA/CDCA mixture at the ratio of carrier or control, or dimethyl sulfoxide (DMSO) for 24 hours. To quantify AKT and FOXO1 phosphorylation subsequent to insulin stimulation, differentiated cells were treated with 100 nmol/L insulin . For glucose uptake assays, BA-treated cells were incubated with and without 100 nmol/L insulin, glucose uptake was quantified using fluorescence, and protein content was determined for normalization of glucose uptake.t tests. Non-normal data are reported as median (IQR) and were analyzed using the nonparametric Mann Whitney U test. Fisher’s exact tests were used for categorical data, 1-way ANOVA was used for analyses of more than 2 groups, and 2-way ANOVA was used for repeated measures. All tests were 2-sided, and performed using GraphPad Prism 9.0. A P value of less than 0.05 was considered significant.All human data were first assessed for normality. Non-normal data were log transformed. Normal data are reported as mean ± SEM and were analyzed using parametric unpaired The human study received approval from the SingHealth Centralized Institutional Review Board. All participants provided written informed consent prior to the study.RRS, MRH, AKG, and HCT conceptualized the study. RRS and HCT acquired funding. RRS, HCT, FK, JLG, RMVD, RST, KAR, and CYC supervised the study. RRS wrote the original draft of the manuscript. SZ, RC, MC, DCM, BJC, KPS, THVD, JP, LJT, SVH, BR, PC, JLG, SC, JF, and XS conducted the investigation and analyses. JLG, AKG, FK, SC, JF, KAR, XS, HCT, and RRS provided resources. SZ, AKG, FK, CD, HCT, and MRH reviewed and edited the manuscript.


Xanthomonas is a genus of gram-negative bacterium containing more than 35 species. Among these pathogenic species, Xanthomonas albilineans  is of global interest, responsible for leaf scald disease in sugarcane. Another notable Xanthomonas species is Xanthomonas sachari (Xsa), a sugarcane-associated agent of chlorotic streak disease.Xanthomonas strains was evaluated by disease index (DI) and Area Under Disease Progress Curve (AUDPC) in the susceptible inoculated plants (GT 46) and clustered into three groups of five highly potent, seven mild virulent, and twelve weak virulent strains. The highly potent strain  and its weak virulent related strain  were sequenced, assembled, and annotated in the circular genomes. The genomic size of JG43 was smaller than that of DD13. Both strains (JG43 and DD13) lacked a Type III secretory system (T3SS) and T6SS. However, JG43 possessed Salmonella pathogenicity island-1 (SPI-1). More pathogen-host interaction (PHI) genes and virulent factors in 17 genomic islands (GIs) were detected in JG43, among which six were related to pathogenicity. Albicidin and a two-component system associated with virulence were also detected in JG43. Furthermore, 23 Xanthomonas strains were sequenced and classified into three categories based on Single Nucleotide Polymorphism (SNP) mutation loci and pathogenicity, using JG43 as a reference genome. Transitions were dominant SNP mutations, while structural variation (SV) is frequent intrachromosomal rearrangement (ITX). Two essential genes (rpfC/rpfG) of the two-component system and another gene related to SNP were mutated to understand their virulence effect. The mutation of rpfG resulted in a decrease in pathogenicity.The virulence of 24 Xanthomonas strains and variations by 23 Xanthomonas strains. We sequenced, assembled, and annotated the circular genomes of Xal JG43 and Xsa DD13, identifying diversity detected by pathogenic factors and systems. Furthermore, complete genomic sequences and sequenced data will provide a theoretical basis for identifying pathogenic factors responsible for sugarcane leaf scald disease.These findings revealed virulence of 24 The online version contains supplementary material available at 10.1186/s12864-022-08900-2. Xanthomonas  is a large genus of gram-negative, yellow-pigmented bacteria associated with plants. The genus, which locates at the base of the Gamma proteobacteria, comprises 27 species that cause severe diseases in ~ 400 plant hosts, including a wide variety of economically important crops, such as rice, citrus, banana, cabbage, tomato, pepper, and bean  × 100.Furthermore, the area under the disease-progress curve (AUDPC) value was calculated , 86.\docyi is the severity of the symptoms at the ith observation; xi—day at the ith observation, and n—the total number of observations.where AUDPC is the area under the disease progress curve, X. albilineans JG43 was sequenced by Oxford Nanopore Technologies (ONT) and assembled using Canu (V1.5) [X. sacchari DD13 was sequenced using SMRT II sequencing technology , and a complete circular bacterial chromosome was assembled using HGAP software [The genome of u (V1.5) . Assemblsoftware . The gensoftware .https://www.repeatmasker.org/) [http://eddylab.org/infernal/) [http://lowelab.ucsc.edu/tRNAscan-SE/) [https://www.ebi.ac.uk/Tools/psa/genewise/) [http://www.pathogenomics.sfu.ca/islandviewer/) [http://phispy.sourceforge.net/) [Assembled genome was analyzed to identify the repeat sequences that were searched against the known repeat sequence database (Repbase) in the bacterial genome using RepeatMasker (V4.0.5) (er.org/) . Non-codfernal/) , while tcan-SE/) . The Gennewise/) . Genomicviewer/) , and proge.net/) .www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/) [https://www.genome.jp/kegg/) [https://www.expasy.org/resources/uniprotkb-swiss-prot) and TrEMBL (http://www.bioinfo.pte.hu/more/TrEMBL.htm) [https://www.ebi.ac.uk/Tools/hmmer/) [https://www.blast2go.com/) [The genes were blasted against the databases of non-redundant proteins (oteins/) , Kyoto Ep/kegg/) , Swiss-PMBL.htm) . Pfam fu/hmmer/) . Functiogo.com/) , 101.http://www.cazy.org/) using HMMER software [http://www.cbs.dtu.dk/services/TMHMM/), Kohgpi (http://gpi.unibe.ch/) [http://www.cbs.dtu.dk/services/SignalP/) [https://card.mcmaster.ca/) using RGI in CARD Database [http://www.mgc.ac.cn/VFs/) [Carbohydrate EnZymes genes were annotated against Carbohydrate Active EnZymes Database (CAZyme) (software . Transmeibe.ch/) , and SigignalP/) . TransmeDatabase . The vircn/VFs/) .Xal JG43 by GATK software (https://gatk.broadinstitute.org/hc/en-us) [X. albilineans strains was sequenced using Illumina Novaseq 6000 platform, with an average coverage of 339 × . The redundant reads (MarkDuplicates) were filtered by Picard software to ensure the detection accuracy of clean reads [X. albilineans, which were used to construct a phylogenetic tree. After removing ambiguous positions, a final dataset of 17,935 SNPs was generated for each sequence, which was aligned through the neighbor-joining method of MEGA7 software, utilizing the bootstrap value of 1000 replicates [Single nucleotide polymorphism (SNP) was called against the reference genome of c/en-us) . The genan reads . A totalplicates , 110.Xal genomes with Xal JG43, and the aligned reads were sorted using Samtools (V1.12) [Minimap2 (V2.17) was used to align the 23 sequenced  (V1.12) . BCFtool (V1.12) . BEDTool (V1.12) . Circos  (V1.12) .The single-base mutation was performed by the PNA-directed PCR clamping , and twopK18mobSacB plasmid to form a recombinant plasmid. The recombinant plasmid was digested, verified, and sequenced. The recombinant plasmid was introduced into the host bacterium by electro-transformation. The upstream and downstream of the target gene underwent single homologous exchange and double homologous exchange with the homologous fragment of the host bacterium. A 10% sucrose was used to screen double homologous exchange. Furthermore, internal and external primers were also used to screen double homologous exchange. Finally, the target gene was deleted , (c) and (d) show leaf scald symptoms after X. albilineans invade sugarcane; (b) Colony of X. albilineans isolated from diseased sugarcane plant; Right side: X. sacchari cause chlorotic streak disease. (a), (c) and (d) show chlorotic streak symptoms after X. sacchari infect sugarcane; (b) Colony of X. sacchari isolated from the diseased sugarcane plant. Fig.S2. Type III secretion system (T3SS) (a), and SPI-1 family (b) of six Xanthomonasspecies. Fig. S3. Type IV secretion system (T4SS) (a), T5SS and T6SS (b) of six Xanthomonas species. Fig. S4. Potential pathogenic factors of six Xanthomonas species, including CRISPR system, Lipopolysaccharide transport system protein, Glycogen, Type III secretion regulators, Two-component system regulators, Three-component system, and TALEs. Fig. S5. Verification of rpfC and rpfH mutations. (a) PCR amplification from the upstream and downstream 500 bp of rpfC. M: 2000 bp; Lane 1: rpfC Gene left arm; Lane 2: rpfC gene right arm. (b) Validation of enzymic fragment ligated with PK18mobsacB, a 500bp upstream and downstream fragment of rpfC gene. M: 5000 bp; Lane 1,2,3：Validation of rpfC recombinant plasmid fragment by enzyme digestion; Lane 4 not included in this experiment. (c) PCR amplified from mutants and its wild type JG43. M:1000 bp; Lane 1, 2, 3; PCR fragment amplified with mutants; Lane 4: PCR fragment amplified with JG43 as template; Lane 5: Water control; Lane 6, 7, 8: Internal primer verification of the target fragment missing in 123, none, which proves the successful deletion of rpfC gene; Lang 9: Internal primer fragment of PCR amplified with JG43 as template. (d)PCR validation of rpfHgene. M:1000 bp; Lane 1:Xcc8004; Lane 2: DD13; Lane 3: JG43; Lane 4: Water control;Lane 5: not included in this experiment. Fig. S6. rpf gene cluster of six Xanthomonas species. Fig. S7. PCR validation of single-base SNPs mutations. (a) PCR validation of single-base SNP mutations of candidate genes in FS 12. M:2000 bp; Lane 1: 1312440-G-C-L; Lane 2: 1312440-G-C-R; Lane 3: 1316566-A-G-L; Lane 4: 1316566-A-G-R; Lane 5: 1316572-A-G-L; Lane 6: 1316572-A-G-R; Lane 7: 1316840-G-A-L; Lane 8: 1316840-G-A-R; Lane 9: 1316855-T-C-L; Lane10: 1316855-T-C-R; Lane 11: 1316974-A-G-L; lane12: 1316974-A-G-R; lane13: 1317164-T-C-L; Lane 14: 1317164-T-C-R; Lane 15: 3055754-G-A-L; Lane 16: 3055754-G-A-R. (b) PCR validation of single-base SNP mutations of candidate genes in FS12 (Lanes 1 and 2), FS25 (Lanes 3 and 4), FS63 (Lanes 5 and 6) and NM10 (Lanes 7 and 8). M:2000 bp; Lane 1: 3055807-G-C-L; Lane 2: 3055807-G-C-R; Lane 3: 2749153-C-T-L; Lane 4: 2749153-C-T-R; Lane 5: 1508978-C-A-L; Lane 6: 1508978-C-A-R; Lane 7:1510223-A-C-L; Lane 8: 1510223-A-C-R. (c) SNP point mutation fusion fragment in FS12. M:2000 bp; Lane 1: 1312440-G-C; Lane 2: 1316566-A-G; Lane 3: 1316572-A-G; Lane 4:1316840-G-A; Lane 5: 1316855-T-C; Lane 6:1316974-A-G; Lane 7: 1317164-T-C. (d) SNP point mutation fusion fragment in FS12. M: 2000 bp; Lane 1:3055754-G-A; Lane 2:3055807-G-C; Lane 3: 3055836-T-G. (e) SNP point mutation fusion fragment in FS25 (Lane 1:2749153-C-T), FS63 (Lane 2: 1508978-C-A), and NM10 (Lane 3: 1510223-A-C). M:5000 bp.Additional file 2: Table S1. Basic information of Xal JG43 and Xsa DD13. Table S2. Repeat contents from genome sequence of Xal JG43 and Xsa DD13. Table S3. Genes in plasmid from the genome of Xal JG43. (.xls ) Table S4. Carbohydrate-active enzymes (CAZys) in Xal JG43 and Xsa DD13. Table S5. Comparative pathogenomics of X. albilineans JG43 and its related X. sacchari DD13. (.xls ) Table S6. Genomic island and prophages of Xal JG43 and Xsa DD13. (.xls ) Table S7. Resequencing of 23 X. albilineans strains. Table S8. Genomic variations (SNPs and SVs) obtained from 23 sequenced X. albilineans strains against JG43. Table S9. SNP mutations in 23 strains of X. albilineans. Table S10. Mutations at the DNA level in 23 strains of X. albilineans. Table S11. List of primers used in this study.Additional file 3.


Scientific Reports 10.1038/s41598-022-18110-1, published online 18 August 2022Correction to: The Funding section in the original version of this Article was incomplete.“Yayasan Universiti Teknologi PETRONAS (YUTP), Cost Center 015LC0-278 received by D. L. C. C.”now reads:“Yayasan Universiti Teknologi PETRONAS (YUTP), Cost Center 015LC0-278 and NCRF 015MCO-030 received by D. L. C. C.”The original Article has been corrected.


Correction: Inflamm Regen 42, 37 (2022)https://doi.org/10.1186/s41232-022-00217-7Following publication of the original article , authorsThe original version was:1*, Endo Yushiro1, Koga Tomohiro1,2, Yoshiura Koh-ichiro3 and Migita Kiyoshi4Atsushi KawakamiThe correct authorship has been updated in this Correction.The original article  has been


Concerns have been raised about ecological momentary assessment (EMA) acceptability among patients with schizophrenia spectrum disorders (SSD), which is of major relevance during the e-Mental health-focused COVID-19 pandemic.To investigate i) the levels of adherence to a passive smartphone-based EMA tool, the Evidence-Based Behavior (eB2), among SSD patients; and ii) putative predictors of this.Sample: SSD (F20-29-ICD10) outpatients, age 18-64, without financial incentives, recruited over 17/06/2019-11/03/2020 at the Hospital Universitario Fundación Jiménez Díaz . Those who accepted the eB2 installation -users- and those who did not -non-users- were compared in sociodemographic, clinical, premorbid adjustment, neurocognitive, psychopathological, insight and metacognitive variables by a multivariable binary logistic regression model.Sample (N=77): n=41 males; age: 47.69±9.76 years, n=24 users (31.2%). n=14 users (70%) had the eB2 installed at follow-up (median=14.50 weeks).2=25.296,df=6,p<0.001. Nagelkerke-R2=44.7%. Correctly classified: 76.9%, users:54.5%, non-users:88.4%.XAcceptability of a smartphone-based EMA application among SSD patients was low. Age (young) and good premorbid adjustment predicted acceptability. e-Mental Health methods need to be tailored for patients with SSD. Otherwise, these highly vulnerable individuals may be neglected by e-health-based services in the post-COVID-19 years ahead.


We assessed cross-reactivity to BA.1, BA.2, and BA.5 of neutralizing antibodies elicited by ancestral, Delta, and Omicron BA.1 SARS-CoV-2 infection in mice. Primary infection elicited homologous antibodies with poor cross-reactivity to Omicron strains. This pattern remained after BA.1 challenge, although ancestral- and Delta-infected mice were protected from BA.1 infection. The SARS-CoV-2 Omicron variant  emerged nearly 2 years after the ancestral strain was identified  of SARS-CoV-2/Australia/Vic/01/20 , SARS-CoV-2/Australia/Vic/18440/2021 (Delta), and SARS-CoV-2/Australia/NSW/RPAH-1933/2021 (Omicron BA.1) strains in 7- to 9-week-old female K18hACE2 transgenic mice (2 TCID50), selected so that the mice would survive primary infection . We mock-infected 15 mice with phosphate-buffered saline (PBS). We collected blood on day 27 after primary infection and then challenged mice with 104 TCID50 of Omicron BA.1 virus. We collected lungs and nasal turbinates (NTs) 2 and 4 days after challenge; we weighed and monitored 5 mice per group for clinical signs for 14 days , survived without weight loss. The control group had mean virus titers of 102.6 (day 2) and 102.7 (day 4) in NTs and 103.7 (day 2) and 103.5 (day 4) TCID50/organ in lungs after Omicron BA.1 challenge. After primary infection, all Omicron BA1–infected mice survived without major weight loss, but 1 ancestral strain–infected and 5 Delta-infected mice died during days 8–13. After challenge with 10Consistent with other reports , panel CThe homologous responses were strongest to ancestral (geometric mean titer [GMT] 709), followed by Delta (GMT 129), and were lowest to BA.1 (GMT 83) . The lowPrimary Omicron BA.1 infection did not induce heterologous neutralizing activity against ancestral, Delta, BA.2, or BA.5 viruses . In contDespite the absence of detectable BA.1 virus in the respiratory tract tissues after secondary infection in mice previously infected with ancestral or Delta , panel Chttps://doi.org/10.1101/2022.01.03.21268582). A boost occurred in preexisting SARS-CoV-2 neutralizing antibodies to ancestral and Delta but not in cross-reactivity to Omicron, probably because more epitopes are shared between ancestral and Delta than between those strains and Omicron. Serologic data from humans suggest that >3 exposures to ancestral strains as infection or vaccination or a combination are needed to induce cross-reactive antibodies to BA.1 (https://doi.org/10.1101/2022.05.10.22274906), we did not detect cross-reactive neutralizing antibodies after primary infection with ancestral and Delta strains. Protection from replication of the Omicron BA.1 strain despite the lack of cross-reactive neutralizing antibodies may be attributable to mucosal immunity or T-cell responses in ancestral strain–infected and Delta-infected mice (Our observations are consistent with BA.1 being antigenically distinct from the ancestral and Delta strains (K. van der Straten K et al., unpub. data, Additional information about SARS-CoV-2 Omicron BA.1 challenge after ancestral or Delta infection in mice.


Panax notoginseng (Burkill) F. H. Chen is not completely known. Morphological traits, N use and allocation, photosynthetic capacity and saponins accumulation were evaluated in two- and three-year-old P. notoginseng grown under different N regimes. The number and length of fibrous root, total root length and root volume were reduced with the increase of N supply. The accumulation of leaf and stem biomass (above-ground) were enhanced with increasing N supply, and LN-grown plants had the lowest root biomass. Above-ground biomass was closely correlated with N content, and the relationship between root biomass and N content was negatives in P. notoginseng (r = −0.92). N use efficiency-related parameters, NUE , NC (N content in carboxylation system component) and Pn (the net photosynthetic rate) were reduced in HN-grown P. notoginseng. SLN (specific leaf N), Chl (chlorophyll), NL (N content in light capture component) increased with an increase in N application. Interestingly, root biomass was positively correlated with NUE, yield and Pn. Above-ground biomass was close negatively correlated with photosynthetic N use efficiency (PNUE). Saponins content was positively correlated with NUE and Pn. Additionally, HN improved the root yield of per plant compared with LN, but reduced the accumulation of saponins, and the lowest yield of saponins per unit area (35.71 kg·hm−2) was recorded in HN-grown plants. HN-grown medicinal plants could inhibit the accumulation of root biomass by reducing N use and photosynthetic capacity, and HN-induced decrease in the accumulation of saponins (C-containing metabolites) might be closely related to the decline in N efficiency and photosynthetic capacity. Overall, N excess reduces the yield of root and C-containing secondary metabolites (active ingredient) in N-sensitive medicinal species such as P. notoginseng.Nitrogen (N) is an important macronutrient and is comprehensively involved in the synthesis of secondary metabolites. However, the interaction between N supply and crop yield and the accumulation of effective constituents in an N-sensitive medicinal plant  Nitrogen (N) is a determinant nutrient for plant biomass or crop yield . Yellow e.g., carboxylation and bioenergetics components), results in a lowed yield of N-excess Brassica campestris L.  R. Br. grown under low N condition (Datura stramonium L. is significantly increased with an increase in soluble sugar and proline content (primary metabolites) under N-excess condition  balance and consequently change the content of C- and N-containing secondary metabolites in the medicinal species . Excessibacum L. . Correspondition . The N-condition . UnexpecPanax notoginseng (Burkill) F. H. Chen (Sanqi in Chineses) is a perennial medicinal plant and a member of the Araliaceae family, which is a typically shade-tolerant and N-sensitive plants  enhances the accumulation of biomass and saponins though optimizing root architecture and N uptake efficiency in P. notoginseng , moderate nitrogen (MN) and high nitrogen (HN). We hypothesized that (i) root biomass of P. notoginseng might be reduced accompanying with HN-driven inhibition on photosynthetic capacity and NUE (N use efficiency); (ii) HN-driven decrease in saponin accumulation might be reflected by the C/N imbalance; (iii) N stress might reduce the yield of P. notoginseng.The present study aimed to shed light on an interaction between N availability and crop yield and saponins accumulation in the medicinal plant 2O) was 6.84, total N content was 0.17%, total phosphorus (P) was 0.23%, the available P content was 11.04 mg·kg−1, total potassium (K) was 0.24%, and the available K content was 127.32 mg·g−1.The study was conducted at the Yunnan Agricultural University teaching and experimental farm in Kunming, China , with an average annual rainfall and average annual temperature of about 1,006.7 mm and 14.5 °C, respectively. The properties of raw soil physical and chemical was determined as described by P. notoginseng, and the full sunlight irradiance is about 10%  provided one-year-old P. notoginseng seedlings. Subsequently, healthy and uniform seedlings were transplanted into a plastic flowerpot (30 cm × 25 cm × 20 cm) with each containing three rootstocks (−2), high nitrogen ) were designed (2O5), calcium superphosphate  and potassium sulfate (52% K2O), respectively. The same amounts of P (225 kg·P2O5·hm−2) and K (450 kg·K2O·hm−2) fertilizers were used in all treatments with the exception of the N fertilizer. Fertilization was applied in four times a year . In each pot, basal doses of P and K at the rates of 0.45 and 0.90 g, respectively , were applied at time while N was applied according to the treatments. N fertilizer rate 0 (LN), 0.45 (MN) and 0.90 (HN) g·pot−1 .A permeable black plastic net was used to create a shade-house for bout 10% . Meanwhibout 10% . Permeabotstocks . There wdesigned , and eacAt November, the two- and three-year-old plants were sampled from the experimental farm and then separated into root , stem and leaf in room. The length, width, and area of leaf were measured by LI-3000 leaf-area meter . Root tuber and stem diameter were measured by vernier caliper. Plant height, grown breadth, the length of main root and total root were determined as described by The samples were dried at 60 °C for 96 h. Dry matter was determined, and these results were used to calculate the percentage of biomass allocation into leaf , stem , roots , as well as root to shoot ratio (RSR). The root yield of per plant and economic yield (root yield of per hectare) were calculated based on root biomass data.P. notoginseng leaves were soaked. A standing period of 3 h was followed by a centrifugation of 3,000 g·min−1 for 10 min. A JASCO V-670 spectrophotometer  was used to measure absorbance at 665 and 649 nm wavelengths. Chl a, Chl b and Chl a/Chl b were analyzed as described by In 15 mL of acetone-ethanol mixture (2:1 v/v), 0.5 g of fresh 2 concentration of 10%, 25 °C, 500 μmol·photons·m−2·s−1 and 400 μmol·CO2·mol−1, respectively. Photosynthetic gas exchange parameters  were collected as previously described in LI-6400XT photosynthesis system  was used to determine photosynthetic gas exchange parameters. Set with a blue light ratio, temperature, photosynthetic photon flux density (PPFD) and COVcmax (maximum carboxylation efficiency), Jmax (maximum electron transfer rate), SLN and Chl contents, NC (N content in carboxylation system component), NB (N content in bioenergetics component) and NL (N content in light-harvesting systems component) were analyzed according to the method described by photo) = NB + NC + NL. Photosynthetic N use efficiency (PNUE) = Pmax (maximum net photosynthetic rate)/SLN.The leaf, stem and root N contents were determined by Kjeldagl method . AdditioP. notoginseng grown under different N regimes. The following equations were used to calculate N uptake and use efficiency (−1) = yield (underground dry weight)/plant N accumulation; NAE (kg·kg−1) = (yield with N application – yield without N application)/N rate; NUPE (kg·kg−1) = above-ground total N content/N rate; RNF (%) = /N rate × 100; NCR (%) = (yield with N application – yield without N application)/yield with N application × 100; NPFP (kg·kg−1) = yield with N application/N rate.Based on the biomass and N contents, N use efficiency (NUE), N agronomic efficiency (NAE), N uptake efficiency (NUPE), recovery of N fertilizer (RNF), N contribution rate (NCR), N partial factor productivity (NPFP) were calculated in ficiency : NUE  were purchased from Yuanye Bio-technology . Kit column  was used for the determination, and the mobile phase was acetonitrile (ACN)-water. Chromatographic conditions: elution with 0–5 min, 17–20% ACN; 5–20 min, 20% ACN; 20–45 min, 20–42% ACN; 45–50 min, 42–100% ACN; set with flow rate, injection volume, monitoring wavelength and column temperature of 1.0 mL·min−1, 10 µL, 203 nm and room temperature, respectively. Total saponins are the sum of Rg1, Rb1, Re, Rd and R1. The HPLC chromatograms of P. notoginseng root grown in different N environments are shown in Dry root samples of 0.3 g were extracted in 100% methanol and sonicated for 30 min. The solution volume was fixed to 25 mL. Saponin contents were determined as described by n = 5 or 7). One-way analysis of variance was used to evaluate the effect of N treatment in a year by T-test using SPSS software (IBM SPSS Statistics). LSD-test was used to compare treatment means, with significant effects having P < 0.05. Plots were made using Origin 2021 and GraphPad 8.0 software. Pearson correlation coefficients were assessed using Origin 2021. Principal component analysis loading factors were assessed using Origin 2021.All data in the tables and figures were mean ± standard deviation (SD) of 5–7 independent biological replicates performed (P. notogisneng between MN and LN conditions (P > 0.05). The stem and leaf N content were higher in two-year-old P. notoginseng compared with three-year-old plants . For two-year-old P. notoginseng, main root length and total root length were increased by 31.24% and 11.10% in MN-grown plants compared with HN-grown P. notoginseng, respectively . The number of fibrous roots, length of fibrous root, total root length, and root volume declined with an increase in N application . Leaf biomass was increased by 145.45% and 125.00% in MN-grown plants compared with LN- and HN-grown P. notoginseng, respectively (P > 0.05). RMF, SMF and LMF were increased by 110.00%, 88.89% and 72.73% in MN-grown plants compared with HN-grown P. notoginsneng, respectively . Leaf biomass was increased by 127.62% and 54.60% in HN-grown plants compared with LN- and MN-grown P. notoginseng, respectively (P > 0.05). SMF and LMF were improved by 27.59% and 28.57% in HN-grown P. notoginseng compared with LN-grown plants . NUE was declined by 62.96% and 34.03% in two- and three-year old P. notoginseng grown under HN condition compared with MN conditions, respectively (P < 0.05). The minimum values of NAE, NUPE, NCR, and NPFP were obtained in the HN-grown P. notoginaseng (P < 0.05). RNF was increased by 29.57% in three-year-old P. notoginseng grown under HN compared with MN condition (There were considerable differences in N efficiency of ondition .P < 0.05), and SLN was higher in two-year-old plants compared with three-year-old plants (P. notoginseng in LN and MN conditions (P < 0.05).SLN increased with an increase in N application . CE (carboxylation efficiency) and Jmax were highest in two-year-old plants under MN condition. CE and Jmax were increased by 57.14% and 57.58% in MN-grown plants comparted with HN-grown P. notoginseng, respectively (Vcmax and Γ* (carbon dioxide compensation point) variables, were not significantly different in two-year-old plants grown under LN and HN conditions , LSP (light saturating point), Vcmax, and Rd (dark respiration rate) were significantly declined in three-year-old plants under LN condition (P < 0.05). For three-year-old plants, the maximum values of Pmax, CE, Γ*, Jmax, Vcmax, and Jmax/Vcmax were obtained in MN plants . HN induces the increase in NL, and NC was reduced by 13.79% in two-year-old plants grown under HN compared with MN  were not significantly different N regimes (P < 0.05). The minimum value of total saponins (%) were recorded in three-year-old plants grown under HN condition (P < 0.05). For three-year-old plants, the LN and HN-grown P. notoginseng showed 32.58% and 28.68% lower saponins yield of per plant than the MN ones  and SLN (r = −0.87). Root biomass was close positively correlated with N use efficiency (as reflected by NCR (r = 0.79), NPFP (r = 0.91) and NUE (r = 1.00)). There was little correlation between root biomass and RNF, plant height and leaf area. Stem and leaf biomass were close negatively correlated with SPAD and PNUE. N application was close negatively correlated with NAE (r = −0.87), NCR (r = −0.85), NPFP (r = −0.91) and NUPE (r = −0.75). NUPE was positively correlated with the root length (r = 0.66) and root tuber diameter (r = 0.59). In addition, Pn was negatively correlated with N application (r = −0.88), leaf area (r = −0.57), Chl contents (r = −0.75), SLN (r = −0.44) and leaf N content (r = −0.45). The relationship between saponins and Pn (r = 0.45), root biomass (r = 0.54) as well as NPFP (r = 0.57) were positive correlation in P. notoginseng. Saponins content was negatively correlated with Chl content (r = −0.67) and SLN (r = −0.64).Pearson correlation coefficients of 27 parameters were evaluated in  regimes . As showP. notoginseng biomass or saponins. In PC1, the weighting coefficients of biomass parameters , yield, NUE, SLN, SPAD and stem N content were larger . The cumulative contribution of PC1, PC2 and PC3 reached 84.80% . Thus, te larger . NUE, yie larger . In PC2,e larger . In PC3,e larger . LCP ande larger .Arabidopsis thaliana L. by increasing the length of total root and fibrous root (r = 0.66) and root tuber diameter  Previouoginseng , 4. HN iativa L. . Overalle.g., Dodonaea viscosa (L.) Jacq., Lolium perenne L. and Betula spp. (P. notoginseng are exposed to HN condition (r = −0.93) and stem (r = −0.72) N content . ula spp. , and shoula spp. . These rondition . It has ondition . However content . It is aMolinia caerulea (L.) Moench , and lower NAE and NCR were obtained in HN-grown P. notoginseng (Many studies have shown that N use is not positively related to N uptake . N conte) Moench . This isce N use , 4. Howeoginseng , Table 3L and Pn were recorded in LN-grown plants  and stem (r = −0.86) biomass  and yield (r = 0.53) were positively correlated with Pn  Fern.-Vill. grown under high N condition  Iljinsk.  is generally used as a traditional Chinese medicine  is recorded in three-year-old P. notoginseng grown HN condition  was recorded in HN-grown plants (Centella asiatica L. and Stevia rebaudiana (Bertoni) Hemsl.  Hemsl. ). Howeve) Hemsl. , 7E, 7F. quality . In shorP. notoginseng (P. notoginseng.A model was proposed to explain the interaction between high N and the accumulation of biomass and C-containing secondary metabolites in a N-sensitive medicinal species, such as oginseng . In conc10.7717/peerj.14933/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14933/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14933/supp-3Supplemental Information 3Click here for additional data file.


Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy. •Airway organoids from different donors display distinct compositions of cell types•Organoid biobank models the spectrum of the epithelium response to pathogens•TSPAN8 is a conserved mediator of infection for SARS-CoV-2 variants Roose and colleagues generated a biobank of 20 airway organoids for modeling variations of airway epithelium response to SARS-CoV-2. They discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection independently of ACE2-Spike interaction. Pre-treatment of airway organoids with a blocking TSPAN8 antibody decreased SARS-CoV-2 infection levels in airway organoids, suggesting TSPAN8 as a therapeutic target for COVID-19. While most SARS-CoV-2-infected individuals develop asymptomatic to mild disease, some develop a severe disease characterized by immune cell dysfunction (trans-membrane serine proteases (TMPRSS2) results in Spike protein activation and viral entry (Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease (COVID-19) with more than half a billion cases worldwide  derived from adult stem cells of different individuals. We used this biobank to first optimize viral infection of AO with H1N1/PR8 influenza, and next performed a comprehensive analysis of SARS-CoV-2 infection with repeat infections. Spectral flow analysis of infected AO was used to assess cellular and functional responses of the epithelial cell compartment. Single-cell RNA sequencing (scRNA-seq) and Spectral flow enabled the discovery of Tetraspanin-8 (TSPAN8) as a conserved mediator of SARS-CoV-2 Ancestral (WA-1)-, Delta-, and Omicron-variant infection. Reductionist HEK293T cell-pseudo-virus approaches showed that TSPAN8 facilitates viral entry independently of the Spike-ACE2 interaction. We show that TSPAN8 is not an alternative entry receptor. Blocking TSPAN8 in airway epithelial organoids prior to infection is associated with a decrease in the viral load of AOs. Based on our TSPAN8 work in the context of cancer , we proplike cells , goblet-like cells  , three populations of cells expressing basal cell markers CD49f+CD271+, CD49fnegCD271+, CD49f+CD271+, and a population of CD49fnegCD271negacTUBAnegMUC5ACnegTSPAN8neg cells. AOs derived from different donors displayed distinct cell-type compositions even though cultured in identical growth factors and environmental conditions  , pre-gobnditions G. Furthenditions H and S1Anditions B. We gennditions C. Spectrnditions D and extnditions E revealenificant I–1K. So,high/CD271−neg ciliated cells and acTUBAneg/MUC5AC+ goblet cells  A. ConfocSpectral flow analysis on five independent SARS-CoV-2 infections of 2522UL show that infections are consistent C and 3D To uncover the rules of infection with SARS-CoV-2, we selected a panel of 12 AOs A that ca+, or CD271+, or CD49fnegCD271neg cells  for Mock and SARS-CoV-2-infected organoids on 21 variables  A. PCA lA showed + cells H upon SA+ cells I–S3L. Th+ cells J, confir+ cells . Surprison rates K and S3Gon rates , and theon rates C motivatWe infected four organoids  with SARS-CoV-2 WA-1 and performed scRNA-seq. Unsupervised clustering analysis based on most variable gene expression across all cells , regardlTSPAN8 mRNA reads were present in 64% of single cells positive for SARS-CoV-2 reads (size of the circle in +TSPAN8−) from pre-goblet cells (MUC5AC−TSPAN8+)  , allowinTSPAN8+) C but decTSPAN8+) A. As we TSPAN8+) J, most STSPAN8+) D, whereaTSPAN8+) E. FurtheTSPAN8+) F and S5BTSPAN8+) K, the prnfection G and S5Cnfection H and 5I.TSPAN8 expression decreases in airway brushes of acute illness in patients caused by non-SARS-CoV-2 respiratory viruses, while airway brushes from COVID-19 patients revealed preservation of TSPAN8 levels  numbers and influences their composition . EV fromjeroen.roose@ucsf.edu.A list of materials used and detailed methods are found on the Supplementary Materials section. Materials are available upon request.GSE211562.Resources: GEO: L.H. and J.P.R.: conceived the study. L.H., S.L., and K.K.: Spectral flow. L.H., K.K., S.L., O.M.G.: organoid biobank. L.H., M.M., and L.R.: BSL3 work. C.A., A.A.R., and A.J.C.: scRNA-seq. J.C.L.: statistical analyses. C.A., L.C.L.: scRNA-seq analyses. L.R.B. and D.J.E.: advice on airway populations. N.K.S. and M.K.: H1N1 virus. L.H., S.L., O.M.G., and K.B.: microscopy. L.H., S.L., K.B.: HEK239T cell line creation. J.Z.L., V.D., S.M., M.M.: patient samples. G.K., D.M.J., M.M., A.J.C.: funding. M.O.: SARS-CoV-2 virus, and SARS-CoV-2 pseudo-viruses. S.L., M.P.: pseudo-virus infections. J.R.K.: surgery airway samples, clinical data discussion. L.H., S.L., J.P.R.: manuscript writing. J.P.R.: funding. G.K.F., D.M.J., M.M., M.O., M.M., A.J.C., D.E., A.N.S., and J.R.K.: edits on the manuscript.


Misgurnus anguillicaudatus), the most widely distributed species of the family Cobitidae, displays a mud-dwelling behavior and intestinal air-breathing, inhabiting the muddy bottom of extensive freshwater habitats. However, lack of high-quality reference genome seriously limits the interpretation of the genetic basis of specialized adaptations of the loach to the adverse environments including but not limited to the extreme water temperature, hypoxic and noxious mud environment.The loach (fos), a regulator of bone development, is positively selected in loach. Knockout of fos (ID: Mis0086400.1) led to severe osteopetrosis and movement difficulties, combined with the comparison results of bone mineral density, supporting the hypothesis that fos is associated with loach mud-dwelling behavior. Based on genomic and transcriptomic analysis, we identified two key elements involved in the intestinal air-breathing of loach: a novel gene (ID: mis0158000.1) and heat shock protein beta-1 (hspb1). The flavin-containing monooxygenase 5 (fmo5) genes, central to xenobiotic metabolism, undergone expansion in loach and were identified as differentially expressed genes in a drug stress trial. A fmo5−/− (ID: Mis0185930.1) loach displayed liver and intestine injury, indicating the importance of this gene to the adaptation of the loach to the noxious mud.This study generated a 1.10-Gb high-quality, chromosome-anchored genome assembly, with a contig N50 of 3.83 Mb. Multiple comparative genomic analyses found that proto-oncogene c-Fos (Our work provides valuable insights into the genetic basis of biological adaptation to adverse environments.The online version contains supplementary material available at 10.1186/s12915-023-01517-1. Misgurnus anguillicaudatus), the most widely distributed species of the family Cobitidae, inhabits in the bottom of lakes, ponds, and other freshwater areas with humus-rich mud ; ugt, UDP-glucuronosyltransferase. Table S5. Primers used in this study. qPCR, quantitative PCR; # indicated T7 promoter sequences. Fos, proto-oncogene c-Fos; fmo5, dimethylaniline monooxygenase [N-oxide-forming] 5 (Mis0185930.1); hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha; atf6, cyclic AMP-dependent transcription factor; eif2ak3, eukaryotic translation initiation factor 2-alpha kinase 3; chop, DNA damage-inducible transcript 3 protein; muc2, mucin-2; Mis0185950.1, Mis0185940.1, Mis0185920.1, Mis0185970.1, Mis0186000.1, Mis0186010.1 (fmo5), dimethylaniline monooxygenase [N-oxide-forming] 5; Mis0115330.1, Mis0115330.1, Mis0135560.1 (ugt2a2), UDP-glucuronosyltransferase 2A2; Mis0072610.1 (ugt2a1), UDP-glucuronosyltransferase 2A1; npr2, atrial natriuretic peptide receptor 2; f9, coagulation factor IX; hspb1, heat shock protein beta-1; hyou1, hypoxia up-regulated protein 1; ptafr, platelet-activating factor receptor; scx, basic helix-loop-helix transcription factor scleraxis; galnt8, polypeptide N-acetylgalactosaminyltransferase 8; krt13, keratin, type I cytoskeletal 13 Cytokeratin-13; gp2, pancreatic secretory granule membrane major glycoprotein; smco3, single-pass membrane and coiled-coil domain-containing protein 3; ccl5, C-C motif chemokine 5; ifi44, interferon-induced protein 44; atf4, cyclic AMP-dependent transcription factor 4; ugt1a1, UDP-glucuronosyltransferase 1-1. Table S6. A summary of sequencing data used in genome assembly and annotation of Misgurnus anguillicaudatus.Additional file 2: Fig S1. Genome-wide Hi-C interaction map. Fig S2. Divergence distributions of four TE sequences predicted by the de novo method (a) and comparison of the distribution of several features in the final gene set for seven fish species (b). TE, transport elements; DNA, DNA transposons; LTR, long terminal repeats; LINE, long interspersed nuclear elements; SINE, short interspersed nuclear element. The seven fish species: Misgurnus anguillicaudatus, Carassius auratus, Cyprinus carpio, Danio rerio, Sinocyclocheilus graham, S. rhinocerous, and Triplophysa tibetana. Fig S3. The construction of maximum likelihood tree of fos gene and generation of the fos (ID: Mis0086400.1) knockout loach (fos−/−) by CRISPR/Cas9 technology. (a) Maximum likelihood tree ) of fos gene in fish species. Different branch colors represent different species. Red words indicate the knockout of fos gene in loach genome. (b) Schematic position of the CRISPR/Cas9 target site for fos gene knockout. (c) The transcription level of fos in livers of wild-type loach (WT) and fos−/− loach. (d) A statistics of survival rate of fertilized eggs of WT loach and heterozygous F1 generation (F1 generation self-crossed). *** extremely significant difference (p < 0.001). hpf, hours post fertilization; fos, Proto-oncogene c-Fos. The gene in red color is a positively selected gene in the loach genome. Fig S4. Identification and expression analysis of air-breathing- and digestion/absorption- related genes in loach Misgurnus anguillicaudatus. (a) Maximum likelihood tree ) of hb gene family. Different color backgrounds represent different genes. Different branch colors and inner circle colors represent different species. hbb, hemoglobin subunit beta; hba, hemoglobin subunit alpha. (b) Expression levels of hbb and hba in posterior intestines of the loach under air exposure. (c) qPCR validation of RNA-seq data of loach posterior intestines under air exposure. (d) Maximum likelihood tree ) of air-breathing-related genes. Different branch colors represent different species. (e) Maximum likelihood tree of digestion/absorption-related genes ). Atp1a, sodium/potassium-transporting ATPase subunit; ryr, ryanodine receptor. Different color backgrounds represent different genes. Different branch colors represent different species. Npr2, atrial natriuretic peptide receptor 2; f9, coagulation factor IX; hspb1, heat shock protein beta-1; hyou1, hypoxia up-regulated protein 1; ptafr, platelet-activating factor receptor; scx, basic helix-loop-helix transcription factor scleraxis; galnt8, polypeptide N-acetylgalactosaminyltransferase 8; krt13, keratin, type I cytoskeletal 13 Cytokeratin-13; gp2, pancreatic secretory granule membrane major glycoprotein; smco3, single-pass membrane and coiled-coil domain-containing protein 3; ccl5, C-C motif chemokine 5; ifi44, interferon-induced protein 44; cldn5, claudin-5 Transmembrane protein deleted in VCFS; hspb1, heat shock protein beta-1; vegfr1 (flt1), vascular endothelial growth factor receptor 1; ctgf, connective tissue growth factor CCN family member 2. Fig S5. Identification of FMO and UGT gene families. (a) Maximum likelihood tree ) of FMO gene family. Different colors represent different FMO gene families and different branch colors represent different species. (b) Maximum likelihood tree ) of UGT gene family. Different colors represent different UGT gene families and different inner circle colors represent different species. Fig S6. qPCR validation of RNA-seq data from the drug stress trial. Fig S7. The expression levels of ugt genes in liver tissues of the loach under five drug stress. Mis115330.1 and Mis0135560.1 (ugt2a2), UDP-glucuronosyltransferase 2A2; Mis0072610.1 (ugt2a1), UDP-glucuronosyltransferase 2A1. Fig S8. Tissue expression analysis and the knockout of fmo5 (ID: Mis0185930.1) gene (fmo5−/−) in loach Misgurnus anguillicaudatus genome. (a) Tissue expression analysis of fmo5 (ID: Mis0185930.1) of the loach. (b) Schematic position of the CRISPR/Cas9 target site for fmo5 gene knockout. (c) The mRNA and protein expression levels of fmo5 in livers of wild-type (WT) and fmo5−/− loach. Different letters above error bars indicate significant difference among different tissues (p < 0.05); *** extremely significant difference (p < 0.001). Fig S9. Survival rates of wild-type (WT) and fmo5deletion (fmo5−/−) loach under five drug stress.


There is an error in reference 5. The correct reference is: Masashi K, Norihiro S, Hidetsugu N.Regular change in spontaneous preparative behaviour on intra-abdominal pressure and breathing during dynamic lifting. Eur J Pppl Physiol. 2014; 114(11):2233–9