Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

RNA-Editing Enzyme ADAR1 p150 Isoform Is Critical for Germinal Center B Cell Response.

Adenosine deaminase acting on RNA (ADAR)1 is the principal enzyme for adenosine-to-inosine editing, an RNA modification-avoiding cytosolic nucleic acid sensor's activation triggered by endogenous dsRNAs. Two ADAR1 isoforms exist in mammals, a longer IFN-inducible and mainly cytoplasm-localized p150 isoform and a shorter constitutively expressed and primarily nucleus-localized p110 isoform. Studies of ADAR1 mutant mice have demonstrated that ADAR1 is essential for multiple physiological processes, including embryonic development, innate immune response, and B and T lymphocyte development. However, it remained unknown whether ADAR1 plays a role in the humoral immune response. In this study, we conditionally delete Adar1 in activated B cells and show that ADAR1-deficient mice have a defective T cell-dependent Ab response and diminished germinal center (GC) B cells. Using various double mutant mice concurrently deficient in ADAR1 and different downstream dsRNA sensors, we demonstrate that ADAR1 regulates the GC response by preventing hyperactivation of the melanoma differentiation-associated protein 5 (MDA5) but not the protein kinase R or RNase L pathway. We also show that p150 is exclusively responsible for ADAR1's function in the GC response, and the p110 isoform cannot substitute for the p150's role, even when p110 is constitutively expressed in the cytoplasm. We further demonstrated that the dsRNA-binding but not the RNA-editing activity is required for ADAR1's function in the GC response. Thus, our data suggest that the ADAR1 p150 isoform plays a crucial role in regulating the GC B cell response.

Mettl14-Mediated m6A Modification Is Essential for Germinal Center B Cell Response.

The germinal center (GC) response is essential for generating memory B and long-lived Ab-secreting plasma cells during the T cell-dependent immune response. In the GC, signals via the BCR and CD40 collaboratively promote the proliferation and positive selection of GC B cells expressing BCRs with high affinities for specific Ags. Although a complex gene transcriptional regulatory network is known to control the GC response, it remains elusive how the positive selection of GC B cells is modulated posttranscriptionally. In this study, we show that methyltransferase like 14 (Mettl14)-mediated methylation of adenosines at the position N 6 of mRNA (N 6-methyladenosine [m6A]) is essential for the GC B cell response in mice. Ablation of Mettl14 in B cells leads to compromised GC B cell proliferation and a defective Ab response. Interestingly, we unravel that Mettl14-mediated m6A regulates the expression of genes critical for positive selection and cell cycle regulation of GC B cells in a Ythdf2-dependent but Myc-independent manner. Furthermore, our study reveals that Mettl14-mediated m6A modification promotes mRNA decay of negative immune regulators, such as Lax1 and Tipe2, to upregulate genes requisite for GC B cell positive selection and proliferation. Thus, our findings suggest that Mettl14-mediated m6A modification plays an essential role in the GC B cell response.

Comparison of vonoprazan bismuth-containing triple therapy with quadruple therapy in Helicobacter pylori-infected treatment-naive patients: a prospective multicenter randomized controlled trial.

BACKGROUND AND AIM: Helicobacter pylori infection is linked to various gastrointestinal conditions, such as chronic active gastritis, peptic ulcers, and gastric cancer. Traditional treatment options encounter difficulties due to antibiotic resistance and adverse effects. Therefore, the aim of this study was to explore the effectiveness of a new treatment plan that combines vonoprazan (VPZ), amoxicillin, and bismuth for the eradication of H. pylori. METHODS: A total of 600 patients infected with H. pylori were recruited for this multicenter randomized controlled trial. Patients treated for H. pylori elimination were randomly assigned at a 1:1 ratio to receive 14 days of vonoprazan-based triple therapy (vonoprazan + amoxicillin + bismuth, group A) or standard quadruple therapy (esomeprazole + clarithromycin + amoxicillin + bismuth, group B). Compliance and adverse effects were tracked through daily medication and side effect records. All patients underwent a 13C/14C-urea breath test 4 weeks after treatment completion. RESULTS: Intention-to-treat (ITT) and per-protocol (PP) analyses revealed no substantial differences in H. pylori eradication rates between groups A and B (ITT: 83.7% vs 83.2%; PP: 90.9% vs 89.7%). However, significant differences were observed in the assessment of side effects (13.7% vs 28.6%, P < 0.001). Specifically, group A had significantly fewer "bitter mouths" than group B did (3.7% vs 16.2%, P < 0.001). CONCLUSION: Triple therapy comprising vonoprazan (20 mg), amoxicillin (750 mg), and bismuth potassium citrate (220 mg) achieved a PP eradication rate ≥90%, paralleling standard quadruple therapy, and had fewer adverse events and lower costs (¥306.8 vs ¥645.8) for treatment-naive patients.

Estrogen lowers triglyceride via regulating hepatic APOA5 expression.

Estrogen had been found to be negatively associated with serum triglyceride (TG) levels. Apolipoprotein A5 (APOA5), a novel member of apolipoprotein family, was reported to have a strong ability to decrease serum concentrations of TG. Clinical data found concentrations of APOA5 were higher in woman than that in men, and the negative relationship between APOA5 and TG levels was more significant in woman. These suggests APOA5 may involve in estrogen actions. Therefore, we hypothesize estrogen up-regulates serum concentrations of APOA5 and subsequently decreases serum TG levels. We will design the following experiments to test this hypothesis. (1) We will treat wild and APOA5-defeted ovariectomized hamster with or without estrogen to examine if estrogen could up-regulate concentrations of APOA5 and decrease TG levels. (2) We will treat HepG2 cells with estrogen and investigate the possible mechanisms.

17ß-estradiol lowers triglycerides in adipocytes via estrogen receptor α and it may be attenuated by inflammation.

BACKGROUND: Estrogen was reported to protect against obesity, however the mechanism remains unclear. We aimed to investigate the impact of 17ß-estradiol (17ß-E2) on triglyceride metabolism in adipocytes with or without lipopolysacchride (LPS) stimulating, providing novel potential mechanism for estrogen action. METHODS: 3T3-L1 adipocytes were cultured and differentiated into mature adipocytes in vitro. The differentiated 3T3-L1 cells were divided into six groups: (i) control group, treated with 0.1% DMSO alone; (ii) 17ß-E2 group, treated with 1, 0.1, or 0.001 µM 17ß-E2 for 48 h; (iii) 17ß-E2 plus MPP group, pre-treated with 10 µM MPP (a selective ERα receptor inhibitor) for 1 h, then incubated with 1 µM 17ß-E2 for 48 h; (iv) 17ß-E2 plus PHTPP group, pre-treated with 10 µM PHTPP (a selective ERß receptor inhibitor), then incubated with 1 µM 17ß-E2 for 48 h; (v) LPS group, pre-treated with 100 ng/mL LPS for 24 h, then cells were washed by PBS for 3 times and incubated with 0.1% DMSO alone for 48 h; (vi) 17ß-E2 plus LPS group, pre-treated with 100 ng/mL LPS for 24 h, then cells were washed by PBS for 3 times and incubated with 1 µM 17ß-E2 for 48 h. The levels of triglyceride and adipose triglyceride lipase (ATGL) in differentiated 3T3-L1 cells and the concentrations of interleukin-6 (IL-6) in culture medium were measured. RESULTS: Comparing with control group, 1 µM and 0.1 µM 17ß-E2 decreased the intracellular TG levels by about 20% and 10% respectively (all P < 0.05). The triglyceride-lowing effect of 17ß-E2 in differentiated 3T3-L1 cells was abolished by ERα antagonist MPP but not ERß antagonist PHTPP. Comparing with control group, the IL-6 levels were significantly higher in the culture medium of the cultured differentiated 3T3-L1 cells in LPS group and 17ß-E2 + LPS group (all P < 0.05). And, the IL-6 levels were similar in LPS group and 17ß-E2 + LPS group (P > 0.05). There was no significant difference in the triglyceride contents of differentiated 3T3-L1 cells among control group, LPS group and 17ß-E2 + LPS group (all P > 0.05). ATGL expression in 17ß-E2 group was significantly higher than control group (P < 0.05), which was abolished by ERα antagonist MPP or LPS. CONCLUSIONS: 17ß-E2 increased ATGL expression and lowered triglycerides in adipocytes but not in LPS stimulated adipocytes via estrogen ERα.

Organocatalytic regioselective asymmetric Michael addition of azlactones to o-hydroxy chalcone derivatives.

The regioselective and enantioselective Michael addition between azlactones and o-hydroxy chalcone derivatives is reported. Enantiomerically enriched N,O-aminals with two continuous stereogenic centers are exclusively obtained in moderate to good yields with excellent diastereoselectivities and good to excellent enantioselectivities. The experimental results show that an o-hydroxy group on the cinnamenyl motif of chalcone derivatives plays a crucial role at the reaction sites for the regioselective Michael addition. In addition, circular dichroism (CD) spectroscopy and density functional theory (DFT) are used to investigate the absolute configuration of N,O-aminals and the corresponding transition-state structures.

Hepatic-targeted gene delivery using cationic mannan vehicle.

The incidence of hepatic diseases continuously increases worldwide and causes significant mortality because of inefficient pharmacotherapy. Gene therapy is a new strategy in the treatment of hepatic diseases, but the disadvantages of insufficient retention in the liver and undesirable side effects hinder its application. In this study, we developed a novel nonviral vehicle targeted to liver. Mannan was cationized with spermine at varying grafted ratios to deliver the gene and was optimized in cytotoxicity and transfection in vitro. A spermine-mannan (SM)-based delivery system was proven to be hepatic targeted and was capable of prolonging the gene retention period in the liver. Moreover, SM at N/P of 20 was confirmed to be less interfered with by the serum. Optimized SM vehicle has relatively high hepatic transfection with almost no toxicity induction in the liver, which highlighted its potential in the treatment of hepatic diseases.

Co-transfection gene delivery of dendritic cells induced effective lymph node targeting and anti-tumor vaccination.

PURPOSE: Successful genetically engineered Dendritic Cell (DC) can enhance DC's antigen presentation and lymph node migration. The present study aims to genetically engineer a DC using an efficient non-viral gene delivery vector to induce a highly efficient antigen presentation and lymph node targeting in vivo. METHODS: Spermine-dextran (SD), a cationic polysaccharide vector, was used to prepare a gene delivery system for DC engineering. Transfection efficiency, nuclear trafficking, and safety of the SD/DNA complex were evaluated. A vaccine prepared by engineering DC with SD/gp100, a plasmid encoding melanoma-associated antigen, was injected subcutaneously into mice to evaluate the tumor suppression. The migration of the engineered DCs was also evaluated in vitro and in vivo. RESULTS: SD/DNA complex has a better transfection behavior in vitro than commercially purchased reagents. The DC vaccine co-transfected with plasmid coding CCR7, a chemokine receptor essential for DC migration, and plasmid coding gp100 displayed superior tumor suppression than that with plasmid coding gp100 alone. Migration assay demonstrated that DC transfected with SD/CCR7 can promote DC migration capacity. CONCLUSIONS: The study is the first to report the application of nonviral vector SD to co-transfect DC with gp100 and CCR7-coding plasmid to induce both the capacity of antigen presentation and lymph node targeting.

The RNA-binding protein hnRNP F is required for the germinal center B cell response.

The T cell-dependent (TD) antibody response involves the generation of high affinity, immunoglobulin heavy chain class-switched antibodies that are generated through germinal center (GC) response. This process is controlled by coordinated transcriptional and post-transcriptional gene regulatory mechanisms. RNA-binding proteins (RBPs) have emerged as critical players in post-transcriptional gene regulation. Here we demonstrate that B cell-specific deletion of RBP hnRNP F leads to diminished production of class-switched antibodies with high affinities in response to a TD antigen challenge. B cells deficient in hnRNP F are characterized by defective proliferation and c-Myc upregulation upon antigenic stimulation. Mechanistically, hnRNP F directly binds to the G-tracts of Cd40 pre-mRNA to promote the inclusion of Cd40 exon 6 that encodes its transmembrane domain, thus enabling appropriate CD40 cell surface expression. Furthermore, we find that hnRNP A1 and A2B1 can bind to the same region of Cd40 pre-mRNA but suppress exon 6 inclusion, suggesting that these hnRNPs and hnRNP F might antagonize each-other's effects on Cd40 splicing. In summary, our study uncovers an important posttranscriptional mechanism regulating the GC response.

The histone H2B ubiquitination regulator Wac is essential for plasma cell differentiation.

Naïve B cells become activated and differentiate into antibody-secreting plasma cells (PCs) when encountering antigens. Here, we reveal that the WW domain-containing adapter protein with coiled-coil (Wac), which is important for histone H2B ubiquitination (ubH2B), is essential for PC differentiation. We demonstrate that B cell-specific Wac knockout mice have severely compromised T cell-dependent and -independent antibody responses. PC differentiation is drastically compromised despite undisturbed germinal center B cell response in the mutant mice. We also observe a significant reduction in global ubH2B in Wac-deficient B cells, which is correlated with downregulated expression of some genes critical for cell metabolism. Thus, our findings demonstrate an essential role of Wac-mediated ubH2B in PC differentiation and shed light on the epigenetic mechanisms underlying this process.

Optimization and internalization mechanisms of PEGylated adenovirus vector with targeting peptide for cancer gene therapy.

We have previously developed a novel adenovirus vector (Adv) that targeted tumor tissues/vasculatures after systemic administration. The surface of this Adv is conjugated with CGKRK tumor homing peptide by the cross-linking reaction of polyethyleneglycol (PEG). In this study, we showed that the condition of PEG modification was important to minimize the gene expression in normal tissues after systemic treatment. When Adv was modified only with PEG-linked CGKRK, its luciferase expression was enhanced even in the liver tissue, as well as the tumor tissue. However, in the reaction with the mixture of non-cross-linking PEG and PEG-linked CGKRK, we found out that the best modification could suppress its gene expression in the liver, without losing that in the tumor. We also studied the internalization mechanisms of CGKRK-conjugated Adv. Results suggested that there is a specific interaction of the CGKRK peptide with a receptor at the cell surface enabling efficient internalization of CGKRK-conjugated Adv. The presence of cell-surface heparan sulfate is important receptor for the cellular binding and uptake of CGKRK-conjugated Adv. Moreover, macropinocytosis-mediated endocytosis is also important in endocytosis of CGKRK-conjugated Adv, aside from clathrin-mediated and caveolae-mediated endocytosis. These results could help evaluate the potentiality of CGKRK-conjugated Adv as a prototype vector with suitable efficacy and safety for systemic cancer gene therapy.

Gene-carried chitosan-linked polyethylenimine induced high gene transfection efficiency on dendritic cells.

A dendritic cell (DC) networking system has become an attractive approach in cancer immunotherapy. Successful DC gene engineering depends on the development of transgene vectors. A cationic polymer, chitosan-linked polyethylenimine (PEI) (CP), possessing the advantages of both PEI and chitosan, has been applied in nonviral transfection of DCs. Physicochemical evaluation showed that CP/DNA complexes could form cationic nanoparticles. Compared with DCs transfected with commercial reagent, Lipofectamine2000, it showed higher transfection efficiency and lower cytotoxicity when DCs were transfected with CP/DNA complexes. A nuclear trafficking observation of CP/DNA complexes by a confocal laser scanning microscope further revealed that the CP could help DNA enter into the cytoplasm and finally into the nucleus of a DC. Finally, vaccination of DCs transfected with CP/DNA encoding gp100 slightly improved resistance to the B16BL6 melanoma challenge. This is the first report that CP polymer is used as a nonviral vector for DC gene delivery and DC vaccine. Essentially, these results might be helpful to design a promising nonviral vector for DC gene delivery.

[A clinical analysis of AIDS-related non-Hodgkin lymphoma in 5 patients and review of literature].

OBJECTIVE: To analyze the clinical characteristics of AIDS-related non-Hodgkin lymphoma (ARL) and review relative literature for the diagnosis and treatment of ARL. METHOD: The clinical data of ARL patients admitted to Peking Union Medical College Hospital from April 2009 to April 2011 were retrospectively analyzed. RESULTS: Five male ARL patients aged 32 to 65 years old were included in this retrospective study. Among them, two patients were found to be HIV-positive for the first time, three were on regular highly active anti-retroviral therapy (HAART) for 7 - 8 months before the emergence of lymphoma-related symptoms. CD(4)(+) T cell count was (69 - 232) × 10(6)/L at presentation. Two patients firstly presented with sore throat and throat ulcer, one with cervical nodules, one with pelvic mass, one with fever and edema in right thigh. Through pathological analysis, four patients had B cell-originated lymphoma, with one Burkitt lymphoma and three diffuse large B cell lymphomas; one patient had T-cell lymphoma. Four patients were treated with chemotherapy, with one complete remission, one relapse, one non-response, and one death. One patient had radiotherapy only and had progressed disease. Bone marrow suppression and gastrointestinal disturbance were the main adverse effects of chemotherapy. CONCLUSIONS: Lymphoma should be considered in any HIV-infected patients presented with unexplainable adenopathy, recurrent sore throat or throat ulcer, or fever of unknown origin. Biopsy should be rigorously carried out. Appropriate chemotherapy, together with HAART, may improve the prognosis greatly.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eµ-Myc transgenic mice and significantly improves their survival.

Adenosine deaminase acting on RNA-1 is essential for early B lymphopoiesis.

Adenosine deaminase acting on RNA-1 (ADAR1) is a ubiquitously expressed RNA deaminase catalyzing adenosine-to-inosine editing to prevent hyperactivated cytosolic double-stranded RNA (dsRNA) response mediated by MDA5. Here, we demonstrate that ADAR1 is essential for early B lymphopoiesis from late pro-B and large pre-B cell stages onward. ADAR1 exerts its requisite role via both MDA5-dependent and -independent pathways. Interestingly, the MDA5-dependent mechanisms regulate early pro-B to large pre-B cell transition by promoting early B cell survival. In contrast, the MDA5-independent mechanisms control large pre-B to small pre-B cell transition by regulating pre-B cell receptor (pre-BCR) expression. Moreover, we show that protein kinase R (PKR) and oligoadenylate synthetase/ribonuclease (OAS/RNase) L pathways are dispensable for ADAR1's role in early B lymphopoiesis. Importantly, we demonstrate that p150 isoform of ADAR1 exclusively accounts for ADAR1's function in early B lymphopoiesis, and its conventional dsRNA-binding, but not the Z-DNA/RNA-binding or the RNA-editing, activity is required for ADAR1's function in B cell development. Thus, our findings suggest that ADAR1 regulates early B lymphopoiesis through various mechanisms.

Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion.

Genes of the Eya family and of the Six1/2 subfamily are expressed throughout development of vertebrate cranial placodes and are required for their differentiation into ganglia and sense organs. How they regulate placodal neurogenesis, however, remains unclear. Through loss of function studies in Xenopus we show that Eya1 and Six1 are required for neuronal differentiation in all neurogenic placodes. The effects of overexpression of Eya1 or Six1 are dose dependent. At higher levels, Eya1 and Six1 expand the expression of SoxB1 genes (Sox2, Sox3), maintain cells in a proliferative state and block expression of neuronal determination and differentiation genes. At lower levels, Eya1 and Six1 promote neuronal differentiation, acting downstream of and/or parallel to Ngnr1. Our findings suggest that Eya1 and Six1 are required for both the regulation of placodal neuronal progenitor proliferation, through their effects on SoxB1 expression, and subsequent neuronal differentiation.

[Study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products].

OBJECTIVE: To study on HPLC fingerprint characteristic analysis of Cordyceps sinensis and its similar products. METHODS: To determinate 13 samples of Cordyceps sinensis and its similar products by HPLC, and analyze the HPLC results with similar appraisal method and graphical methods of multivariate sample in two dimensional plane such as the methods of profile, radar chart and constellation graph. RESULTS: The similar appraisal method might synthesize the similar degree in quantification, while the graphical methods such as profile graph, radar chart and constellation graph could show more details about the classification and the characteristic of varieties directly. CONCLUSIONS: We recommend the combined application of similar appraisal method and the graphical methods due to its advantages on the judgment and characteristic analysis of fingerprint.

Xenopus cadherin-6 regulates growth and epithelial development of the retina.

Cadherins are crucial for tissue cohesion, separation of cell layers and cell migration during embryogenesis. To investigate the role of classical type II Xcadherin-6 (Xcad-6), we performed loss-of-function studies by morpholino oligonucleotide injections. This resulted in severe eye defects which could be rescued with murine cadherin-6. In the absence of Xcadherin-6, morphological alterations and a decrease in cell proliferation were observed with eye cup formation. Eye field transplantations of Xcadherin-6 depleted donors yielded grafts that failed to form a proper neuroepithelium in a wildtype environment. At later developmental stages Xcadherin-6 deficient eyes showed lamination defects in the outer neural retina, a reduced thickness of the ganglion cell layer (GCL) and a fragmented retina pigment epithelium (RPE). Thus, Xcadherin-6 is essential early in eye development for structural organization and growth of the neuroepithelium before it differentiates into neural retina and RPE.

The study on the chromatographic fingerprint of Fructus xanthii by microwave assisted extraction coupled with GC-MS.

The chromatographic fingerprint of Fructus xanthii, a kind of Traditional Chinese Medicines (TCMs), was studied by microwave assisted extraction (MAE) coupled with gas chromatography-mass spectrometry (GC-MS). The optimized conditions of MAE were examined. The method of MAE was evaluated in contrast to heat reflux extraction (HRE) method and by the validation tests of precision and repeatability. The relative standard deviations (RSDs) of retention time and peak area of each component were less than 0.2% and 6%, respectively. Twenty-five different batches of samples collected from different producing areas and the toasting process of F. xanthii were studied. The characteristic differences in the producing areas and the chemical variances in the toasting process were obtained and studied by principal components analysis (PCA) and similarity analysis. The trends of main varying components were attempted to be described in order to specify the related pharmacology and toxicology in crude and toasted samples. The results suggest that the chromatographic fingerprint developed by MAE coupled with GC-MS provides useful information to reveal the quality of F. xanthii and evaluate the quality changes in the producing process.