SERPINA3, FGA, AGP1, ITIH3 and SAA1 as novel biomarkers for eosinophilic granulomatosis with polyangiitis diagnosis and activity assessment.

OBJECTIVE: To identify novel biomarkers for diagnosis and prediction of active eosinophilic granulomatosis with polyangiitis (EGPA) through data-independent acquisition (DIA) analysis. METHODS: Plasma from 11 EGPA patients and 10 healthy controls (HCs) were analyzed through DIA to identify potential biomarkers. The results were validated in 32 EGPA patients, 24 disease controls (DCs), and 20 HCs using enzyme-linked immunosorbent assay (ELISA). The receiver operating characteristic (ROC) curve was used to assess the diagnostic value of candidate biomarkers. RESULTS: Thirty-five differentially expressed proteins (DEPs) (24 upregulated and 11 downregulated) were screened between EGPA and HC groups. Five proteins, including serine proteinase inhibitor A3 (SERPINA3), alpha-fibrinogen (FGA), alpha-1 acid glycoprotein 1(AGP1), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), and serum amyloid A1 (SAA1), were significantly upregulated in EGPA compared with HCs. Apart from SAA1, all proteins were also higher in EGPA patients compared with DCs. Furthermore, a panel of SERPINA3 and SAA1 exhibited potential diagnostic value for EGPA with an area under the curve (AUC) of 0.953, while a panel of SERPINA3, FGA, AGP1, and ITIH3 showed good discriminative power to differentiate EGPA from DCs with AUC of 0.926. Moreover, SERPINA3, FGA, and AGP levels were significantly higher in active EGPA and correlated well with disease activity. A combination of SERPINA3 and AGP1 exhibited an excellent AUC of 0.918 for disease activity assessment. CONCLUSION: SERPINA3, FGA, AGP1, ITIH3 and SAA1 were identified as potential biomarkers for EGPA diagnosis and disease activity assessment. Among them, as a single biomarker, SERPINA3 has the best diagnostic performance.

Exploration of molecular signatures associated with different clinical features of Takayasu arteritis based on a prospective cohort study.

Takayasu arteritis (TAK) is complicated disorder without reliable biomarkers. Here, we aimed to explore TAK-associated factor panels and their changes after biologic treatment. Five factor panels were identified: 1. systemic inflammation: C3, ESR, CRP, PLT, IL-6, C4, and IgG; 2. vascular inflammation: YKL40, IL-16, PTX3, and CCL2; 3. immune regulation panel: IL-10, IFN-γ, CCL5, and MMP1; 4. angiogenesis and fibrosis: FGF, PDGFAB, and VEGF; and 5. vascular remodeling: CD19+ B cell ratio, MMP3, and leptin. Panel 1 parameters were closely related to disease activity, while Panel 5 parameters, particularly CD19+ B cell ratio and leptin, were significantly higher in ischemic patients. After treatment, tocilizumab had a stronger inhibitory effect on Panel 1 parameters, PTX3, and YKL-40, while adalimumab led to an increase in IL-16, CCL2, and leptin levels. Altogether, these data expanded our knowledge regarding molecular background in TAK development and shed light on precise treatment in future studies.

Single-cell profiling of ineffective erythropoiesis in a mouse model of ß-thalassaemia intermedia.

Beta-thalassaemia is an inherited haemoglobin disorder characterised by ineffective erythropoiesis (IE). The detailed pathogenesis of IE remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to examine IE in Th3/+ ß-thalassaemic mice. The results showed that the erythroid group was remarkably expanded, and genes involved in biological processes such as iron metabolism, haeme synthesis, protein folding, and response to heat were significantly upregulated from erythroid progenitors to reticulocytes in ß-thalassaemic mice. In particular, we identified a unique cell population close to reticulocytes, named ThReticulocytes, characterised by a high level of heat shock protein 70 (Hsp70) expression and dysregulation of iron metabolism and haeme synthesis signalling. Treatment of ß-thalassaemic mice with the haeme oxygenase inhibitor tin-mesoporphyrin effectively improved the iron disorder and IE, and the ThReticulocyte population and Hsp70 expression were significantly suppressed. This study revealed in detail the progression of IE at the single-cell level and possibly provided clues to find therapeutic targets in thalassaemia.

A microRNA sponge, LINC02193, promotes neutrophil activation by upregulating ICAM1 and is correlated with ANCA-associated vasculitis.

OBJECTIVE: To investigate the pathogenic role and underlying mechanisms of lncRNAs in antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). METHODS：: RNA-sequencing (RNA-seq) was applied to screen the expression profile of lncRNAs in peripheral leukocytes from 5 AAV patients and 5 healthy controls (HC). Candidate lncRNAs were preliminarily verified in peripheral leukocytes from 46 AAV patients and 35 HC by qRT-PCR. Then, the identified LINC02193 was further validated in peripheral neutrophils from 67 AAV patients, 45 HC and 64 disease controls. Correlation between LINC02193 levels and disease activity was analyzed. Then, a loss-of-function study was conducted to investigate the role of LINC02193 in neutrophils activation. Furthermore, bioinformatics analysis, dual luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to explore the mechanism of LINC02193 regulating neutrophils activation. RESULTS: A total of 467 upregulated and 412 downregulated lncRNAs were identified in AAV patients. From top 5 upregulated lncRNAs, an elevation of LINC02193 was validated in a larger sample of AAV patients, and positively correlated with disease activity. Knockdown of LINC02193 inhibited ROS and NO production, NETs release and adhesion to endothelial cells of differentiated human promyelocytic leukaemia HL­60 cells (dHL-60), whereas overexpression of ICAM1 counteracted these effects. Mechanistic analysis demonstrated that LINC02193 acted as a miR-485-5p sponge to relieve the repressive effect of miR-485-5p on ICAM1, thus promoting ICAM1 expression. CONCLUSION: LINC02193, a novel lncRNA identified in AAV could function as competing endogenous RNAs (ceRNA) for miR-485-5p to promote ICAM1 expression and neutrophils activation, suggesting its potential as a therapeutic target of AAV.

Treatment efficacy and safety of adalimumab versus tocilizumab in patients with active and severe Takayasu arteritis: an open-label study.

OBJECTIVE: This study aimed to compare the efficacy and safety of adalimumab (ADA) versus tocilizumab (TCZ) in patients with Takayasu arteritis (TAK). METHODS: This was a randomized, controlled, open-label study. Forty patients with active and severe TAK were enrolled. They were treated with ADA (n = 21) combined with glucocorticoids (GCs) and methotrexate (MTX) or TCZ (n = 19) combined with GCs and MTX. The planned follow-up duration was 12 months. The primary end point was the efficacy rate (ER) at 6 months. The secondary endpoints included ER at 9 and 12 months, relapse rate, GC tapering, adverse effects, and life quality changes during treatment. RESULTS: In the intention-to-treat (ITT) population, the ER at 6 months was higher in the ADA group (85.71% vs 52.63%, P= 0.02). A similar direction of effect was noted in the per-protocol set (89.47% vs 62.50%, P= 0.06). The percentages of patients who achieved a GC dose of ≤ 10 mg/day at 6 months were similar between the ADA and TCZ groups (47.37% vs 43.75%, P= 0.83). The ERs at 9 and 12 months were similar between the two groups (P> 0.05). During the first 12 months of treatment, the relapse rate and adverse event incidence were comparable between the two groups (ADA vs TCZ: 9.52% vs 10.53%, P= 0.96; 38.10% vs 47.37%, P= 0.55, respectively). CONCLUSION: ADA combined with GCs and MTX may be more efficacious than TCZ combined with GCs and MTX among patients with active and severe TAK. TRIAL REGISTRATION: Clinicaltrials.gov; NCT04300686.

PET vascular activity score for predicting new angiographic lesions in patients with Takayasu arteritis: a Chinese cohort study.

OBJECTIVE: To investigate the ability of 18F-fluorodeoxyglucose PET/CT to predict new lesions in Takayasu arteritis. METHODS: Eighty-two Chinese patients with newly diagnosed Takayasu arteritis were recruited. Their clinical characteristics, serum biomarkers and imaging results were recorded at baseline and every visit. They were followed up for at least 2 years. New angiographic lesions were evaluated by magnetic resonance angiography. Baseline PET vascular activity scores (PETVAS) for predicting new lesions were evaluated. RESULTS: At baseline, a moderate correlation was observed between PETVAS and ESR (r = 0.74, P < 0.01) and CRP level (r = 0.69, P < 0.01). Overall, 18 (22%) patients showed new lesions on imaging during a median follow-up time of 36 months. The median time to the first occurrence of new lesions was 18 months. Compared with patients without new lesions, the patients with new lesions included more female patients (67.2% vs 94.4%, P = 0.03), patients with higher ESR values (20 vs 49, P = 0.02) and patients with active disease (62.5% vs 94.4%, P < 0.01). Multivariate Cox regression analysis revealed PETVAS was an independent risk factor for new angiographic lesions (PETVAS ≥8, hazard ratio = 7.56; 95% CI 2.20, 26.01, P < 0.01) with adjustment of age, sex, chest pain, ESR and Physician Global Assessment. Furthermore, patients with PETVAS ≥8 at baseline were more likely to experience adverse events including arterial ischaemic events during the follow-up. CONCLUSION: PETVAS showed good performance in predicting new lesions in Takayasu arteritis.

Heme oxygenase 1 in erythropoiesis: an important regulator beyond catalyzing heme catabolism.

Heme oxygenase 1 (HO-1), encoded by the HMOX-1 gene, is the main heme oxygenase that catalyzes the degradation of heme into iron, carbon monoxide, and biliverdin. HMOX-1 gene expression is stimulated by oxidative stress and regulated at transcriptional and post-transcriptional levels. After translation, subcellular location and protein stability of HO-1 are also altered by different extracellular and intracellular stimuli. HO-1 plays a key role in regulating iron homeostasis and cell protection and has become a new target for disease treatment. Erythropoiesis is a tightly controlled, iron-dependent process that begins with hematopoietic stem cells and maturates to red blood cells. HO-1 is expressed in hematopoietic stem/progenitor cells, hematopoietic niche cells, erythroblasts, and especially erythroblastic island and phagocytic macrophages. HO-1 functions importantly in the entire erythroid development process by influencing hematopoietic stem cell proliferation, erythroid lineage engagement, terminal erythroid differentiation, and even senescent RBC erythrophagocytosis. HO-1 is also related to stress erythropoiesis and certain red blood cell diseases. Elucidation of HO-1 regulation and function in erythropoiesis will be of great significance for the treatment of related diseases.

Circular RNA expression profiles and identification of hsa_circ_0028381 as a potential biomarker of anti-neutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVES: Accumulating evidence indicates the role of dysregulated circRNAs in autoimmune diseases. In this study, we investigated their role in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) by analysing the expression profiles of circRNA in plasma of AAV patients and exploring their potential as biomarkers of AAV. METHODS: RNA-sequencing (RNA-seq) was performed to identify the plasma circRNA and mRNA expression profiles from five AAV patients and five healthy controls (HCs). Quantitative reverse-transcription (qRT)-PCR confirmed that hsa_circ_0028381 was confirmed to be significantly upregulated in a validation cohort of 51 AAV patients and 30 HCs and was further verified in other connective tissue diseases (CTDs). The diagnostic value of hsa_circ_0028381 was assessed by receiver operating characteristic (ROC) curve analysis. RESULTS: RNA expression profiles revealed aberrant expression of 143 circRNAs (62 upregulated and 81 downregulated) and 304 mRNAs (151 upregulated and 153 downregulated) in AAV patients compared to HCs. qRT-PCR verification suggested that hsa_circ_0028381 levels were significantly increased in plasma from AAV patients compared to those in HCs and other CTDs. ROC curve analysis showed has_circ_0028381 had good diagnostic value for distinguishing AAV patients from controls (HCs and other CTDs) with an area under the curve (AUC) of 0.81. In addition, hsa_circ_0028381 was associated with renal involvement. Most importantly, increased baseline levels of hsa_circ_0028381 had predictive value for progression to end-stage renal disease (ESRD). CONCLUSIONS: RNA-seq revealed that circRNAs are aberrantly expressed in the plasma of AAV patients. Hsa_circ_0028381 was implicated as a potential biomarker for AAV diagnosis and renal prognosis.

Single-Cell RNA-Sequencing Reveals Peripheral T Helper Cells Promoting the Development of IgG4-Related Disease by Enhancing B Cell Activation and Differentiation.

Abnormal B cell differentiation plays a critical role in IgG4-related disease (IgG4-RD), but the underlying mechanism remains largely unknown. We investigated the cell landscape from three IgG4-RD retroperitoneal tissues and three control tissues using single-cell RNA-sequencing. Critical cell type or markers were further validated in the peripheral blood from the patients with IgG4-RD and healthy controls via flow cytometry as well as in the IgG4-RD and control tissue via immunofluorescence staining. The increases in B cells, plasma cells, and CD4+ T cells were found in IgG4-RD retroperitoneal tissue. Importantly, among CD4+ T cells, an increase in CD4+CXCR5-PD1hi peripheral T helper (Tph) cells with a high expression of IL-21 and TIGIT was discovered in IgG4-RD tissue, which was further validated in peripheral blood of the patients with IgG4-RD. The Tph cell and TIGIT+ Tph cell proportion were remarkably higher in active IgG4-RD patients and correlated with disease activity. Moreover, TIGIT+CD4+ cells were able to promote B cell differentiation via IL-21. Our study revealed that Tph cells are increased in IgG4-RD and probably play critical roles in B cell differentiation through TIGIT-IL-21 axis. Peripheral Tph cell and TIGIT+Tph cell are potential markers for IgG4-RD disease activity.

Ferric Single-Site Catalyst Confined in a Zeolite Framework for Propane Dehydrogenation.

Non-oxidative dehydrogenation of propane is a highly efficient approach for industrial preparation of propene that is commonly catalyzed by noble Pt or toxic Cr catalysts and suffers from coking. In this work, ferric catalyst confined in a zeolite framework was synthesized by a hydrothermal procedure. The isolated Fe in the framework formed distorted tetrahedra, which were beneficial for the selective dehydrogenation of propane and reached over 95 % propene selectivity and over 99 % total olefins selectivity. This catalyst had a silanol-free structure and was oxygen tolerant, hydrothermally stable, and coke free, with a deactivation constant of 0.01 h-1 . This study provided guidance for the synthesis of structural heteroatomic zeolite and efficient propane non-oxidative dehydrogenation over early transition metals.

The addition of oral iron improves chemotherapy-induced anemia in patients receiving erythropoiesis-stimulating agents.

Although many studies have shown that supplementation with iron and erythropoiesis-stimulating agents (ESA) is frequently used for managing chemotherapy-induced anemia (CIA), optimal combination therapy using these agents together to ameliorate anemia is not well characterized. To assess the effects of ESA combined with oral or intravenous (IV) iron on relieving CIA, PubMed, Cochrane Library, Embase and China National Knowledge Infrastructure (CNKI) were searched for articles. Data collected in the articles were meta-analyzed using RevMan 5.3 software with a random-effects model. Our comprehensive search yielded 1666 potentially relevant trials. A total of 41 trials randomizing 4200 patients with CIA fulfilled inclusion criteria, including 34 Chinese articles and 7 English articles. Meta-analysis showed that treatment with both ESA and iron more effectively improved CIA relative to iron supplementation alone, with increased hemoglobin, hematocrit, red blood cell count and hematopoietic response rate. Subgroup analyses revealed iron administration, both oral and IV iron, improved anemia in ESA-treated cancer patients with CIA. Our analysis demonstrates that iron supplementation combined with ESA more effectively ameliorates CIA relative to iron supplementation alone, without regard to whether IV or oral iron was used. Together, our findings may contribute to the clinical treatment of CIA using iron therapy with or without ESA.

Treatment efficacy and safety of tofacitinib versus methotrexate in Takayasu arteritis: a prospective observational study.

OBJECTIVE: To compare the treatment efficacy and safety of tofacitinib (TOF) versus methotrexate (MTX) in Takayasu arteritis (TAK). METHODS: Fifty-three patients with active disease from an ongoing prospective TAK cohort in China were included in this study. Twenty-seven patients were treated with glucocorticoids (GCs) and TOF, and 26 patients were treated with GCs with MTX. The observation period was 12 months. Complete remission (CR), inflammatory parameter changes, GCs tapering and safety were assessed at the 6th, 9th and 12th month. Vascular lesions were evaluated at the 6th and 12th month, and relapse was analysed during 12 months. RESULTS: The CR rate was higher in the TOF group than in the MTX group (6 months: 85.19% vs 61.54%, p=0.07; 12 months: 88.46% vs 56.52%, p=0.02). During 12 months' treatment, patients in the TOF group achieved a relatively lower relapse rate (11.54% vs 34.78%, p=0.052) and a longer median relapse-free duration (11.65±0.98 vs 10.48±2.31 months, p=0.03). Average GCs dose at the 3rd, 6th and 12th month was lower in the TOF group than that in the MTX group (p<0.05). A difference was not observed in disease improvement or disease progression on imaging between the two groups (p>0.05). Prevalence of side effects was low in both groups (3.70% vs 15.38%, p=0.19). CONCLUSION: TOF was superior to MTX for CR induction, a tendency to prevent relapse and tapering of the GCs dose in TAK treatment. A good safety profile for TOF was also documented in patients with TAK.

ELAVL1 is transcriptionally activated by FOXC1 and promotes ferroptosis in myocardial ischemia/reperfusion injury by regulating autophagy.

AIMS: Myocardial ischemia is the most common form of cardiovascular disease and the leading cause of morbidity and mortality. Understanding the mechanisms is very crucial for the development of effective therapy. Therefore, this study aimed to investigate the functional roles and mechanisms by which ELAVL1 regulates myocardial ischemia and reperfusion (I/R) injury. METHODS: Mouse myocardial I/R model and cultured myocardial cells exposed to hypoxia/reperfusion (H/R) were used in this study. Features of ferroptosis were evidenced by LDH activity, GPx4 activity, cellular iron, ROS, LPO, and GSH levels. The expression levels of autophagy markers (Beclin-1, p62, LC3), ELAVL1 and FOXC1 were measured by qRT-PCR, immunostaining and western blot. RIP assay, biotin-pull down, ChIP and dual luciferase activity assay were employed to examine the interactions of ELAVL1/Beclin-1 mRNA and FOXC1/ELAVL1 promoter. CCK-8 assay was used to examine viability of cells. TTC staining was performed to assess the myocardial I/R injury. RESULTS: Myocardial I/R surgery induced ferroptosis and up-regulated ELAVL1 level. Knockdown of ELAVL1 decreased ferroptosis and ameliorated I/R injury. Si-ELAVL1 repressed autophagy and inhibition of autophagy by inhibitor suppressed ferroptosis and I/R injury in myocardial cells. Increase of autophagy could reverse the effects of ELAVL1 knockdown on ferroptosis and I/R injury. ELAVL1 directly bound with and stabilized Beclin-1 mRNA. Furthermore, FOXC1 bound to ELAVL1 promoter region and activated its transcription upon H/R exposure. CONCLUSION: FOXC1 transcriptionally activated ELAVL1 may promote ferroptosis during myocardial I/R by modulating autophagy, leading to myocardial injury. Inhibition of ELAVL1-mediated autophagic ferroptosis would be a new viewpoint in the treatment of myocardial I/R injury.

Pulmonary findings on high-resolution computed tomography in Takayasu arteritis.

OBJECTIVE: This study aimed to describe pulmonary high-resolution CT (HRCT) findings in Takayasu arteritis (TA) and to determine possible causes. METHODS: A total of 243 TA patients were enrolled from a prospective cohort after excluding patients with other pulmonary disorders or incomplete data. Patients were divided into two groups: those with normal lung HRCT and those with abnormal lung HRCT. Clinical characteristics were compared between groups and binary logistic regression analysis was applied to identify possible causes of the lung lesions. Follow-up HRCT (obtained in 64 patients) was analysed to study changes in pulmonary lesions after treatment. RESULTS: Of the 243 patients, 107 (44.0%) had normal lung HRCT while 136 (56.0%) had abnormal lung HRCT, including stripe opacity (60.3%), nodules (44.9%), patchy opacity (25.0%), pleural thickening (15.4%), pleural effusion (10.3%), ground-glass opacity (8.1%), pulmonary infarction (6.6%), mosaic attenuation (4.4%), bronchiectasis (3.7%) and pulmonary oedema (2.2%). Patients with abnormal HRCT were significantly more likely to have type II arterial involvement (25% vs 12.2%, P = 0.04), pulmonary arterial involvement (PAI; 21.3% vs 5.6%, P < 0.001), pulmonary hypertension (20.6% vs 8.4%, P = 0.01) and abnormal heart function (27.9% vs 7.6%, P < 0.001). Logistic regression analysis demonstrated that PAI, worsened heart function and age were associated with presence of pulmonary lesions. Pulmonary infarction, pleural effusion and patchy opacities improved partially after treatment. CONCLUSION: Pulmonary lesions are not rare in patients with TA. Age, PAI and worsened heart function are potential risk factors for presence of pulmonary lesions in TA.

The value of ultrasonography combined with clinical features for predicting carotid imaging progression of Takayasu's arteritis: a prospective cohort study.

OBJECTIVES: To identify valuable ultrasonography findings combined with clinical markers for predicting carotid progression of Takayasu's arteritis (TAK) on imaging during a 1-year follow-up period. METHODS: From May 2016 to June 2019, 77 Chinese TAK patients with carotid artery involvement were enrolled in the present study. The patients' clinical characteristics and serological test and carotid ultrasonography results were recorded at baseline and each visit. Carotid progression was evaluated by ultrasonography every 3 months during the 1-year follow-up. Baseline clinical characteristics and ultrasonography results for predicting progression on imaging were identified. RESULTS: Sixteen (20.8%) patients presented with carotid progression on imaging during the 1-year follow-up period. The patients in the progressive group were younger (23.4±3.7 vs. 32.3±9.8 years, p<0.01) than those in the non-progressive group. At baseline, the vessel wall was thicker in the progressive group than in the non-progressive group (2.4±0.8 vs. 1.9±0.5 mm, p=0.041). Furthermore, the proportion of patients with refractory disease (87.5% vs. 16.4%, p<0.01) was higher in the progressive group than in the non-progressive group. Patients with a thickened carotid wall (≥1.9 mm), refractory disease, and younger age (≤30 years) might be at a high risk of carotid progression on imaging (75%, AUC: 0.93, sensitivity: 75%, specificity: 93.4%). CONCLUSIONS: Younger patients with early vascular structural changes at baseline as well as refractory disease seemed more likely to show carotid progression on imaging.

GDF11 contributes to hepatic hepcidin (HAMP) inhibition through SMURF1-mediated BMP-SMAD signalling suppression.

Hepcidin (HAMP) synthesis is suppressed by erythropoiesis to increase iron availability for red blood cell production. This effect is thought to result from factors secreted by erythroid precursors. Growth differentiation factor 11 (GDF11) expression was recently shown to increase in erythroid cells of ß-thalassaemia, and decrease with improvement in anaemia. Whether GDF11 regulates hepatic HAMP production has never been experimentally studied. Here, we explore GDF11 function during erythropoiesis-triggered HAMP suppression. Our results confirm that exogenous erythropoietin significantly increases Gdf11 as well as Erfe (erythroferrone) expression, and Gdf11 is also increased, albeit at a lower degree than Erfe, in phlebotomized wild type and ß-thalassaemic mice. GDF11 is expressed predominantly in erythroid burst forming unit- and erythroid colony-forming unit- cells during erythropoiesis. Exogeneous GDF11 administration results in HAMP suppression in vivo and in vitro. Furthermore, exogenous GDF11 decreases BMP-SMAD signalling, enhances SMAD ubiquitin regulatory factor 1 (SMURF1) expression and induces ERK1/2 (MAPK3/1) signalling. ERK1/2 signalling activation is required for GDF11 or SMURF1-mediated suppression in BMP-SMAD signalling and HAMP expression. This research newly characterizes GDF11 in erythropoiesis-mediated HAMP suppression, in addition to ERFE.

Knockdown of spliceosome U2AF1 significantly inhibits the development of human erythroid cells.

U2AF1 (U2AF35) is the small subunit of the U2 auxiliary factor (U2AF) that constitutes the U2 snRNP (small nuclear ribonucleoproteins) of the spliceosome. Here, we examined the function of U2AF1 in human erythropoiesis. First, we examined the expression of U2AF1 during in vitro human erythropoiesis and showed that U2AF1 was highly expressed in the erythroid progenitor burst-forming-unit erythroid (BFU-E) cell stage. A colony assay revealed that U2AF1 knockdown cells failed to form BFU-E and colony-forming-unit erythroid (CFU-E) colonies. Our results further showed that knockdown of U2AF1 significantly inhibited cell growth and induced apoptosis in erythropoiesis. Additionally, knockdown of U2AF1 also delayed terminal erythroid differentiation. To explore the molecular basis of the impaired function of erythroid development, RNA-seq was performed and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that several biological pathways, including the p53 signalling pathway, MAPK signalling pathway and haematopoietic cell lineage, were involved, with the p53 signalling pathway showing the greatest involvement. Western blot analysis revealed an increase in the protein levels of downstream targets of p53 following U2AF1 knockdown. The data further showed that depletion of U2AF1 altered alternatively spliced apoptosis-associated gene transcripts in CFU-E cells. Our findings elucidate the role of U2AF1 in human erythropoiesis and reveal the underlying mechanisms.

Vapor-Phase Strategy to Pillaring of Two-Dimensional Zeolite.

Two-dimensional (2D) layered zeolites are new forms of 3D zeolite frameworks. They can be pillared to form more open porous structures with increased access for reactants that are too big for the micropores of zeolites. The current pillaring procedure, however, requires intercalation of pillaring precursors by dispersing 2D zeolite in an alkoxide liquid and hydrolizing entrapped alkoxide to form inorganic oxide pillars in an aqueous alkaline solution. Both steps use excess solvents, generate significant waste, and require multiple synthesis and separation steps. Here we report a vapor-phase pillarization (VPP) process to produce pillared zeolites from 2D layered zeolite structures. The VPP process has ∼100% efficiency of alkoxide usage in the intercalation step, requires less (and, in some cases, zero) water addition in the hydrolysis step, does not require separation for product recovery, and generates no liquid waste. Furthermore, synthesis of pillared zeolites via the VPP process can be accomplished within a single apparatus with one-time operation. The pillared zeolite prepared by the VPP method preserved the zeolitic layered structure as well as acidity and showed enhancement in catalytic alkylation of mesitylene with benzyl alcohol compared to 2D layered zeolite without pillarization treatment.

Decreasing TfR1 expression reverses anemia and hepcidin suppression in ß-thalassemic mice.

Iron availability for erythropoiesis and its dysregulation in ß-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in ß-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in ß-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that ß-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective ß-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in ß-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with ß-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to ß-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in ß-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during ß-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in ß-thalassemic mice.

Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis.

Terminal erythroid differentiation is tightly coordinated with cell-cycle exit, which is regulated by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain to be fully defined. We show here that p19INK4d, a member of CDKI family, is abundantly expressed in erythroblasts and that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, and led to increased apoptosis and generation of abnormally nucleated late-stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather, it led to decreased expression of GATA1 protein. Importantly, the differentiation and nuclear defects were rescued by ectopic expression of GATA1. Because the GATA1 protein is protected by nuclear heat shock protein family (HSP) member HSP70, we examined the effects of p19INK4d knockdown on HSP70 and found that p19INK4d knockdown led to decreased expression of HSP70 and its nuclear localization. The reduced levels of HSP70 are the result of reduced extracellular signal-regulated kinase (ERK) activation. Further biochemical analysis revealed that p19INK4d directly binds to Raf kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1, which in turn led to reduced ERK activation. Thus we have identified an unexpected role for p19INK4d via a novel PEBP1-p-ERK-HSP70-GATA1 pathway. These findings are likely to have implications for improved understanding of disordered erythropoiesis.