[Analysis of clinical features and genetic variant in a neonate with Au-Kline syndrome due to a de novo variant of the HNRNPK gene].

OBJECTIVE: To explore the clinical phenotype and genetic basis of a neonate with Au-Kline syndrome (AKS). METHODS: Clinical data and result of genetic testing of a neonate with AKS who was admitted to the Affiliated Provincial Children's Hospital of Anhui Medical University in January 2021 were retrospectively analyzed. Relevant literature was searched from the Wanfang Data Knowledge Service Platform, China National Knowledge Infrastructure and PubMed databases using key words "Au Kline syndrome", "Au-Kline syndrome", "HNRNPK" and "AKS". The research period was set as from January 1, 2000 to December 31, 2020. RESULTS: The male newborn has manifested feeding difficulties, hypotonia, absence of the upper jaw to the uvula and facial dysmorphism. Trio-whole exome sequencing revealed that he has harbored a frameshift c.478dupA (p.Ile160AsnfsTer7) variant of the HNRNPK gene, which was varified by Sanger sequencing to have a de novo origin. The variant has not been included in the databases. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was rated as pathogenic (PVS1+PS2+PM2_Supporting). Literature retrieval has identified 14 children with AKS and de novo mutations of the HNRNPK gene. Their clinical manifestations have included growth and motor retardation, various degree of mental retardation, facial dysmorphism and a high frequency of congenital heart malformations. CONCLUSION: The AKS in this child may be attributed to the c478dupA frameshifting variant of the HNRNPK gene. Diagnosis of AKS should be suspected for children with mental retardation and multiple congenital malformation syndromes including Kabuki syndrome.

Auriculocondylar syndrome 2 caused by a novel PLCB4 variant in a male Chinese neonate: A case report and review of the literature.

BACKGROUND: Auriculocondylar syndrome (ARCND) is a rare congenital craniofacial developmental malformation syndrome of the first and second pharyngeal arches with external ear malformation at the junction between the lobe and helix, micromaxillary malformation, and mandibular condylar hypoplasia. Four subtypes of ARCND have been described so far, that is, ARCND1 (OMIM # 602483), ARCND2 (ARCND2A, OMIM # 614669; ARCND2B, OMIM # 620458), ARCND3 (OMIM # 615706), and ARCND4 (OMIM # 620457). METHODS: This study reports a case of ARCND2 resulting from a novel pathogenic variant in the PLCB4 gene, and summarizes PLCB4 gene mutation sites and phenotypes of ARCND2. RESULTS: The proband, a 5-day-old male neonate, was referred to our hospital for respiratory distress. Micrognathia, microstomia, distinctive question mark ears, as well as mandibular condyle hypoplasia were identified. Trio-based whole-exome sequencing identified a novel missense variant of NM_001377142.1:c.1928C>T (NP_001364071.1:p.Ser643Phe) in the PLCB4 gene, which was predicted to impair the local structural stability with a result that the protein function might be affected. From a review of the literature, only 36 patients with PLCB4 gene mutations were retrieved. CONCLUSION: As with other studies examining familial cases of ARCND2, incomplete penetrance and variable expressivity were observed within different families' heterozygous mutations in PLCB4 gene. Although, motor and intellectual development are in the normal range in the vast majority of patients with ARCND2, long-term follow-up and assessment are still required.

Influence of Abutment Screw-Tightening Methods on the Screw Joint: Immediate and Long-Term Stability.

Objective: To evaluate the influence of screw-tightening methods on the immediate and long-term stability of dental implant screw joints. Methodology. A total of 150 implants of three different implant systems with different diameters were used in this study. Each group was divided into three subgroups (n = 5), according to the tightening methods (A-tightening with recommended torque and retorque after 10 min; B-tightening with recommended torque, then loosening and immediate retorque; C-tightening with recommended torque only once). The operating time of tightening the assemblies was recorded. Ten minutes later, the immediate removal torque (IRT) (Ncm) was measured. After retightening the assemblies, a dynamic load between 20 and 200 N was applied for 105 cycles, and the postloading removal torque (PRT) (Ncm) was measured. Scanning electron microscopy (SEM) was used to observe the surface topography of the screws. Results: For different types of implants, the IRTs were 11.92 ± 1.04-34.12 ± 0.36 Ncm for method A, 11.64 ± 0.57-33.96 ± 0.29 Ncm for method B, and 10.30 ± 0.41-31.62 ± 0.52 Ncm for method C, and the IRTs of methods A and B were 6.28%-21.58% higher than that of method C (P ≤ 0.046). The PRTs were 4.08 ± 0.77-29.86 ± 0.65 Ncm for method A, 4.04 ± 0.40-29.60 ± 0.36 Ncm for method B, and 2.98 ± 0.26-26.38 ± 0.59 Ncm for method C, and the PRTs of methods A and B were 11.77%-44.87% higher than that of method C (P ≤ 0.016). The removal torque loss rates of methods A (12.49% ± 0.99%-65.88% ± 4.83%) and B (12.84% ± 0.96%-65.35% ± 1.95%) were 3.04%-7.74% lower than that of method C (16.58% ± 0.56%-71.10% ± 1.58%) (P ≤ 0.017). The operating time of method A was much longer than those of methods B and C (P < 0.001). The structural integrity disruption of the screw thread was observed according to the SEM results in all postloading groups. Conclusions: Method B (torquing and then loosening and immediate retorquing) increases the screw joint immediate stability by 6.28%-21.58% and the long-term stability by 11.77%-44.87% compared with method C (torquing only once), has comparable screw joint stability compared with method A (retorquing after 10 min), saves time and is recommended in clinical settings.

A composite hydrogel loaded with the processed pyritum promotes bone repair via stimulate the osteogenic differentiation of BMSCs.

Tissue engineering shows promise in repairing extensive bone defects. The promotion of proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by biological scaffolds has a significant impact on bone regeneration outcomes. In this study we used an injectable hydrogel, known as aminated mesoporous silica gel composite hydrogel (MSNs-NH2@GelMA), loaded with a natural drug, processed pyritum (PP), to promote healing of bone defects. The mechanical properties of the composite hydrogel were significantly superior to those of the blank hydrogel. In vitro experiments revealed that the composite hydrogel stimulated the osteogenic differentiation of BMSCs, and significantly increased the expression of type I collagen (Col 1), runt-related transcription factor 2 (Runx 2), alkaline phosphatase (ALP), osteocalcin (OCN). In vivo experiments showed that the composite hydrogel promoted the generation of new bones. These findings provide evidence that the composite hydrogel pyritum-loaded holds promise as a biomaterial for bone repair.

Accuracy of the preparation depth in mixed targeted restorative space type veneers assisted by different guides: An in vitro study.

PURPOSE: Most veneers are mixed targeted restorative space (MTRS)-type restorations that are partially within the original tooth and require inconsistent preparation depths. This study aimed to evaluate the accuracy of the preparation depth for MTRS veneer preparation. METHODS: MTRS veneer preparation models were developed using the twisted maxillary central incisor (MCI) as the original tooth and the standard MCI as the waxing. Veneer preparations were performed using freehand (MF), silicone (MS), thermoplastic (MT), 3D-printed uniform (MD), and auto-stop (MA) guides. The prepared and original MCI were scanned and superimposed using a custom-made base. The mean absolute differences (MADs) were measured to evaluate the accuracy of the preparation depth. Statistical analysis was performed using the multivariate analysis of variance (MANOVA) test (α=0.05). RESULTS: The accuracy of the preparation depth was 0.237±0.090, 0.191±0.099, 0.149±0.078, 0.093±0.050, and 0.059±0.040 mm in MF, MS, MT, MD, and MA, respectively. The MADs between the groups were significant (P<0.05). The accuracy of the trial restoration was 0.140±0.081 mm in the MS, and the accuracy of the guiding tube was 0.055±0.033, 0.036±0.011, and 0.033±0.010 mm in the MT, MD, and MA, respectively. CONCLUSIONS: In MTRS veneer preparation for MCI, tooth preparation guides improved the accuracy of the preparation depth by visualizing the TRS profile and providing clear measurement points. The accuracy of the guide is influenced by its flexibility, and the accuracy of the preparation depth is affected by the accuracy of the measurement points.

Enhancing cell adhesive and antibacterial activities of glass-fibre-reinforced polyetherketoneketone through Mg and Ag PIII.

Glass-fibre-reinforced polyetherketoneketone (PEKK-GF) shows great potential for application as a dental implant restoration material; however, its surface bioinertness and poor antibacterial properties limit its integration with peri-implant soft tissue, which is critical in the long-term success of implant restoration. Herein, functional magnesium (Mg) and silver (Ag) ions were introduced into PEKK-GF by plasma immersion ion implantation (PIII). Surface characterization confirmed that the surface morphology of PEKK-GF was not visibly affected by PIII treatment. Further tests revealed that PIII changed the wettability and electrochemical environment of the PEKK-GF surface and enabled the release of Mg2+ and Ag+ modulated by Giavanni effect. In vitro experiments showed that Mg/Ag PIII-treated PEKK-GF promoted the proliferation and adhesion of human gingival fibroblasts and upregulated the expression of adhesion-related genes and proteins. In addition, the treated samples inhibited the metabolic viability and adhesion of Streptococcus mutans and Porphyromonas gingivalis on their surfaces, distorting bacterial morphology. Mg/Ag PIII surface treatment improved the soft tissue integration and antibacterial activities of PEKK-GF, which will further support and broaden its adoption in dentistry.

Physical and biological implications of accelerated aging on stereolithographic additive-manufactured zirconia for dental implant abutment.

PURPOSE: This study aimed to comparatively investigate the effects of accelerated aging on the physical and biological features of zirconia manufactured by digital light processing (DLP) and conventional subtractive manufacturing (SM) with similar composition. METHODS: Both the DLP- and SM-fabricated zirconia samples (7 mm × 7.5 mm × 1.5 mm) were grouped according to aging (134 °C, 0.2 MPa, 100% humidity) times, including 0 h, 5 h, and 10 h. Phase assemblage and surface topography of zirconia manufactured by different technologies were evaluated before and after aging. The biological effects of zirconia on human gingival fibroblast (HGF) cell events, including cell viability, proliferation, morphology and adhesion, were also evaluated by live/dead viability assay, cck-8 assay, scanning electron microscopy and confocal laser scanning microscopy respectively. RESULTS: The DLP-fabricated zirconia showed a higher initial cubic phase content and rate of phase transformation than the SM-fabricated zirconia. Among the different aging time-based groups, the 5 h-aged group exhibited significantly lower sub-micron scale surface roughness compared with the other groups. Aging did not significantly alter cellular behavior in any zirconia type, except for minor changes in adhesive cell numbers recorded in an aging time/culturing time-dependent manner. In addition to small differences in cell alignment patterns and overall cell morphology, the two zirconia types presented comparable biological performance before and after aging. CONCLUSION: Although the microstructure and surface characteristics of DLP-fabricated zirconia can be affected by autoclave aging, this newly manufactured zirconia is likely to maintain desirable long-term biocompatibility as an implant abutment material.

Effects of ionic strength and particle size on transport of microplastic and humic acid in porous media.

As an emerging pollutant, the transport behavior of colloidal microplastic particles (CMPs) in saturated porous media may be affected by the simultaneous presence of other substances in the natural environment. In this study, colloidal polystyrene microplastic particles (PSMPs) were selected as the representative of CMPs to investigate the cotransport behaviors of CMPs in the presence of humic acid (HA) under varied environmental conditions (ionic strength: 1, 100 mM KCl; HA concentration: 0, 5, 10, 20 mg⋅L-1) in porous media. The presence of HA with different concentrations was found to increase the mobility of 1.0-µm and 0.2-µm CMPs in porous media in a non-linear and non-monotonic manner. Furthermore, the HA-facilitated transport of CMPs occurred under both electrostatically unfavorable and favorable attachment conditions (limited to the conditions examined in this study, corresponding to 1 and 100 mM KCl, respectively). The transport behavior of the smaller-sized CMPs (0.2-µm CMPs) was more sensitive to the change of ionic strength and the presence of HA than that of the larger-sized CMPs (1.0-µm CMPs). The cotransport process of CMPs and HA was affected by many factors. Modeling results showed that a small amount of competitive blocking occurred during the cotransport process. Moreover, both the presence of HA and change in ionic strength could affect the surface properties of CMPs. Thus, the cotransport behavior of CMPs with HA was different from the transport of individual CMPs in porous media. Experimental results revealed that HA induced complexity in the transport behavior of CMPs in the aqueous environment. Therefore, undeniably, a lot more systematic explorations are further demanded to better comprehend the CMPs cotransport mechanism in the presence of other substances.

Cyclosporine A Promotes Bone Remodeling in LPS-Related Inflammation via Inhibiting ROS/ERK Signaling: Studies In Vivo and In Vitro.

In some inflammatory diseases of bone, osteogenesis and osteoclasis are uncoupled and the balance is usually tipped resulting in bone destruction. The underlying mechanism of osteogenic dysfunction in inflammation still needs further study. This study is aimed at investigating the effects of cyclosporine A (CsA) on bone remodeling in lipopolysaccharide- (LPS-) related inflammation. In vivo, an alveolar bone defect model was established using 10-week-old C57BL/6J mice. The mice were divided into phosphate-buffered saline (PBS), LPS, and LPS+CsA groups. After 3 weeks, micro-CT analysis and histomorphometric evaluation were conducted. In vitro, murine osteoblasts were treated with vehicle medium, LPS, LPS+CsA, LPS+extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor (LPS+PD98059), and LPS+antioxidant (LPS+EUK134). Cell proliferation, osteogenic behaviors, oxidative stress, and ERK signaling were determined. By these approaches, LPS inhibited bone remodeling and promoted oxidative stress accumulation in alveolar bone defects. When animals were treated with CsA, all LPS-induced biochemical changes ameliorated with a marked protective effect. In vitro, the reactive oxygen species (ROS) levels in mitochondria increased in LPS-treated osteoblasts, with decreased expression of osteogenic differentiation genes. The CsA, PD98059, and EUK134 presented remarkable protective effects against LPS treatment. CsA effectively enhanced bone remodeling and attenuated oxidative stress caused by LPS via inhibiting ROS/ERK signaling. Taken together, the protective effect of CsA and the inhibitory effect of ERK signaling on the maintenance of mitochondrial function and reduction of ROS levels hold promise as a potential novel therapeutic strategy for inflammatory diseases in bones.

[Precise tooth preparation technique guided by 3D printing guide plate with quantitative hole].

The minimum amount of tooth preparation that can be fully controlled is crucial in achieving long-term, stable, and effective aesthetic restoration, which is also a major difficulty in aesthetic restoration. The tooth preparation can be imple-mented efficiently and accurately through digital technology based on the fixed-deep hole guiding technology. Prior the actual tooth preparation, the technology first designs the virtual contour, layering, and virtual occlusion of the prosthesis on the computer. Then, virtual tooth preparation is carried out by cutting back according to the virtual prosthesis. Next, the virtual drilling operation plan is designed according to the shape of the virtual tooth preparation and the contour of the abutment tooth. Finally, the tooth preparation guide plate is designed and printed in 3D. It realizes the whole process of quantitative and precise guidance of dental preparation, visualizes the restoration space, reduces the clinical operation time, and guarantees the quality of dental preparation. It also promotes the improvement of the teaching quality of digital practical exercises.

Improved therapeutic efficacy against murine carcinoma by combining honokiol with gene therapy of PNAS-4, a novel pro-apoptotic gene.

PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. Recent studies have indicated that honokiol can induce apoptosis, inhibit angiogenesis, and suppress tumor growth. In the present study, we investigated whether mouse PNAS-4 (mPNAS-4) could augment the apoptosis of tumor cells induced by honokiol in vitro, and whether the antiangiogenic activity of honokiol and induction of apoptosis by mPNAS-4 could work cooperatively to improve the antitumor efficacy in vivo. In vitro, mPNAS-4 inhibited proliferation of murine colorectal carcinoma CT26 and Lewis lung carcinoma LL2 cells through induction of apoptosis, and significantly augmented the apoptosis of CT26 and LL2 cells induced by honokiol. Compared with treatment with mPNAS-4 or honokiol alone, in vivo systemic administration of an expression plasmid encoding mPNAS-4 and low-dose honokiol significantly suppressed tumor growth through the enhanced induction of apoptosis and the augmented inhibition of angiogenesis. Our data suggest that the combined treatment with mPNAS-4 plus honokiol augments antitumor effects in vitro and in vivo, and that the improved antitumor activity in vivo may be associated with enhanced induction of apoptosis and augmented inhibition of angiogenesis. The present study may provide a novel and effective method for the treatment of cancer.

TEGDMA releasing in resin composites with different filler contents and its correlation with mitochondrial mediated cytotoxicity in human gingival fibroblasts.

Compositions of resin composite exhibit cytotoxicity, especially Triethylene-glycol-dimethacrylate (TEGDMA), yet the underlying mechanisms and its relationship with filler content are poorly understood. Here, specimens of five composites (VITA LC, VITA ZETA, Z350, Filtek P60, and AP-X), containing different filler size and weight, were immersed into culture medium for 72 h. After TEGDMA quantification, the resin composite eluates were used to incubate HGFs. Cellular viability was evaluated. Total reactive oxygen species (ROS) and mitochondrial ROS were detected to assess oxidative stress. Adenosine triphosphate and cytochrome c oxidase (CcO) activity, mitochondrial membrane potential and morphology, mitochondrial biogenesis regulators were analyzed to evaluate mitochondrial functions. Results showed that TEGDMA release negatively correlated to filler size and weight of tested composites. Although cell viability reduction was not significant, total and mitochondrial ROS production showed a positive relationship with the amount of TEGDMA in composite eluates. Furthermore, the expression of mitochondrial biogenesis markers and mitochondrial fusion protein, were markedly elevated in TEGDMA rich eluates, especially in VITA-LC group, shown as elongated mitochondrial morphology and aberrant mitochondrial functions. Overall, TEGDMA could elute easier from those resin composites with less filler content and cause oxidative stress in HGFs via mitochondria dysregulation. These data can be instructive to optimize the synthesis of resin composites from the perspective of biocompatibility. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1132-1142, 2019.

Minimal invasive microscopic tooth preparation in esthetic restoration: a specialist consensus.

By removing a part of the structure, the tooth preparation provides restorative space, bonding surface, and finish line for various restorations on abutment. Preparation technique plays critical role in achieving the optimal result of tooth preparation. With successful application of microscope in endodontics for >30 years, there is a full expectation of microscopic dentistry. However, as relatively little progress has been made in the application of microscopic dentistry in prosthodontics, the following assumptions have been proposed: Is it suitable to choose the tooth preparation technique under the naked eye in the microscopic vision? Is there a more accurate preparation technology intended for the microscope? To obtain long-term stable therapeutic effects, is it much easier to achieve maximum tooth preservation and retinal protection and maintain periodontal tissue and oral function health under microscopic vision? Whether the microscopic prosthodontics is a gimmick or a breakthrough in obtaining an ideal tooth preparation should be resolved in microscopic tooth preparation. This article attempts to illustrate the concept, core elements, and indications of microscopic minimally invasive tooth preparation, physiological basis of dental pulp, periodontium and functions involved in tool preparation, position ergonomics and visual basis for dentists, comparison of tooth preparation by naked eyes and a microscope, and comparison of different designs of microscopic minimally invasive tooth preparation techniques. Furthermore, a clinical protocol for microscopic minimally invasive tooth preparation based on target restorative space guide plate has been put forward and new insights on the quantity and shape of microscopic minimally invasive tooth preparation has been provided.

[Minimal invasive microscopic tooth preparation based on endodontic, periodontal and functional health].

Tooth preparation is the primary and core operation technique for dental esthetic restoration treatment, due to its effect of providing restoration space, bonding interfaces and marginal lines for dental rehabilitation after tooth tissue reduction. The concept of microscopic minimal invasive dentistry put forward the issue of conducting high-quality tooth preparation, conserve tooth-structure, protect vital pulp and periodontal tissue simultaneously. This study reviewed the concepts, physiology background, design and minimal invasive microscopic tooth preparation, and in the meantime, individualized strategies and the two core elements of tooth preparation (quantity and shape) are listed.

[Application of 3D printing in aesthetic oral rehabilitation].

3D printing technology can be used in prosthodontics to obtain detailed structures. The technique offers a possible supersession for the most conventional restorations technologies. Contemporary aesthetic restorations encounter difficulties in the consistency between the analysis and design stages and the clinical implementation stage. 3D printing transfers aesthetic designs to customize the finial restoration fabrication, which could be an appropriate optimization to the aformentioned problem. Meanwhile, 3D printing technology can be employed to manufacture target restoration space guide (TRS guide), which is a blueprint for the aesthetic ceramic restorations and presents a general functional and aesthetic situation of patients. The guidance provided by TRS guide ensures precision and minimal invasive operation in aesthetic restorations. These new digital technologies have revolutionized aesthetic rehabilitation. This paper introduces the application of 3D printing in aesthetic oral rehabilitation.

Polydimethylsiloxane (PDMS)-Based Flexible Optical Electrodes with Conductive Composite Hydrogels Integrated Probe for Optogenetics.

Optical electrodes are important tools for optogenetic research. Flexible optical electrodes represent a refinement over traditional fiber-based electrodes because they contact with target cells gently by reducing mechanical mismatch, thereby enhancing their long-term, stable signal acquisition capability. Until now, little attention has been paid to flexible intracortical optical electrodes. Here, we reported a novel flexible penetrating optical electrode with a probe made of composite hydrogels. We used polydimethylsiloxane (PDMS), a kind of transparent material, to fabricate waveguide by capillary assembly method with two tungsten wires inside providing mechanic support. Then one tungsten wire was withdrawn out and the microchannel was filled with hydrogel composed of polyvinyl alcohol (PVA), multi-walled carbon nanotubes (MWCNT), poly(3,4-ethylenedioxythiophene) (PEDOT), and polystyrene sulfonate (PSS) as an electrical recording and stimulation probe. With PDMS as the waveguide and PVA/MWCNT/PEDOT/PSS hydrogel as the electroprobe, the optical electrode becomes a flexible package. The morphology observed by scanning electron microscopy showed that the PVA/MWCNT/PEDOT/PSS hydrogel had a loose surface structure, which would allow the effective adhesion to target neurons. A buckling test showed that our electrode maintained bending strength comparable to that of previously reported flexible penetrating electrodes. Finally, the electrical properties showed a lower impedance and higher charge capacity after PEDOT/PSS modification. The flexible penetrating optical electrode we developed may be used for long-term in vivo optogenetics studies.

Engineering the heart: evaluation of conductive nanomaterials for improving implant integration and cardiac function.

Recently, carbon nanotubes together with other types of conductive materials have been used to enhance the viability and function of cardiomyocytes in vitro. Here we demonstrated a paradigm to construct ECTs for cardiac repair using conductive nanomaterials. Single walled carbon nanotubes (SWNTs) were incorporated into gelatin hydrogel scaffolds to construct three-dimensional ECTs. We found that SWNTs could provide cellular microenvironment in vitro favorable for cardiac contraction and the expression of electrochemical associated proteins. Upon implantation into the infarct hearts in rats, ECTs structurally integrated with the host myocardium, with different types of cells observed to mutually invade into implants and host tissues. The functional measurements showed that SWNTs were essential to improve the performance of ECTs in inhibiting pathological deterioration of myocardium. This work suggested that conductive nanomaterials hold therapeutic potential in engineering cardiac tissues to repair myocardial infarction.

A novel PEGylated liposome-encapsulated SANT75 suppresses tumor growth through inhibiting hedgehog signaling pathway.

The Hedgehog (Hh) pathway inhibitors have shown great promise in cancer therapeutics. SANT75, a novel compound we previously designed to specially inhibit the Smoothened (SMO) protein in the Hh pathway, has greater inhibitory potency than many of commonly used Hh inhibitors. However, preclinical studies of SANT75 revealed water insolubility and acute toxicity. To overcome these limitations, we developed a liposomal formulation of SANT75 and investigated its antitumor efficacy in vitro and in vivo. We encapsulated SANT75 into PEGylated liposome and the mean particle size distribution and zeta-potential (ZP) of liposomes were optimized. Using the Shh-light2 cell and Gli-GFP or Flk-GFP transgenic reporter zebrafish, we confirmed that liposome-encapsulated SANT75 inhibited Hh activity with similar potency as the original SANT75. SANT75 encapsulated into liposome exerted strong tumor growth-inhibiting effects in vitro and in vivo. In addition, the liposomal SANT75 therapy efficiently improved the survival time of tumor-bearing mice without obvious systemic toxicity. The pathological morphology and immunohistochemistry staining revealed that liposomal SANT75 induced tumor cell apoptosis, inhibited tumor angiogenesis as assessed by CD31 and down-regulated the expression of Hh target protein Gli-1 in tumor tissues. Our findings suggest that liposomal formulated SANT75 has improved solubility and bioavailability and should be further developed as a drug candidate for treating tumors with abnormally high Hh activity.

Plasmid DNA containing multiple CpG motifs triggers a strong immune response to hepatitis B surface antigen when combined with incomplete Freund's adjuvant but not aluminum hydroxide.

Adjuvants are important components of recombinant protein vaccines which are often poorly immunogenic. For decades, the search for new vaccine adjuvants has been predominantly empirical. In addition, combinations of more than one adjuvant plus antigen have been systematically studied. Plasmid DNA containing additional oligodeoxynucleotides with unmethylated CpG motifs (CpG ODN) entrapped in liposomes has been used as an adjuvant for DNA vaccines and has shown powerful immunostimulatory functions. In our study, the combination of plasmid DNA containing 16 additional CpG ODNs (pv-16CpG) and aluminum hydroxide (AL) or incomplete Freund's adjuvant (IFA) was used as an adjuvant for a hepatitis B surface antigen (HBsAg) vaccine to immunize C57BL/6J mice. ELISA and ELISPOT assays were used to analyze the immunological effects of the novel vaccine. A significant enhancement of the anti-HBs titer and seroconversion was observed when the CpG plasmid was combined with IFA, but not with AL. In addition, anti-HBs antibody isotype analysis revealed that the combination of CpG plasmid and IFA induced a strong HBsAg-specific IgG2a response. Moreover, the ELISPOT assays suggested that pv-16CpG suspended in IFA evoked a strong T helper 1 (Th1) immune response and high IFN-γ production. These results demonstrate that pv-16CpG suspended in IFA is able to induce cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant for use in protein vaccines.