The role of cGAMP via the STING pathway in modulating germinal center responses and CD4 T cell differentiation.

Germinal center (GC) responses are essential for establishing protective, long-lasting immunity through the differentiation of GC B cells (BGC) and plasma cells (BPC), along with the generation of antigen-specific antibodies. Among the various pathways influencing immune responses, the STING (Stimulator of Interferon Genes) pathway has emerged as significant, especially in innate immunity, and extends its influence to adaptive responses. In this study, we examined how the STING ligand cGAMP can modulate these key elements of the adaptive immune response, particularly in enhancing GC reactions and the differentiation of BGC, BPC, and follicular helper T cells (TFH). Employing in vivo models, we evaluated various antigens and the administration of cGAMP in Alum adjuvant, investigating the differentiation of BGC, BPC, and TFH cells, along with the production of antigen-specific antibodies. cGAMP enhances the differentiation of BGC and BPC, leading to increased antigen-specific antibody production. This effect is shown to be type I Interferon-dependent, with a substantial reduction in BPC frequency upon interferon (IFN)-ß blockade. Additionally, cGAMP's influence on TFH differentiation varies over time, which may be critical for refining vaccine strategies. The findings elucidate a complex, antigen-specific influence of cGAMP on T and B cell responses, providing insights that could optimize vaccine efficacy.

Accuracy of customized abutment data superimposition according to the extent of scanning area.

PURPOSE: To evaluate the accuracy of superimposition of customized abutment library data onto scanned abutment data according to the extent of the scanning area. MATERIALS AND METHODS: A patient model was fabricated by a 3D printer (Probo, DIO Implant), and a customized abutment was fabricated using a four-axis milling machine (ARUM 4X-100, Doowon). The customized abutment library data were generated using a laboratory scanner (E3, 3Shape) for superimposition after intraoral scanning. A cone-shaped structure was embedded into the library data at the center of the connection part. The customized abutment was placed on the model, and the model was scanned using a laboratory scanner to produce reference data. Three different test group datasets were generated using intraoral scanner and computer-aided design software: (1) fully scanned customized abutment; (2) insufficiently scanned proximal surface; and (3) insufficiently scanned margin, assuming challenging intraoral conditions. The library data were superimposed onto each test group; thereafter, the distance and angle between the reference and test group data were analyzed by using the embedded cone. Statistical analysis was performed using one-way analysis of variance followed by post hoc Tukey test for multiple comparisons. RESULTS: There were no statistically significant differences between the mean distance and angle of the test group data (with three different scanning areas) and the reference data. CONCLUSION: The superimposition technique can be used clinically, not only when the scan is complete, but also when the proximal surface and margin of the customized abutment have been scanned incompletely.

Publisher Correction: Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

The originally published version of this Article contained an error in the spelling of the author Da-eun Kim, which was incorrectly given as Da-Eun Kim. Furthermore, in Figure 1a, the Cas9 protein was positioned incorrectly during typesetting. These errors have now been corrected in both the PDF and HTML versions of the Article.

Targeted Base Editing via RNA-Guided Cytidine Deaminases in Xenopus laevis Embryos.

Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

The bacteria-derived clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are powerful tools for genome engineering. Recently, in addition to Cas protein engineering, the improvement of guide RNAs are also performed, contributing to broadening the research area of CRISPR-Cas9 systems. Here we develop a fusion guide RNA (fgRNA) that functions with both Cas9 and Cpf1 proteins to induce mutations in human cells. Furthermore, we demonstrate that fgRNAs can be used in multiplex genome editing and orthogonal genome manipulation with two types of Cas proteins. Our results show that fgRNAs can be used as a tool for performing multiple gene manipulations.

Extracellular ATP induces apoptotic signaling in human monocyte leukemic cells, HL-60 and F-36P.

Extracellular adenosine 5'-triphosphate (ATP) affects the function of many tissues and cells. To confirm the biological activity of ATP on human myeloid leukemic cells, F-36P and HL-60, cells were treated with a variety of concentrations of ATP. The stimulation with extracellular ATP induced the arrest of cell proliferation and cell death from the analysis of Annexin-V staining and caspase activity by flow cytometry. The Annexin-V positive cells in both cell lines were dramatically increased following ATP stimulation. The expression of P2 purinergic receptor genes was confirmed, such as P2X1, P2X4, P2X5, P2X7 and P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y11 in both leukemic cell lines. Interestingly, ATP induced intracellular calcium flux in HL-60 cells but not in F-36P cells, as determined by Fluo-3 AM staining. Cell cycle analysis revealed that ATP treatment arrested both F-36P and HL-60 cells at G1/GO. Taken together, these data showed that extracellular ATP via P2 receptor genes was involved in the cell proliferation and survival in human myeloid leukemic cells, HL-60 and F-36P cells by the induction of apoptosis and control of cell cycle. Our data suggest that treatment with extracellular nucleotides may be a novel and powerful therapeutic avenue for myeloid leukemic disease.

Fracture Strength After Fatigue Loading of Lithium Disilicate Pressed Zirconia Crowns.

PURPOSE: The aim of this study was to evaluate the fracture resistance of fatigued lithium disilicate pressed zirconia crowns versus other ceramic crowns. MATERIALS AND METHODS: Lithium disilicate pressed zirconia, fluorapatite pressed zirconia, monolithic lithium disilicate, and monolithic zirconia crowns were fabricated. Cyclic and static loadings were applied to the mesiobuccal cusp tip after thermocycling and fracture strengths were recorded. RESULTS: Fatigued lithium disilicate pressed zirconia crowns were found to have a fracture resistance of 9,117.81 ± 727.04 N, compared with 9,240.61 ± 887.21 N for monolithic zirconia crowns, 3,030.18 ± 1,505.83 N for fluorapatite pressed zirconia crowns, and 4,173.94 ± 877.46 N for monolithic lithium disilicate crowns (P = .001). CONCLUSION: This in vitro study shows that fatigued lithium disilicate pressed zirconia and monolithic zirconia crowns have better fracture resistance than fluorapatite pressed zirconia and monolithic lithium disilicate crowns.

The stability of vocational interests from early adolescence to middle adulthood: a quantitative review of longitudinal studies.

The present meta-analysis examined the stability of vocational interests from early adolescence (age 12) to middle adulthood (age 40). Stability was represented by rank-order and profile correlations. Interest stability remained unchanged during much of adolescence and increased dramatically during the college years (age 18-21.9), where it remained for the next 2 decades. Analyses of potential moderators showed that retest time interval was negatively related to interest stability and that rank-order stability was less stable than profile stability. Although cohort standings did not moderate stability, interests of the 1940s birth cohort were less stable than those of other cohorts. Furthermore, interests reflecting hands-on physical activities and self-expressive/artistic activities were more stable than scientific, social, enterprising, and clerical interests. Vocational interests showed substantial continuity over time, as evidenced by their higher longitudinal stability when compared with rank-order stability of personality traits. The findings are discussed in the context of psychosocial development.

Marginal and Internal Fit of Conventional Metal-Ceramic and Lithium Disilicate CAD/CAM Crowns.

This study aimed to evaluate and compare the marginal and internal gap widths of lithium disilicate computer-aided design / computer-assisted manufacture (LDC) crowns and conventionally produced porcelain-fused-to-metal (PFM) crowns. A convenience sample of 21 patients treated with a single restoration was selected. PFM and LDC crowns were fabricated for each selected abutment tooth, following traditional crown preparation. Silicone replicas were produced, and internal gaps and marginal gaps were measured. Internal gaps were significantly larger for the axial and occlusal surfaces of LDC crowns than for those of PFM crowns (P < .001). Marginal gaps were not significantly different (P > .05). Both LDC crowns and PFM crowns showed clinically acceptable marginal fit.

Phenotype of peroxisome proliferator-activated receptor-alpha(PPARalpha)deficient mice on mixed background fed high fat diet.

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-alpha PPARalpha) on obesity. Previous reports demonstrated that PPARalpha is a critical modulator of lipid homeostasis, but the overt, obese phenotypic characterization in the strain of PPAR deficient (PPARalpha-/-) mice is influenced by other factors, including diet and genetics. Therefore, it is necessary to establish the phenotypic characterization of PPARalpha-/- mice prior to the obesity-related study. In this study, we observed phenotype of PPARalpha-/- mice on mixed genetic background (C57BL/6Nx129/Sv) fed a high fat diet for 16 weeks. PPARalpha-/- mice, regardless of sex, raised body growth rate significantly comparing with wild type and showed male-specific fatty change in the liver. They were shown to lack hepatic induction of PPARalpha target genes encoding enzymes for fatty acid beta-oxidation.

Implant overdenture using a locator bar system by drill and tapping technique in a mandible edentulous patient: a case report.

Various options have been introduced for the selection of implant overdenture attachments. Attachment wear due to the repeated insertion and removal of dentures has caused problems such as decreased retention and the requirement for suprastructure remanufacturing. In these cases, a Locator bar system was applied using the drill and tapping technique to achieve total retrievability. In a 55-year-old female patient who showed three degrees of mobility in most of her teeth due to severe alveolar bone loss, a complete denture in the maxilla and an implant supported type overdenture in the mandible were planned after extracting all the remaining teeth. Six implants were placed from canine region to the distal molar region, and the locator was connected to the milled bar using the drill and tapping technique. For a 61-year-old female edentulous patient who complained of poor retention with old denture, a complete denture in the maxilla and an implant-tissue supported type overdenture in the mandible were planned. Four implants were placed in front of mental foramen, and the Locator was also connected to the Hader bar using the drill and tapping technique. With this technique, female parts can be easily replaced, and retention can be continuously maintained.

Synergistic therapeutic effects of cytokine-induced killer cells and temozolomide against glioblastoma.

The presence of active immunity within the brain supports the possibility of effective immunotherapy for glioblastoma (GBM). To provide a clinically-relevant adoptive immunotherapy for GBM using ex vivo expanded cytokine-induced killer (CIK) cells, the treatment capability of CIK cells, either alone or in combination with temozolomide (TMZ) were evaluated. Human CIK (hCIK) cells were cultured from PBMC using activating anti-CD3 antibody and IL-2, which were 99% CD3+, 91% CD3+CD8+ and 29% CD3+CD56+. In vitro, hCIK cells showed tumor-specific cytotoxicity against U-87MG human GBM cells. When hCIK cells were injected into tail veins of immune-compromised mice bearing U-87MG tumors in their brains, numerous CIK cells infiltrated into the brain tumors. CIK treatments (1x10(5), 1x10(6) or 1x10(7), once a week for four weeks) inhibited the tumor growth significantly in a dose-dependent manner; 44, 54 and 72% tumor volume reduction, respectively, compared with the control group (P<0.05). Moreover, hCIK cells (1x10(7), once a week for four weeks) and TMZ (2.5 mg/kg, daily for 5 days) combination treatment further increased tumor cell apoptosis and decreased tumor cell proliferation and vessel density (P<0.05), creating a more potent therapeutic effect (95% reduction in tumor volume) compared with either hCIK cells or TMZ single therapy (72% for both, P<0.05). Taken together, CIK cell-immunotherapy and TMZ chemotherapy have synergistic therapeutic effects and could be combined for a successful treatment of GBM.

Signal crosstalk between estrogen and peroxisome proliferator-activated receptor alpha on adiposity.

Peroxisome proliferator-activated receptor alpha and estrogen are believed to be involved in metabolic changes leading to obesity. To test this relationship, we divided female wildtype and PPAR alpha-deficient mice fed on a high fat diet into the following groups: mock-operated, ovariectomized (OVX), and E(2)-treated. The visceral white adipose tissue and plasma cholesterol levels were increased significantly in wild type OVX and decreased in the E(2)-treated group, but interestingly not in PPAR alpha-deficient mice. The mRNA levels of lipoprotein lipase in adipose tissue were also increased in only wild type OVX and decreased significantly in E(2)-treated mice. These novel results suggest the possibility of signaling crosstalk between PPAR alpha and E2, causing obesity in vivo.

Extracellular ATP is involved in the induction of apoptosis in murine hematopoietic cells.

Extracellular nucleotides have multiple biological actions in processes such as proliferation, differentiation, chemotaxis, and cytokine secretion through P2X receptors on the cell surface. To determine the biological activity of adenosine triphosphate (ATP) and the expression of P2 nucleotide receptors in murine bone marrow-derived hematopoietic cells and stem cells/progenitor cells, we investigated the effects of ATP in assays of cell proliferation and cell death in vitro. Our results demonstrated that several subtypes of P2X receptors were expressed on hematopoietic cells and that P2X7, in particular, was partially expressed in hematopoietic stem cells/progenitor cells. In addition, stimulation of hematopoietic cells with high concentrations of ATP caused severe inhibition of cell proliferation despite the presence of cytokine stimulation. We analyzed the apoptotic effects of stimulation with several different dosages of ATP and confirmed the enhanced apoptotic activity in hematopoietic cells and progenitor cells. Antagonists, against P2X receptors and ATP, suramin and oxidized ATP, inhibited the induction of cell death for murine hematopoietic cells. Our data suggest that extracellular nucleotides may provide a novel and powerful tool for regulating the cell fate of hematopoietic stem cells.