Development and validation of a machine learning model to predict prognosis in HIV-negative cryptococcal meningitis patients: a multicenter study.

PURPOSE: To predict prognosis in HIV-negative cryptococcal meningitis (CM) patients by developing and validating a machine learning (ML) model. METHODS: This study involved 523 HIV-negative CM patients diagnosed between January 1, 1998, and August 31, 2022, by neurologists from 3 tertiary Chinese centers. Prognosis was evaluated at 10 weeks after the initiation of antifungal therapy. RESULTS: The final prediction model for HIV-negative CM patients comprised 8 variables: Cerebrospinal fluid (CSF) cryptococcal count, CSF white blood cell (WBC), altered mental status, hearing impairment, CSF chloride levels, CSF opening pressure (OP), aspartate aminotransferase levels at admission, and decreased rate of CSF cryptococcal count within 2 weeks after admission. The areas under the curve (AUCs) in the internal, temporal, and external validation sets were 0.87 (95% CI 0.794-0.944), 0.92 (95% CI 0.795-1.000), and 0.86 (95% CI 0.744-0.975), respectively. An artificial intelligence (AI) model was trained to detect and count cryptococci, and the mean average precision (mAP) was 0.993. CONCLUSION: A ML model for predicting prognosis in HIV-negative CM patients was built and validated, and the model might provide a reference for personalized treatment of HIV-negative CM patients. The change in the CSF cryptococcal count in the early phase of HIV-negative CM treatment can reflect the prognosis of the disease. In addition, utilizing AI to detect and count CSF cryptococci in HIV-negative CM patients can eliminate the interference of human factors in detecting cryptococci in CSF samples and reduce the workload of the examiner.

Reversely immortalized mouse salivary gland cells presented a promising metabolic and fibrotic response upon BMP9/Gdf2 stimulation.

The submandibular gland (SMG) and the sublingual gland (SLG) are two of the three major salivary glands in mammals. In mice, they are adjacent to each other and open into the oral cavity, producing saliva to lubricate the mouth and aid in food digestion. Though salivary gland dysfunction accompanied with fibrosis and metabolic disturbance is common in clinic, in-depth mechanistic research is lacking. Currently, research on how to rescue salivary function is challenging, as it must resort to using terminally differentiated acinar cells or precursor acinar cells with unknown differentiation. In this study, we established reversely immortalized mouse primary SMG cells (iSMGCs) and SLG cells (iSLGCs) on the first postnatal day (P0). The iSMGCs and iSLGCs grew well, exhibited many salivary gland characteristics, and retained the metabolism-related genes derived from the original tissue as demonstrated using transcriptome sequencing (RNA-seq) analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these two cell lines, which overlapped with those of the SMG and SLG, were enriched in cysteine and methionine metabolism. Furthermore, we investigated the role of bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), on metabolic and fibrotic functions in the SMG and SLG. We demonstrated that iSMGCs and iSLGCs presented promising adipogenic and fibrotic responses upon BMP9/Gdf2 stimulation. Thus, our findings indicate that iSMGCs and iSLGCs faithfully reproduce characteristics of SMG and SLG cells and present a promising prospect for use in future study of salivary gland metabolism and fibrosis upon BMP9/Gdf2 stimulation.

BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/ß-catenin signalling activity.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/ß-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down ß-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down ß-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

Differential gene expression in HaCaT cells may account for the various clinical presentation caused by anthropophilic and geophilic dermatophytes infections.

BACKGROUND: Despite the worldwide prevalence of dermatophyte infections, only a few genes are reported to be related to dermatophyte infections. In addition, the mechanism by which different ecological dermatophytes infection leads to varying intensity of inflammation remains unclear. OBJECTIVES: To investigate the mechanism of varying intensity of skin inflammation caused by different ecological dermatophytes infection. METHODS: We infected HaCaT cells with anthropophilic and geophilic dermatophytes to mimic various ecological dermatophyte infections. RNA-sequencing (RNA-seq) was employed to identify the change in the gene expression of HaCaT cells. To verify the expression of differentially expressed genes (DEGs), we selected 18 HaCaT cells genes to conduct qPCR experiments. In addition, immunoblotting was conducted to validate key genes from the MAPK signalling pathway. RESULTS: After HaCaT cells were infected with the anthropophilic Trichophyton rubrum (T rubrum) and the geophilic Microsporum gypseum (M gypseum), 118 and 619 differentially expressed genes were identified in HaCaT cells, respectively. These genes may provide a clue as to how keratinocytes respond to anthropophilic and geophilic dermatophytes. We also found that JUN may play a critical role in keratinocytes infected with M gypseum. CONCLUSIONS: Differential gene expression in HaCaT cells may account for the various clinical presentation caused by anthropophilic and geophilic dermatophytes infections. In addition, the intense inflammatory reaction of M gypseum infection may be triggered by activating the JNK-JUN signalling pathway.

Matrigel Scaffolding Enhances BMP9-induced Bone Formation in Dental Follicle Stem/Precursor Cells.

Bone tissue engineering requires a combination of cells, efficient biochemical and physicochemical factors, and biocompatible scaffolds. In this study, we evaluated the potential use of injectable Matrigel as a scaffold for the delivery of rat dental follicle stem/precursor cells (rDFSCs) transduced by bone morphogenetic protein (BMP) 9 to enhance osteogenic differentiation in vitro and promote ectopic bone formation in vivo. Recombinant adenovirus was used to overexpress BMP9 in rDFSCs. Alkaline phosphatase activity was measured using a histochemical staining assay and a chemiluminescence assay kit. Quantitative real-time polymerase chain reaction was used to determine mRNA expression levels of bone-related genes including distal-less homeobox 5 (DLX5), osteopontin (OPN), osterix (Osx), and runt-related transcription factor 2 (Runx2). Matrix mineralization was examined by Alizarin Red S staining. rDFSCs proliferation was analyzed using the Cell Counting Kit-8 assay. Subcutaneous implantation of rDFSCs-containing Matrigel scaffolds was used, and micro-computed tomography analysis, histological evaluation, and trichrome staining of implants extracted at 6 weeks were performed. We found that BMP9 enhanced alkaline phosphatase activity and mineralization in rDFSCs. The expression of bone-related genes (DLX5, OPN, Osx, and Runx2) was also increased as a result of BMP9 stimulation. Micro-computed tomography analysis and histological evaluation revealed that the bone masses retrieved from BMP9-overexpressing rDFSCs were significantly more pronounced in those with than in those without Matrigel. Our results suggest that BMP9 effectively promote osteogenic differentiation of rDFSCs, and Matrigel facilitate BMP9-induced osteogenesis of rDFSCs in vivo.

Bone Regeneration of Canine Peri-implant Defects Using Cell Sheets of Adipose-Derived Mesenchymal Stem Cells and Platelet-Rich Fibrin Membranes.

PURPOSE: Insufficient bone volume compromises the success rate and osseointegration of immediate implantation. The objective of the present study was to engineer bone tissue by using adipose-derived stem cell (ASC) sheets and autologous platelet-rich fibrin (PRF) to enhance new bone formation and osseointegration around dental implants. MATERIAL AND METHODS: The proliferation and osteogenic potential of ASCs treated with autologous PRF were evaluated with CCK-8 assays, alkaline phosphatase staining, and real-time quantitative polymerase chain reaction. A 3-wall bone defect around each immediate implant was generated in the mandible and randomly treated with ASC sheets plus PRF (group A), ASC sheets only (group B), PRF only (group C), or no treatment (group D). Micro-computed tomography, biomechanical tests, fluorescent bone labeling, and histologic assessments were performed to evaluate bone regeneration capacity. RESULTS: The proliferation and osteogenic potential of canine ASCs were markedly enhanced by PRF. Group A exhibited considerably more new bone formation and re-osseointegration (41.17 ± 1.44 and 55.06 ± 0.06%, respectively) than did the other 3 groups. Fluorescent labeling showed that the most rapid bone remodeling activity occurred in group A (P < .05). CONCLUSION: These results suggest that sheets of ASC combined with autologous PRF could be a promising tissue-engineering strategy for bone formation in immediate implantation.

iTRAQ-Based Proteomic Analysis Exploring the Influence of Hypoxia on the Proteome of Dental Pulp Stem Cells under 3D Culture.

Hypoxic preconditioning is commonly applied to enhance mesenchymal stem cells (MSCs) therapeutic effect before transplantation. Elucidating the effect of hypoxic preconditioning would be beneficial for improved application. However, the influence of hypoxia on dental tissue derived MSCs cultured in 3D was unknown. Thus, the present study is to investigate gene expression and proteome of dental pulp stem cells (DPSCs) after hypoxic preconditioning. DPSCs were isolated, cultured in a 3D system under the normoxic and hypoxic conditions. The gene expression was examined with reverse transcription polymerase chain reaction, and the proteome was analyzed using iTRAQ-based mass spectrometry. The expressions of HIF-1α, VEGFA, KDR at mRNA level was upregulated while BMP-2 was downregulated. Two thousand one hundred and fifteen proteins were identified and 57 proteins exhibited significant differences after hypoxic preconditioning (30 up-regulated, 27 down-regulated). Bioinformatic analysis revealed the majority of up-regulated proteins are involved in cellular process, angiogenesis, protein binding and transport, regulation of response to stimulus, metabolic processes, and immune response. Increased IL-6 and decreased TGF-1ß protein expression under hypoxic condition were verified by ELISA. Hypoxic preconditioning partly affected the gene and protein expression in DPSCs under 3D culture and may enhance the efficacy of MSCs transplantation.

Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

PURPOSE: The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. METHODS: Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. RESULTS: Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. CONCLUSIONS: Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

Increased inter-hemispheric resting-state functional connectivity in acute lacunar stroke patients with aphasia.

Aphasia is a devastating neurological condition affecting a person's ability to communicate and reintegrate into the society. It may occur in 20% or more of patients after stroke. The recovery of language function is accompanied by brain reorganization, and identifying the inter-hemispheric interaction post-stroke will conduce to more targeted treatments. Previous studies suggested that robust homotopic resting-state functional connectivity is a key characteristic of the brain's intrinsic functional architecture, and communication between the left and right cerebral hemispheres is important for language processing. In this study, voxel-mirrored homotopic connectivity (VMHC) was used to examine the inter-hemispheric resting-state functional connectivity (RSFC) differences between 37 patients with acute lacunar stroke in the left hemisphere and 28 healthy controls. Besides, correlation analyses were carried out to investigate the relationship between VMHC values of brain regions showing abnormal inter-hemispheric RSFC and clinical variables [i.e., aphasia quotient (AQ) scores, National Institutes of Health Stroke Scale (NIHSS) and Mini-Mental State Examination of patients]. Compared with healthy controls, patients showed significantly increased VMHC in the pars orbitalis of the inferior frontal gyrus, anterior part of the superior temporal gyrus (STG) and lingual gyrus. No brain region showed decreased VMHC in the patient group than in the healthy control group. The AQ scores were negatively correlated with VMHC values in the STG. NIHSS scores were positively correlated with VMHC values in the lingual gyrus. We hope these results could shed new insights into the pathology of aphasia in patients with acute lacunar stroke.

An ultrasound-triggered injectable sodium alginate scaffold loaded with electrospun microspheres for on-demand drug delivery to accelerate bone defect regeneration.

Biological processes, such as bone defects healing are precisely controlled in both time and space. This spatiotemporal characteristic inspires novel therapeutic strategies. The sustained-release systems including hydrogels are commonly utilized in the treatment of bone defect; however, traditional hydrogels often release drugs at a consistent rate, lacking temporal precision. In this study, a hybrid hydrogel has been developed by using sodium alginate, sucrose acetate isobutyrate, and electrospray microspheres as the base materials, and designed with ultrasound response, and on-demand release properties. Sucrose acetate isobutyrate was added to the hybrid hydrogel to prevent burst release. The network structure of the hybrid hydrogel is formed by the interconnection of Ca2+ with the carboxyl groups of sodium alginate. Notably, when the hybrid hydrogel is exposed to ultrasound, the ionic bond can be broken to promote drug release; when ultrasound is turned off, the release returned to a low-release state. This hybrid hydrogel reveals not only injectability, degradability, and good mechanical properties but also shows multiple responses to ultrasound. And it has good biocompatibility and promotes osteogenesis efficiency in vivo. Thus, this hybrid hydrogel provides a promising therapeutic strategy for the treatment of bone defects.

Stabilization of Pin1 by USP34 promotes Ubc9 isomerization and protein sumoylation in glioma stem cells.

The peptidyl-prolyl cis-trans isomerase Pin1 is a pivotal therapeutic target in cancers, but the regulation of Pin1 protein stability is largely unknown. High Pin1 expression is associated with SUMO1-modified protein hypersumoylation in glioma stem cells (GSCs), but the underlying mechanisms remain elusive. Here we demonstrate that Pin1 is deubiquitinated and stabilized by USP34, which promotes isomerization of the sole SUMO E2 enzyme Ubc9, leading to SUMO1-modified hypersumoylation to support GSC maintenance. Pin1 interacts with USP34, a deubiquitinase with preferential expression and oncogenic function in GSCs. Such interaction is facilitated by Plk1-mediated phosphorylation of Pin1. Disruption of USP34 or inhibition of Plk1 promotes poly-ubiquitination and degradation of Pin1. Furthermore, Pin1 isomerizes Ubc9 to upregulate Ubc9 thioester formation with SUMO1, which requires CDK1-mediated phosphorylation of Ubc9. Combined inhibition of Pin1 and CDK1 with sulfopin and RO3306 most effectively suppresses orthotopic tumor growth. Our findings provide multiple molecular targets to induce Pin1 degradation and suppress hypersumoylation for cancer treatment.

Phosphocreatine promotes epigenetic reprogramming to facilitate glioblastoma growth through stabilizing BRD2.

Glioblastoma (GBM) exhibits profound metabolic plasticity for survival and therapeutic resistance, while the underlying mechanisms remain unclear. Here, we show that GBM stem cells (GSCs) reprogram the epigenetic landscape by producing substantial amounts of phosphocreatine (PCr). This production is attributed to the elevated transcription of brain-type creatine kinase (CKB), mediated by Zinc finger E-box binding homeobox 1 (ZEB1). PCr inhibits the poly-ubiquitination of the chromatin regulator bromodomain containing protein 2 (BRD2) by outcompeting the E3 ubiquitin ligase SPOP for BRD2 binding. Pharmacological disruption of PCr biosynthesis by cyclocreatine leads to BRD2 degradation and a decrease in its targets' transcription, which inhibits chromosome segregation and cell proliferation. Notably, cyclocreatine treatment significantly impedes tumor growth and sensitizes tumors to a BRD2 inhibitor in mouse GBM models without detectable side effects. These findings highlight that high production of PCr is a druggable metabolic feature of GBM and a promising therapeutic target for GBM treatment.

The fibroblast growth factor-Klotho axis at molecular level.

Klotho is a recently discovered protein that has positive effects on all systems of the body, for example, regulating calcium and phosphorus metabolism, protecting nerves, delaying aging and so on. Fibroblast growth factors (FGFs) are a group of polypeptides that function throughout the body by binding with cell surface FGF receptors (FGFRs). Endocrine FGFs require Klotho as a co-receptor for FGFRs. There is increasing evidence that Klotho participates in calcium and phosphorus regulation and metabolic regulation via the FGF-Klotho axis. Moreover, soluble Klotho can function as a separate hormone to regulate homeostasis on various ion channels and carrier channels on the cell surface. This review mainly explains the molecular basis of the membrane signaling mechanism of Klotho.

The influence of conceptual concreteness on the reading acquisition and integration of novel words into semantic memory via thematic relations.

Plenty of studies have been conducted to reveal neurocognitive underpinnings of conceptual representation. Compared with that of concrete concepts, the neurocognitive correlates of abstract concepts remain elusive. The current study aimed to investigate the influence of conceptual concreteness on the reading acquisition and integration of novel words into semantic memory. We constructed two-sentence contexts in which two-character pseudowords were embedded as novel words. Participants read the contexts to infer the meaning of novel words which were either concrete or abstract, and then performed a lexical decision task and a cued-recall memory task. In lexical decision task, primed by the learned novel words, their corresponding concepts, thematically related or unrelated words as well as unlearned pseudowords were judged whether they were words or not. In memory task, participants were presented with the novel words and asked to write down their meaning. The contextual reading and memory test can demonstrate the modulation of conceptual concreteness on novel word learning and the lexical decision task can reveal whether concrete and abstract novel words are integrated into semantic memory similarly or not. During contextual reading, abstract novel words presented for the first time elicited a larger N400 than concrete ones. In memory task, the meaning of concrete novel words was recollected better than abstract novel words. These results indicate that abstract novel words are more difficult to acquire during contextual reading, and to retain afterwards. For lexical decision task behavioral and ERPs were graded, with the longest reaction time, the lowest accuracy and the largest N400s for the unrelated words, then the thematically related words and finally the corresponding concepts of the novel words, regardless of conceptual concreteness. The results suggest that both concrete and abstract novel words can be integrated into semantic memory via thematic relations. These findings are discussed in terms of differential representational framework which posits that concrete words connect with each other via semantic similarities, and abstract ones via thematic relations.

Single-Cell RNA-Seq Analysis Identifies Angiotensinogen and Galanin as Unique Molecular Markers of Acinar Cells in Murine Salivary Glands.

The submandibular gland (SMG) and sublingual gland (SLG) are two of three major salivary glands in mammals and comprise serous and mucous acinar cells. The two glands share some functional properties, which are largely dependent on the types of acinar cells. In recent years, while ScRNA-seq (single-cell sequencing) with a 10 × platform has been used to explore molecular markers in salivary glands, few studies have examined the acinar heterogeneity and unique molecular markers between SMG and SLG. This study aimed to identify the molecular markers of acinar cells in the SLG and SMG. We performed ScRNA-seq analyses in 4-week-old mice and verified the screened molecular markers using reverse transcription-quantitative real-time PCR, immunohistochemistry, and immunofluorescence. Our results showed prominently heterogeneous acinar cells, although there was great similarity in the cluster composition between the two glands at 4 weeks. Furthermore, we demonstrated that Agt is a specific marker of SMG serous acinar cells, whereas Gal is a specific marker of SLG mucous acinar cells. Trajectory inference revealed that Agt and Gal represent two types of differential acinar cell clusters during late development in adults. Thus, we reveal previously unknown specific markers for salivary acinar cell diversity, which has extensive implications for their further functional research.

A Dual-Antioxidative Coating on Transmucosal Component of Implant to Repair Connective Tissue Barrier for Treatment of Peri-Implantitis.

Since the microgap between implant and surrounding connective tissue creates the pass for pathogen invasion, sustained pathological stimuli can accelerate macrophage-mediated inflammation, therefore affecting peri-implant tissue regeneration and aggravate peri-implantitis. As the transmucosal component of implant, the abutment therefore needs to be biofunctionalized to repair the gingival barrier. Here, a mussel-bioinspired implant abutment coating containing tannic acid (TA), cerium and minocycline (TA-Ce-Mino) is reported. TA provides pyrogallol and catechol groups to promote cell adherence. Besides, Ce3+ /Ce4+  conversion exhibits enzyme-mimetic activity to remove reactive oxygen species while generating O2 , therefore promoting anti-inflammatory M2 macrophage polarization to help create a regenerative environment. Minocycline is involved on the TA surface to create local drug storage for responsive antibiosis. Moreover, the underlying therapeutic mechanism is revealed whereby the coating exhibits exogenous antioxidation from the inherent properties of Ce and TA and endogenous antioxidation through mitochondrial homeostasis maintenance and antioxidases promotion. In addition, it stimulates integrin to activate PI3K/Akt and RhoA/ROCK pathways to enhance VEGF-mediated angiogenesis and tissue regeneration. Combining the antibiosis and multidimensional orchestration, TA-Ce-Mino repairs soft tissue barriers and effector cell differentiation, thereby isolating the immune microenvironment from pathogen invasion. Consequently, this study provides critical insight into the design and biological mechanism of abutment surface modification to prevent peri-implantitis.

Protein sumoylation in normal and cancer stem cells.

Stem cells with the capacity of self-renewal and differentiation play pivotal roles in normal tissues and malignant tumors. Whereas stem cells are supposed to be genetically identical to their non-stem cell counterparts, cell stemness is deliberately regulated by a dynamic network of molecular mechanisms. Reversible post-translational protein modifications (PTMs) are rapid and reversible non-genetic processes that regulate essentially all physiological and pathological process. Numerous studies have reported the involvement of post-translational protein modifications in the acquirement and maintenance of cell stemness. Recent studies underscore the importance of protein sumoylation, i.e., the covalent attachment of the small ubiquitin-like modifiers (SUMO), as a critical post-translational protein modification in the stem cell populations in development and tumorigenesis. In this review, we summarize the functions of protein sumoylation in different kinds of normal and cancer stem cells. In addition, we describe the upstream regulators and the downstream effectors of protein sumoylation associated with cell stemness. We also introduce the translational studies aiming at sumoylation to target stem cells for disease treatment. Finally, we propose future directions for sumoylation studies in stem cells.

Different grinding speeds affect induced regeneration capacity of human treated dentin matrix.

Human-treated dentin matrix (hTDM) is a biomaterial scaffold, which can induce implant cells to differentiate into odontoblasts and then form neo-dentin. However, hTDM with long storage or prepared by high-speed handpiece would not to form neo-dentin. In this research, we developed two fresh hTDM with different grinding speeds, which were low-speed hTDM (LTDM) with maximum speed of 500 rpm and high-speed hTDM (HTDM) with a speed of 3,80,000 rpm. Here, we aim to understand whether there were induced regeneration capacity differences between LTDM and HTDM. Scanning electron microscope showed that DFCs grew well on both materials, but the morphology of DFCs and the extracellular matrix was different. Especially, the secreted extracellular matrixes on the inner surface of LTDM were regular morphology and ordered arrangement around the dentin tubules. The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the dentin markers DSPP and DMP-1 were about 2× greater in DFCs induced by LTDM than by HTDM, and osteogenic marker BSP was about 2× greater in DFCs induced by HTDM than by LTDM. Histological examinations of the harvested grafts observed the formation of neo-tissue were different, and there were neo-dentin formed on the inner surface of LTDM and neo-cementum formed on the outer surface of HTDM. In summary, it found that the induction abilities of LTDM and HTDM are different, and the dentin matrix is directional. This study lays a necessary foundation for searching the key factors of dentin regeneration in future.

Comparison of the biomechanical differences in the occlusal movement of wild-type and BMP9 knockout mice with apical periodontitis.

Apical periodontitis is a common clinical disease caused by bacteria; bacterial metabolites can cause an imbalance in bone homeostasis, bone mass reduction, and tooth loss. Bone resorption in apical periodontitis causes a concentration of stress in the tooth and periodontal tissues during occlusion, which aggravates the disease. Emerging evidence indicates that bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), may play an important role in tooth and dentoalveolar development. Herein, we investigated the role of BMP9 in the development of apical periodontitis and its effects on the biomechanics of dentoalveolar bone. Apical periodontitis models were established in five BMP9 knockout (KO) mice and five C57BL/6 WT (wild-type) mice. At baseline and 14, 28, and 42 days after modeling, in vivo micro-computed tomography analysis and three-dimensional (3D) reconstruction were performed to evaluate the apical lesion in each mouse, and confirm that the animal models were successfully established. Finite element analysis (FEA) was performed to study the stress and strain at the alveolar fossa of each mouse under the same vertical and lateral stress. FEA revealed that the stress and strain at the alveolar fossa of each mouse gradually concentrated on the tooth cervix. The stress and strain at the tooth cervix gradually increased with time but were decreased at day 42. Under the same lingual loading, the maximum differences of the stress and strain at the tooth root in KO mice were greater than those in WT mice. Thus, these findings demonstrate that BMP9 could affect the biomechanical response of the alveolar fossa at the tooth root in mice with apical periodontitis. Moreover, the effects of BMP9 on the biomechanical response of the alveolar bone may be site-dependent. Overall, this work contributes to an improved understanding of the pathogenesis of apical periodontitis and may inform the development of new treatment strategies for apical periodontitis.

[Evolution of hepatitis B virus quasispecies during antiviral therapy in one chronic hepatitis B patient].

OBJECTIVE: To investigate the evolution of hepatitis B virus (HBV) quasispecies in one patient during lamivudine (LAM) monotherapy and switching to entecavir (ETV) rescue treatment. METHODS: Serum samples were taken at seven different time points during antiviral therapy (0, 24, 48, 60, 72, 96, 152 weeks, respectively), the HBV DNA polymerase gene was amplified, cloned and sequenced to analyze the amino acid substitutions within HBV DNA polymerase gene and distribution of virus quasispecies. Quantitative detection of the HBV wild strains and total virus was performed by amplification refractory mutation system real-time PCR (ARMS-PCR). RESULTS: Three mutation patterns detected during antiviral therapy in the patient: rtM204V, rtM204V+rtL180M and rtM204I. The HBV quasispecies were found always in dynamic variation. The HBV populations were completely replaced with the LAM-resistant variants when the viral breakthrough was encountered during LAM monotherapy. Interestingly, the wild-type variants presented gradually dominant (79.3%) with the decline of HBV DNA load after switching to ETV rescue administration. ARMS-PCR results showed that the wild-type variants account ed for 68.55% of the HBV populations at baseline and this proportion declined to 0.21% when the viral breakthrough emerged under LAM therapy. The wild-type variants gradually increased from week 24 after switching to ETV rescue therapy and the proportion of HBV wild-type variants in the population fluctuated between 16.01% to 26.93%. CONCLUSIONS: The distribution of virus quasispecies were always in dynamic variation during sequential therapy with nucleotide analogs in chronic hepatitis B patients. Different patterns of dynamic HBV quasispecies may have different contribution in ETV resistance in LMV refractory patients with ETV administration.